DESC:FIELD OF INVENTION:
The present invention discloses an easy-to-use, point-of-care (PoC), portable device for detection and quantification of bio-analytes. The present invention particularly relates to point-of-care (PoC), portable device for detection and quantification of bio-analytes such as free Human chorionic gonadotropin (ß-hCG) and Pregnancy-associated plasma protein A (PAPP-A) in maternal biological fluid. The present invention also relates to a device comprising a test strip or a biosensor comprising of the bio analyte specific electrochemistry, a read-out device for analysing the analogue signal and converting it into a digital read-out.
BACKGROUND OF INVENTION:
Down syndrome (trisomy 21) is one of the most common causes of intellectual disability among live born children. The recommended protocol by Indian College of Obstetricians & Gynaecologists-Federation of Obstetric and Gynaecological Societies of India (ICOG-FOGSI) and other international guidelines for identifying such anomalies is a “combined” prenatal screening test, to be conducted precisely between 11th and 13th weeks of pregnancy which includes a specialized blood test (Dual marker test) to quantify levels of biochemical analytes also referred herein as bio-analytes or biomarkers or maternal markers (free ß-hCG and PAPP-A) for screening of pregnant women at risk of carrying abnormal/anomalous fetus. However, these blood tests are available only in limited advanced labs and is confined generally to metro cities. For smaller cities, blood samples are collected and transported to nearest metro city for testing, making these tests expensive and time consuming . The local availability of a Point of Care (PoC) device for testing these biomarkers would make screening of risk group women easy and convenient and would drastically improve testing compliance in risk groups.
Sachdeva et al. 2021, discloses application of polymer base electronic devices as Point-of-Care Testing (POCT) devices. The document also discloses application of metal nanoparticle and polymer nanocomposite in lateral flow strip designs. Sachdeva et al. further discloses application of organic polymers on top of a photosensitive porous substrate such as silica. However, it does not disclose application of PANI and or Ag-PANI nanocomposite in fabrication of the devices and applicability of the devices in ß-hCG or PAPP-A screening in maternal serum.
Wong et al. 2015, discloses PoC devices for the detection of hCG, wherein nanogold and silver enhancements are used and wherein software is used for the quantitative analysis of bio-analytes. The document further discloses PoC devices capable of providing instant testing results on-site. Further discloses amine-polymer composite addition after the surface activation steps. However, it does not disclose application of PANI and or Ag-PANI nanocomposite in fabrication of the device and applicability of the device in PAPP-A screening in maternal serum.
Osuna et al. 2022, which discloses application of PANI combined with metallic nanoparticles for electrochemical device fabrication. It also discloses detection of glucose in blood serum sample. However, the document does not disclose application of the devices in detection of beta-hCG and PAPP-A in maternal serum.
Hence, their remains an unmet need for Point-of-Care Testing (POCT) or point-of-care (PoC), device for screening and quantification of bio-markers or bio-analytes. Specifically there is need to detect and quantify bio-analytes related to Down syndrome, Edward syndrome, Patau Syndrome and other related anomalies viz. free Human chorionic gonadotropin (ß-hCG) and Pregnancy-associated plasma protein A (PAPP-A) in maternal serum during 11th and 13th weeks of pregnancy.
OBJECTIVES OF THE PRESENT INVENTION:
The prime objective of this invention is to provide a device for detecting a bio-analyte.
The second objective of the present invention is to provide a device detecting a bio-analyte for prenatal screening.
The third objective of the present invention is to provide a device detecting a bio-analyte from maternal serum or blood.
The fourth objective of the present invention is to provide a low-cost, user-friendly, stable, sensitive, compact, portable, point-of-care and standalone device for detecting a bio-analyte.
The fifth objective of the present invention is to provide a device for detection, screening and quantification of bio-analytes or bio-markers related to Down syndrome, Edward syndrome, Patau Syndrome and other related anomalies viz. free Human chorionic gonadotropin (ß-hCG) and Pregnancy-associated plasma protein A (PAPP-A) in maternal serum during 11th and 13th weeks of pregnancy.
The sixth objective of the present invention is to provide a process for fabrication of a biosensor for a device for detecting a bio-analyte.
The seventh objective of the present invention is to provide a method of detection of a bio-analyte by a device for detecting a bio-analyte.
SUMMARY OF THE PRESENT INVENTION:
This summary is provided to introduce a selection of concepts, in a simplified format, that are further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention nor is it intended to determine the scope of the invention.
The present invention provides a solution to availability of a device for prenatal screening limited to advanced labs and confined generally to metro cities. The present invention provides a low-cost, user-friendly, stable, sensitive, compact, portable, point-of-care and standalone device for detecting a bio-analyte. In particular, the present invention provides a device for detecting a bio-analyte from maternal serum or blood.
In one of the embodiments, the present invention provides a device (A) for detecting a bio-analyte (12), the device comprising;
a) a biosensor (1) fabricated on a substrate (8) coated with a functionalized conductive polymer (7), wherein the functionalized conductive polymer is immobilized with a plurality of antibodies (6, 13) specific to the bio-analyte (12), wherein the biosensor (1) is adapted to change electrical property (11) by interaction with the bio-analyte (12),
one or more electrodes (2,3,4) attached to the substrate (8), wherein the one or more electrode (2,3,4) is adapted to simultaneously collect and deliver the change in electrical property; and
b) a read-out device (10) adapted to display the change in electrical property, wherein the read-out device comprises a circuit assembly, and a potentiostat or a galvanostat (10).
