Title of Invention	"AFFINITY BIOSENSOR FOR DOPAMINE"
Abstract	A novel process for the development of a biosensing method having a receptor protein as sensing element for a Surface Plasmon Resonance SPR based affinity biosensor for detection of dopamine DA comprising the incubation of receptor D-RC in the concentration range of 20-30 ug/ml, with DA in the concentration range 0.1 ng/ml -700 ng/ml, followed by injection over DA-BSA conjugate and duration of injection in the range of 3 to 7 minutes preferably 5 minutes leading to a shift of resonance angle by 0.020° to 0.040°, the decreases in the resonance angle shift being in proportion to the concentration of DA; the sensor surfaces being fabricated by physical adsorption of the conjugates on to the transducer surfaces, a microscopic glass slide coated with a ~5 nm layer of chromium and ~ 50nm layer of gold serving as transducer surface attached to the prism of the surface plasmon resonance instrument, wherein the sensor surface is functionalized with a protein conjugate of dopamine and bovine serum albumin with phosphate buffered saline (PBS) 0.1M, pH 7.2 as carrier solution.

Title of Invention

"AFFINITY BIOSENSOR FOR DOPAMINE"

Abstract

A novel process for the development of a biosensing method having a receptor protein as sensing element for a Surface Plasmon Resonance SPR based affinity biosensor for detection of dopamine DA comprising the incubation of receptor D-RC in the concentration range of 20-30 ug/ml, with DA in the concentration range 0.1 ng/ml -700 ng/ml, followed by injection over DA-BSA conjugate and duration of injection in the range of 3 to 7 minutes preferably 5 minutes leading to a shift of resonance angle by 0.020° to 0.040°, the decreases in the resonance angle shift being in proportion to the concentration of DA; the sensor surfaces being fabricated by physical adsorption of the conjugates on to the transducer surfaces, a microscopic glass slide coated with a ~5 nm layer of chromium and ~ 50nm layer of gold serving as transducer surface attached to the prism of the surface plasmon resonance instrument, wherein the sensor surface is functionalized with a protein conjugate of dopamine and bovine serum albumin with phosphate buffered saline (PBS) 0.1M, pH 7.2 as carrier solution.

