Title of Invention

AN APPARATUS AND METHOD FOR REGULATING FLUID FLOW IN MICRO STRUCTURES

Abstract The invention discloses an apparatus for regulating fluid flow in microstructures in a programmable manner comprising a first substrate such as herein described comprising a plurality of first fluidic components (110); a second substrate such as herein described comprising a plurality of second fluidic components (111) corresponding to the first fluidic components; a material layer (105) such as herein described separating the plurality of first fluidic components from the plurality of second fluidic components; and electromagnetic generating means for generating an electromagnetic radiation for directing onto the layer of material at a position corresponding to a portion of the layer located between at least a pair of corresponding fluidic components from the plurality of first fluidic components and the plurality of second fluidic components said electromagnetic radiation causing perforation of the material layer at the position allowing fluid communication between at least a pair of fluidic components. The invention also discloses a method for regulating fluid flow in microstructures using the said apparatus.
Full Text AN APPARATUS AND METHOD FOR REGULATING FLUID FLOW IN MICRO
STRUCTURES
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the priority of U.S. Provisional Application No. 60/430,792
filed December 4, 2002 entitled "DEVICES AND METHODS FOR PROGRAMMABLE
MICROSCALE MANIPULATION OF FLUIDS" the whole of which is herby incorporated
by reference herein.
FIELD OF THE INVENTION
The present invention relates to the field of microfluidic circuits for chemical,
biological, and biochemical processes or reactions. More specifically, it discloses devices
and methods for regulating fluid flow in microstructures in a programmable manner.
BACKGROUND OF THE INVENTION
In recent years, the pharmaceutical, biotechnology, chemical and related industries
have increasingly adopted micro-chamber and channel structures for performing various
reactions and analyses. The benefits of these structures include miniaturization, reduction in
space and cost of reagents and enable one to perform a large number of reactions without
human intervention, either in parallel or in series (i.e., one after the other).
Microfluidic devices are by far the most promising candidates to realize a micro-
TAS (Micro Total Analysis System). In general, all attempts in this direction can be
characterized in two ways: according to the forces responsible for the fluid transport and
according to the mechanism used to direct the flow of fluids. The former are referred to as
motors. The latter are referred to as valves, and constitute logic or analogue actuators,
essential for a number of basic operations such as volumetric quantitation of fluids, mixing
of fluids, connecting a set of fluid inputs to a set of fluid outputs, sealing containers (to gas
or to liquids passage according to the application) in a sufficiently tight manner to allow
fluid storage, regulating the fluid flow speed.
As motors, the prior art disclose a variety of solutions, including electro-kinetic and
electro-osmotic transport, mechanical micro-pumps, external pressure, acoustic energy, and
the centripetal force. The present invention is concerned primarily, but not exclusively, with
the category of centripetal devices. Accordingly, a summary of some of the prior art related
to centripetal devices includes: Yamaji et al. (EP00392475A2) and Takase et al.
(EP00417305A1) disclose a liquid sample analyser based on a rotating disk; Kellogg et al.
(US 6,063,589/WO0187485A2) and Mian et al. (US 6,319,469, US21055812A1) disclose
devices and methods for using centripetal acceleration to drive fluid movement in a
microfluidic system; Kopf-Sill et al. (US 5,160,702) teaches an analyser with improved
rotor structure; and Gordon (US 5,892,577, US 6,256,088, US 6,339,473) teaches an
apparatus and method for carrying out analysis of samples.
Devices with the ability to regulate fluid flow through the use of valves are known in
the prior art, and differ in their ability to provide real-time control and analogue adjustment
of the fluid flow. As an example, some valves have the ability to regulate fluid flow in an
analogue manner, like hot water taps, some valves switch between on-off states and vice
versa, like irrigation actuators, some valves have a single on-off transition, like electrical
safety switches, or off-on-transition, like safety valves in pressurized circuits.
Prior art microfluidic valve devices suffer from the drawback of high cost per valve,
as well as the integration scale and complexity that can be achieved. Unfortunately, the
reliability of most prior art devices within the meso-scale range is suspect. Further,
alteration of sample material by the components of the valve and function of the valve has
contributed to their unreliable nature and failure to produce a micro-analytical device with
reproducible results. The design of prior art valving devices makes their manufacturing cost
and complexity unsuitable for their cost effective use within micro-analytical devices diat
are "throw-away" and mass produced.
A summary of some of the prior art valve devices is as follows: Unger et al. US
Patent No. 6,408,878 (Unger) teaches elastomeric valve and pump systems wherein a
second elastomeric layer is bonded onto a top surface of the first elastomeric layer such that
a control channel forms in the second recess between the first and second elastomeric layers
and the first elastomeric layer is positioned on top of a planar substrate such that a flow
channel forms in the first recess between the first elastomeric layer and the planar substrate.
Unfortunately, Unger suffers from complexity of design and cost of manufacturing. In
addition to the complexity of the valve, a control system based on pneumatic actuators has
to be connected to the various valves through multiple independent lines, and its
multiplexing (required in order to have fewer control lines than actual valves on the
devices) has impact on the circuit design and requires accurate pressure control.
A patent to Kellogg et al. US Patent No. 6,302,134 (Kellogg) teaches a heat-
activated wax valve in a microfluidics array. This heat-activated wax valve within
microsystem platforms requires numerous microfluidics components such as resistive
heating elements, temperature sensing elements, mixing structures, to form these heat
activated wax sacrificial valves. Apart from a significant occupancy of surface on the
microfluidic circuit, the valve of Kellogg further requires an electronic spindle designed
rotor capable of transferring electrical signals to and from the microsystem platforms. The
requirements and complexity of the Kellogg valve make it impractical to use within micro-
analytical systems. Further, the waste from valve actuation can contaminate samples of
interest. In addition, the heat is transferred to the wax initially clogging the capillary by heat
conduction. In this manner, the heat is also unavoidably transferred to the chip and to the
fluids by conduction and convection. This is undesired in most biological applications
where the samples could be significantly degraded by heat.
A further prior art valving systems can be found in Kellogg et al. U.S. Patent No.
6,143,248 (Kellogg '248). Kellogg 248 teaches a capillary microvalve that requires
centripetal acceleration to drive fluid in micro-fluid system. The valving device of Kellogg
'248 can only be used in a device having centripetal acceleration and also suffers from
difficulty in its manufacture.
Another prior art device Kellogg et al. US2002/0097632A1 (Kellogg Application)
discloses a bi-directional flow centrifugal microfluidic devices. The valve within the
Kellogg Application particularly provides microsystem platforms for achieving efficient
mixing of one or a plurality of fluids on the surface of the platform when fluid flow is
motivated by centripetal force produced by rotation. This bi-directional flow system is
restricted in its use to mixing systems within centripetally driven micro-analytical systems.
Numerous other prior art devices have attempted to improve upon valving devices
for micro-analytical platforms, such as Onishi et al. (US 5,547,472) that teaches a catheter
with medicament injection pores; Derand et al. (WO00102737A1) (Derand), which teaches
polymer valves. An important feature of the polymers used in the valves of the Derand is
that they switch from a swelled state to a contracted state or vice versa in a reversible
manner, making the choice of the polymer (and its biocompatibility) restricted to a specific
class of materials. In addition, the plug is foreseen to be within a capillary, making the
manufacturing of the device more expensive and less suitable for mass production since
each valve has to be manufactured and positioned within the circuit.
Larsson et al. (W099/58245) discloses a microfluidic device where the flow of
fluids is controlled by different surfaces of the device having different surface
characteristics; McNeely et al. (US 2002/0033193) discloses remote valving for
microfluidic flow control, Williams (US 2001/0054702A1) teaches a valve for use in
microfluidic structures and Parce et al. (US 6,379,974) teaches microfluidic devices and
systems utilizing electrokinetic material transport systems to selectively control and direct
the transport of materials. Unfortunately, all suffer from complexity of their control
systems, design, reliability, high manufacturing costs and application limited to given type
of fluids.
Another approach within prior art devices is shown in Limon et al, US Patent No.
5,869,002 (Limon) where an analysis card containing two mutually separate chambers
separated by a frangible partition that is arranged within the analysis card and made of an
absorbent and preferably plastic material for absorbing light energy having at least a
predetermined wavelength and converting it into heat energy capable of removing the
frangible partition thus causing fluid communication between the chambers. Unfortunately,
Limon suffers from several deficiencies. The valve of Limon is restricted to a certain
configuration that is not adaptable to numerous micro-analytical platforms. More
importantly the light energy required within Limon is of such intensity and duration that
alteration occurs to the fluids or sample of interest within the adjoining chambers. To
overcome the alteration, Limon et al. teach the use of cavities around the frangible partition,
to preserve the liquid or liquids circulating in the analysis card from any premature or
excessive heating. The valving device of Limon also suffers from its inflexibility in
configuration and lack of adaptability to various micro-analytical platforms such as rotating
disks or meso-scale devices. Unfortunately, the configuration required by Limon is not
adaptable to an economical manufacturing process.
Another drawback of prior art microfluidic circuits has been the difficulty to
reconcile flexibility, in the form of fully programmable and configurable devices, with
simplicity, in the form of manufacturing and operation. To regulate the flow of fluids
through a microfluidic circuit, valves were provided. Prior art methods either rely on active
components that can only be provided in limited numbers for reasons of cost and ease of
manufacturing, or on passive components that cannot be actuated independently and
additionally may depend on characteristics of fluid or the sample of interest. Many active
valve systems in the prior art; are also characterized by a control system that has to be
physically connected to the device, which is often not miniaturized (like the pressure control
assembly of the Topaz Crystallizer by Fluidigm Corporation, San Francisco, CA) and
therefore increases significantly the device complexity, system integration and portability.
A significant drawback of prior art microfluidic circuits has been the difficulty in the
handling of biological samples. Prior art devices suffer from valving components that may
contaminate the sample of interest, alter or destroy such sample.
Some of the prior art micro-valves integrated in a microfluidic circuit occupy a large
surface of the chip. This is at the expense of the other functional components of the device,
making the circuit integration (number of components per unit surface) smaller and
therefore the chips more expensive. This need to occupy a large surface detracts from their
use within a microfluidic circuit.
Another drawback of prior art microfluidic circuits has been the reliability of
valving components. Prior art devices suffer from occasional failure and most importantly
the lack of feedback controls to recognize such failures. While this aspect may be neglected
in chips with a moderate number of valves, (e.g. chips of small complexity), the need of
high integration of microfluidic devices requires a higher reliability than the prior art offers
of the basic functional components and in particular of valving devices.
A further drawback of prior art microfluidic valves consists in narrow manufacturing
tolerances on geometry, surface properties, choice of materials, and complexity of
production process. Increasing the integration scale (number of valves in a device) for a
manufacturing process which is either complex or has tight tolerances or both results in a
high production failure rate, further driving up the cost of production.
Another aspect particular to microfluidics consists in the required disposability of
the valves and the overall circuitry. It is well known in the art that the surface-to-volume
ratio increases with decreasing volumes. Since a large fraction of the sample is in contact
with the chip and valve surfaces, it also means that the fluids contamination is a bigger issue
than in the macro-scale world. To avoid contamination of sample, a valve should be
preferably used with a single type of sample, and possibly only once to avoid changes in the
sample concentration. A valving method relying on reusable valves is therefore less
attractive in most microfluidic applications.
The present invention meets the need for a flexible, reliable and yet a simple means
to regulate fluid flow, as well as a variety of other needs.
SUMMARY OF THE INVENTION
The present invention is directed towards a microfluidic circuit in which fluid flow
is regulated by putting two microfluidic components that are initially separated into fluid
communication. Both the time at which the two components are connected and the position
of such fluid communication are arbitrary and can be determined externally. Accordingly,
the invention describes an infinite number of preferably irreversible valves, all of which are
initially in the closed state, but may be opened at any time and in any order.
When a valve is closed, the fluid is contained in a first microfluidic component. As
soon as the valve is opened, fluid communication is enabled to at least one or more
additional microfluidic components. Whether the fluid will flow into the additional
components, to what extent and at which speed, depends on the forces acting on the fluid
and the impediments to fluid flow through valving components.
In microfluidic circuits, fluid transport may be achieved through the use of
mechanical micropumps, electric fields, application of acoustic energy, external pressure, or
centripetal force. A valve according the invention is independent of the mechanism for
fluid transport and is therefore compatible with, but not limited to, any of the above means
for fluid transport. In general, the inventive valves may be opened, but preferably not
closed. This feature is relatively less important for microfluidic circuits where the direction
of the force is irreversible, such as is the case for devices exploiting the centripetal force,
and can be overcome in most other cases by a suitable design of the circuit and of its basic
components.
It is contemplated within the scope of the invention that in order to "close" the
inventive valve a two-component "glue" can be distributed to the opposite sides of the
opened valve. The glue is chosen among those that do not require mixing of the two
components, and have a reasonably fast setting time in order to seal the valve opening. The
glue can be acrylic glue commercially available as V5004 by Permabond, which has good
flowing characteristics and is non-stringing. It is further contemplated within the scope of
the invention that another "glue" having the advantage of biological compatibility, such as
fibrin glue or the like may be used to seal an opened valve. A fibrin glue such as tissuecol
is contemplated within the scope of the invention. The fibrin glue contains two components
that include a fibrin protein on one side and thrombin on the other side. Their contact
produces a coagulant reaction that seals the valve.
It is also contemplated within the scope of the invention that fluid passage can be
arrested by opening a valve allowing fluid to enter one branch of a Tesla valve. The latter is
a fluidic diode or a valvular conduit, that allows easy flow in one direction but in the other
the flow gets hung up forming eddies, or counter-currents that stop the flow as surely as if a
mechanical valve were moved into the shut position. With this configuration, opening a
valve according to the present invention allows fluid to enter in one of the branches and
thereby stop fluidic flow, which is the same functionality achieved by the action of closing a
valve.
As another example, the inventive valve can be used to distribute a fluid changing
significantly the surface properties of a channel (for example, making it more or less
hydrophobic). This could produce, as a net effect, the consequence that another fluid (for
example water) does not enter the output channel anymore, and the output channel could be
considered as "closed" for what concerns the passage of water.
Accordingly, in one aspect of the present invention, an apparatus for processing
biological or chemical fluids includes a first substrate comprising a plurality of first meso-
scale fluidic components, a second substrate comprising a plurality of second meso-scale
fluidic components corresponding to the first meso-scale fluidic components. It is
contemplated within the scope of the invention that the inventive apparatus may further
comprise additional substrate layers. According to the invention, these additional substrate
layers can contain a plurality of fluidic channels, chambers and manipulative components
such as lenses and filters.
Between each substrate layer, a material layer or perforation layer separates the
plurality of first meso-scale fluidic components from the plurality of second meso-scale
fluidic components or additional nano-scale or meso-scale components. The structure of the
material layer could be homogeneous or heterogeneous, for example including multilayer
and coatings. According to the invention the material layer or perforation layer is
comprised of a polymeric compound such as Poly(methyl methacrylate), hereafter referred
to as PMMA. It is contemplated within the scope of the invention that other materials such
as Low Density Polyethylene (LDPE), Linear Low Density Polyethylene (LLDPE), High
Density Polyethylene (HDPE), Polyethylene Teraphathalate (PET), Polyethylene (PE),
polycarbonate (PC), Polyethylene Terephthalate Glycol (PETG) , Polystyrene (PS), Ethyl
Vinyl Acetate (EVA), polyethylene napthalate (PEN) or the like can be used. These
polymers can be used singularly or in combination with each other. The use of polymers is
preferred because of its ease of use and manufacturing. It is clear that other options, for
example metallic foils with or without additional surface treatment, are possible, the choice