In another embodiment, the present invention provides a device for detecting a bio-analyte wherein the bio-analytes are maternal markers free ß-hCG and PAPP-A.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the bio-analytes are alpha fetoprotein (AFP) and Placenta growth factor (PIGF).
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the substrate comprises a conductive substrate or a non-conductive substrate.
In yet another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the non-conductive substrate comprises glass, silicon dioxide, or plastic.
In yet another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the conductive substrate comprises gold, silver, or platinum or modified Carbon or Graphene.
In one of the preferred embodiments, the present invention provides a device for detecting a bio-analyte, wherein the substrate is silicon dioxide.
In one of the preferred embodiments, the present invention provides a device for detecting a bio-analyte, wherein the substrate is gold.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the one or more electrodes are gold or silver electrodes.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the conductive polymer of the biosensor comprises polyaniline (PANI), polypyrrole or polythiophene.
In one of the embodiments, the present invention provides a device for detecting a bio-analyte, wherein the conductive polymer of the biosensor comprises silver nanoparticles (AgNPs) and Polyaniline nanocomposite (AgNPs-PANI).
In one of the embodiments, the present invention provides a device for detecting a bio-analyte, wherein the silver nanoparticles (AgNPs) to Polyaniline (PANI) of the Polyaniline nanocomposite (AgNPs-PANI) is in a ratio of 1:1300.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the conductive polymer is functionalized by a linker selected from a group consisting of Glutaraldehyde, 1-Ethyl-3-diaminopropyl carbodiimide and N-hydroxysuccinimide (EDC-NHS), Streptavidin, or Avidin.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the plurality of antibodies specific to the bio-analyte are monoclonal capture antibodies from mouse or rabbit and whole, monoclonal or polyclonal detection antibodies from mouse, goat, chicken or sheep.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the change in electrical property is directly proportional to the concentration of the bio analyte.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the device detection range for free beta hCG is 20 to 200 ng/mL.
In another embodiment, the present invention provides a device for detecting a bio-analyte,, wherein the device detection range for PAPP-A is 1 to 32 µg/mL.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the read-out device is configured to convert analogue signal into a digital read-out.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the read-out device is read-out device comprises an optical signal source coupled with a circuit assembly and a resistance meter for the electrical read-outs.
In another embodiment the present invention provides a device for detecting a bio-analyte, wherein the optical source is a LED.
In another embodiment the present invention provides a device for detecting a bio-analyte, wherein the device is configured to utilize a statistical data for calculation and normalization of electrical read-outs.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the device is compact, portable, point-of-care and standalone.
In another embodiment, the present invention provides a process for fabrication of a biosensor, the process comprising;
- selecting a substrate as a base of the biosensor;
- coating a conductive polymer layer on the substrate;
- functionalizing the conductive polymer coating on the substrate with a linker;
- immobilizing a plurality of antibodies specific to a bio-analyte on the functionalized conductive polymer of the substrate;
- blocking the non-specific active regions of the conductive polymer layer with a bovine serum albumin (BSA);
- fabrication of one or more electrode attached to the surface of the functionalized conductive polymer of the substrate to obtain the biosensor.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the substrate comprises a conductive substrate or a non-conductive substrate.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the non-conductive substrate comprises glass, silicon dioxide, or plastic.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the conductive substrate comprises gold, silver or platinum or modified Carbon or Graphene.
In one of the preferred embodiments, the present invention provides a process for fabrication of a biosensor, wherein the substrate is silicon dioxide.
In one of the preferred embodiments, the present invention a process for fabrication of a biosensor, wherein the substrate is gold.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the conductive polymer of the biosensor comprises polyaniline (PANI) , polypyrrole or polythiophene.
In one of the embodiment, the present invention provides a process for fabrication of a biosensor, wherein the conductive polymer of the biosensor comprises silver nanoparticles and Polyaniline nanocomposite (AgNPs-PANI).
In one of the embodiment, the present invention provides a process for fabrication of a biosensor, wherein the silver nanoparticles (AgNPs) to Polyaniline (PANI) of the Polyaniline nanocomposite (AgNPs-PANI) is in a ratio of 1: 1300.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the linker are selected from a group consisting of Glutaraldehyde, 1-Ethyl-3-diaminopropyl carbodiimide and N-hydroxysuccinimide (EDC-NHS), Streptavidin, or Avidin.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the plurality of antibodies specific to the bio-analyte are monoclonal capture antibodies from mouse or rabbit and whole, monoclonal or polyclonal detection antibodies from mouse, goat, chicken or sheep.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the one or more electrodes are gold or silver electrodes.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the the bio-analytes are maternal markers free ß-hCG and PAPP-A.
In yet another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, the method comprising;
a) obtaining a biosensor with a unique antibody directed against a distinct antigenic determinant present on the bio-analyte;
b) adding a sample containing the bio-analyte to the biosensor;
c) capturing the change in electrical property before and after sample addition;
d) measuring the change in electrical property by one or more electrodes;
e) converting change in electrical property into concentration;
displaying the change in electrical property in terms of concentration of bio-analyte by a readout device;
wherein the change in electrical property is directly proportional to the concentration of the bio analyte.
In another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, wherein the bio-analytes are maternal markers free ß-hCG and PAPP-A.