Full Text	Field of Invention: This invention relates to the development of a D 3 dopamine receptor based, sensing element for a "surface plasmon resonance" based affinity biosensor for highly sensitive and selective detection of dopamine. Background of the Invention; Dopamine is a substance of prime importance for optimal regulation of human nervous system. In the biomedical field, to detect the presence and to monitor the level of dopamine is of immense importance for the clinicians to identify the CNS related disorders as well as to decide the dose estimation of external supplement. Various analytical methods including HPLC, capillary electrophoresis, fluorescence and amperometry have been demonstrated for detection of dopamine. Reviewing the wide number of reports, it can be realized that the selective detection of dopamine remains a challenge because of the presence of large excess of ascorbic acid, uric acid and other analogous endogenous compounds with dopamine in biological samples, which limits their application to invivo analysis. Moreover, they often involve time consuming and complicated procedures. Various strategies have been employed in electrochemical based sensors for alleviation of this selectivity problem such as, coupling with chromatographic separation, surface pretreatment and using perm-selective membranes. However the problem of selective detection of dopamine remains unsolved. Biosensors are analytical devices comprising a biological or biologically derived sensing element immobilized at a physicochemical transducer to measure one or more analytes. The sensing element could be enzymes, microorganism, tissue slices, biomimetic catalysts, antibodies, nucleic acids, receptor proteins or synthetic receptors. In contrast to the most common enzyme based biosensors, the affinity based biosensing element (antibodies, receptors) do not usually catalyze chemical transformations and rather undergo a physical transformation while interacting with corresponding antigen/ligand which can be detected by physical transducer. Most advanced Surface Plasmon Resonance (SPR) based optical transducers are now well established technology capable of monitoring antibody-antigen based immuno-reactions or receptor-ligand based affinity reactions. The binding between immobilized receptor and ligand changes the refractive index, leading to the change in SPR angle, which can be monitored in real time. The magnitude of the change in SPR signal is directly proportional to the mass bound to the surface even at nanogram levels. It is necessary to develop an appropriate biosensor which can measure dopamine independent of presence of antibodies against it, as antibody production is a reaction phenomenon of body in response to entrance/presence of antigen. There is no biosensor yet developed to measure the presence of dopamine invivo or invitro conditions. Objects of the Invention; The object of this invention is to develop a receptor based affinity biosensor for highly selective and sensitive detection of dopamine. Other objective is to develop a sensor capable of performing invivo estimation of dopamine without prior separation in medical diagnostics. Another object of this invention is to use Surface Plasmon Resonance based optical transducer for dopamine sensing. Yet another object is to use receptor for developing biosensor which is a natural target for analyte as they possess high affinity and specificity refined by evolutionary process. Other object of this invention is to avoid the use of antibody as recognizing element which involves challenging the experimental animals. Another object is to develop the stability of receptor subunits by using immobilization conditions that closely related to natural environment. Yet another object is to develop a multiple analysis system of hundreds of binding events from very small sample volumes. Detailed Description of the Invention; The principle of the invention is that a biosensor to sense qualitative and quantitative presence of dopamine invitro and invivo can be developed using receptors instead of antibodies. Inventors have established here receptor based sensing element for a SPR based affinity biosensor for highly sensitive and selective detection of dopamine (DA), employing a D3 dopamine receptor and a home made DA-bo vine serum albumin (DA-BSA) conjugate. The new methodology presented here using receptor as a recognition element for dopamine detection based on Surface Plasmon Resonance (SPR) technique proved to be a potential tool for highly sensitive and selective detection of dopamine with good reliability and reproducibility. The sensing idea is simple and the high affinity molecular recognition of the dopamine receptor (D-RC) provided remarkable specificity for dopamine (DA) against ascorbic acid (AA), uric acid (UA) and other dopamine analogues viz., 3,4 dihydroxyphenyl acetic acid (DOPAC) and 3-(3,4 dihydroxyphenyl)-alanine (DOPA). The proposed method provides a new and promising route for reliable and economic biochemical diagnosis of risk of dopamine related disorders. The sensor surfaces were fabricated by physical adsorption of the conjugates (analyte-carrier protein) onto the transducer surfaces. A microscopic glass slide coated with a ~5 nm layer of chromium and ~50 nm layer of gold served as transducer surface and was attached to the prism of the surface plasmon resonance instrument (Model SPR-670, Nippon Laser and Electronics, Japan). The sensor surface was functionalized with a protein conjugate of dopamine and bovine serum albumin (BSA). Phosphate buffered saline (PBS, 0.1 M, pH 7.2) was used as the carrier solution. A pepsin solution, glysin -HCI buffer, pH 2 was used for regeneration of sensor surface. The sensitive and selective interactions of the dopamine receptors (D-RC) with dopamine-bo vine serum albumin conjugate (DA-BSA) were monitored using SPR technique. Detection of dopamine (DA) was carried out using the principle of indirect competitive inhibition, which is a promising protocol for sensitive detection of small molecules in techniques like SPR where molecular interaction is being observed. The developed affinity biosensor is remarkably sensitive showing, detection limit 85 pg/ml (ppt, parts per trillion) and highly selective with a response time of 5 minutes. Monitoring dopamine concentration through present "Affinity Sensor" technique will be helpful as an early marker rather than as a diagnosis of CNS-related disorders. The two main novelties lie in the use of SPR as optical transducer and the use of a natural receptor as bio-recognizing element in the development of affinity biosensor for dopamine. We Claim; 1. A novel process for the development of a biosensing method having a receptor protein as sensing element for a Surface Plasmon Resonance SPR based affinity biosensor for detection of dopamine DA comprising the incubation of receptor D-RC in the concentration range of 20-30 ug/ml, with DA in the concentration range 0.1 ng/ml -700 ng/ml, followed by injection over DA-BSA conjugate and duration of injection in the range of 3 to 7 minutes preferably 5 minutes leading to a shift of resonance angle by 0.020° to 0.040°, the decreases in the resonance angle shift being in proportion to the concentration of DA; the sensor surfaces being fabricated by physical adsorption of the conjugates on to the transducer surfaces, a microscopic glass slide coated with a ~5 nm layer of chromium and ~ SOnrn layer of gold serving as transducer surface attached to the prism of the surface plasmon resonance instrument, wherein the sensor surface is functionalized with a protein conjugate of dopamine and bovine serum albumin with phosphate buffered saline (PBS) 0.1M, pH 7.2 as carrier solution. 2. A novel process for the development of a biosensing method as claimed in claim 1, wherein the immobilised assay format could be reused by simple regeneration of its original state by injecting 2-10 jig/ml pepsin solution (glysin -HC1 buffer, pH 2.0) for 10-60 s. 3. The novel process for the development of bio sensing method as claimed in claim 1 wherein the biosensor is providing remarkable sensitivity with a detection limit of 85 pg/ml and a response time of 5 minutes. 4. The process for the development of a biosensing method as claimed in claim 1, wherein the sensing idea is simple and the high affinity molecular recognition of the D-RC provided remarkable specificity for DA against interference from coexisting compounds such as ascorbic acid, uric acid and other DA analogues. 5. The process for the development of a biosensing method as claimed in claim 1 wherein almost similar resonance angle shift for the interaction of D-RC with DA-BSA conjugate is detected after multiple analysis cycles to be reused for nearly 20 cyles of measurements. 6. The process for the development of a biosensing method as claimed in claim 1, wherein monitoring dopamine concentration is used as an early marker rather than diagnosis. 7. The process for the development of a biosensing method as claimed in claim 1, wherein affinity interaction based biosensor is used for the first time for dopamine detection using receptor as the bio recognition element. 8. The process for the development of a biosensing method as claimed in claim 1, wherein the SPR is used for the first time as physical transducer for sensing dopamine.