being connected with the type of electromagnetic radiation generating means used in the
application.
The material layer may further comprise optical dye or other like material or layers
having adsorptive properties of pre-selected electromagnetic radiation. The absorption can
occur through known modifications as those used in absorbing light filters, for example
including metallic foils or modifying the surface optical characteristics (n refraction index
and k extinction coefficient) or by means of other surface properties like roughness, in such
a way that a sufficient amount of pre-selected electromagnetic energy is absorbed with the
consequence of perforation. Other technologies can make use of light absorbing globules,
for example carbon-black particles, dye emulsions, nanocrystals. In addition, reflective
layers, polarization changing layers, wavelength shifting layers could be used to enhance
the effective absorption of electromagnetic energy.
An advantage of the current invention consists in the extreme compactness of the
valve in the microfluidic circuit that allows maximizing the surface used for fluid storage,
incubation and reactions to occur. The valve size, by tuning the optical system position,
power and pulse duration of the electromagnetic radiation generating means, can be also
adapted to the circuit in a wide range of dimensions, down to the diffraction limit or below.
When laminar flow is desired within the microfluidic circuit, the valve cross section should
approximately match the cross section of the capillaries that are interconnected. When
mixing is desired, valves with, a cross section largely different from the fluidic cross section
of the circuit are preferable, in order to allow turbulences to act as active mixing agents.
The valves of the present invention are intrinsically low-cost; in particular they have
null marginal cost since the cost of the device does not depend on how many valves are
implemented on the circuit itself.
The valves according to the invention have a dead volume that can be neglected in
microfluidics applications, and is smaller than most other valve designs in the prior art. The
inventive valve is typically easy to open, to implement and to build, both in laboratory
devices and industrial manufacturing production.
The inventive valve can be extremely fluid tight, by choosing a material layer
having low permeability. This allows using the inventive valve as seals for storage of
chemicals. Many valving systems, for example those based on hydrophobic breaks, or
based on calibrated capillary tension forces on the fluid, or based on pressure actuated
modifications of the polymer shape, do not provide enough tightness to vapours and liquids
to make the storage possible.
The valve of the invention could be also used for the storage of lyophilised
molecules, and for example proteins. In fact, the partial permeability of the material layer
to water vapour, could be used in order to control the loss of water vapour by sublimation of
a frozen compound, when the disk is heated in vacuum. This would allow long-term
storage of molecules, for example pharmaceutical compounds, in very small volumes and
ready for use. The molecules could be collected by having a solvent, through a valve
opened in the material layer, dissolving the molecules and then exiting the storage cavity by
means of a second valve on the layer.
According to the invention, an electromagnetic generating means for generating
electromagnetic radiation for directing onto the material layer or perforation layer at a
position corresponding to a portion of the material layer located between at least a pair of
corresponding meso-scale fluidic components from the plurality of first meso-scale fluidic
components and the plurality of second meso-scale fluidic components. The
electromagnetic generating means allows perforation of the material layer at a position
allowing fluid communication between the pair of meso-scale fluidic components. The
perforation of the material layer occurs in a defined manner that is dependent on the
wavelength and intensity of the radiation, which is applied to the device within a limited
space and time, thereby avoiding any substantial alteration of fluids or samples of interest.
In another aspect of the present invention, an apparatus for multiplexing biological
or chemical fluids includes a first substrate comprising a set of input capillaries, a second
substrate comprising a set of output capillaries corresponding to the set of input capillaries,
a layer of material positioned between the first substrate and the second substrate forming
an interface between each of the input capillaries and each of the output capillaries
corresponding thereto and electromagnetic radiation generating means for generating
electromagnetic radiation for directing onto the material layer at the interface between a first
input capillary of the plurality of input capillaries and a corresponding first output capillary
of the plurality of output capillaries. The electromagnetic generating means allows
perforation of the material layer at the interface allowing first input capillary and the first
output capillary to be in fluid communication without damage or substantial alteration to the
biological sample or fluids within the microfluidic network.
The multiplexing capability addresses the need of a flexible, programmable fluid
handling. The choice of the fluids involved in a reaction, for example, can be made in real-
time during protocol execution.
In yet another aspect of the present invention, an apparatus for volumetric
quantitation of a liquid in a centripetal device includes a first meso-scale fluidic component
containing a liquid for quantitation, a second meso-scale fluidic component and fluid
communication means for placing the first and the second fluidic components in fluid
communication at a first position. Upon a centripetal force or other force being placed on
the liquid, a first amount of the liquid left in the first fluidic component or a second amount
of liquid transferred to the second fluidic component is determined by the choice of the first
position.
In another aspect of the present invention, a method for volumetric quantitation of a
liquid in a centripetal device includes loading a liquid in a first meso-scale fluidic
component, enabling fluid communication at a first position between the first fluidic
component and a second meso-scale fluidic component, spinning the centripetal device to
cause a portion of the liquid to be transferred from the first fluidic component to the second
fluidic component and determining a first amount of liquid left in the first fluidic
component or a second amount of liquid transferred to the second fluid component.
This method has the advantage of an arbitrary volume to be quantified, without
being bound to discrete steps in the dosing volume.
In another aspect of the present invention, a method for separating a fluid into its
fractions makes use of the centrifugation occurring during the rotation to separate the
medium into its constituting fractions by determining the choice of the perforation position.
In still yet another aspect of the present invention, a method of moving a liquid
sample in a centripetal device from an outer radial position to an inner radial position
includes loading a buffer liquid in a first meso-scale fluidic component, loading a liquid
sample in a second meso-scale fluidic component, enabling gas-tight fluid communication
between the first meso-scale fluidic component and the second meso-scale component
across a fluidic circuit sealed on one end by the buffer liquid and on the other end by the
liquid sample, enabling the buffer liquid to exit the first fluidic component and spinning the
centripetal device to cause the buffer liquid to exit the first fluidic component. Movement
of the buffer liquid exiting the first fluidic component forces the liquid sample from an outer
radial position to an inner radial position.
In a further aspect of the present invention, a method of performing a washing step
in a centripetal device by moving a liquid sample from an outer radial position to an inner
radial position includes loading a buffer liquid in a first meso-scale fluidic component,
loading a liquid sample in a second meso-scale fluidic component, enabling gas-tight fluid
communication between the first meso-scale fluidic component and the second meso-scale
component across a fluidic circuit sealed on one end by the buffer liquid and on the other
end by the liquid sample, enabling the buffer liquid to exit the first fluidic component and
spinning the centripetal device to cause the buffer liquid to exit the first fluidic component.
Movement of the buffer liquid exiting the first fluidic component forces the liquid sample
from an outer radial position to an inner radial position.
In still yet another aspect of the present invention, a method for determining a polar
position and a radial position of a pickup in a reference frame of a rotating device includes
detecting a first marker on the device by means of a pickup, detecting a second marker on
the device by means of the pickup, wherein an angular distance from the first marker to the
second marker is a continuous or discontinuous , derivable or non-derivable, non-constant
function of a radial position of the pickup, recording the time elapsed between the detection
of the first marker and the second marker, determining a radial position of the pickup from
the elapsed time and a rotation period of the rotating device and determining a polar
position of the pickup a first time using the difference between the first time and a second
time corresponding to the detection of a marker and the rotation period of the rotating
device.
In another aspect of the present invention, a method for determining a polar position
and a radial position of a pickup in a reference frame of a rotating device includes recording
a first time at which a pickup detects a first marker on a rotating device, recording a second
time at which the pickup detects a second marker on the device, wherein an angular distance
from the first marker to the second marker is a continuous or discontinuous, derivable or
non-derivable non-constant function of a radial position of the pickup, determining the
radial position of the pickup from the difference in time between the second time and the
first time and a rotation period of the rotating device and determining a polar position of the
pickup at a third time using the difference between the third time and a fourth time
corresponding to the detection of a marker and the rotation period.
In yet another aspect of the present invention, a method for processing biological or
chemical fluids includes providing a first substrate comprising a plurality of first meso-scale
fluidic components, providing a second substrate comprising a plurality of second meso-
scale fluidic components corresponding to the first meso-scale fluidic components,
providing a layer of material separating the plurality of first meso-scale fluidic components
from the plurality of second meso-scale fluidic components, directing electromagnetic
radiation onto the layer at a position corresponding to a portion of the layer located between
at least a pair of corresponding meso-scale fluidic components from the plurality of first
meso-scale fluidic components and the plurality of second meso-scale fluidic components
and perforating the material layer at the position, wherein the perforation of the material
layer allows fluid communication between the pair of meso-scale fluidic components
without damage or substantial alteration of any fluid or sample of interest within such
microfluidic network.
In another aspect of the present invention, a processing disk for processing
biological or chemical fluids includes a first substrate comprising a plurality of first meso-
scale fluidic components, a second substrate comprising a plurality of second meso-scale
fluidic components corresponding to the first meso-scale fluidic components, and a layer of
material separating the plurality of first meso-scale fluidic components from the plurality of
second meso-scale fluidic components.
These and other advantages, objects and features of the invention will be apparent
through the detailed description of the embodiments and the drawings attached hereto. It is
also to be understood that both the foregoing general description and the following detailed
description are exemplary and not restrictive of the scope of the invention.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The foregoing and other features and advantages of the present invention will be
more fully understood from the following detailed description of illustrative embodiments,
taken in conjunction with the accompanying drawing in which:
FIG. 1A illustrates the components comprising the disk according to the invention;
FIG. 1B illustrates the components comprising the disk according to the invention
microfluidic components on both sides of the material layer are shown in a possible
configuration;
FIG. 2A illustrates a section of the inventive disk wherein microfluidic components
within each top and bottom side are separated by a material layer;
FIG. 2B illustrates a section of the inventive disk wherein microfluidic components
within each top and bottom side are separated by a material layer and the bottom side
microfluidic component contains a fluid or sample;
FIG. 2C illustrates a section of the inventive disk wherein microfluidic components
within each top and bottom side are separated by a material layer and the bottom side
microfluidic component contains a fluid or sample and the material layer is perforated by
electromagnetic radiation;
FIG. 2D illustrates a section of the inventive disk wherein microfluidic components
within each top and bottom side are separated by a material layer and the bottom side
microfluidic component contains a fluid or sample and the material layer is perforated by
electromagnetic radiation and the sample moves through centripetal forces from the bottom
microfluidic chamber to the top microfluidic chamber;
FIG. 2E illustrates a section of the inventive disk wherein microfluidic components
within each top and bottom side are separated by a material layer and the bottom side
microfluidic component contains a fluid or sample and the material layer is perforated by
electromagnetic radiation and the sample moves through centripetal forces from the bottom
microfluidic chamber to the top microfluidic chamber;
FIG. 3 A illustrates a geometrical layout of the multiplexing nature of the inventive
valve;
FIG. 3B illustrates the combined use of multiplexing units and dosimeters in an
embodiment where three different fluids are dosed and collected into different reactors.
This illustrative embodiment graphically describes the process control of an enzymatic
assay where the inhibition of a drug compound on the enzyme activity on a specific
substrate is tested in an homogeneous way,
FIG. 4 illustrates a half section of the inventive disk containing the self metering
embodiment according to the invention;
FIG. 5A illustrates the self metering embodiment according to the invention wherein
a sample to be metered is within the sample metering chamber;
FIG. 5B illustrates the self metering embodiment according to the invention wherein
a sample to be metered within the sample metering chamber is valved in a manner allowing
the fluid to exit;
FIG. 5C, 5D, 5E, 5F and 5G illustrate the self metering embodiment according to the
invention wherein a sample to be metered is with sample metering chamber containing a
meniscus allowing for the metering of a known quantity within a subsequent sample
chamber,
FIG. 6 is a schematic depiction of the reflow embodiment according to the
invention;
FIG. 7 is a schematic that illustrates the optical feedback according to the invention;
FIG. 8 is a graphic depiction of transmittance of energy passing through after
perforation of the material layer;
FIG. 9 depicts the nanostack structure of the laser diode upon a sample object;
FIG. 10 depicts perforation entrance holes of a laser beam shot upon a sample
object;
FIG. 11 depicts perforation exit holes of a laser beam shot upon a sample object;
FIG. 12 is a graphic depiction of the wavelength absorption spectrum of an infrared
dye incorporated into the perforation layer;
FIG. 13 depicts the distribution of an infrared dye within the material layer,
FIG. 14 depicts a microfluidic chip used within biological experiments;
FIG. 15 is a graphic representation of chip rinsing data within the biological
experiments;
FIG. 16 is a graphic representation of the ratio between unexposed drops and
exposed drops;
FIG. 17 is a graphic representation of the results of the drop experiments; and
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides centrifugal rotors and microsystems and in
particular nano-scale or meso-scale microfluidic valve technology platforms as well as a
number of its applications for providing centripetally-motivated fluid micromanipulation.
For the purpose of illustration, the drawings as well as the description will generally refer to
centripetal systems. However, the means disclosed in this invention are equally applicable
in microfluidic components relying on other forces to effect fluid transport.
For the purposes of this specification, the term "sample" will be understood to
encompass any fluid, solution or mixture, either isolated or detected as a constituent of a
more complex mixture, or synthesized from precursor species. A sample may farther be
comprised of a suspension or an emulsion containing beads, nanoparticles, globules, cells,
or the like.
For the purposes of this specification, the term "in fluid communication" or "fluidly
connected" is intended to define components that are operably interconnected to allow fluid
flow between components. In illustrative embodiments, the micro-analytical platform
comprises a rotatable platform, such as a disk, or experimental microfluidic chips whereby
fluid movement on the disk is motivated by centripetal force upon rotation of the disk and
fluid movement on the experimental chip is motivated by pumps and fluid communication
is achieved by the perforation of a material layer.
For the purposes of this specification, the term "material layer" or "perforation
layer" is intended to define components that separate various micro fluidic components such
as chambers, channels and other microfluidic elements and upon perforation by
electromagnetic irradiation bring such micro fluidic components in fluid communication
with each other.
For the purposes of this specification, the term "biological sample", "sample of
interest" or "biological fluid sample" will be understood to mean any biologically-derived
or synthesized analytical sample, including but not limited to blood, plasma, serum, lymph,
saliva, tears, cerebrospinal fluid, urine, sweat, plant and vegetable extracts, semen, or any
cellular or cellular components of such sample.
For the purposes of this specification, the term "perforation" is intended to define
the dissolution of a portion of any such material layer or perforation layer either by
decomposition or phase change (into a different solid aggregation, liquid, gas or plasma
states) or chemical uncoupling of any such material forming such perforation or material
layer. Such perforation is achieved by electromagnetic irradiation having an energy and
wavelength meant to be absorbed by such material layer or additives contained within or
adjacent to such material layer with the consequence of creating a passing through hole in
the layer.
For the purposes of this specification, the term "ablation" specifically refers to a
rapid process where the heat wave ejects the material that is vaporised into plasma.
For the purposes of this specification, the term "meso-scale", or "nano-scale" will be