In another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, wherein the bio-analytes are AFP and PIGF.
In another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, wherein the sample is maternal serum or blood.
In another embodiment, the present invention provides a device for detecting a bio-analyte, that can select relevant data set and a statistic called the “multiple of the median” (MoM) calculation to normalize the electrical readout.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Illustrates schematic representation of on-point device and its components, in accordance with an implementation of the present invention.
Figure 2A illustrates process flow of the device fabrication and the final architecture, in accordance with an implementation of the present invention.
Figure 2B illustrates schematic representation for functionalization, in accordance with an implementation of the present invention.
Figure 3A illustrates a mechanism of functioning of the biosensor.
Figure 3B illustrates a schematic of a biosensor development.
Figure 4A illustrates schematic representation of a biosensor test strip, in accordance with an embodiment of the present invention.
Figure 4B illustrates schematic representation of on-point device and its components, in accordance with an embodiment of the present invention.
Figure 5 illustrates A. setup for electrochemical polymerization by using cyclic voltameter, B: uncoated and PANI coated gold electrode.
Figure 6 illustrates UV-Vis analysis of Aniline and PANI.
Figure 7 illustrates FTIR analysis of Aniline and PANI.
Figure 8 illustrates DSC curve of A: Aniline, B: PANI.
Figure 9 illustrates Cyclic voltammograms for 6mm electrodes.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION OF THE INVENTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
The term “some” as used herein is defined as “none, or one, or more than one, or all.” Accordingly, the terms “none,” “one,” “more than one,” “more than one, but not all” or “all” would all fall under the definition of “some.” The term “some embodiments” may refer to no embodiments or to one embodiment or to several embodiments or to all embodiments. Accordingly, the term “some embodiments” is defined as meaning “no embodiment, or one embodiment, or more than one embodiment, or all embodiments.”
The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the spirit and scope of the claims or their equivalents.
More specifically, any terms used herein such as but not limited to “includes,” “comprises,” “has,” “consists,” and grammatical variants thereof do NOT specify an exact limitation or restriction and certainly do NOT exclude the possible addition of one or more features or elements, unless otherwise stated, and furthermore must NOT be taken to exclude the possible removal of one or more of the listed features and elements, unless otherwise stated with the limiting language “MUST comprise” or “NEEDS TO include.”
Whether or not a certain feature or element was limited to being used only once, either way it may still be referred to as “one or more features” or “one or more elements” or “at least one feature” or “at least one element.” Furthermore, the use of the terms “one or more” or “at least one” feature or element do NOT preclude there being none of that feature or element, unless otherwise specified by limiting language such as “there NEEDS to be one or more . . . ” or “one or more element is REQUIRED.”
Unless otherwise defined, all terms, and especially any technical and/or scientific terms, used herein may be taken to have the same meaning as commonly understood by one having an ordinary skill in the art.
Reference is made herein to some “embodiments.” It should be understood that an embodiment is an example of a possible implementation of any features and/or elements presented in the attached claims. Some embodiments have been described for the purpose of illuminating one or more of the potential ways in which the specific features and/or elements of the attached claims fulfil the requirements of uniqueness, utility and non-obviousness.
Use of the phrases and/or terms such as but not limited to “a first embodiment,” “a further embodiment,” “an alternate embodiment,” “one embodiment,” “an embodiment,” “multiple embodiments,” “some embodiments,” “other embodiments,” “further embodiment”, “furthermore embodiment”, “additional embodiment” or variants thereof do NOT necessarily refer to the same embodiments. Unless otherwise specified, one or more particular features and/or elements described in connection with one or more embodiments may be found in one embodiment, or may be found in more than one embodiment, or may be found in all embodiments, or may be found in no embodiments. Although one or more features and/or elements may be described herein in the context of only a single embodiment, or alternatively in the context of more than one embodiment, or further alternatively in the context of all embodiments, the features and/or elements may instead be provided separately or in any appropriate combination or not at all. Conversely, any features and/or elements described in the context of separate embodiments may alternatively be realized as existing together in the context of a single embodiment.
Any particular and all details set forth herein are used in the context of some embodiments and therefore should NOT be necessarily taken as limiting factors to the attached claims. The attached claims and their legal equivalents can be realized in the context of embodiments other than the ones used as illustrative examples in the description below.
Embodiments of the present invention will be described below in detail with reference to the accompanying drawings.
The present invention discloses an easy-to-use, point-of-care (PoC), portable device for quantification of the bio-analytes, free Human chorionic gonadotropin (??-hCG) and Pregnancy-associated plasma protein A (PAPP-A) in maternal serum or blood. The device comprises a biosensor test strip comprising of the analyte specific electrochemistry, a readout device for analysing the analogue signal and converting it into a digital read-out and a software for calculating the Multiples of Median (MoM) to normalize the test result. The device is based on the principle of Electrochemical immunosensing for analyte quantification.
In one of the embodiments, the present invention provides a device (A) for detecting a bio-analyte (12), the device comprising;
a) a biosensor (1) fabricated on a substrate (8) coated with a functionalized conductive polymer (7), wherein the functionalized conductive polymer is immobilized with a plurality of antibodies (6, 13) specific to the bio-analyte (12), wherein the biosensor (1) is adapted to change electrical property (11) by interaction with the bio-analyte (12),
one or more electrodes (2,3,4) attached to the substrate (8), wherein the one or more electrode (2,3,4) is adapted to simultaneously collect and deliver the change in electrical property; and
b) a read-out device (10) adapted to display the change in electrical property, wherein the read-out device comprises a circuit assembly, and a potentiostat or a galvanostat (10).