Full Text

Field of Invention:
This invention relates to the development of a D 3 dopamine receptor based, sensing element for a "surface plasmon resonance" based affinity biosensor for highly sensitive and selective detection of dopamine.
Background of the Invention;
Dopamine is a substance of prime importance for optimal regulation of human nervous system. In the biomedical field, to detect the presence and to monitor the level of dopamine is of immense importance for the clinicians to identify the CNS related disorders as well as to decide the dose estimation of external supplement.
Various analytical methods including HPLC, capillary electrophoresis, fluorescence and amperometry have been demonstrated for detection of dopamine. Reviewing the wide number of reports, it can be realized that the selective detection of dopamine remains a challenge because of the presence of large excess of ascorbic acid, uric acid and other analogous endogenous compounds with dopamine in biological samples, which limits their application to invivo analysis. Moreover, they often involve time consuming and complicated procedures. Various strategies have been employed in electrochemical based sensors for alleviation of this selectivity problem such as, coupling with chromatographic separation, surface pretreatment and using perm-selective membranes. However the problem of selective detection of dopamine remains unsolved.

Biosensors are analytical devices comprising a biological or biologically derived sensing element immobilized at a physicochemical transducer to measure one or more analytes. The sensing element could be enzymes, microorganism, tissue slices, biomimetic catalysts, antibodies, nucleic acids, receptor proteins or synthetic receptors.
In contrast to the most common enzyme based biosensors, the affinity based biosensing element (antibodies, receptors) do not usually catalyze chemical transformations and rather undergo a physical transformation while interacting with corresponding antigen/ligand which can be detected by physical transducer. Most advanced Surface Plasmon Resonance (SPR) based optical transducers are now well established technology capable of monitoring antibody-antigen based immuno-reactions or receptor-ligand based affinity reactions. The binding between immobilized receptor and ligand changes the refractive index, leading to the change in SPR angle, which can be monitored in real time. The magnitude of the change in SPR signal is directly proportional to the mass bound to the surface even at nanogram levels.
It is necessary to develop an appropriate biosensor which can measure dopamine independent of presence of antibodies against it, as antibody production is a reaction phenomenon of body in response to entrance/presence of antigen.
There is no biosensor yet developed to measure the presence of dopamine invivo or invitro conditions.