understood to mean any volume, able to contain fluids, with dimensions preferably in the
sub-micron to millimetre range.
Representative applications of centripetal systems (e.g., centrifuge) employ circular
devices, with the rotation axis at their centre. For the purpose of illustration, the drawings,
as well as the description, will generally refer to such devices. Other shapes, including
elliptical and rectangular devices, irregular surfaces and volumes, and devices for which the
rotation axis does not pass through the centre, may be beneficial for specific applications.
The microfluidic device used for illustrative purposes in the present invention will
be referred to as a disk which, in some embodiments, is rotating around a given axis. The
operations that may be performed depend on the shape, the material composition and the
complexity of the disk. The microfluidic system may include, in addition to the disk, one or
more than one external moiety designed to perform operations on the disk, including but not
limited to the loading of chemical, biological or biochemical fluids, the optical readout of
signals, the detection of radioactivity, the analysis of assays, detection of compounds of
interest, injection of samples from the disk to a chromatographer or mass spectrometer,
exposure of the disk to x-ray or gamma or neutron beams, transfer of fluids to or from the
disk, transfer of fluids from a disk to another disk.
In an illustrative embodiment of the present invention, the external moiety includes a
pickup, a device capable of focusing a substantial amount of electromagnetic radiation onto
a point in the disk, and a spinning device. The disk and the pickup are designed to interact
primarily by means of electromagnetic radiation, at a pre-selected preferred wavelength or
spectrum of wavelengths. Hereafter, this wavelength or spectrum of wavelengths will be
referred to as the "pickup wavelength" or "pre-selected wavelength".
In one aspect of the present invention, a novel system for valve implementation in a
microfluidic circuit is presented. It represents a fully programmable (active) solution where
the fluid flow is controlled by means of a distributed valve system, meaning that the
position of a given valve is arbitrary and the valves themselves, extdend to the full
microfluidic circuit. The described valves are typically limited to a closed-to-open
transition, even if schemes that restore a valve state from open to close are possible and here
commented. Another significant advantage of the system is the large number of valves that
may be integrated in a circuit.
THE DISK
The preferred embodiment of the disk includes a circular microfluidic device.
Rectangular disks, rotating around an axis that preferably does not intersect the disk
volume, offer specific advantages. To achieve compatibility with commercial products
related to compact disk technology, the disks may have similar or identical dimensions
thereto. Likewise, rectangular disks, with a footprint equal to that of micro well plates or of
credit cards, are particularly suited for automatic handling and storage of compounds,
including the transfer of fluids between disks and the transfer of fluids from disks to
standard well plates used in the chemical and biochemical industry, as well as from standard
well plates to disks.
As shown in FIG. 1 A, the internal structure of the disk 100 in one illustrative
embodiment comprises a sandwich of at least three layers: a top side 101, a bottom side
103, and a material layer 105 separating the two sides 101,103. To achieve higher densities
in a single disk, the sandwich structure can be replicated to produce a multi-base sandwich.
In such a configuration, sides 101,103 are contained between at least two material layers
105 and have microfluidic components on both surfaces, and possibly comprise additional
microfluidic elements putting into fluid communication the components at their respective
surfaces.
Turning to FIG 1B the disk according to the invention is shown wherein the top side
101 contains a microfluidic structure 110 (which is a dosimeter reservoir described below)
and the bottom side 103 contains a corresponding microfluidic structure 111 (which is the
capillary outlets of the dosimeter). Numerous microfluidic structures can be incorporated
within the top side 101 and bottom side 103. These microfluidic structures are separated by
the material layer 105 and can be place in fluid communication with each other by the
perforation of the material layer 105. The microfluidic structures 110, 111 contained within
the respective top and bottom sides 101,103 may be mirror images of each other or they
may be dissimilar structures separated by the material layer 105 having an integrated
function with each other upon perforation of the material layer 105.
A. MATERIAL LAYER
A variety of materials are suitable for the material layer 105 or perforation layer,
including but not limited to thin polymer foils and metallic foils. The thickness in
microfluidic applications typically varies between about 0.5 to about 100 microns,
depending on the material properties and on the characteristics of the pickup.
In a first illustrative embodiment infrared absorbing polymer foils are used since
they can be easily perforated with simple and economical means. These polymer foils are
comprised of a polymeric compound such as Poly(methyl methacrylate), Low Density
Polyethylene (LDPE), Linear Low Density Polyethylene (LLDPE), High Density
Polyethylene (HDPE), Polyethylene Teraphathalate (PET), Polyethylene (PE),
polycarbonate (PC), Polyethylene Terephthalate Glycol (PETG), Polystyrene (PS), Ethyl
Vinyl Acetate (EVA), polyethylene napthalate (PEN) or the like can be used.
It is contemplated within the scope of the invention that these polymeric compounds
may be used singularly or in combination with each other. In a further illustrative
embodiment copper foils with a thickness in the order of about 2 microns are can be used.
Copper foils are routinely produced for applications in the electronic industry, specifically
for printed circuit boards. Thin metallic foils, such as copper, exhibit a natural absorption
in the domain of ultraviolet light wavelength, which is of interest in some of the illustrative
embodiments.
It is further contemplated within the scope of the invention that additional materials
may be used for the material layer 10S such as waxes, because of their low melting point,
and polysaccharides such as cellulose, or the like. It is also contemplated within the scope
of the invention that liquid crystal polymers may be used for the material layer.
The choice of material layer 105 is dictated by the requirement for large light
absorption at the pickup wavelength. Except for the choice of material, large absorption
may be achieved by modifying the optical properties of any such material using dyes,
coatings, surface treatments or by an appropriate multi-layer construction to exploit light
interference processes. It is contemplated within the scope of the invention that dyes having
desired optical properties such as ADS905AM, an infrared dye from American Dye Source
Inc. of USA, whose chemical formulation is C62 H96 N6 Sb F8 or the Epolight 2057, an
infrared dye from Epolin Inc. of USA whose absorption spectrum is suitable to near infrared
sources, can be loaded into the material layer. It is further contemplated that infrared
absorbing solutions such as Epolight 2180, Epolight 2189, and Carbon black loading, either
in homogeneous dispersion or in heterogeneous suspension or emulsion (globules or
particles) can be used. It is also contemplated within the scope of the invention that other
absorption spectrums below or above infrared may be used to match the material layer 105
with any form of electro-magnetic radiation.
In one illustrative embodiment a material layer 105 is formed from PMMA and
loaded with the infrared dye ADS905AM in a weight percent of approximately 0.5%. The
dye is suspended within the PMMA film in non-uniform globules. While the suspension of
such dye is not distributed uniformly throughout the material layer, it is sufficiently
distributed to produce a desired absorption of the pre-selected wavelength.
It is further contemplated within the scope of the invention that other dyes such as
Epolight 2057, Epolight 2180, Epolight 2189 or the like may be used to achieve a desired
spectral absorption. It is also contemplated within the scope of the invention that other
compounds other than dyes such as Carbon black, or the like, having light absorption
properties may be used to achieve a desired spectral absorption.
Additional requirements for the material layer 105 depend on the application, and
relate specifically to the interaction of fluids with neighbouring materials. Examples of
additional requirements are resistance to corrosion, prevention of fluid contamination,
absence or presence of catalytic reaction, the build-up of electrical charge and/or the
presence of electrical currents, biocompatibility.
Procedures to achieve the permanent connection between the material layer 105 and
the two sides 101,103 include bonding or gluing as known in the art (lamination, hot
bonding, UV bonding, plasma treatment of the surfaces, solvent bonding, pressure adhesive,
heat adhesives). The bonding procedure may exploit polymer foils treated with thermoset
films on both sides. Such foils are commercially available and currently produced for
printed circuit boards. In addition, various materials suitable as a base are available off-the-
shelf, including carbon-black loaded polyester and black Mylar. In a first illustrative
embodiment, the material layer 105 shows no internal structure, obviating the need for any
alignment of the material layer 105 with respect to the sides 101,103.
B. SIDES
With continuing reference to FIG. 1, the sides 101,103 comprise the microfluidic
components of the disks, containing the fluids. Depressions within one surface of the
substrate forming the sides 101,103 form the microfluidic components. Meso-scale
components and channels, also referred to as capillaries or micro-capillaries, may be
provided by a variety of techniques known in the art, including engraving, fluoric acid wet
etching, embossing, hot embossing, micro-machining, laser ablation, mechanical machining
or polymer moulding.
It is contemplated within the scope of the invention that microfluidic components
such as chambers and channels may be formed by the printing of such components upon a
substrate, wherein such printed components and substrate form the sides 101, 103 of the
inventive disk. The printing of microfluidic structures can be achieved by silk screening
printing techniques or other printing techniques known in the art.
Each meso-scale component includes a volume, able to contain fluids, with
dimensions preferably in the sub-micron to millimetre range. In an illustrative embodiment,
the meso-scale components are understood as open components engraved or printed on the
surface of the sides 101,103 and facing the material layer 105. The sides 101,103 may
further comprise additional fluid connections and components, including dedicated input
and output ports to allow the fluids to reach the meso-scale network, instruments, batteries,
electrical connections and other instrumentation. Suitable materials for the sides 101,103
include glass, quartz, monomers, silicon, polymers, acrylic plastics, and polycarbonates,
cyclic olefin copolymers (COC). It is contemplated within the scope of the invention that
the sides 101,103 may integrate optical and electrical components, including motors,
conductors, chips, lenses and. prisms. It is also contemplated within the scope of the
invention that the surfaces in contact with the material layer 105 may be modified, in
particular to have different optical properties allowing for the focusing of the pickup onto
the material layer 105.
It is contemplated within the scope of the invention to have disks with sides totally
sealed, where gases in contact with fluids cannot exit from the device. This characteristic
can be exploited to handle high toxicity chemicals or radioactive fluids., and allows
performing operations with disks in vacuum, or in general when the external pressure is
different from the internal pressure (pressurized reactions).
For the purpose of the present invention, the sides 101,103 forming the disk are
substantially transparent or selected portions thereof are transparent to wavelengths of light
corresponding to the pickup wavelength. According to the invention optical lenses and
optical components may be embedded within the sides 101,103 to direct a light wavelength
to a desired valving area within the microfluidic network. It is contemplated within the
scope of the invention that there is a different refraction index of the side with respect to the
material layer 105, in order to allow optical detection of the interface surface by reflection
of the pickup light emission or for a better compatibility with the fluids present in the
microfluidic components.
Additional requirements of the sides 101,103 depend on the application, including
interaction or contamination of the fluids in the device and optical properties affecting the
study of the fluids in the device and their reactions. In addition, cost and ease of mass
production are also considered.
THE PICKUP
The pickup includes an optical means to irradiate the material layer or layers of the
disk at the pickup wavelength. In one illustrative embodiment, a laser source whose tight
emission is focussed, by optical elements (for example) and through one side of the disk,
onto a focal point located inside the material layer or on the material layer near to it is
contemplated. According to the invention, a requirement for the pickup is the ability to
concentrate or focus a sufficient amount of electromagnetic energy on a sufficiently small
surface area of the base. Accordingly, a basic operation of the present invention is the
perforation of the material layer at a specific time and position, induced by irradiation
provided by the pickup. The preferred wavelengths for the emission are in the infrared,
visible, and ultra-violet parts of the spectrum. Wavelengths within the infrared region are
desirable since most biological samples - including cells - and fluids used in the biological
domain do not absorb near infrared radiation and therefore are not substantially affected by
infrared irradiation.
The laser emission may be achieved by means of compact and low-cost laser diodes,
with commercially available diodes spanning a large range of frequencies, starting from and
above 375 nm. The largest power available in current laser diodes for commercial compact
disk readers is about 200 mW. The highest intensity achieved by this technology is in the
near infrared region. In an illustrative embodiment the laser diode used is an OSRAM
PL90_3, made by Osraxn. In yet a further illustrative embodiment the laser diode used is a
JDS SDL-6380A made by JDS Uniphase. While the JDS diode has a lower peak power
than the OSRAM diode, its better heat dissipation, smaller slit size, narrower far field
emission and larger maximum pulse width allows for better performance. It is
contemplated within the scope of the invention that other sources such as q-switched lasers,
diode pulsed solid state lasers (D.PSS), carbon dioxide lasers, Titanium Sapphire lasers fibre
lasers, excimer lasers, flash lamps, gas discharge or the like may be used.
According to the invention, the laser diode operates in a substantially pulsed mode.
The pulse geometry is selectable to deliver desired energies to intended targets and allows
the instantaneous power to be significantly larger, provided that the operational duty cycle
is short enough to allow appropriate cooling of the laser junction. Commercial laser diodes
with peak power output reaching up to about 70W are available and even higher peak rates
are possible with more expensive solutions.
An aspect of using very short pulses is that the minute energy deposited in the base
is hardly transferred to the samples and to the surroundings area. The heat wave propagates
away from the perforation spot with a finite speed. During a short pulse with high intensity,
the outgoing energy flow can be smaller than the ingoing energy flow, the energy remains
therefore concentrated in a limited spot, with a steep rise in localized temperature rapidly
producing perforation with efficacy.
Optical focusing is typically achieved by a single optical system composed of few
optical elements. To achieve optimal collimation and alignment of the beam onto the base,
one element may be moved in different directions, for instance by means of electric coils
immersed in a magnetic field. Optimisation of the optical path has been made by taking
into account the disk exposure requirements. The side's thickness can introduce significant
coma and astigmatism, which is sometimes difficult to correct when small spots are
envisaged.
In an illustrative embodiment, the optical system is comprised of a f=6mm MG
GLC001 collector lens, a LiteOn CD pickup (NA=0.45) by LiteOn, with a real focus in
between the two systems at 25 mm from the CD lens front face. This particular
configuration collects onto the base a light intensity of about 16 µJ in about 10 us exposure.
The effective power density of this configuration has proven to be perfectly sufficient for
perforation of various material layers. By means of a pinhole and a pyrometer PEM 100 by
Lasertechnik Berlin of Germany, it was possible to estimate and optimize the amount of
optical energy effectively collimated onto the focal spot onto the base.
The above configuration provides that the pickup structure implemented in
commercially available CD devices, with applications in audio, video and computer data
storage, may be applied to the present invention, and that focusing the laser onto the base is
possible by processing the fraction of light reflected at the base surface with an appropriate
optical system.
In another illustrative embodiment, the pickup may contain two or more light
sources, only one of which is used to perforate the base. The focusing of the lens and the
determination of the pickup position is obtained through a different source which can be a
low power, continuous or quasi-continuum (QCW) wave emission. The use of multiple
light sources allows choosing a base that absorbs the radiation meant to perforate the base,
and reflect the radiation meant to determine the base position through the same optical
system.
The pickup device may further include an optical system for determining if the
focussing of the electromagnetic radiation onto the base is achieved. For example, partial
reflectivity of the base may be used as an optical feedback mechanism if analysed through a
Foucault (astigmatic) focussing system. Such a system has been implemented in
commercially available optical readers for CD and DVD media.
The pickup in the present invention is similar to devices used to manipulate
microscopic objects by means of a focussed light beam. This operation, also known as
tweezing, allows one to hold and move single objects using the electromagnetic forces
generated by a light wave front, which is substantially convergent or divergent.
It is contemplated within the scope of the invention mat the pickup of the present
invention may be a device serving one or more distinct purposes including, for example, the
control of the fluidic process by perforation of a material layer, tweezing of particles and
optical analysis of the samples contained in the microfluidic components. It should be also
noted that the pickup does not have contact with the microfluidic device. This potential can
be exploited in those applications where contamination has to be absolutely avoided, for