In another embodiment, the present invention provides a device for detecting a bio-analyte wherein the bio-analytes are maternal markers free ß-hCG and PAPP-A.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the bio-analytes are alpha fetoprotein (AFP) and Placenta growth factor (PIGF).
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the substrate comprises a conductive substrate or a non-conductive substrate.
In yet another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the non-conductive substrate comprises glass, silicon dioxide, or plastic.
In yet another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the conductive substrate comprises gold, silver, or platinum or modified Carbon or Graphene.
In one of the preferred embodiments, the present invention provides a device for detecting a bio-analyte, wherein the substrate is silicon dioxide.
In one of the preferred embodiments, the present invention provides a device for detecting a bio-analyte, wherein the substrate is gold.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the one or more electrodes are gold or silver electrodes.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the conductive polymer of the biosensor comprises polyaniline (PANI), polypyrrole or polythiophene.
In one of the embodiments, the present invention provides a device for detecting a bio-analyte, wherein the conductive polymer of the biosensor comprises silver nanoparticles (AgNPs) and Polyaniline nanocomposite (AgNPs-PANI).
In one of the embodiments, the present invention provides a device for detecting a bio-analyte, wherein the silver nanoparticles (AgNPs) to Polyaniline (PANI) of the Polyaniline nanocomposite (AgNPs-PANI) is in a ratio of 1:1300.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the conductive polymer is functionalized by a linker selected from a group consisting of Glutaraldehyde, 1-Ethyl-3-diaminopropyl carbodiimide and N-hydroxysuccinimide (EDC-NHS), Streptavidin, or Avidin.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the plurality of antibodies specific to the bio-analyte are monoclonal capture antibodies from mouse or rabbit and whole, monoclonal or polyclonal detection antibodies from mouse, goat, chicken or sheep.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the change in electrical property is directly proportional to the concentration of the bio analyte.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the device detection range for free beta hCG is 20 to 200 ng/mL.
In another embodiment, the present invention provides a device for detecting a bio-analyte,, wherein the device detection range for PAPP-A is 1 to 32 µg/mL.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the read-out device is configured to convert analogue signal into a digital read-out.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the read-out device is read-out device comprises an optical signal source coupled with a circuit assembly and a resistance meter for the electrical read-outs.
In a preferred embodiment, the present invention provides a device for detecting a bio-analyte, wherein the optical source is a LED.
In another embodiment the present invention provides a device for detecting a bio-analyte, wherein the optical source is a LED.
In another embodiment the present invention provides a device for detecting a bio-analyte, wherein the device is configured to utilize a statistical data for calculation and normalization of test results.
In another embodiment, the present invention provides a device for detecting a bio-analyte, wherein the device is compact, portable, point-of-care and standalone.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, the process comprising;
- selecting a substrate as a base of the biosensor;
- coating a conductive polymer layer on the substrate;
- functionalizing the conductive polymer coating on the substrate with a linker;
- immobilizing a plurality of antibodies specific to a bio-analyte on the functionalized conductive polymer of the substrate;
- blocking the non-specific active regions of the conductive polymer layer with a bovine serum albumin (BSA);
- fabrication of one or more electrode on the surface of the functionalized conductive polymer of the substrate to obtain the biosensor.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the substrate comprises a conductive substrate or a non-conductive substrate.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the non-conductive substrate comprises glass, silicon dioxide, plastic.
In yet another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the conductive substrate comprises gold, silver or platinum or modified Carbon or Graphene.
In one of the preferred embodiments, the present invention provides a device for detecting a bio-analyte, wherein the substrate is silicon dioxide.
In one of the preferred embodiments, the present invention provides a device for detecting a bio-analyte, wherein the substrate is gold.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the conductive polymer of the biosensor comprises polyaniline (PANI) or polypyrrole or polythiophene.
In one of the embodiment, the present invention provides a process for fabrication of a biosensor, wherein the conductive polymer of the biosensor comprises silver nanoparticles and Polyaniline nanocomposite (AgNPs-PANI).
In one of the embodiment, the present invention provides a process for fabrication of a biosensor, wherein the silver nanoparticles (AgNPs) to Polyaniline (PANI) of the Polyaniline nanocomposite (AgNPs-PANI) is in a ratio of 1: 1300.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the linker are selected from a group consisting of Glutaraldehyde, 1-Ethyl-3-diaminopropyl carbodiimide and N-hydroxysuccinimide (EDC-NHS) , Streptavidin, or Avidin.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the plurality of antibodies specific to the bio-analyte are monoclonal capture antibodies from mouse or rabbit and whole, monoclonal or polyclonal detection antibodies from mouse, goat, chicken or sheep..
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the one or more electrodes are gold or silver electrodes.
In another embodiment, the present invention provides a process for fabrication of a biosensor, wherein the the bio-analytes are maternal markers free ß-hCG and PAPP-A.
In yet another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, the method comprising;
a) obtaining a biosensor with a unique antibody directed against a distinct antigenic determinant present on the bio-analyte;
b) adding a sample containing the bio-analyte to the biosensor;
c) capturing the change in electrical property before and after sample addition;
d) measuring the change in electrical property by one or more electrodes;
e) converting change in electrical property into concentration;
displaying the change in electrical property in terms of concentration of bio-analyte by a readout device;
wherein the change in electrical property is directly proportional to the concentration of the bio analyte.
In another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, wherein the bio-analytes are maternal markers free ß-hCG and PAPP-A.
In another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, wherein the bio-analytes are AFP and PIGF.
In another embodiment, the present invention provides a method of detection of a bio-analyte by a device for detecting a bio-analyte, wherein the sample is maternal serum or blood.
In another embodiment, the present invention provides a device for detecting a bio-analyte, that can select relevant data set and a statistic called the “multiple of the median” (MoM) calculation to normalize the test result.
In one of the embodiment, the present invention comprises a device (A) as illustrated in Figure 1 comprises a biosensor (1) also referred as biosensor strip or sensor or test strip or strip is fabricated on a substrate (8) coated with a functionalized conductive polymer (7), wherein the functionalized conductive polymer (7) is immobilized with a plurality of antibodies specific(6, 13) to the bio-analyte (12), wherein the biosensor (1) is adapted to change electrical property (11) by interaction with the bio-analyte (12) , one or more electrodes (2, 3, 4) attached to the substrate, wherein the one or more electrodes is adapted to simultaneously collect and deliver the change in electrical property and a read-out device adapted to display the change in electrical property, wherein the read-out device comprises a circuit assembly, and a potentiostat or galvanostat(10).
In one of the embodiment, the present invention comprises a device as illustrated in Figure 4 A illustrates schematic representation of an embodiment of the biosensor test strip, Figure 4 B illustrates schematic representation of on-point device and its components, in accordance with an embodiment of the present invention, wherein the read-out device comprises an optical signal source coupled with a circuit assembly and a resistance meter, wherein the optical signal source is aligned on top of the biosensor as an optical conduit for the bio-analyte.
Principal or mechanism of functioning of the device for detecting a bio-analyte.
The biosensor of the device for detecting a bio-analyte works on the principle of a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). The assay system utilizes unique antibodies directed against a distinct antigenic determinant present on the bio-analyte (free b-hCG and PAPP-A) molecules. Antibodies specific for free-b-hCG and PAPP-A were used as capture antibody for immobilization on the biosensor surface. When the test sample containing bio-analyte or antigen is added on the biosensor strip, the distinct antigenic determinants present on the bio-analyte binds to the specific sites on the capture antibody causing change in electrical property before and after sample addition. Next, the free-b-hCG and PAPP-A specific detection antibody tagged with antibody-enzyme (horseradish peroxidase) when added on the strip, further initiates a redox potential resulting in additional change in electrical property which is measured using a read-out device comprising a potentiostat. The concentration of the bio-analyte is directly proportional to the intensity of the electrical property generated after addition of the test sample and is used for quantification. The sample taken is maternal serum or blood Figure 3A illustrates a mechanism of functioning of the biosensor. Figure 3B illustrates a schematic of a biosensor development.. The detection range of the device for free ??-hCG: 20 ng/mL to 200 ng/mL and for PAPP-A: 1 µg/mL to 32 µg/mL.
In an embodiment for such biosensor fabrication, gold electrodes as a substrate is used herein. Further, conducting polymer or nanocomposite material in the form of a thin film may be chosen as a matrix for immobilization of antibodies which allow interaction with the biological sample. Inventors of present application have synthesized and optimized a Polyaniline (PANI) and or Silver-Polyaniline nano-composite (AgNPs-PANI) to attain excellent conductivity, which is important to exploit the the electro-chemical principle with high sensitivity and specificity of detection. The variation in concentration of the bio-analyte influences the charge transfer from the conducting surface and results in a distinct change in the electrical property.
The schematic representation of the process flow of the device fabrication and the final architecture, in accordance with an implementation of the present invention is depicted in Figure 2A.
The major stages involved in development are given below:
1) Development of biosensor strip: The test strip comprises an electrochemical-based biosensor for the accurate quantification of two maternal biomarkers, free ß-hCG and PAPP-A. The biosensor is fabricated on a substrate coated with a functionalized conductive polymer and or nanocomposite for immobilization of antibodies.
The major steps involved in development of biosensor or test strip are as follows:
a. Selection of circuit components: Selection of coating materials based on conductivity, stability, functionalization capacity and electrical impedance, coating substrate on the basis of flexibility & transparency, electrode on the basis of signal quality and cost, optical source on the basis of wavelength and electrical conductivity and or impedance.
b. Synthesis of conductive coating material: A Polyaniline (PANI) and or Silver-Polyaniline nano-composite (AgNPs-PANI) are synthesized and ratio of silver nanoparticles to PANI is optimized to obtain excellent conductivity which is important to exploit the electro-chemical principle with high sensitivity and specificity of detection.
c. Characterization of synthesized coating material and nanoparticles: The X-ray powder diffraction (XRD) patterns of the as-prepared coating material and nanoparticles are characterized by using UV-Visible spectroscopy, DSC, FTIR powder X-ray diffractometer,. Scanning electron microscope (SEM) is employed to characterize the morphologies and size of the synthesized samples. Similarly, a Zeta potential analyser is used to characterize the electrokinetic properties of the synthesized conductive nanoparticles.
d. Coating substrate with conductive layer and its functionalization: The synthesised Polyaniline (PANI) and or Silver-Polyaniline nano-composite (Ag-PANI) are coated on substrate. The coating on substrate is functionalized with a linker addition.
e. Immobilization of antibodies on Functionalized coating: After functionalization of coating, the covalent bond based attachment leads to immobilization of bio-analyte specific antibodies. Non-specific active regions of the biosensor are blocked by treatment with albumin. The schematic representation for the whole process is given in Figure 2.
f. Electrode fabrication on test strip: Further, one or more electrodes selected from Gold or aluminium Al electrodes are fabricated on the biosensor surface for conductivity and or impedance property measurements.
2. Microelectronic circuit assembly for the Read-out: The read-out device comprises a circuit assembly and a potentiostat or galvanostat for the electrical read-outs. Real-time collection of bio-electric signal is achieved by mounting the biosensor test strip in the sample slot of device whereby the change in electrical property before and after sample addition is recorded, converted and displayed as concentration of bio-analyte on the screen of device.
3. Integration of Biosensor Test strip with read-out device: Coupling of test strip electrodes to a potentiostat or galvanostat in the read-out device to measure change in electrical property after sample addition to quantify the biomarker level (change in electrical property as a function of concentration of biomarker) followed by digital display of results.
4. Software: Currently, a statistic called the “multiple of the median” (MoM) is used to normalize the test result. The MoM is a measure of how far an individual test result deviates from the median (middle) value of a large set of results obtained from unaffected pregnancies. The calculation will include factors like Maternal age, Nuchal Translucency, Nasal Bone and serum biomarker level to calculate the MoM. Based on the value of MoM, the risk prediction for pregnancy at risk of carrying foetus with aneuploidies is given. The MoM calculation requires data base based on the testing method and ethnicity. Hence, it will be critical to select / develop software with relevant data set and MoM calculation.
The biosensor for detection and quantification of free ??-hCG and PAPP-A comprises the following layers:
1. Substrate: It is the bottom-most layer of the biosensor. It can be made of inert (non-conductive) materials like Glass, Silicon dioxide, plastic, etc. or conductive electrodes made up of gold, silver, platinum, carbon, graphene etc. This layer helps create a base for the biosensor and holds the other layers on it. Gold electrode is used as the metallic substrate in this study.
2. Conductive layer: This layer is created by deposition of an optimized coating of conductive polymers like polyaniline (PANI), polypyrrole, or polythiophene etc. This conductive layer provides the desired functional groups to bind and immobilize the antibody layer on top of this polymeric layer. Further, it also improves the electrical properties of the biosensor.
3. Linker: Linkers like Glutaraldehyde, EDC-NHS, Streptavidin, Avidin are chemicals which create a bridge between the amine functional groups of the conductive polymer with the carboxyl groups of the linker.
4. Primary or capture Antibody layer: The primary antibody (1°Ab) layer was immobilized above the conductive layer using the linker activated -COOH groups at the Fc region of 1°Ab. Antibodies specific to the bio-analytes, hCG and PAPP-A are monoclonal antibodies from mouse or rabbit.
5. Blocking layer: The Bovine serum albumin (BSA) was added to the immobilized layer of antibody to block any unreacted free functional groups of linker via the amine functional groups of BSA.
Once the biosensor is ready, test sample containing the analyte is added on Biosensor, followed by addition of the secondary or detection antibody and the change in electrical property before and after sample addition is recorded.
Optimized Protocols for biosensor design.
Step 1: Method for coating of PANI on metallic (Gold) substrate.
1. Cleaning the metallic (Gold) Electrode: The gold working electrode was cleaned thoroughly to remove any surface contaminants that may interfere with the electrochemical polymerization process. This was achieved by rinsing the electrode with deionized water, ethanol and methanol and followed by drying.
2. Preparation of Polyaniline Electrolyte Solution: Aniline monomer 0.5 M was dissolved in 0.5 M Perchloric acid.
3. Electrochemical Polymerization: The gold working electrode along with counter and reference electrodes (one or more electrodes) were immersed in aniline electrolyte solution and an electrochemical polymerization process began by using a potentiostat or a galvanostat. The potential range for the electrochemical polymerization was from -0.5 V to +1.5 V vs Ag/AgCl, and the scan rate was between 50-150 mV/s. The electrode was exposed to a total of 5-25 scans after which the gold electrode was removed followed by washing with deionized water to remove any residual electrolyte solution. Figure 5 illustrates A: Setup for electrochemical polymerization by using cyclic voltameter, B: uncoated and PANI coated gold electrode.
Characterization of the PANI coating.
1. UV-Vis spectroscopy.
The synthesized PANI was analysed by UV-Vis spectroscopy for qualitative analysis. Appropriate amount of Aniline monomer and PANI were taken in solvent (NMP or DMA) at a concentration between 0.1-0.3 % w/v to obtain a colloidal dispersion of these samples. The UV-Vis spectra in the range of 200-800 nm was recorded for determination of the characteristics peaks. Figure 6 illustrates UV-Vis analysis of Aniline and PANI.
Results and discussion.
UV-Vis spectrum of Aniline and PANI is illustrated in Figure 6. Aniline exhibited a strong absorption band at 290 nm and PANI exhibits two bands around 340-360 nm and 650 nm, Moreover, the disappearance of the 290 nm absorption band in the UV-Vis spectra implies 100% conversion of aniline to PANI. The UV-Visible absorption spectra of PANI depend on the type and level of doping, the extent of conjugation, and the nature of the solvent. PANI exhibits two bands around 340-360 nm and 650 nm. The band around 340-360 nm are attributed to the p-p* transition in the aromatic rings. The absorption bands around 650 nm correspond to the intramolecular electronic transition between quinoid and benzenoid units. The spectra provide evidence of successful formation of PANI from its monomer Aniline.
2. Fourier transform infrared spectroscopy (FTIR).
The synthesized PANI was also characterized by FTIR. FTIR-400 spectrophotometer was used for the determination of IR spectra of synthesized material by using potassium bromide (KBr) to make a sample disc. Figure 7 illustrates FTIR analysis of Aniline and PANI.
Result and discussion.
FTIR peaks at 1400 cm-1 and 1492 cm-1 are attributed to C-C bonds for aniline and PANI, respectively as shown in Figure 7 . Stretching C-N has different spectra of aniline and PANI, the PANI spectrum being at wavenumber 1159 cm-1 and the aniline spectrum being at wavenumber 1149 cm-1. Stretching -N-H also contains information about the differences in spectra between aniline and PANI. Aniline, stretching -N-H showed two peaks at wavenumbers 3340 and 3360 cm-1, whereas PANI showed a slightly shifting peak towards 3250 cm-1. This is so because PANI includes a combination of primary and secondary amines, whereas aniline exclusively contain primary -NH2. Aniline and PANI spectras both had similarities and differences, which supported the formation of PANI.
3. Differential Scanning Calorimetry (DSC).
DSC sample analysis was performed using METTLER STARe SW 12.10. About 2-5 mg of the samples were weighed and added to the pan. The pan was sealed and loaded into the DSC sample holder. The DSC was carried out for all samples in the range of 0 – 140°C at a heating rate of 10°C/min under a continuous flow of dry nitrogen gas (10 ml/min). Figure 8 illustrates DSC curve of A: Aniline, B: PANI.
Result and discussion.
The DSC thermograms with change in enthalpy of Aniline and PANI is shown in Figure 8. The heat transition of PANI was observed by monitoring the plot of heat flow against temperature. Aniline showed an endothermic peak at 174.20°C and after polymerization, its endothermic peak shifted to 103.15°C PANI. Broad peaks are a sign of a polymer that is partially crystalline.
Step 2: Site directed immobilization of primary or capture Antibody on PANI coated gold electrodes.
For the immobilization, the carboxyl group of the 1°Ab is activated by using (3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). EDC-NHS activated 1°Ab is captured on the NH2-functionalized PANI coated electrode.
For the activation of COOH- group of 1°Ab, EDC/NHS reagent was prepared by dissolving EDC (20mM) and NHS (50mM) in acetate buffer (pH 5.1). 500 µL of 1°Ab of free ß hCG or PAPP-A was added in the EDC/NHS solution and incubated at room temperature (RT). After the incubation period, 15 µL of 2-mercaptoethanol was added to quench the reaction and vortexed at RT. The EDC/NHS activated 1°Ab was then added on the PANI coated gold electrodes and again incubated at room temperature to immobilize the 1°Ab on the functionalized surface. Subsequently, the electrodes were washed off by acetate buffer (pH 5.1) using dip method and air-dried. Monoclonal antibody for hCG and PAPP-A were used.
Step 3: Treatment of antibody immobilized PANI coated electrodes with bovine serum albumin.
1°Ab immobilized electrodes were treated with 15 µL of bovine serum albumin (BSA) to block the unoccupied spaces between the 1°Ab to prevent the nonspecific binding of analyte. After the incubation period of blocking, electrodes were then washed off using PBS buffer (pH 7.4) by dip method and kept for air-drying.
Step 4: Addition of Analyte or antigen.
BSA-treated coated electrodes were treated with varying concentrations of antigen [free beta hCG (20-200 ng/ml) and PAPP-A(5-32 µg/ml)] and incubated at RT. After the incubation period of antigen, the electrodes were washed with PBS buffer using the dip method and air-dried.
Step 5: Addition of Secondary or detection Antibody
AG-BSA-1°Ab-treated PANI coated electrodes were incubated with HRP conjugated Secondary antibody (2°Ab) of free beta hCG or PAPP-A for the period of 15 minutes and washed with PBS buffer using dip method before undergoing evaluation. The 2°Ab or detection antibody was a monoclonal antibody tagged with HRP for free beta hCG and PAPP-A.
Testing of the biosensor by CV measurement:
The designed biosensors were evaluated after biosensor coating using cyclic voltammetry testing. Cyclic voltammetry (CV) measurements were recorded over a voltage range spanning from -0.1 to 1.0 V. Two cycles of 20 scans are recorded and the average of second cycle is noted as the oxidation and reduction peak.
Results and Discussion:
CV measurement for testing of biosensor with varying concentration of analytes (free beta hCG) :
The effect of various concentrations of analyte on the redox peaks of 6 mm PANI coated gold electrode was evaluated by CV analysis. For this, we selected three varying concentrations of free beta hCG (20 ng/ml, 100 ng/ml and 200 ng/ml).
The study of different concentrations of analyte revealed that the biosensor electrodes treated with low concentration of analyte exhibited much higher (around 6 fold) oxidation values (Peak) than without analyte treated (PBS treated) electrodes. Additionally, enhancement in oxidation and reduction values was observed when concentration of analyte was increased from 20 to 200 ng/ml. Interestingly, a shift and decrease in reduction peak (dual peaks) was observed with 200 ng/ml analyte treated electrodes with O/R ratio around 1.40. This could indicate that there is a major loss of electrons during the oxidation reaction which could lead to formation of more than one oxidation state conformation on working electrodes due to saturation of the available functional groups on 1°Ab resulting in drop of signal intensity.
Table 1: CV testing data of varying concentrations of free beta hCG antigen (20ng/ml, 100ng/ml and 200ng/ml) added on Biosensor.
Samples Oxidation peak (µA) Reduction peak (µA) Ratio (O/R)
PBS 256±79 442±160 0.59
20ng/ml free ß-hCG 1530±322 2216±840 0.77
100ng/ml free ß-hCG 2504±316 4262±227 0.59
200ng/ml free ß-hCG 3083±333 2243±351 1.41

CV measurement for testing of biosensor with varying concentration of analytes (PAPP-A) :
The effect of various concentrations of analyte on the redox peaks of 6mm PANI coated gold electrode was evaluated by CV analysis. For this, we selected three varying concentrations of PAPP-A (5 µg/ml, 15 µg/ml and 30 µg/ml).
The CV study for PAPP-A revealed a small difference between the presence and absence of analyte. Additionally, the observed oxidation-reduction peak ratio ranged from 0.7 to 0.9, suggesting a near-balanced redox state. Further investigations are on-going to improve the resolution and sensitivity of the biosensor for detection of PAPP-A.
Table 2: CV testing data of varying concentrations of PAPP-A antigen (5 µg/ml, 15 µg/ml and 30 µg/ml) added on Biosensor.
Samples Oxidation peak (µA) Reduction peak (µA) Ratio (O/R)
PBS 833±34 1086±338 0.9
5µg/ml PAPP-A 1152±345 1648±237 0.69
15µg/ml PAPP-A 1064±42 1217±65 0.87
30µg/ml PAPP-A 1021±64 1457±156 0.7 ,CLAIMS:1. A device (A) for detecting a bio-analyte (12), the device comprising;
a) a biosensor (1) fabricated on a substrate (8) coated with a functionalized conductive polymer (7), wherein the functionalized conductive polymer is immobilized with a plurality of antibodies (6, 13) specific to the bio-analyte (12), wherein the biosensor (1) is adapted to change electrical property (11) by interaction with the bio-analyte (12),
one or more electrodes (2,3,4) attached to the substrate (8), wherein the one or more electrode (2,3,4) is adapted to simultaneously collect and deliver the change in electrical property; and
b) a read-out device (10) adapted to display the change in electrical property, wherein the read-out device comprises a circuit assembly, and a potentiostat or a galvanostat (10).
2. The device as claimed in claim 1, wherein the bio-analytes are maternal markers free ß-hCG and PAPP-A.
3. The device as claimed in claim 1, wherein the substrate comprises a conductive substrate or a non-conductive substrate, wherein the non-conductive substrate comprises glass, silicon dioxide, plastic wherein the conductive substrate comprises gold, silver, platinum, carbon or graphene.
4. The device as claimed in claim 1, wherein the one or more electrodes are gold or silver electrodes.
5. The device as claimed in claim 1, wherein the conductive polymer of the biosensor comprises polyaniline (PANI), polypyrrole, or polythiophene.
6. The device as claimed in claim 1, wherein the conductive polymer of the biosensor comprises silver nanoparticles (AgNPs) and Polyaniline nanocomposite (AgNPs-PANI).
7. The device as claimed in claim 1, wherein the conductive polymer is functionalized by a linker selected from a group consisting of Glutaraldehyde, 1-Ethyl-3-diaminopropyl carbodiimide and N-hydroxysuccinimide (EDC-NHS), Streptavidin, or Avidin.
8. The device as claimed in claim 1, wherein the change in electrical property is directly proportional to the concentration of the bio analyte.
9. The device as claimed in claim 1, wherein the device detection range for free beta hCG is 20 to 200 ng/mL, wherein the device detection range for PAPP-A is 1 to 32 µg/mL.
10. The device as claimed in claim 1, wherein the device is compact, portable, point-of-care and standalone.
11. A process for fabrication of a biosensor, the process comprising;
- selecting a substrate as a base of the biosensor;
- coating a conductive polymer layer on the substrate;
- functionalizing the conductive polymer coating on the substrate with a linker;
- immobilizing a plurality of antibodies specific to a bio-analyte on the functionalized conductive polymer of the substrate;
- blocking the non-specific active regions of the conductive polymer layer with a bovine serum albumin (BSA);
- fabrication of one or more electrode on the surface of the functionalized conductive polymer of the substrate to obtain the biosensor.
12. A method of detection of a bio-analyte by a device for detecting a bio-analyte, the method comprising;
a) obtaining a biosensor with a unique antibody directed against a distinct antigenic determinant present on the bio-analyte;
b) adding a sample containing the bio-analyte to the biosensor;
c) capturing the change in electrical property before and after sample addition;
d) measuring the change in electrical property by one or more electrodes;
e) converting change in electrical property into concentration;
f) displaying the change in electrical property in terms of concentration of bio-analyte by a readout device;
wherein the change in electrical property is directly proportional to the concentration of the bio analyte.
13. The method as claimed in claim 12, wherein the sample is maternal serum or blood.