Objects of the Invention;
The object of this invention is to develop a receptor based affinity biosensor for highly selective and sensitive detection of dopamine.
Other objective is to develop a sensor capable of performing invivo estimation of dopamine without prior separation in medical diagnostics.
Another object of this invention is to use Surface Plasmon Resonance based optical transducer for dopamine sensing.
Yet another object is to use receptor for developing biosensor which is a natural target for analyte as they possess high affinity and specificity refined by evolutionary process.
Other object of this invention is to avoid the use of antibody as recognizing element which involves challenging the experimental animals.
Another object is to develop the stability of receptor subunits by using immobilization conditions that closely related to natural environment.
Yet another object is to develop a multiple analysis system of hundreds of binding events from very small sample volumes.

Detailed Description of the Invention;
The principle of the invention is that a biosensor to sense qualitative and quantitative presence of dopamine invitro and invivo can be developed using receptors instead of antibodies.
Inventors have established here receptor based sensing element for a SPR based affinity biosensor for highly sensitive and selective detection of dopamine (DA), employing a D3 dopamine receptor and a home made DA-bo vine serum albumin (DA-BSA) conjugate.
The new methodology presented here using receptor as a recognition element for dopamine detection based on Surface Plasmon Resonance (SPR) technique proved to be a potential tool for highly sensitive and selective detection of dopamine with good reliability and reproducibility. The sensing idea is simple and the high affinity molecular recognition of the dopamine receptor (D-RC) provided remarkable specificity for dopamine (DA) against ascorbic acid (AA), uric acid (UA) and other dopamine analogues viz., 3,4 dihydroxyphenyl acetic acid (DOPAC) and 3-(3,4 dihydroxyphenyl)-alanine (DOPA). The proposed method provides a new and promising route for reliable and economic biochemical diagnosis of risk of dopamine related disorders.
The sensor surfaces were fabricated by physical adsorption of the conjugates (analyte-carrier protein) onto the transducer surfaces. A microscopic glass slide coated with a ~5 nm layer of chromium and ~50 nm layer of gold served as transducer surface and was attached to the prism of the surface plasmon resonance instrument (Model SPR-670, Nippon Laser and Electronics, Japan). The sensor surface was

functionalized with a protein conjugate of dopamine and bovine serum albumin (BSA). Phosphate buffered saline (PBS, 0.1 M, pH 7.2) was used as the carrier solution. A pepsin solution, glysin -HCI buffer, pH 2 was used for regeneration of sensor surface.
The sensitive and selective interactions of the dopamine receptors (D-RC) with dopamine-bo vine serum albumin conjugate (DA-BSA) were monitored using SPR technique. Detection of dopamine (DA) was carried out using the principle of indirect competitive inhibition, which is a promising protocol for sensitive detection of small molecules in techniques like SPR where molecular interaction is being observed. The developed affinity biosensor is remarkably sensitive showing, detection limit 85 pg/ml (ppt, parts per trillion) and highly selective with a response time of 5 minutes.
Monitoring dopamine concentration through present "Affinity Sensor" technique will be helpful as an early marker rather than as a diagnosis of CNS-related disorders. The two main novelties lie in the use of SPR as optical transducer and the use of a natural receptor as bio-recognizing element in the development of affinity biosensor for dopamine.