example forensic analysis of samples (contamination from the outside to the inside) or
handling toxic or radioactive fluids (contamination from the inside to the outside).
MATERIAL LAYER PERFORATION TO OPEN A VALVE
Microfluidic handling in the present invention is regulated and performed through
the dynamic, real-time configuration of a microfluidic network. This configuration is
achieved by putting into fluid communication microfluidic components within sides of the
disk on opposite sides of a material layer. This connection may be used either by a fluid
moving from one microfluidic component to a second one or by two fluids coming into
contact in specific places. The former is referred to as a flow valve, the second as contact
establishment.
Turning to FIG 2A-2E, the connection of two or more microfluidic components is
shown. To achieve fluid communication, the following operations are performed: The
pickup (not shown) or the disk 200 is positioned such that an electromagnetic radiation
emission 206 is directed at a position of the material layer 205 where perforation is to occur.
This may be achieved by moving either the disk 200, or the pickup, or both; The focusing
system, if present, is adjusted to minimize the spot size and concentrate the energy onto the
material layer 205 in the position of perforation; Electromagnetic radiation of sufficient
intensity is generated by the pickup and directed onto the material layer 205 that is
positioned between microfluidic components within the top side 201 and bottom side 203 of
the disk 200. The intensity of such radiation, its limited time duration and its limited spatial
application prevents or substantially avoids alteration of fluids 207 (or samples) within the
microfluidic network. The energy deposition, specifically, the fraction of energy absorbed
by the material layer 205, causes perforation (also referred to as drilling) of the material
layer 205.
The forces acting on the fluid 207, in a preferred embodiment a centripetal force,
cause the fluid 207 to flow from the microfluidic component in one side to the microfluidic
component in the other side through the point of perforation 208. The point of perforation
208 and the resulting opening is referred to as a virtual laser valve (VLV). This typically
allows the fluid 207 to access the next step or merge with the adjoining fluidic chamber or
channel in the microfluidic network.
The drilling of the base or perforation occurs via different physical phenomena,
including ablation and melting, or through the breaking or relaxation of molecular bonds.
Their relative importance depends on the energy density, the pickup wavelength, the time
duration of the pulse, the composition of the material layer, the polarization of the
electromagnetic radiation, phenomena of heat dissipation in an irradiated body, the
development of plasma waves, and the presence of materials neighbouring the point of
perforation zone. Ablation specifically refers to the rapid process where the heat wave
ejects the material that is vaporised into plasma. Melting occurs through an intermediate
state of liquid phase, unavoidably leading to partial transfer of heat from the illuminated
area to the sides.
Both ablation and melting may produce a gas, like CO2, for example, when hitting a
polymer, with additional small solid deposits. Both processes are industrially used for a
number of commercial applications, including micro-mechanical devices (MEMS), polymer
laser drilling and cutting, metal drilling and cutting, and surface treatment by ablation. A
substantial and growing experience with excimer lasers, for example by Lambda-Physik,
shows that the potential of ultra-violet laser emission may allow achievement of a high
quality perforation by direct breakage of the molecular bonds. This type of drilling achieves
high resolution and high quality drilling, which may be of substantial benefit to the present
invention to achieve a large integration scale of the microfhiidic components onto a disk.
Because the perforation volume is small, compared to the dimensions of the
microfhiidic components in the sides, the overall amount of material scattered by opening a
valve is negligible and does not substantially impact or alter fluids in the microfluidic
components. The pickup is protected from the blast of material since it occurs within the
sides. As described here, the perforation process is generally irreversible: the material layer
205 is removed upon perforation when opening the valve. According to the invention, the
material layer 205 generally cannot be recovered to restore the valve to its closed state.
Nevertheless, the present invention may be applied to configurations where the valve may
be closed. One such configuration includes a case where a polymer in the Uquid phase,
which can allow the flow of gases, polymerizes at the position of perforation or in another
position connected to the circuit in a gas tight manner to impede gas flow or fluid
movement. A similar result can be achieved by thermoset materials and fibrin glue, or other
2-components sealing agents. A different implementation of an open-to-close transition
makes use of a Tesla valve, which can be opened by opening a valve. The Tesla valve
increases the impedance of the fluid flow, effectively achieving the result of blocking the
fluid flow in one given direction.
The optical characteristics of the material layer 205 and the sides 201,203 determine
both the modality of energy deposition and the requirements of the electromagnetic
radiation provided by the pickup. Material layers 205 formed from polymers are
advantageous because of their low enthalpy: the energy required to transform a polymer
from the solid to the liquid state is typically smaller than that required in the case of metals.
Consequently, smaller energy density is sufficient for perforation. Conversely, the sides
201, 203 should be as transparent as possible at the pickup wavelength, with optical
properties such that the focussed pickup emission is not scattered before arriving at the base
surface nor absorbed leading to heating up of the side material or adjacent fluids. Effects to
be considered include bi-refringence, optical quality of the surface, and uniformity of the
optical thickness. Various polymers, including the polycarbonates used in compact disk
applications, are substantially transparent in the entire visible spectrum as well as the near
infrared, and in addition, show good optical quality of the surfaces.
Components of the invention such as disks, chambers, channels, filters and their
respective optical characteristics are advantageously provided having a variety of
composition and surface coatings appropriate for a particular application. Component
composition will be a function of structural requirements, manufacturing processes, and
reagent compatibility/chemical resistance properties, including biocompatibility.
Specifically, components of the invention such as sides are provided that are made
from inorganic crystalline or amorphous materials, e.g. silicon, silica, quartz, or from
organic materials such as plastics, for example, poly(methyl methacrylate) (PMMA),
acetonitrile-butadiene-styrene (ABS), polycarbonate, polyethylene, polystyrene, polyolefin,
polypropylene, fluoropolymer and metallocene. Thermoset materials, like SUS and PDMS
are a viable solution. Surface properties of these materials may be modified for specific
applications. Surface modification can be achieved by methods known in the art that
include but are not limited to silanization, ion implantation and chemical treatment with
inert-gas plasmas (i.e., gases through which electrical currents are passed to create
ionization). Similar processes could be applied to the material layer for a complete
treatment of the surfaces in contact with the fluids.
It is contemplated within the scope of the invention that components of the disk may
be made of composites, co-polymers or combinations of these materials, for example,
components manufactured of a plastic material having embedded therein an optically
transparent glass surface comprising for example the detection chamber of the disk or lenses
and mirrors for directing electromagnetic radiation to a valving area within the material
layer in order to perforate such layer.
Disks of the invention and their respective components are preferably fabricated
from thermoplastics such as Teflon, polyethylene, polypropylene, methylmethacrylate and
polycarbonates, among others, due to their ease of moulding, stamping and milling.
Alternatively, such components can be made of silica, glass, quartz, or thermoset materials.
A micro analytical fluid handling system according to the invention is fabricated by
sequential application of one or more of these materials laid down onto the thermoplastic
substrate. Disks of the invention may be fabricated with an injection moulded, optically-
clear base layer or sides having optical pits in the manner of a conventional compact disk
(CD). It is contemplated within the scope of the invention that other methods of fabrication
or manufacture known in the art may be used. It is further contemplated that microfluidic
chips using the inventive valving may be manufactured by this same sequential application
of materials.
At the time of perforation of the material layer, fluid may be present adjacent to or in
contact with the material layer above, below, or on both sides. In that case, energy
deposited by or generated during the perforation process may be transferred to the fluid.
Except for very rare configurations, the energy transfer is negligible compared to the heat
capacity of the fluid. It is contemplated within the scope of the invention that a valve can be
opened with an optical energy of less than 16 µJ. If all energy that goes into the base
ablation were absorbed by one microlitre of water, its temperature would increase by about
0.002 degrees Celsius only.
According to the invention, by changing the time duration of the pulse or the
focusing properties of the pickup, the valve diameter can be modified, and this feature can
be effectively used in applications where fluid flow regulation is necessary, for example in
mixture control, or where fluidic motion resistance (including its mixing) is affected
differently by valves of different size.
It is contemplated within the scope of the invention that for microfluidic
configurations and applications where sample or fluids are adversely impacted by thermal
changes, heat sinks or cooling faces of heat pumps may be incorporated into the
microfluidic network to compensate for any such thermal changes.
MULTIPLEXING OPERATIONS
In one aspect of the present invention, the arbitrary position of the valve is exploited
to implement logic for the flow of fluids. This can be implemented at any moment during
the process as performed in the microfluidic apparatus, a feature referred to as real-time
capability. Of particular interest is the logic scheme depending on the outcome of a
previous operation. A microfluidic component that can perform this operation in the most
generic way is hereafter denominated a multiplexer, analogous to the component in digital
electronics with equivalent functionality.
FIG. 3A illustrates one embodiment of the multiplexer, which includes a matrix of N
input capillaries 303 on one side of the disk facing M output capillaries 305 on the other
side of the disk. In the following, two fluidic components are considered to be facing one
another when they are separated by the base only, in at least one position. The multiplexer
therefore allows one or more of a set of inputs to be in fluid communication with one or
more of a set of outputs. A set of capillaries is to be understood as two or more capillaries.
The simplest case is shown in FIG. 3 A, where the opening of a valve in a first
position 301 connects a first input capillary 306 with a first output capillary 308. After fluid
communication has been enabled, the actual passage of fluid from the input capillary 306 to
the output capillary 308 may be achieved by applying a force on the fluid. Examples of
such force include spinning a centripetal device, exerting an overpressure in the input
capillary line 306, or exerting an underpressure in the output capillary line 308. A proper
venting design (not shown in the figure) ensures adequate escape for air contained in the
fluidic components downstream from the moving fluid.
The extension of multiplexing to higher levels of complexity may be achieved using
a multiplicity of valves at the crossing points in the matrix of facing capillaries. The valves
enable fluid communication between the desired input and output capillaries.
Except to provide connections between a number of inputs to a number of outputs,
the multiplexer may equally well be used to connect a number of inputs to a single output or
a single input to a number of outputs. Since such connections do not by itself guarantee
homogenous mixing in the first case or uniform distribution in the second case, the possible
limitation may be somewhat mitigated by adjusting the time at which the different valves
involved are operated, by preceding the multiplexing stage by appropriate volumetric
quantitation, or by introducing intermediate multiplexing networks.
The multiplexer is primarily intended for use in a programmable device (such as the
one described in the present invention) and to be an interconnecting network between
different components of a microfluidic device. If a large number of input channels are to be
connected to a large number of output channels, though not in all possible combinations, the
physical size of the multiplexing network may be reduced by breaking it down in different
stages with relatively lower integration.
In an illustrative embodiment, the combined functionality of the dosimeter and of
the multiplexer is shown with reference to Fig. 3B. In this embodiment, a disk according to
the invention is equipped with input wells 312,313,314. The input wells 312, 313, 314 are
in fluid communication with a multiplexing matrix layer 316. The multiplexing matrix
layer 316 is comprised of a grid of fluidic channels that are fluidly connected to dosimeter
units 317 (here schematically represented as all having the same dimensions). The
dosimeter units 317 are in fluid communication with segmented (separated) multiplexing
units 318 that are in fluid communication with reactor chambers 310,311.
In this scheme, for clarity of explanation, the venting lines are not drawn, even if
they are an essential requirement in order to allow the fluid moving into a component filled
with gas (air or any other inert gas sealed in the device at manufacturing).
This illustrative embodiment represents a generic microfluidic layout to perform a
homogeneous assay. This assay can be used for the measurement of compound inhibition in
an enzymatic reaction on a substrate, where the reaction kinetics (inhibition) is measured in
real-time, by detecting the fluorescence emission polarization of convenient dyes. It is
contemplated within the scope of the invention that many other assays known in the art may
be used without substantial modification to the device configuration.
With continuing reference to FIG. 3B, an enzyme is pipetted into the input well 314,
a substrate is pipetted into the input well 313 and a compound of interest is inserted into the
input well 312. Knowledge of the pipetted amount is not required, and the choice of the
input wells is totally arbitrary. The opening of valve 320 and valve 321 according to the
invention allows the distribution the compound of interests into one dosimeter arbitrarily
chosen. At the same time, more dosimeters can be used in order to perform parallel
operation, and for example the compound is also diverted by opening valve 322 into another
dosimeter connected to a different multiplexing segment in layer 317. With a similar
operation, the appropriate substrate contained in the input well 313 is diverted into another
dosimeter by opening valves 323 and 324, and the enzyme from input well 314 is taken to
the dosimeter layer by opening valves 325 and 326.
The correspondence between input wells and dosimeters is defined by the user in
real time, and allows matching in optimal way the dosimeter to the reagents according to the
amount of fluid expected to be quantitated and the dosimeter volume.
The purging of the dosimeters occurs by directing into a waste reservoir the filled
dosimeters opening valves 322, 330,331 on the dosimeter layer, valves 341, 346, 349 on
the segmented multiplexing layer, and valve 347 that determines the reactor 311 to be the
waste reactor where fluids are consequently collected. It is contemplated within the scope
of the invention that the inventive microfluidic disk can be configured so that no fluid exits
from the microfluidic structure.
The actual assay is performed by dispensing the required amount of substrate in
reactor 310, the amount being determined by the choice of the position of valve 340 and the
choice of the reactor determined by the opening of valves 342 and 343. At any moment
decided by the user, the compound and the enzyme can be added to the same reactor by
opening, with the same logic, 348 and 350, 344 and 345.
It is evident that - by opening other valves - another reactor in the reactor layer
could be filled by different amounts of the same reagents, or the reagents (for example the
compound or the substrate) could be replaced by others stored in the input wells to have
different reactions tested and measured. Even the reaction protocol (order of dispensing for
example) could be different for different reactors.
Despite the apparent complexity, all operations have been reduced to a single
process, which is the opening of a valve on the material layer at a desired location.
The procedure is independent of the type of fluids involved, and is constantly in
control of the user at any time during the process. For example, the addition of a stopping
agent after the reaction has started could be decided according to the data from the
fluorescent readout.
VOLUMETRIC QUANTITATION
In another aspect of the present invention, an arbitrary position of the valve is
exploited to perform volumetric quantitation ofliquids in centripetal microfluidic
components. The corresponding fluidic component will hereafter be referred to as a
dosimeter 400.
FIG. 4 depicts an illustrative embodiment for the dosimeter 400. It consists of an
elongated reservoir 401 with the longest axis substantially positioned in the radial direction
of the centripetal device. Along this axis, a portion of the device closer to the rotation axis
will be referred to as upper, and the portion further from the centre of rotation will be
referred to as lower. The shape and volume of the dosimeter are designed according to an
amount of fluid to be quantitated and a desired resolution in the quantitation process.
According to the invention, fluid is filled into the reservoir 401 through an inlet in the upper
part (not shown), and a venting line 402 is present to allow the fluid to enter.
Turning to FIG. 5A to 5G, when the device is rotated, the centripetal force causes
the fluid to move into the lower part of the reservoir 501 as shown in FIG 5A. The
capillaries indicated as 503 and 504 constitute two different outputs of the dosimeter 500.
One of the capillary outputs, 503 in the specific case, is used as a purge line. Output lines
are situated in the side opposite to the side containing the dosimeter, separated from it only
by the material layer. The overall number of output lines depends on the specific
application or implementation.
The first operation of the quantitation process consists in evacuating part of a liquid
505 to be metered into the purge line 503. This evacuation is achieved by opening a valve
510 by perforating the material layer in first position 510, and spinning the centripetal
device to achieve this result. Any liquid 505 above the level defined by the valve 510, as a
consequence of the applied centripetal force, flows into the purge line 503 and eventually
into a first purging chamber 512. The liquid 505 remaining within the reservoir 501 forms a
meniscus 514 as depicted in FIG 5C.
The extraction of a defined volume of liquid 505 from the reservoir 501 may be
performed by creating a second valve 515 in correspondence of the second capillary 504
line that puts into fluid communication the reservoir 501 with the second capillary line 504.
The liquid contained in the dosimeter between the first valve 510 and a second valve 515 is
extracted in the second capillary line 504 by spinning the centripetal device and applying
centripetal force. The position of the valve in relation to the meniscus 514 within the
reservoir 501 and the knowledge of the reservoir's 501 geometrical shape, allows the
determination of the volume of liquid extracted into second capillary line 504 which flows
into a second metered chamber 516.
As highlighted in position 514 of the figure, liquids in meso-scale fluidic
components generally display a. significant meniscus 514, i.e. the upper level of the liquid
will not typically be flat. This behaviour varies among liquids and depends on surface
tension, hydrophilicity and hydrophobicity of materials, thus making the shape of the
meniscus unpredictable. It is a benefit of the present invention that the volumetric
quantitation is substantially independent of knowledge concerning the meniscus shape. The
same liquid, in the same geometrical conditions and in contact with the same materials, will
exhibit the same meniscus shape, rendering the quantitation process independent of the
meniscus shape.
A single dosimeter may be used for various liquid extractions on the same output
line. FIG. 5F shows how opening an additional valve, in third position 520, allows the
extraction of a second volume of liquid into the next step of the microfluidic network. The
second metered amount is sent to the metered chamber 516 as shown in FIG. 5G. This is
called the multi-spill capability of the dosimeter.
Another aspect of the present invention is the multi-output capability of a single
dosimeter. The same dosimeter may serve various output lines, so that the same liquid may
be sent to an output by opening a valve in an appropriate position connecting the dosimeter
to the mentioned output line.
Quantitation may occur in real-time at the same time as fluid processing. For
example, the multi-output capability of the dosimeter may be used to keep the pH of a
reaction constant, by subsequent extraction of an acid or of a base, according to feedback
provided by the reaction itself while the reaction occurs.
Another application of the dosimeter is in the phase separation of fluids by means of
centrifugation. For example, blood can be separated into its components (plasma,
lymphocytes and erythrocytes) by means of centrifugation and possibly additional reagents
(sucrose or Ficoll by Amersham for example) inside a dosimeter. The opening of valves
near or at the separation interfaces allows the separation into different outputs of the
dosimeter of the various components, in an adaptive way. Similar separation can be
performed on fluids containing cells or lysates, on emulsions or on suspensions of particles.
The capability of dispensing amounts of fluids when desired by the user has also an
active role in mixing. For example, the dissolution of a solid phase chemical entity into a
solvent depends on its concentration in the liquid phase. It is possible to dispense an amount
of fluid into a "siphon" shaped capillary where the exceeding liquid exits from a position
located at an inner radius. The amount of liquid can be left in contact with the solid phase
solute for an amount of time sufficient to allow diffusion to occur. Then, by means of the
valve, an additional amount of fluid can displace the previous solvent, remaining in place
for additional dissolution of the solid phase but with a reduced initial solute concentration.
This operation can be repeated various times for repetitive dilution of the solid phase
chemical entity.
REDIRECTION IN A CENTRIPETAL DEVICE
A common problem in centripetal devices, especially if complex processes are
implemented, is related to the uni-directionality of the centripetal force. Given a fixed
rotation axis, the fluids move only from inner to outer positions, and the process terminates
when the position of the fluid reaches the outer edge of the centripetal device. This feature
excludes the use of centripetal devices in cases where the process to be accomplished
comprises a large number of steps. Here, as in the following, the "radial position of a mass
of liquid" denotes the radial position of the centre of mass of the liquid.
In one aspect of the present invention, the above limitation is overcome by an
appropriate arrangement of the inventive valves within a microfluidic circuit. The process
of moving a sample liquid from an outer to an inner radial position by means of the
centripetal force itself is referred to as reflow. The required energy is obtained at the
expense of the potential energy of another mass of fluid, hereafter called buffer fluid, whose
only purpose is to provide energy for the reflow process. The mass of buffer fluid can be
placed at any radius on the disk, and the buffer fluid can have any density. Overall energy
conservation constraints link the characteristics of buffer and sample liquids, specifically
the respective volume, density, initial radial position, and final radial position of buffer and
sample liquids. Another possibility for reflow consists in making use of additional sources
of energy like differences of pneumatic pressure or chemical energy. In a way similar to the
one described in the next section "fluid transport activated by a valve", a bottle could be
actuated in order to push or pull the liquid towards an inner part of the disk. The pneumatic
overpressure or underpressure, for example, could be generated by the centripetal force
itself by having a mass of liquid, compressing or decompressing a gas volume. In this case,
the energy could be stored by avoid the fluid to move back when the centripetal force is
reduced, for example by having a Tesla valve or similar functional device onto the path of
the liquid. The stored energy can then be recollected in a later moment when the centripetal
acceleration has been reduced, and used to reflow the sample fluid.
An illustrative embodiment of the reflow method consists of the following steps, as
illustrated in Fig. 6: The buffer fluid 601 is loaded in a reservoir 602. To the extent that the
buffer fluid 601 does not participate in any reaction or process, the liquid used is
independent of the use of the disk. Accordingly, buffer loading may be performed at the
disk-manufacturing phase. An important requirement is for the reservoir 602 to be gas-
tight, i.e. sealed to prevent air or gas to freely enter or exit. The sample liquid 610 flows
into a sample reservoir 612 through position 613. For this operation, the sample reservoir
612 requires a venting line 614. In these conditions, the sample liquid 610 generally cannot
flow through the capillary 615 since the presence of air - trapped below the liquid mass -
impedes the flow of the liquid into it even if valve 616 would be already open.
The opening of a first valve 603 along the fluid communication circuit enables fluid
communication between reservoirs 602 and 612 if valves 617 and 618 are already open
Where appropriate, an additional reservoir 622 referred to as a trap, can serve to collect the
sample liquid 610. When fluid communication is enabled, it does not by itself lead to a
movement of the fluids, because the potential energy is at a local minimum, with the buffer
fluid 601 prevented from flowing into a second capillary 604. The buffer fluid 601 and the
sample fluid 610 are integral elements to guarantee the gas-tightness of the fluid
communication circuit, and the reservoirs 602, 612 are designed to maintain this gas-
tightness up to the end of the process. Opening a second valve in position 605 enables the
reflow operation. The reflow operation is initiated by spinning the centripetal device,
thereby exerting on buffer liquid 601 and sample liquid 610 a force proportional to their
mass and to the acceleration a=w2*r, where omega is the angular velocity of the device and r
the radial position of the liquid, neglecting Coriolis forces.
The movement of the buffer fluid 601 into capillary 604 causes a reduction in the
gas pressure in the fluid communication circuit. For appropriate kinematical conditions,
this results in a suction force, pulling the sample liquid 610 from position 615 into the trap
capillary 623, and moving the gas contained in the trap 622 into the reservoir 602 through a
reservoir capillary 624. Suction is the process whereby a force is exerted upon a liquid
body by reason of reduced gas pressure over a portion of its surface.
When the sample liquid 61.0 reaches the trap 622, the centripetal force causes it to
move towards the bottom part of the trap 622. The suction of the reservoir capillary 624 is
not exerted on the sample liquid 610, but on the gas above the liquid mass, so that the
sample liquid 610 contained in the trap 622 does not enter into reservoir capillary 624.
When the entire sample liquid 610 has been sucked into the trap 622, the
communication circuit is no longer gas-tight and atmospheric pressure, through the venting
line 614 or through the input line 613, enters into reservoir 612, trap capillary 623, trap 622,
reservoir capillary 624, and reservoir 602. At this moment, with the centripetal device still
spinning, the buffer fluid moves completely into the exit capillary 604 and the final state of
the reflow operation consists of the sample liquid 610 having moved from reservoir 612 to
the trap reservoir 622.
The reflow operation allows longer processes to be performed in a given centripetal
device. With the trap reservoir 622 at a smaller radial position than the sample reservoir
612, a long process may be broken down as follows: the first series of steps may be
performed by moving liquids from an inner to an outer radial position, comparable to
sample reservoir 612, reflow is then applied to bring the liquid to the trap reservoir 622, at
which point the remainder of the process may be performed, moving again from an inner to
an outer radial position. The number of reflow operations is generally limited only by the
amount of buffer liquid that is loaded onto the disk and its radial position.
The relative radial positions of the sample reservoir 612 and buffer reservoir 602,
and of the trap reservoir 622, are arbitrary. However, a given set of relative radial positions
will determine the minimum mass of buffer liquid for a given mass of sample liquid. The
choice of radial positions may be driven by the required configuration of input and output
ports. For example, input ports may be distributed in a rectangular shape array covering the
top side of the disk, and buffer liquid reservoirs may be used to reflow the input liquids into
traps located at the minimal radial positions available on the disk. Typically, the minimal
radius accessible corresponds to the circumference around the spindle support. The output
of the process, generally available at maximal radial positions, may be transported with the
same reflow procedure into an array uniformly distributed on the top surface of the device,
including the same array used as input.
A functionality similar to reflow, that can be assimilated to reflow, consists in
washing steps during a biological or chemical protocol. The washing procedure is
performed by aspiration of the liquid contained in a reservoir of appropriate shape towards
the inner part of the disk, so that the reservoir, after washing, can still be filled with other
liquids that do not flow out. This procedure is particularly relevant for the execution of
heterogeneous assays, and can be performed by a variant of the reflow method already
explained. It is contemplated within the scope of the invention that the buffer fluid 601 can
be either liquid or gas.
POSITIONING SYSTEM OF THE PICKUP
One aspect of the present invention concerns the setting and knowledge of the
pickup position, at a given time, with respect to the disk reference frame. This position may
be decomposed as a focussing position, a polar position and a radial position. These
directions are the cylindrical coordinates of the pickup head in the reference frame of the
rotating disk, with the rotation axis corresponding to the cylinder axis.
The focussing movement of the pickup head, relative to the base surface, has already
been described and can be achieved by "voice coil" movement of the focussing optics or of
one of the light sources or of any other optical element. Indeed, the focussing mechanism
employed in standard compact disk drivers performs this operation, together with a fine-
tuning movement of the lens in the radial direction.
Radial positioning of the pickup may be achieved by means of the previously
mentioned voice coil, together with a coarse displacement of the pickup assembly.
Different types of motors, including linear motors, DC motors, servomotors, and stepper
motors, may achieve this displacement. Rotating the disk around its axis performs polar
positioning at a given moment of the pickup.
One conventional solution includes using high-resolution optical encoders: a rotary
encoder for the polar position and a linear encoder for the radial position. In addition,
digitally encoded information in the radial and polar directions, stored onto the disk, may be
used to determine where the spot is directed following art, each of which is incorporated by
reference. Gordon (US 6,327,031, US 22085202A1) teaches an apparatus and method for
carrying out analysis of samples; Virtanen (US 6,030,581) teaches a laboratory in a disk;
and
Mian et al. (US2001/0055812A1) teaches devices and methods for using centripetal
acceleration to drive fluid movement in a microfluidics system with on-board informatics.
In one illustrative embodiment of the present invention a method to determine the
radial and polar position of the pickup head is provided. Specifically, in the reference frame
of the disk, the radial and polar position are determined using timing information, as
measured from the occurrence of a signal from the pickup induced by markers, of the
rotating device. The pickup measures (as in commercial CD driver pickups) the light
reflected from the scanned surface;.
A marker generally is a line on the base with specific optical properties, with a polar
position varying as a function of the radius. Examples of specific optical properties are
higher or lower reflectivity compared to the area around the marker. The marker may also
be situated on a side, and may include a capillary filled with a liquid with peculiar optical
properties - defined as including reflectivity, absorption or fluorescent emission. The
change in reflectivity may be detected and provides a signal whose time may be recorded.
This is referred to as the time of a marker signal in the present invention.
If the rotational speed of the device is constant over at least one rotation period, the
marker signal offers a precision measurement of the rotational period of the disk and
therefore of its instantaneous rotational speed. The time elapsed from the occurrence of a
marker signal divided by the rotation period, is in itself the measurement of the polar
position of the pickup with respect to the disk. According to the invention, a solution for a
simpler transformation into polar coordinates is therefore a marker which is a straight line,
where all the points have fixed polar coordinates (polar angle equal to zero) and the
previously mentioned ratio multiplied by two (2) times % indicates me polar angle position
at a given moment.
The addition of a second marker allows the measurement of the radial position,
provided the polar angle difference between the two is a non-constant function of the radial
position. An example non-constant function is the following:
Polar coordinate = radial coordinate * Constant 1 + Constanr2.
Other specific shapes, also non derivable and non-continuous or with zigzag shape,
can.be envisaged typically in order to occupy a limited angular sector of the disk, while
keeping the necessary polar and radial coordinate resolution on the instantaneous pickup
position. With knowledge of the rotation period and the time difference between the two
markers a polar position of the second marker, with respect to the first one, may be
determined. Given the shape of the two markers, the difference in polar position is then
used to determine the radial position of the pickup in the disk reference frame.
According to the invention, the properties of the second marker differ from those of
the first marker such that the two markers may be distinguished on the basis of the signal
produced by the pickup. Suitable properties include reflectivity, width, structure, line
duplication, and the like.
This method assumes that the disk rotates around a fixed and known axis, defining
the origin for the radial and polar coordinates. In practical cases, a removable disk is
subject to misalignments when mounted on a spindle support, and the actual rotation axis
does not necessarily coincide with the expected one. Additional markers may be provided
to determine the actual rotation axis of the disk to address this issue. More specifically, the
measurement of the time difference between markers may be used to verify the assumed
axis position. With more than two markers of known shape, the time difference between
them contains information on the axis position. The axis position may be inferred by
minimizing the difference between measured time differences and time differences expected
on the basis of a given axis position.
This method may also be applied to devices that rotate around an axis lying outside
the device's perimeter. In the case of rectangular shape disks, not only may the relative
position of the pickup head with respect to the disk be determined, but also the disk position
with respect to the rotation axis (including rotation) may be determined on the basis of a
sufficient number of markers on the disk. The number of markers required depends on the
accuracy needed.
TEMPERATURE MONITORING AND CONTROL
Due to the structure of the disk, its temperature may be controlled by application of
an external heat or cooling source. The sides may have transparent or absorbing properties
to thermal radiation and, in particular, to electromagnetic radiation in the infrared or
microwave light spectrum. It is recognized that other heat exchange mechanisms other than
radiation could be used, including convective fluid flow, resistive heating and conduction.
For integrated microfluidic devices, it is often useful to have a means to determine the local
temperature. In particular determining local temperature is useful for rapidly varying
thermal cycles such as those required for the polymerase chain reaction (PCR).
The two-layer structure of the disk may also allow for two facing reservoirs: one
used for the sample fluid whose temperature requires monitoring, and the second one
containing a thermometric liquid. In a preferred embodiment, the thermometric liquid is
based on water or alcohol. Because of the material layer thickness, there is generally a large
thermal conductivity between the two fluid masses, so the temperature of the thermometric
liquid may be approximated as the temperature of the sample liquid. The thermometric
liquid temperature may be monitored as classical thermometers by measuring the (relative)
expansion coefficient of the fluid, with respect to the volume it has at a reference
temperature. Accordingly, the liquid contained in the capillary moves according to the
volumetric expansion of the liquid inside the reservoir, and the determination of its position
provides temperature monitoring.
Alternatively, the pickup light itself may be used for local heating of a fluid. By off-
focusing the pickup light to irradiate a large area of the material layer, the material layer
absorption - or the thermometric fluid absorption if the liquid is chosen accordingly -
dissipates the energy as heat in the sample fluid, producing an increase of its temperature.
Furthermore, the pickup itself may be used to monitor the position of the
thermometric fluid meniscus in a capillary, by evaluating the change of reflectivity
corresponding to the air-liquid interface in the capillary. This evaluation may be performed
by means of the focusing feedback mechanism described above.
ELECTRICAL CONNECTIONS
According to the invention, the base may be used to distribute electrical connections
to different parts and positions of the microfiuidic circuit. Provided the base is an insulator,
various techniques are available to deposit thin layers of conductive materials, including
metals, conductive polymers, conductive inks and graphite. Some of the techniques (for
example electroless chemical deposition of metals) also allow, through photolithographic
techniques, to deposit the conductor in a specific shape and partem, generating electrical
distribution lines. These electrical lines may be used to generate electric fields, for example
for electrophoresis, or to provide electrical power to components present on the disk. The
electrical connections may be powered on the disk itself (micro batteries) or may exploit the
presence of a magnetic field that, because of the disk rotation, induces an electric field on
the conductor generating an electrical potential difference. The magnetic field, in particular,
can be used to induce an apparent electric field onto a rotating disk, the magnetic field being
used or for the generation of electrical currents or to generate an apparent electric field, for
example required in protocols like patch clamp, voltage sensitive probe dyes and
electrophoresis.
Alternatively, the conductors can have an electrical connection on the spindle
support, typically by mechanical contact, which is subsequently connected to the fixed part
of the apparatus by means of brush contacts, through conductors coaxial to the rotation axis
or by conductive liquid connections.
DETECTION DEVICES
One aim of the present invention is to perform programmable, flexible and
automated manipulation of fluids.. In most applications, the detection of reaction products,
meaning (in general) any detection of observable quantities consequent to a process, is of
importance for a practical use of the device.
In the present apparatus, it is possible to use the pickup itself for various operations,
by making use of the readout present in the device to perform the focussing onto the base.
The reflectivity information of the materials present on the focal point of the pickup may be
used not only for the scope of valves and disk operation, but also to produce data
concerning the fluidic process.
In another illustrative embodiment of the invention, the reflected light may be
associated with the position (in space) of the pickup, to generate images by using the pickup
as a con-focal microscope. Bi-dimensional images may be easily constructed by varying
the radial position of the pickup during spinning, and collecting the data from the pickup,
for example, by digitisation. Three-dimensional images (via the con-focal properties of the
optics) may be constructed using the focusing movement of the pickup, and varying the
distance of me pickup from the base. Due to the low depth of focus achievable by con-focal
optical systems, three-dimensional images of the fluids (and of the objects contained in the
fluid that have a size and optical properties which are detectable) may be collected and
stored for analysis purposes. Counting methods, for example applied to cells present in the
fluid, are possible and profit significantly from volume scanning to increase the statistical
significance of the data present in a small volume sample.
In yet another embodiment of the present invention, the disk is essentially a flat,
typically transparent, thin substrate containing fluids which may include additional devices.
These additional devices may be used to collect information on the fluids contained in the
disk. These devices could be biosensors, transducers or arrays of tissues, cells and
molecules. Standard well plate reader scanners, for example, may collect information on
the optical properties of the fluids contained in the disk in a vast range of the
electromagnetic spectrum, with the aim of colorimetric analysis, fluorescence detection, and
radioactive emission measurement.
In yet another embodiment, the disk can be used as an optical medium where the
light is collected and transferred by internal reflection, possibly using prisms, lenses or other
micro optical components integrated onto the surface.
Another possible readout technology relies on the loading, during the manufacturing
process, of the sides or of the base material with scintillating dyes. A radioactive activity
related to the assay is converted into light signals inside the scintillating material, and the
light intensity is used as a measurement of the radioactivity of the sample. The same result
can be obtained by loading liquid scintillator in a microfluidic component facing the
sample, and separated by it only by the base.
It is contemplated within the scope of the invention that off-board detection may be
utilized. Examples include mass spectrometers, irradiation with gamma, x-ray or neutron
beams, and chromatography. Removable components within the microfluidic network, such
as peel away MALDI targets foils or the like can be incorporated into the sides of the
inventive disk. These peel away target surfaces can be advantageously positioned so that
they form sides of chambers used for collection of a sample or samples of interest.
MIXING
In microfluidic devices, the fluid dynamics are typically dominated by laminar flow.
In this sense, mixing - which is natural in the macroscopic world because of different
phenomena like convection or turbulent motion - constitutes a critical issue. According to
the invention, various mixing methods may be used. Magnetic beads may be transported in
the fluids through capillaries, and can be agitated from the outside by the presence of a
static magnetic field when the disk is spinning (or via a variable magnetic Field). Another
method makes use of material layer's elasticity; having the material layer facing a vibrating
reservoir. The vibration may be achieved in different ways: mechanically or induced by
external electric or magnetic fields.
Another method according to the invention makes use of variation of the angular
speed and direction of the rotating disk, including the generation of vibrational and torsional
modes at resonant rotation frequencies.
A further method consists in the use of the Coriolis force to generate turbulent flow
inside the channels of the disk.
Alternatively, the fluid may be circulated, by changing the rotational speed of the
disk, in alternated directions along the capillary. The reverse direction may be easily
obtained by a pneumatic force generated from air (or gas) compressed into a reservoir that,
when the rotational speed of the disk is reduced, returns the stored energy to the fluid.
Alternatively, simple diffusion can be very effective for appropriate geometry of the
microfluidic components (for example, in capillaries).
The valve could also have an active role in mixing. Taking alternatively small
amounts of two fluids to be mixed into the same reservoir or capillary increases the surface
of the interface and therefore mixing by diffusion. Short plugs of multiple fluids could be
alternated inside a capillary for the purpose of improved mixing efficiency by using the
output of the dosimeters.
FLUID TRANSPORT ACTIVATED BY A VALVE
The valves of the present invention have the remarkable feature to withstand large
differences of pressure and to be gas tight. Therefore it is conceivable to have a pneumatic
overpressure or underpressure on one side, which is followed by a sudden gas flow when
the valve is opened.
A pneumatic overpressure can be easily made by means of a closed reservoir
containing a volatile liquid or alternatively, by a chemical reaction between one or more
components releasing gas, for example carbon dioxide. In another embodiment, the
pressure could be generated by means of the centripetal force, compressing a liquid mass
over a confined gas volume. In the last case, it is possible to store the energy for a time
longer than the duration of the centripetal force by having the fluid entering into a Tesla
valve that limits the motion of the fluid backward when the centripetal force is released.
Such a system is hereafter called bottle. The volatile liquid, for example water, can be
heated by means of the laser light in order to produce - by radiative energy transfer - a
given amount of vapour. The connection of the bottle to another circuit, by opening a valve,
will generate a pressure transient in the second circuit. With a multiplexer connection, a
bottle could be put in pneumatic connection with one among a number of circuits. Once a
valve is opened, the bottle is exhausted.
The liquid in the second circuit can be connected to a calibrated capillary - called
output nozzle- exiting from the surface of the chip. By opening the valve, the liquid is
forced to flow through the nozzle, and calibrating the amount of vapour produced it is
possible to avoid the "spraying" of the chemical. The outcome is a collimated liquid jet
exiting from the surface of the chip.
The sample disk can be piled above another one, called receptor disk, which has an
input nozzle in correspondence of the output nozzle of the sample disk. The input nozzle is
a hole connected to a capillary and able to collect the liquid. As an alternative, another
bottle on the receptor disk could be used to suck the liquid by Venturi effect into a capillary,
or by having a vacuum bottle with an underpressure aspirating the liquid into the device.
The same method could be used in order to transfer fluids to and from devices with a
different shape and purpose, such as microtitre plates, microfluidic devices with different
functionality, analytical instruments or any device meant to modify the fluid properties (for
example the fluid temperature). It should be noted that the technique of storing energy by
means of a valve, energy that can be released in a controlled and desired manner, could be
used in order to generate transient fluid flows with the consequence of mixing.
EXAMPLES
The following examples are provided to illustrate the methods and products of the
present invention with particular choices for the several components and particular values
for the several variables described above. As described above, many variations on these
particular examples are possible. These examples are merely illustrative and not limiting of
the present invention.
Example 1
The optical feedback for focusing according to the present invention has been
implemented to assess the correct positioning onto a material layer 701, as depicted in FIG
7. Turning to FIG. 7, the optical feedback makes use of a simple glass 702 (about 0.199
mm thickness) that intercepts a few percent of the light reflected from the material layer 701
(through the same optical system used for the light incident onto the base). The light from
the material layer 701 is imaged through a 48 mm focal length objective 705 onto a CCD
706. The CCD 706 records the actual shape of the laser spot onto the material layer 701,
and can even image the material layer surface and - for example - beads floating in the
fluid nearby the material layer.
It is contemplated within the scope of the invention that optical feedback can be
achieved by the use of Astigmatic focusing. It is further contemplated within the scope of
the invention that the laser junction image can be magnified or demagnified according to the
ratio of the condenser focal length in the setup (currently 3.1 mm) and the CCD objective
(48 mm). The CCD 706 also records images from the material layer 701 with a
magnification of about 20X, a region of about 200 x 150 microns with a pixel resolution of
0.3 urn. It is contemplated within the scope of the invention that the CCD 706 can be
replaced with a diode (probably a matrix of 2x2 diodes to perform also astigmatic focusing
with the same system) because essentially of a speed issue related to the focusing feedback
(lock and tracking of the focus).
When working with a microfluidic platform such as a disk or chip, it is evident that
three surfaces can be detected by this feedback method: the external surface of the side
where the platform is in contact with atmosphere, the internal surface of the side 708 where
the platform is in contact with the Quid (gas or liquid) contained in the capillaries (or
reservoirs), an interface 710 between fluid and the material layer 701. This interface 710 is
used and detected for focusing. It should be noted that - due to the very limited
transparency (0.02% is the measured transmission for a material layer thickness of 10
micrometer and an Epolight 2057 dye concentration of 1% by weight in PMMA) of the
material layer at the laser wavelength - whatever is behind the material layer 701 does not
affect reflections of the laser light, but it is only detectable in transmission mode.
Working at low fluence, it was verified that the gradual base melting, and material
layer 701 modifications, can be imaged by the same system, so to assess empirically the
light density and temperature of the various parts of the elliptic spot.
Extending this concept, it was verified that it is possible and easy to detect if a valve
according to the invention has been opened or not. When a laser spot was correctly
focused, the entire region exposed to the laser radiation was ablated, and there was no
material left, in focus, to reflect the light through the feedback optical system. If the
material was not fully ablated, then the polymer forming the material layer 701 remaining in
the optical path produced a reflection that was easily measured.
It was determined in real time if the inventive valve was correctly opened or if it had
not been opened, and possibly repeating the opening step (for example, at the next turn of
the disk) if necessary. It was found that the valve reproducibility, was better than 1/1000,
meaning that less than one over a thousand valves has possible problems in the fluid
passage (verified by optical inspection). The optical feedback, allows for quality assurance
of the operation of the inventive valve.
It was found that instead of fixing the energy of a shot, and perforating for a
corresponding fixed time at a given power, it was possible to modulate the laser emission
according to the feedback. The laser light was maintained up to the moment reflected light
from the material layer disappeared, and then the laser was switched off. Advantageously,
optical feedback allowed the laser irradiation to be reduced to a minimum, therefore
reducing the amount of energy going into the system minimizing destruction or alteration of
sample. Using optical feedback, the laser MTTF was improved significantly, as it is related
to the temperature of the laser junction that increases significantly with the exposure time.
Using optical feedback, it was possible to increase peak power of the laser, reducing the
average shot pulse length. This allowed reducing even further the size of the heat transport
region (whose diameter goes with the square root of the pulse time length); it also
guaranteed that a valve had been correctly opened..
Example 2
Performance of the optical setup according to the invention can be characterized by
the following example. The optical configuration is such, the energy of the beam after the
CD lens and integrated all over its pupil amount to 16 uJ released in 10 us, corresponding to
1.6W optical power. As expected, the original laser diode power of 6.2W was reduced
because of collimation, matching and reflections in the optical setup.
When an 8 µm material layer of PMMA from Microchem loaded with Epolight 2057
was put on the CD lens focus, and a first shot was made, only approximately 7.6 µJ
emerged from the base onto a pyrometer positioned behind the material layer. Neglecting
reflections, which were expected to be about 4%, the remaining 8.4 microjoule were
therefore deposited into the sample. As a reference, if the energy was uniformly deposited
into a 1 microlitre water sample, its temperature would rise only approximately 0.0018 C
degrees. However, the energy was sufficient enough to fuse the polymer volume
corresponding to the valve area (3 pL), computed at 7.5 uJ.
A second shot on the same position, showed that all the beam energy was measured
on the pyrometer at the rear of the material layer. This measurement indicated that all the
light was concentrated onto the valve surface and that increasing the shot duration did not
release energy into the sample because the material layer was not absorbing it anymore
since the light was passing through the base valve.
As shown in FIG. 8 these above results are compared with data corresponding to a
DVD configuration where a DVD optical pickup was used. In this case, the optical
configuration was not optimized and, because of misalignments, aberrations and coma, part
of the laser energy was not collimated onto the material layer in a concentrated spot. In this
case, the full beam energy was not restored since it was still hitting the material layer that
did not vaporize at low energy density.
Example 3
Performance of the laser according to the invention can be further understood with
reference to the following example. The source of Laser emission used was the OSRAM
SPL PL90_3 diode, having nanostack technology. The nanostack technology consists in the
"vertical" or epitaxial integration of a number of discrete emitters on a semiconductor chip,
and this produces a two to three times increase in maximum power. The specific diode
exhibits an aperture of 200x10 microns, from three overlapping emitters that reach an
optical output of approximately 75 W when limited to 100 ns pulse-lengths. The diodes
were pulsed by means of a. DEI PCX 7410 diode laser driver, from Directed Energy Inc.
capable of covering the regime 20 ns to 1 (is at 10A, and 5 A in CW mode. To access the
regime above 10A, a DEI PCO 7120 hybrid OEM driver was employed. The pulse voltage
and current was monitored by a Tektronix TDS2014 to reconstruct the electrical power onto
the diode, and extrapolate its optical output on the basis of the diode specifications.
Both the condenser and the objective can be chosen among aspheric lenses (as those
used in optical disks systems) and glass multiplets optimized to operate in the near infrared
region (700-1100 nm). The incident beam was monitored by means of a Melles Griot (MG)
wincamD CCD triggered by the laser diode driver, which intercepts the beam by means of a
semi-reflective window. The beam was collimated into the objective and tuned by means of
various Logitech QC 4000 Pro CCDs, which monitored the beam spot size onto the
objective lens, the impact point onto the sample and the light reflected backward from the
sample.
In a preferred configuration, the objective is a CD lens with its voice-coil actuator
that can be moved along two axes by means of current control. This configuration allowed
optimizing optical conditions and permitted shot by shot verification that the laser beam is

in optimal configuration and also permitted prints of the laser diode emitter showing the
nanostack structure onto the sample as shown in FIG. 9. The nanostack structure is visible
as three overlaid narrow slits 901, 902 and 903, which correspond to the three emitting
junctions of the PL90-3 laser diode used in the experiment. The sample under test was a
polyethylene (PE) film from a commercial disposal bag having a thickness of approximately
20 µm. The film was characterized by a high loading of carbon-black. The sample was
exposed with a 6.5 mm objective (NA=0.615) MG 06GLC001 and a 25.6 mm condenser
(NA=0.156) MG 06GLC004 showing evidence of base perforation. The diode laser
parameters were 1=10A with a pulse-length of 100 microseconds, the focusing was
performed by observing diffraction rings in the forward direction without direct observation
of the laser spot on the base. The estimated optical energy of the pulse was below 3µJ,
which is an upper limit that depends on the diode temperature and power degradation that is
expected to occur for this diode in this particular pulse-length regime. It was found that
most of the light was detected behind the base and was not absorbed by the sample material.
The perforation entrance holes 1001 are depicted in FIG 10. The perforation exit
holes 1101 in FIG. 11. The pitch between the entrance and exit holes is approximately 174
microns allowing for an estimation of about 52 microns for the minor axis and 57 microns
for the major axis on average.
Example 4
A solution of PMMA from Microchem of having a molecular weight of
approximately 950,000 Dalton was dissolved at 11% in anisole and spin-coated onto a
silicon substrate treated for detachment of the resulting film. The film was dried at about 20
degrees for approximately 24 hours. The spin coating technique resulted in a film having
thickness homogeneity of approximately 1 micron over a 4 inch wafer. The surface
roughness, measured by alphastep, was approximately 39.6 nm average roughness value
and approximately 53.8 nm root mean square roughness. These mechanical properties of
the PMMA film were matched by its total transparency to infrared light, so that its exposure
to the laser emission did not lead to any observable effects.
A further PMMA film was produced with a loading of approximately 0.5% by
weight of ADS905AM, an infrared dye for American Dye Source Inc., whose absorption
spectrum is shown in FIG. 12. The film was optically perfect to visual inspection but a
microscopic analysis revealed that the dye was not uniformly dissolved into the PMMA.
Upon microscopic analysis it was found that the dye was in sort of an "emulsion" form or
dispersed as non-uniform globules 1301 as depicted in FIG. 13. Despite this lack of
uniformity, no observable laser light was transmitted through the dye loaded film.
Upon exposure to a single laser shot of 100 ns, at 40A, the dye loaded film produced
energy loss into the dye globules. While the non-uniform globules 1301 at the surface often
exploded, no penetration at this exposure occurred.
Upon exposure to multiple laser shots, having a configuration as set forth in example
3, at a frequency of 1 KHz, visible penetration was observed. Such penetration did not
achieve the sample depth of 20 microns; however, the laser light starts to be transmitted
through the polymer foil. This transmission possibly indicates degradation of the dye
absorption in the irradiated region. Without being bound by any particular theory, this
effect is thought to be due to heat generation and subsequent thermal degradation of the dye
molecule (C62H96N6SbF6).
It was found that single laser shots of 10 us duration and 10A current produce
openings that are passing through only when accurately focused. The laser setup used to
produce the laser shots as within this example made use of a commercial CD pickup
objective that required manual focusing at small power and exploited a laboratory grade
25.6 mm condenser as used in the laser setup. The hole diameter was about 20-25 microns
(minor axis) by about 30 microns (major axis). The hole configuration exhibited memory of
the laser aperture shape on the entrance side. This memory of the laser shape is neither a
problem nor limitation. It was found that when the laser focusing was not perfect, the holes
are often not passing-through. It was also found that by increasing the laser shots to 20
microseconds would be enough to effectuate a passing through. It was concluded that a 10
(is laser shot at 10A is sufficient for perforation in these conditions provided the laser is
correctly focused, the optical dye is evenly dispersed and the material layer is about 8
microns.
Example 5
The following experiments were designed in order to maximize the damage to
different biological samples induced by the VLV (Virtual Laser Valve) opening. This was
accomplished by the following strategy: Maximize the number of valves in a given volume
of fluid; increase the laser shot energy to a value which is significantly higher than the one
expected for the prototype/product; minimize the amount of biological sample used in the
experiment which is not exposed/affected by the VLV damage; and having different means
(redundancy, calibration samples, and statistical tests) to assess the correctness of the
assumptions and to validate the experiment consistency.
The first objective implied a large VLV density, and the capability to open a large
number of VLV in a short time (below 30 minutes). Matrices from 100 VLV/mm2 to 600
VLV/mm2 were produced, the last value corresponding to the condition where the base is
destroyed (cut) by the laser within known parameters.
The Laser parameters were kept, during all the experiments, equal to 100 us shot
producing .160 µJ of optical energy, largely sufficient for perforation with good margins.
Fluorescent beads, mixed at a known concentration in the samples, were used for a
quantitative check of the dilutions and sample recovery efficiency in most of the
experiments.
The samples were exposed in two main configurations: inside an experimental chip
and in the so-called "drop" configuration. In all cases, the material layer was identical in
thickness and dye loading.
The data was presented in a unique manner: the calibration samples were averaged
and renormalized to 1 (100%), independently for beads and the biological measurement on
the calibration sample. Every result was expressed as the relative amount of material
collected when the sample is exposed to the valves divided by the corresponding reference
unexposed sample. The term "loss" corresponded to the relative difference (REF-
VLV)/REF, which was a positive number in case of a biological loss or damage, and a
negative number in case the exposed sample had more material than the reference sample.
Chip experiments description
With the exception of the pro-insulin exposures, one micrometer diameter YC
carboxylated fluorescent beads (Polybeads from Polysciences, Warrington, PA, USA) were
added to sample in order to achieve, after final dilution, a concentration of 50 beads/µL.
The beads were used also to verify in qualitative terms the sample inside the chip, and to
monitor the chip rinsing by means of a fluorescent microscope. Calibration samples, as
well as negative samples, were produced by standard dilution techniques.
An experimental chip 1400 used according to this example is represented in FIG 14.
The misalignment of the two sides and the fact one side is tilled with fluorescent beads
helps in disentangling the configuration in depth. As shown in FIG 14, a sample was filled
into an inlet 1401 by means of a peristaltic pump (not shown, from Ismatec) operated
around 5 µL/min through a Tygon tubing of 0.19 or 0.25 mm inner diameter. Previous
experiments had shown that the tube did not induce damage to our samples. In each
experiment new tubes were used to avoid cross-contamination. In most cases, the chip
1400 was filled with fluid without bubbles; in the few cases where these were present, the
actual fluid volume was corrected by means of measurements taken on images taken with a
camera mounted on the microscope. Only one side of the chip 1400, in this case a first side
1402 was filled with fluid.
An important requirement of the filling procedure consisted in being confident that
all fluid (sample) was removed from the inlet 1401 and an outlet 1404 of the chip 1400.
This was accomplished by pipetting and subsequent inspection with fluorescent
microscopy that no beads were present in the connection reservoirs. If fluid was detected,
it was pipetted out of the reservoir up to the absence of fluorescent beads. The
determination of the nominal volume of the chip 1400 was, by itself, very difficult.
Because of the design of the chip 1400, the reservoir filled with liquid had a nominal
volume of approximately 3000 x 1400x50 µm.
As a result of the chip 1400 design, two undesired phenomena occur: on one side,
the pressure generated by the pump on the fluid can be very large (the ratio between the
reactor section and the input capillary cross section is about 80X, meaning that the force
applied onto the base in the reactor is SOX larger with respect to the base in the capillary).
In addition, a movement of the material layer by 10 µm in depth determines a change of
20% in the nominal volume of the reservoir. It was determined that the base was moving
during the filling process when the inlet pressure was applied. Because of this base
movement, a large pressure was applied onto the material layer, so to have it touching the
opposite side surface and determining an absolute reservoir volume of about twice the
nominal one. Care was taken to treat the samples identically in the two filling operations.
When the chip 1400 was not exposed to the Virtual laser Valve (VLV), the rinsing
procedure consisted of removing the sample by transporting an amount of buffer between
50 and 400 µL into the chip 1400. A buffer was inserted by applying a positive pressure to
the inlet 1402 and collecting the fluids from the outlet 1404. The rinsing process was
typically done by a series of these operations, separated by few minutes when the chip
1400 was completely empty of fluids. The rinsing speed was limited to about 50 micro
litres per minute because of the capillaries' cross section. In the capillaries, this flow
corresponds to a fluid speed of 0.3 m/s (1 km/h). The fluid speed inside the chip 2200
hardly achieved 0.4 cm/s, and this explained the long rinsing process (from 30 minutes to 1
hour) required in order to observe one order of magnitude drop in the beads content of the
fluid - and similarly for the biological sample concentration.
The chip filling for the exposure was kept as similar as possible to the calibration
filling, and the same criteria in the analysis of the beads content applied.
The VLV exposure was performed in some cases "downstream" of the laser
direction, and in some cases "upstream".
The rinsing of the chip 1400 exposed to the VLV differed from the one previously
explained since all four inlets were in fluidic communication. The outlet 1404 connection
was connected to the peristaltic pump (independent channels) and fed with the buffer. The
fluid speed was kept from 10 µL per minute to about 40 µL per minute.
In experiments where beads were present, 2 drops of 1 µL from each final
eppendorf tube were taken and deposited onto a labelled microscope slide. The drop was
allowed to gently evaporate, with the consequence that all the beads contained in the drop
were collected onto the flat surface of the glass inside a small perimeter. An image of the
beads was taken, and beads were blindly counted by means of Scion Software package.
Both drops were systematically used in the analysis order to check possible errors in the
process.
Drop experiments description
The analysis of the previous experimental procedure has indicated the possibility to
eliminate systematic errors by a simplified experiment consisting of sample preparation,
fluid handling double drop preparation, single drop valves exposure, double drop collection,
bead measurement and biological processing.
The drop exposure was often performed together with the chip experiments, to
validate the result through consistency analysis. For this reason, the sample preparation was
identical, with the precautions of "equalizing" the expected results (similar final
concentrations).
The double-drop preparation consisted of putting a 1 µL drop onto a piece of
material layer of about 4x4 mm2 suspended onto a glass frame by its corners. The comers
were attached to the glass by means of a minute drop of water, which generated attraction
between the two surfaces. The sample drop had a circular shape and was deposited with a
tip. Using this method, the drop never reached the edges of the material layer nor any other
material. It was noticed that all drops made with the same buffer/sample were similar and
attached to the material layer with a consistent contact angle determining its shape.
However, it was found that the biological sample, its concentration and the buffer fluid
introduced a large variability on the drop shape. The drop had a contact area of about 2
mm2 in the case of pro-insulin in water buffer at 7 µg per µL, and a contact area of about 1
mm2 for the E-coli culture medium with a concentration of about 1 E-6 E. coli per uL.
The single drop exposure consisted of choosing, randomly, one of two drops and
exposing it to laser radiation. Since the drop surface was substantially proportional to the
drop volume (tested with drops of 1-2-3 µL and camera images) there was an overall
constraint in the maximum number of valves that could be opened given by the available
area.
The double drop collection consisted of removing the material layer samples with
the drop from the glass frame, and putting each sample into an eppendorf tube. The
material layer was rinsed inside the tube by means of an inox clamp and accurately cleaned
before each exposure to avoid cross-contaminations. The final eppendorf tube contained a
buffer volume between 50 and 400 µL, where the base was immersed.
The experimental procedure was tested with beads, and it was noticed that after a
VLV opening, the fluid entered into the aperture and filled the VLV volume. Surface
tension avoided that the fluid wets the surface opposite to the drop.
Using the above procedures, an experiment aimed to test the viability of ampicillin
resistant Escherichia Coli {E-Coli) bacteria exposed to the effects of the virtual laser valve.
Bacteria were provided in solution for testing purposes and returned for evaluation also in
solution. Triplicate plating, at different dilutions, was performed for each of the returned
samples. The original bacteria concentration was kept at about SE5 E-Coli/µL, in a culture
buffer and the sample was mixed with beads in the same buffer at a concentration of SO
beads per µL.
The experimental chip, similar to chip 1400 as shown in FIG. 14, had a 2000 x 2000
urn nominal reactor. The chip 1400 was filled without evidence of clogging and
substantially no bubbles were evident in any of the relevant steps. The rinsing strategy
consisted in 4 rinsing steps of 100 µL each, in order to observe and measure the beads/E-
coli content in the samples.
The drop experiment was performed in quadruplicate and the material layer was
removed from the eppendorf before it was sent for analysis. Two negative samples, as well
as two calibration samples were present in the dataset. The calibration samples were diluted
in order to generate the same colony counting of the chips experiment, in. the hypothesis of
310 nL volumes (nominal) and perfect rinsing.
As shown in table 1 below the data was renormalized in both columns to the average
of calibration and calibration II. The corresponding numbers are 181.5 colonies and 43.3
beads respectively, in line with expectations.
The colonies were plated in duplicate at different concentration and both plates were
consistent. The same parameters applied to the beads.
Both the calibration and negative samples were fully compatible regarding beads
and colony counting. Since beads counting resulted from the average of two drops
containing approximately 50 beads, the statistical error of 10% was expected.
The NOVLV drops could be considered as an additional calibration sample. In
terms of the colonies the average was 89%, while in terms of beads the average was 101%.
The chip rinsing data is depicted in FIG 15. The samples refer to 100 µL data points
that translated into a rinsing decay constant of 157 µL. There was no striking difference
between beads and colonies suggesting that the rinsing is essentially independent from the
type of particle to be rinsed. Less, than 7% of the samples were expected to remain in the
chip and therefore since this amount was less than the experimental error there was no
correction as a result.
The physical chip volume was estimated by imaging the chip and was found to be
approximately 520 nL. This volume included capillaries and base bending onto the side.
The volume corresponded to 167%. The integral counts from the chips are as follows:
Integral reference Data 185.67%
Integral Reference Beads 173.41%
Integral VLV Data 145.45%
Integral VLV Beads 124.86%
Well matching the expectation for the NOVLV data. The measurement of the loss
comes from the VLV/NOVLV ratio and corresponded to a loss of 28% for the bead and
22% of the bacteria. The reduction of the beads and bacteria viability suggested that the E-
Coli were damaged by the VLV similar to the beads. This result was independently verified
by the drop experiments. The ratio between unexposed drops and exposed drops is
graphically depicted in FIG. 16, both for E-Coli and beads.
Data showed a consistent loss of 10% of the beads when the drop was exposed. E-
Coli loss was equivalent in the sense that on average 15% less E-Coli were present in the
exposed drops with respect to the unexposed drops. It was concluded that every 10X valve
appeared to reduce the sample by less than 0.7 nL inside the chip and less than 0.9 nL in the
drop experiments. Therefore there was a loss of 0.83 nL per valve for E. Coli and 0.79 nL
per valve for the beads.
Example 5
The resistance of DNA plasmids coding ampicillin resistance to damage by the
virtual laser valve was explored. Tins resistance was determined by measuring the
resistance of cells to ampicillin after being transfected with the same material. Sample
DNA at high concentration was provided in a TE buffer and the sample was mixed with
beads at a concentration of 50 beads per microlitre. The chip used in this example was a
2000 x 2000 uM nominal reactor and the rinsing strategy consisted of two rinsing steps of
400 µL each. The drop experiment was performed in triplicate and the base was removed
from the Eppendorf before it was sent for analysis. All samples used were diluted into a
400 µL buffer volume.
A negative sample as well as a calibration sample was included in the dataset. The
calibration sample was diluted to generate the same colony counting of the chip experiment
and the hypothesis of 310 nL volume (nominal) and perfect rinsing.
The following data as shown in table 2 below was renormalized to the calibration
sample according to the general procedure set forth in the above examples. The
corresponding counting for the calibration samples is 336 colonies of transfected cells.
Transfection was performed in duplicate and in the case of "Calibration", NOVLV Chipl",
"VLV Drop 1" and NOVLV Drop 2", the transfection in duplicate was repeated a second
time.
Table 2
Calibration 100.0%
Negative sample 0.3%
NOVLV Chip 1 304.5%
NOVLV Chip 2 7.7%
VLV Chip 1 188.1%
VLV Chip 2 12.8%
VLV Drop 1 83.3%
NO VLV Drop 1 81.3%
VLV Drop 2 67.6%
NOVLV Drop 2 66.4%
VLV Drop 3 44.0%
NOVLV Drop 3 51.2%
The negative sample matched expectations. Additionally, the rinsing of the chip
was performed with 400 µL volumes instead of 100 µL and the data for the second rinsing
step matched the expectation from previous data.
The physical chip volume was estimated by imaging the chip to be 1.65% in the scale
expressed in table 2 and the integral counts fro the chips were as follows:
Integral Reference Data 312.20%
Integral VLV data 200.89%
Notably, the amount of DNA extracted from the unexposed chip was about a factor
twice as large as expected. The hypothesis of a change in the transfection efficiency was
dismissed by the repetition of the transfection step, done in the same time for the calibration
sample and for the dominant point (NOVLV Chip 1).
The face value ration between the exposed and the unexposed chip points to a 35%
loss of DNA, however, the exposed sample has a counting rate of colonies compatible with
expectation from the geometrical volume of the chip.
The results of the drop experiments are depicted in FIG. 17. The average result
suggests a 3% loss.
Example 6
This experiment consisted in producing one VLV drop, one NOVLV drop and one
microlitre calibration sample of human pro-insulin at 7 microgram per microlitre. The
drops on the base were immersed in a 50 µL buffer. Some problems were experienced with
the behavior of the protein with the tips used, since the affinity of the fluid with the material
of the tip was significantly higher man expected. The drop exposure was modified in order
to avoid possible contacts of the backside of the drop with parafilm. The final eppendorf
tubes were sent for analysis still containing the base samples exposed. No beads were used
in the process.
Approximately 1.3 µL of the sample solution was used for HPLC injection and
HPLC was performed in triplicate for each eppendorf. The peaks shape was analyzed and
no evidence of differences between the three samples was found. It appeared that no
alteration or modification of the sample protein occurred. Further analysis was performed
using quadrupole MS and confirmed the HPLC conclusion.
Example 7
Base loading optimization has been studied by varying the shot energy by means of
a different laser shot pulse length, with the aim to find the perforation limit for bases of
different materials, thickness ana absorption properties. The setup is substantially
optimized for efficient light collection and focusing precision (CCD imaging of the focusing
spot by analysis of the epi-reflected light from the base). Once these limit conditions are
found, an absorption measureme t of the light for the various samples has been made, using
the same laser light source but in a smaller intensity, and measuring the transmitted energy
by means of a PEM 100 pyrometer by Lasertechnik Berlin, Germany. The data is reported
in the following tables, including the minimum laser duration (in the same conditions) that
was necessary to observe the film perforation. It is visible that the minimum laser
perforation conditions are in qualitative agreement with the expectation based on the base
absorptivity and the laser energy, the laser spot size being identical in all experiments.
The following table shows the correlation between absorption properties and
perforation conditions. Different materials and different dyes, both in type and
concentration, have been subject to decreasing intensity of radiation by reducing the pulse
duration of laser emission in identical conditions. Once found the minimum impulse time
for perforation, a transmission measurement has been performed by comparing the laser
intensity (accurately reduced in order to avoid perforation of the layer or damage of the dye)
with identical material once loaded with a dye and without dye loading. It is evident from
the table below that both materials and dye loading affect the perforation limits in identical
irradiation conditions.
Although the inventive valving device is described with a rotating platform
dependant upon centripetal forces, it will be appreciated by those skilled in the art that such
a valve may be used on any microfluidic device were it is desirable to use valving
components. Likewise, it will be further appreciated that the inventive valving device can
be adapted to larger scale analytical devices, with an increase of the overall laser intensity
for larger valves, for example by means of laser diode bars. Likewise, it will be understood
by those skilled in the art that this valving technology can be applied even for smaller scale
devices, in the domain of nanotechnology. In fact, it is evident that the electromagnetic
emission can be reduced down to diff action limited spots, and the valves could be a
fraction of the illuminated spot. Valves in the nanometer range are possible, compatibly
with the molecular structure of the material layer involved.
Although the material layer within the inventive device utilizes dyes having certain
spectral qualities, it will be appreciated by those skilled in the art that other compounds or
particles having desirable absorption properties may be used to capture electromagnetic
radiation in order to perforate the material layer. Likewise, it will be further appreciated
that films or layers having desirable absorption properties may also be used to capture
electromagnetic radiation.
Although the electromagnetic radiation is used within the inventive device to
perforate a material layer, it will be appreciated by those skilled in the art that such
electromagnetic radiation can be used to sublime or melt crystalline structures used for
valving purposes.
Although the inventive valve used within the specification and examples concern the
valving of fluids, it should be appreciated by those skilled in the art that the inventive valve
may be used to valve gases or gaseous fluids. Likewise, it will be further appreciated that,
many applications, for example fuel cells, thrust control in aerospace application, mixture
control for combustion or the like, can utilize the inventive valving technology.
Having now described a few embodiments of the invention, it should be apparent to
those skilled in the art that the foregoing is merely illustrative and not limiting, having been
presented by way of example only. Numerous modifications and other embodiments are
within the scope of ordinary skill in the art and are contemplated as falling within the scope
of the invention as defined by the appended claims and equivalents thereto. The contents of
any references cited throughout this application are hereby incorporated by reference. The
appropriate components, processes, and methods of those documents may be selected for
the present invention and embodiments thereof.
We Claim :
1. An apparatus for regulating fluid flow in microstructures comprising:
a first substrate such as herein described comprising a plurality of first
fluidic components;
a second substrate such as herein described comprising a plurality of second
iluidic components corresponding to the first fluidic components;
a material layer such as herein described separating the plurality of first
fluidic components from the plurality of second fluidic components and
electromagnetic generaling means for generating an electromagnetic
radiation for directing onto the layer of material at a position corresponding to a
portion of the layer located between at least a pair of corresponding fluidic
components from the plurality of first fluidic components and the plurality of
second fluidic components said electromagnetic radiation causing perforation of
the material layer at the position allowing fluid communication between at least
a pair of fluidic components.
2. The apparatus as claimed in claim 1. wherein said electromagnetic radiation is selected
from the group consisting of infrared, visible and ultra-violet spectrum.
3. The apparatus as claimed in claim 1. wherein said electromagnetic radiation generating
means is selected from the group consisting of a laser, compact disk drive pickup and digital
versatile disk drive pickup.
4. The apparatus as claimed in claim 1, wherein said material layer has a thickness from
0.5µM to 100 µM.
5. The apparatus as claimed in claim 1. wherein said material layer is selected from the
group consisting of polymer foils and metallic foils.
6. The apparatus as claimed in claim 1, wherein said material layer is a foil formed from a
material selected from the group consisting of polymers, copolymers, monomers, metals, waxes.
polysaccharides and liquid crystal polymers.
7. The apparatus as claimed in claim 1, wherein said material layer is formed of a
polymeric material loaded with a dye.
8. The apparatus as claimed in claim 7, wherein said dye has optical properties that are
substantially matched to said electromagnetic radiation.
9. The apparatus as claimed in claim 1, wherein said material layer is treated to
substantially absorb said electromagnetic radiation, said treatment is selected from the group
consisting of dye loading, chemical surface treatment, chemical loading, optical interference and
optical polarization.
10. The apparatus as claimed in claim 1, wherein said materia] layer is formed of
multiple layers having selected absorption properties and. wherein said absorption properties are
responsive to said radiation.
11. The apparatus as claimed in claim 1. wherein said material layer is formed of a
polymeric material selected from the group consisting of Poly(methyl methacrylate) (PMMA),
Low Density Polyethylene (LDPE), Linear Low Density Polyethylene (LLDPE), High Density
Polyethylene (HDPE), Polyethylene Teraphathalate (PET). Polyethylene (PE), polycarbonate
(PC), Polyethylene Terephthalate Glycol (PETG) . Polystyrene (PS), Ethyl Vinyl Acetate (EVA),
and polyethylene napthalate (PEN).
12. The apparatus as claimed in claim 1. wherein said first and second substrate are in
the form of a disk wherein rotation of said apparatus produces a centripetal force on a fluid
contained in one or more of said first and second fluidic components, said centripetal force
causing fluids to move to an outer radial position.
13. The apparatus as claimed in claim 1, wherein the substrate materials are selected
from the group consisting of polymers, monomers, co-polymers, resins, ceramic, glass, quartz
and silicon.
14. The apparatus as claimed in claim 1. wherein said first and second substrate
optionally contain optical components selected from the group consisting of lenses, mirrors and
prisms.
15. The apparatus as claimed in claim 1. optionally containing at least one more
additional substrate and at least one more material layer.
16. The apparatus as claimed in claim 1, wherein said radiation has a desired intensity
from 1 4J to 100 4J and exposure time from 1 microsecond to 100 µM.
17. The apparatus as claimed in claim 16. wherein said desired intensity and exposure
time is 10 µJ and 10 us respectively.
18. The apparatus as claimed in claim 16, wherein said radiation has a pulse geometry.
19. The apparatus as claimed in claim 18. wherein said pulse geometry and said desired
exposure time does not substantially alter a sample of interest.
20. The apparatus as claimed in claim 1. wherein at least one substrate has an 5 optical
window allowing for on board sample detection.
21. The apparatus as churned in claim 1, wherein at least one substrate has a removable
portion allowing for off board sample detection.
22. The apparatus as claimed in claim 21. wherein said removable portion is a MALD1
foil.
23. The apparatus as claimed in claim 1, wherein a portion of said electromagnetic
radiation directed upon said material layer is reflected or transmitted into a means for allowing
confirmation of perforation.
24 A method regulating fluid flow in microstructures by using the apparatus as claimed
in claim 1 comprising:
providing a first substrate such as herein described comprising a plurality of first
fluidic components;
providing a second substrate such as herein described comprising a plurality of second
fluidic components corresponding to the first fluidic components;
providing a material layer separating the plurality of first fluidic components from the
plurality of second fluidic components: and
directing electromagnetic radiation onto said material layer in at least one position
corresponding to at least one selected position between at least a pair of corresponding fluidic
components from the plurality of first fluidic components and the plurality of second fluidic
components, said electromagnetic radiation causing perforation in at least one selected
position thereby allowing fluid communication between at least one pair of fluidic
components
25. The method of processing fluids as claimed in claim 24, wherein said material
layer contains a compound having absorptive properties that absorb said electromagnetic
radiation causing perforation.
26. The method of processing fluids as claimed in claim 25, wherein said compound is
an optical dye.
27. The method as claimed in claim 24. wherein said electromagnetic radiation is
selected from the group consisting of infrared, visible and ultra-violet spectrum.
28. The method as claimed in claim 24, wherein said material layer includes a thickness
from 0.5 µM to 100 µM.
29. The method as claimed in claim 24, wherein said material layer is selected from the
group consisting of polymer foils and metallic foils.
30. The method as claimed in claim 24, wherein said material layer is a (oil formed from
a material selected from the group consisting of polymers, copolymers, monomers, metals,
waxes, polysaccharides and liquid crystal polymers.
31. The method as claimed in claim 24, wherein said material layer is formed of a
polymeric material loaded with a dye.
32. The method as claimed in claim 31, wherein said dye has optical properties and said
optical properties are substantially matched to said selected radiation.
33. The method as claimed in claim 24, wherein said material layer is treated to
substantially absorb said selected radiation, said treatment is selected from the group consisting
of dye loading, chemical surface treatment, chemical loading, optical interference and optical
polarization.
34. The method as claimed in claim 24, wherein said material layer is formed of multiple
layers having selected absorption properties wherein said absorption properties are responsive to
said selected radiation.
35. The method as claimed in claim 24, wherein said material layer is formed of a
polymeric material selected from the group consisting of Poly(methyl methacrvlate) (PMMA).
Low Density Polyethylene (LDPE), Linear Low Density Polyethylene (LLDPL), High Densit)
Polyethylene (HDPE), Polyethylene Terephthalate (PET). Polyethylene (PL), polycarbonate
(PC), Polyethylene Terephthalate Glycol (PETG), Polystyrene (PS), Ethyl Vinyl Acetate (LVA),
and polyethylene napthalate (PEN).
36. The method as claimed in claim 24 further comprising the step of:
detecting said electromagnetic radiation in an optical feedback system wherein said
perforation of said material layer is signaled to said optical feedback system thereby controlling
said electromagnetic radiation means to substantially stop.
37. A disk lor processing fluids comprising:
a first substrate such as herein described comprising a plurality of first fluidic
components;
a second substrate such as herein described comprising a plurality of second fluidic
components corresponding to the first fluidic components; and
a material layer such as herein described separating the plurality of first fluidic components from
the plurality of second fluidic components said material layer having means for absorbing
radiation at a selected portion wherein said absorption perforates said material layer at said
selected portion allowing for fluid communication between said first fluidic components and said
second fluidic components.
The invention discloses an apparatus for regulating fluid flow in microstructures in a
programmable manner comprising a first substrate such as herein described comprising a
plurality of first fluidic components (110); a second substrate such as herein described
comprising a plurality of second fluidic components (111) corresponding to the first fluidic
components; a material layer (105) such as herein described separating the plurality of first
fluidic components from the plurality of second fluidic components; and electromagnetic
generating means for generating an electromagnetic radiation for directing onto the layer of
material at a position corresponding to a portion of the layer located between at least a pair
of corresponding fluidic components from the plurality of first fluidic components and the
plurality of second fluidic components said electromagnetic radiation causing perforation
of the material layer at the position allowing fluid communication between at least a pair of
fluidic components.
The invention also discloses a method for regulating fluid flow in microstructures using
the said apparatus.

Documents:

1117-KOLNP-2005-CORRESPONDENCE.pdf

1117-KOLNP-2005-FORM 27.pdf

1117-KOLNP-2005-FORM-27.pdf

1117-kolnp-2005-granted-abstract.pdf

1117-kolnp-2005-granted-assignment.pdf

1117-kolnp-2005-granted-claims.pdf

1117-kolnp-2005-granted-correspondence.pdf

1117-kolnp-2005-granted-description (complete).pdf

1117-kolnp-2005-granted-drawings.pdf

1117-kolnp-2005-granted-examination report.pdf

1117-kolnp-2005-granted-form 1.pdf

1117-kolnp-2005-granted-form 18.pdf

1117-kolnp-2005-granted-form 3.pdf

1117-kolnp-2005-granted-form 5.pdf

1117-kolnp-2005-granted-gpa.pdf

1117-kolnp-2005-granted-reply to examination report.pdf

1117-kolnp-2005-granted-specification.pdf


Patent Number 226392
Indian Patent Application Number 1117/KOLNP/2005
PG Journal Number 51/2008
Publication Date 19-Dec-2008
Grant Date 17-Dec-2008
Date of Filing 10-Jun-2005
Name of Patentee SPINX, INC
Applicant Address 1209 ORANGE STREET, WILMINGTON, DE 19801
Inventors:
# Inventor's Name Inventor's Address
1 ZUCCHELLI PIERO 5 LOTISSEMENT LES RENARDES, F-01210 VERSONNEX
2 VAN DE VYVER, BART 4 PLACE DU CIRQUE, CH-1204 GENEVA
PCT International Classification Number B01L
PCT International Application Number PCT/US2003/038629
PCT International Filing date 2003-12-04
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/430,792 2002-12-04 U.S.A.