We Claim;
1. A novel process for the development of a biosensing method having a receptor protein as sensing element for a Surface Plasmon Resonance SPR based affinity biosensor for detection of dopamine DA comprising the incubation of receptor D-RC in the concentration range of 20-30 ug/ml, with DA in the concentration range 0.1 ng/ml -700 ng/ml, followed by injection over DA-BSA conjugate and duration of injection in the range of 3 to 7 minutes preferably 5 minutes leading to a shift of resonance angle by 0.020° to 0.040°, the decreases in the resonance angle shift being in proportion to the concentration of DA; the sensor surfaces being fabricated by physical adsorption of the conjugates on to the transducer surfaces, a microscopic glass slide coated with a ~5 nm layer of chromium and ~ SOnrn layer of gold serving as transducer surface attached to the prism of the surface plasmon resonance instrument, wherein the sensor surface is functionalized with a protein conjugate of dopamine and bovine serum albumin with phosphate buffered saline (PBS) 0.1M, pH 7.2 as carrier solution.
2. A novel process for the development of a biosensing method as claimed in claim 1, wherein the immobilised assay format could be reused by simple regeneration of its original state by injecting 2-10 jig/ml pepsin solution (glysin -HC1 buffer, pH 2.0) for 10-60 s.

3. The novel process for the development of bio sensing method as
claimed in claim 1 wherein the biosensor is providing
remarkable sensitivity with a detection limit of 85 pg/ml and a
response time of 5 minutes.
4. The process for the development of a biosensing method as
claimed in claim 1, wherein the sensing idea is simple and the
high affinity molecular recognition of the D-RC provided
remarkable specificity for DA against interference from
coexisting compounds such as ascorbic acid, uric acid and
other DA analogues.
5. The process for the development of a biosensing method as
claimed in claim 1 wherein almost similar resonance angle shift
for the interaction of D-RC with DA-BSA conjugate is detected
after multiple analysis cycles to be reused for nearly 20 cyles of
measurements.
6. The process for the development of a biosensing method as
claimed in claim 1, wherein monitoring dopamine concentration
is used as an early marker rather than diagnosis.
7. The process for the development of a biosensing method as
claimed in claim 1, wherein affinity interaction based biosensor
is used for the first time for dopamine detection using receptor
as the bio recognition element.

8. The process for the development of a biosensing method as claimed in claim 1, wherein the SPR is used for the first time as physical transducer for sensing dopamine.

Documents:

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Patent Number

271054

Indian Patent Application Number

2774/DEL/2006

PG Journal Number

06/2016

Publication Date

05-Feb-2016

Grant Date

29-Jan-2016

Date of Filing

22-Dec-2006

Name of Patentee

J.N.V. UNIVERSITY

Applicant Address

JODHPUR-342003.

Inventors:

#	Inventor's Name	Inventor's Address
1	SUNITA KUMBHAT	J.N.V.UNIVERSITY,DEPARTMENT OF CHEMISTRY JODHPUR-342003. INDIA
2	NORIO MIURA	ART,SCIENCE AND TECHNOLOGY CENTER FOR COOPERATIVE RESEARCH,KYUSHU UNIVERSITY,KASUGA-SHI,FUKUOKA, 816-8580.
3	VINOD JOSHI	DIVISON OF VIROLOGY AND MOLECULAR BIOLOGY,DESERT MEDICINE RESEARCH CENTER,(INDIAN COUNCIL OF MEDICAL RESEARCH),JODHPUR-342003.
4	DHESINGH RAVI SHANKARAN	ART,SCIENCE AND TECHNOLOGY CENTER FOR COOPERATIVE RESEARCH,KYUSHU UNIVERSITY,KASUGA-SHI,FUKUOKA, 816-8580.
5	K. VENGATAJALABATHY GOBI	ART,SCIENCE AND TECHNOLOGY CENTER FOR COOPERATIVE RESEARCH,KYUSHU UNIVERSITY,KASUGA-SHI,FUKUOKA, 816-8580.

PCT International Classification Number

G02B

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA