DESC:
FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
Title of invention:
A SYSTEM AND METHOD FOR DETECTING ANALYTES FROM BIOFLUIDS USING BIOSENSOR
APPLICANT:
MYLAB DISCOVERY SOLUTIONS PRIVATE LIMITED
An Indian entity having address as:
PLOT NO 99-B, LONAVALA INDUSTRIAL CO-OPERATIVE ESTATE LTD, NANGARGAON, LONAVALA, PUNE – 410401 MAHARASHTRA, INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed.
CROSS REFERENCE TO RELATED APPLICATION AND PRIORITY
The present application claims priority from Indian Patent Application no. 202121055370 filed on 30 November 2021, incorporated herein by a reference.
TECHNICAL FIELD
The present disclosure relates to a system and a method for detecting analytes from biofluids using biosensor. More particularly, it relates to use of pyrolytic graphite sheet electrode as extended gate of Field Effect Transistor (FET) for detection of analytes from biofluids.
BACKGROUND
The subject matter discussed in the background section should not be assumed to be prior art merely as a result of its mention in the background section. Similarly, a problem mentioned in the background section or associated with the subject matter of the background section should not be assumed to have been previously recognized in the prior art. The subject matter in the background section merely represents different approaches, which in and of themselves may also correspond to implementations of the claimed technology.

The increase in globalization has led to the spread of infectious diseases, such as influenza, Covid-19 and the likes, which spread across continents in a span of few months, leading to death of millions of individuals, making monitoring of human health for early detection of diseases an imperative need. Also, in developing countries, there is shortage of medical infrastructure because of which a large segment of population does not have access to doctors and/or diagnostic labs. Globally, chemical measurement or analytical techniques have seen a surge in demand with the increasing requirement of improved environmental monitoring, health care and food processing. There is an increased need for high throughput, label free, multiplexed sensors for chemical and biological sensing. These analyses have traditionally been carried out in specialized laboratories under the supervision of skilled personnel, with the aid of highly sophisticated analytical instruments. This route of investigation is prone to delays in measurement, sample mishandling and deterioration while transfer. The development of sensors with high selectivity, ease of use and point of care (POC) is the key to the problems encountered by macro scale analytical methods.

In the conventional art, the FET based sensors are capable of miniaturization which have great potential in the world of nanotechnology. Miniaturisation is useful for sensing of recognition elements due to their improved electrochemical, photonic, and magnetic properties, and moreover have an ease and economics of manufacturing. FET sensors are label free sensors i.e., there is no requirement of coloured, fluorescent, enzymatic tags or secondary antibodies or redox mediators. Hence, along with their exceptional sensitivity, the FET based sensors are considered as ideal for point of care diagnostics. In spite of the multitudes of advantages, application of FETs is dependent on the concentration of the solution or buffers that they are immersed in. High electrolyte concentration in the vicinity of FET gate renders it prone to unreliable outcomes as ions presented in the electrolytes can trap at the gate interface, thereby reducing the overall sensitivity or functionality of biosensor. Therefore, segregating the gate from the main FET by a physical extension of the gate electrode is advantageous for biosensing.

FET based sensors are sensitive to the screening length – the distance between the electrode and the site of the reaction, which is impacted by concentration of ions or electrolytes in the measuring medium such as buffers or body fluids. Body fluids have high ionic strength which affects the Debye length and therefore the sensitivity of the sensor. In general, as the Debye length for a solution of physiological buffer (equivalent to 1X PBS) is close to 0.7nm, the Debye length screening is circumvented by conducting the electrical measurements in diluted buffer solutions like 0.1X and 0.01X which correspond to a Debye length of ~3 nm and 7nm respectively. Therefore, it must be understood that when the concentration of body fluid is more, then, the distance between the electrode and the site of reaction affects the sensitivity of the sensor.

In existing extended gate FET based sensors, gold electrodes are typically used. In general, surface of the gold electrode is modified using self-assembled monolayer assemblies (SAMs) with multi-functional moieties such as carboxylate group or amino group which could then be further linked with other recognition elements such as antibodies or thiolated aptamers or DNA probe molecules. Moreover, subsequent modifications of self-assembled monolayers with carboxyl and amino coupling chemistries are routinely performed to covalently attach specific antibodies to detect presence of antigens. Such interaction of recognition elements such as antibodies with test elements such as antigens, causes a change in the current with respect to a change in the voltage between the gold electrode and the reference electrode. Here, it must be noted that surface modification using self-assembled monolayer with linker molecules such as thiols increases the distance between electrode and site of reaction and thereby reducing sensor sensitivity.

Furthermore, graphene-based FET (GFET) sensors use a graphene electrode which is rather challenging to isolate or grow on a substrate, which increases the cost of manufacturing of the sensor. Manufacturing and use of graphene is limited also due to its low thickness making its handling difficult.

Therefore, there is an utmost need of an improved biosensor system which addresses the above-mentioned problems and while maintaining highest sensitivity as well as specificity of detection. Thus, an improved biosensor system and a method for biosensing is needed which enables the system to detect analytes with high sensitivity and specificity, as well as a means to easily quantify a change in the circuit parameters to achieve accurate results.
SUMMARY
This summary is provided to introduce concepts related to a system and a method for detecting analytes from biofluids using biosensor and the concepts are further described below in the detailed description.
Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure. The present disclosure has been made in order to solve the above problems, and it is the object of the present disclosure to provide a system and a method for detecting analytes from biofluids using biosensor.
The present disclosure relates to the system for detecting analytes from biofluids using biosensor. The system may comprise a Field Effect transistor (FET), a trans-impedance amplifier, a signal conditioning circuit, a data acquisition system, a processing and control circuit. The gate of the FET is extended as an encapsulated electrode with an open reaction area. The encapsulated electrode and a reference electrode may be exposed to body fluid placed in a buffer solution and electronically communicated with the FET. The encapsulated electrode may be made up of Pyrolytic Graphite Sheet and configured to immobilize recognition elements on top of Pyrolytic Graphite Sheet. The FET may be configured to detect voltage across the encapsulated electrode and results in change in current of the FET. The trans-impedance amplifier may be configured to receive current from the FET and convert into a voltage signal and further amplify the voltage signal. The signal conditioning circuit may be configured to receive the voltage signal from the trans-impedance amplifier in order to increase signal to noise ratio. The data acquisition system may be configured to convert the voltage signal from the signal conditioning circuit into the digital data. The processing and control circuit may be configured to process digital data and to generate the output data in a user format.
The present disclosure relates to the method of detecting analytes from biofluids using biosensor, which may comprise steps of detecting, via a Field Effect Transistor (FET), voltage across an encapsulated electrode which results in change in current of the FET. The gate of the FET is extended as an encapsulated electrode with an open reaction area. The encapsulated electrode and a reference electrode may be exposed to body fluid placed in a buffer solution and electronically communicated with the FET. The encapsulated electrode may be made up of Pyrolytic Graphite Sheet and configured to immobilize recognition elements on top of Pyrolytic Graphite Sheet. The method may further comprise step for receiving, via a trans-impedance amplifier, current from the FET and convert into a voltage signal and further amplify the voltage signal. The method may further comprise step for receiving, via a signal conditioning circuit, voltage signal from the trans-impedance amplifier in order to increase signal to noise ratio. The method may further comprise step for converting, via a data acquisition system, the voltage signal from the signal conditioning circuit into the digital data. The method may comprise step for processing, via a processing and control circuit, the digital data and to generate the output data in a user format.
BRIEF DESCRIPTION OF DRAWINGS
The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the drawings to refer like features and components.
Figure 1 illustrates a system (100) for detecting analytes from biofluids using biosensor, in accordance with an embodiment of the present subject matter.
Figure 2 illustrates a schematic diagram of the experimental setup, in accordance with an embodiment of the present subject matter.
Figure 3 illustrates a flowchart of method for detecting analytes from biofluids using biosensor, in accordance with an embodiment of the present subject matter.
Figure 4 illustrates a graphical representation of an output current of FET with respect to time for three different viral loads of SARS CoV-2 (hereafter referred to as Covid) infected biological samples and a negative control, in accordance with the first exemplary embodiment of the present subject matter.
DETAILED DESCRIPTION
Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

The present disclosure discloses a system (100) for detecting analytes from biofluids using biosensor. Now referring to Figure 1, the system (100) may comprise a Field Effect Transistor (FET) (105), a trans-impedance amplifier (106), a signal conditioning circuit (109), a data acquisition system (110), a processing and a control circuit (114). The gate of the FET (105) may be extended as an encapsulated electrode (102) with the open reaction area.
Now referring to figure 2, a schematic diagram of the experimental setup of a biosensor, is illustrated in accordance with an embodiment of the present disclosure. Now referring to Figure 1 and 2, the biosensor may be made up of the encapsulated electrode (102), reference electrode (104), a copper tape (101). The encapsulated electrode (102) (also referred as “working electrode”) and a reference electrode (104) may be exposed to body fluid placed in a buffer solution and electronically communicated with the FET (105). In one exemplary embodiment, the encapsulated electrode (102) is made up of pyrolytic graphite sheet and configured to immobilize recognition elements (103) on top of pyrolytic graphite sheet. The thickness of pyrolytic graphite sheet (102) may be 100 µm. In another embodiment, the encapsulated electrode may be made up of carbon materials like carbon coating graphite deposition etc. In one embodiment, recognition elements (103) may be selected from a group of peptides, proteins, nucleic acid, aptamers, antibodies, antibiotics, chemical moieties or any other such similar molecules which bind to the analyte of interest. In one embodiment, the encapsulated electrode (102) may) be connected to FET (105) as a disposable entity by USB connection, PCB edge connectors, relimate (JSK) connector, pogo pins, connector clips etc. The extended gate FET biosensor may be integrated to a handheld measurement system.
The encapsulated electrode (102) may be fabricated using pyrolytic graphite sheet using following steps:
At step 1, pyrolytic graphite sheet of 100 µm thickness may be taken. Further, pyrolytic graphite sheet are commercially available sheets of various thickness, therefore removes the need of deposition. Furthermore, pyrolytic graphite sheets can be cut with a cutting tool or any other such mechanical or physical cutting means, therefore, there is ease of mass manufacturing and eliminating the need for expensive clean room equipment and dedicated specialized laboratories for making electrodes out of materials like gold, platinum, ITO or even screen-printed electrodes or deposition of Carbon nanotubes and the likes.
At step 2, the strips of the pyrolytic graphite sheet may be cut with the help of scissors into desired size.
At step 3, the strips of the pyrolytic graphite sheet (102) may be then fixed using a conductive adhesive such as a conductive copper tape (101) for providing contact to the external world and also to provide a sturdy base to the thin sheet. The copper tape (101) may be further configured to increase the contact area and thereby improve the electrical conductivity of the biosensor.
At step 4, the pyrolytic graphite sheet may be then electrochemically oxidized in at least one of sulphuric acid (H2SO4), potassium permanganate, sodium nitrite or a combination there of electrolyte for a pre-defined time. The surface of the sheet may change from hydrophobic to hydrophilic due of anodic oxidation. The sheets are thoroughly washed with water and used for further modification.
At step 5, oxidized pyrolytic graphite sheet may be then incubated in a mix of (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form intermediate reactive surface carboxylic groups to which the amino groups of the antibodies can bind later. In another embodiment, oxidized pyrolytic graphite sheet is incubated with at least one of the linkers selected from (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Sulfo-NHS, (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC), (N,N’-carbonyldiimidazole) (CDI). In yet another embodiment, oxidized pyrolytic graphite sheet may be decorated with suitable nanomaterials like metal nanoparticles/nanorods, conductive polymers, branched polymers and the likes and further modified for attachment of recognition molecules.
At step 6, the specific antibody solution in the optimized buffer (such as phosphate buffered saline (PBS), Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)) concentration at pH of 7.4 may be then incubated on to the active area of the pyrolytic graphite sheet. Thorough washing with the said buffer is carried out.
At step 7, the unreacted carboxylic groups may be capped with ethanolamine or any other appropriate capping agents in 2-(N-Morpholino)ethanesulfonic acid (MES) buffer at an appropriate pH.
At step 8, the antibody immobilized pyrolytic graphite sheet may be incubated in a solution of bovine serum albumin (BSA) for suppression of non-specific binding. The encapsulated electrode may be thoroughly washed with an appropriate buffer.
At step 9, the fabricated electrode may be subjected to biomaterial stabilizing solutions.
The pyrolytic graphite sheet has high electrical conductivity, therefore the quality of electrode may not be compromised. Further, pyrolytic graphite sheets being commercially available sheets of various thicknesses, eliminates the need for deposition.
The binding of biomolecules or biomaterial or recognition elements like antibodies close to the encapsulated electrodes surface via zero length linkers, eliminates the Debye length effects in bodily fluids. The body fluids are generally, high ionic strength fluids, which affect the Debye length and therefore the sensitivity of the biosensor.
In some embodiments of the present disclosure, recognition elements/biomolecules are attached by zero length linkers, the binding reaction of analyte from the clinical sample or body fluids occurs close to the encapsulated electrode, which in turn gives a good sensitivity to the biosensor. Further, the sample may not require any dilution. Further, the system can be directly used with the clinical sample/body fluid and does not require any sample pre-processing outside in the lab.

Further, an electrode through which gate bias is applied to the test/buffer solution forms the reference electrode (104). In one exemplary embodiment, the reference electrode (104) may be made up of platinum or carbon or Ag/AgCl or any non-polarizable material. In another exemplary embodiment, the reference electrode (104) may be made up of carbon manufactured by depositing, printing, or using a cut metal sheet. In yet another embodiment, the reference electrode (104) may be made up of carbon or Ag/AgCl or any non-polarizable material. In one embodiment, a pulsed gate voltage may be applied to the reference electrode, with the source drain of MOSFET being probed with a constant DC bias. The voltage drop in the test solution reaches the MOSFET dielectric through the extended gate where it drops and modulates the drain current.

The pulsed voltage applied as gate voltage. In one exemplary embodiment, the pulse duration is 125 milli-seconds and the OFF time is 8 seconds, with an predefined amplitude, preferably 0.2 V. The source drain voltage is kept constant at a predefined Volt, preferably 1V.

Now referring back to Figure 1, the FET (105) may be configured to detect voltage across the encapsulated electrode and results in change in current of the FET. In one exemplary embodiment, FET (105) may be a Metal-oxide-semiconductor field-effect transistor. The FET (105) may have the threshold voltage as close to zero as possible. The ratio of change in voltage of the biosensor to change in output current is dependent on the output characteristic of FET.

Referring back to Figure 1 again, the trans-impedance amplifier (106) may be configured to receive current from the FET and convert into a voltage signal with an amplification factor and amplify the voltage signal. The amplification factor may be controlled by an amplification resistor (107) and a filter capacitor (108) arranged in parallel to reduce output noise. In one embodiment, the amplification factor may be dependent on the saturation voltage of the data acquisition system (110). The trans-impedance amplifier may be built from an operational amplifier with low input bias current or with FET in combination with an operational amplifier. The output biasing of the FET stage may be controlled by reference voltage VDS that can be set by the manufacturer according to the output characteristic of FET.
The output of the trans-impedance amplifier (106) may be connected to the signal conditioning circuit (109). The signal conditioning circuit (109) may be configured to receive voltage signal from the trans-impedance amplifier in order to increase signal to noise ratio (S/N) of the system (100). The signal to noise ratio at input of the system (100) is 7.959dB. The S/N ratio at output at 1kHz is 27.74dB. The S/N ratio at output at 10kHz is 51.82dB.
The data acquisition system (110) may be configured to convert the voltage signal from the signal conditioning circuit (109) into the digital data. In one embodiment, the data acquisition system (110) may be a single-ended or differential analog to digital converter or an acquisition module from a different company according to the specification required for the system (100).

The reference voltage source (113) may be configured to generate the reference voltage required to keep the reference electrode (104) at constant potential according to the digital information received from the processing and control circuit (114). The reference voltage may be dependent on the input characteristic of the FET (105) and the open circuit potential generated by different biological samples. The reference voltage source may be a digital to analog converter or a data acquisition system from a different manufacturer. The output voltage of the reference electrode (104) may be then passed through a filter (112) to improve the signal-to-noise ratio of the reference voltage. A unity gain amplifier (111) may be configured to act as a buffer between the reference electrode (104) and the reference voltage source.

The processing and control circuit (114) may be configured to process digital data and to generate the output data in a user format. The processing and control circuit (114) may be made up of different processors with controlling peripherals, microcontrollers from different vendors, or a computer depending on the user’s requirement. In one embodiment, the processing and control circuit (114) may be configured to fetch and execute computer-readable instructions stored in the memory.

Referring to Figure 3, a stepwise flowchart of a method (300) for detecting analytes from biofluids using biosensoris illustrated, in accordance with an embodiment of the present subject matter.

At step 301, the FET (105) may be configured to detect voltage across an encapsulated electrode which results in change in current of the FET.

At step 302, a trans-impedance amplifier (106) may be configured for receiving current from the FET (105) and convert into the voltage signal and amplify the voltage signal.

At step 303, a signal conditioning unit (109) may be configured for receiving voltage signal from the trans-impedance amplifier in order to increase signal to noise ratio.

At step 304, a data acquisition system (110) may be configured for converting the voltage signal from the signal conditioning circuit (109) into digital data.

At step 305, a processing and control circuit (114) may be configured for processing digital data and to generate the output data in a user format.

In first exemplary embodiment, a 10ul volume of Viral Transport Medium is introduced on the biosensor and the measurement is switched on in pulse mode. After 30 seconds of baseline measurements being carried out, 2ul of Covid infected biological sample (nasal swab) having Ct values ranging from 15-30 and negative samples are introduced respectively on the sensor surfaces and the change in drain current with time is measured.

Now referring to Figure 4, a graphical representation of an output current of FET with respect to time for three different viral loads of SARS CoV-2 (Covid-19) infected biological samples and one negative Covid-19 sample in VTM is shown, in accordance with the first exemplary embodiment of the present subject matter. The output current of FET for a negative sample remains almost constant with respect to time, which is indicative of zero viral load. For virus infected positive samples, the magnitude of change in current with time varies with the viral load present in the sample. For example, samples with lower Ct value (i.e. Ct 20), the change in the output current of FET is far more than the output current change for a lower Ct value sample (i.e., Ct 30). Therefore, it has been observed that high output current of FET is indicative of high viral load and low output current is FET indicative of low viral load. The signal output for a sample with low viral loads as low as Ct values of 30, is far higher than the signal for the negative samples, resulting in a high sensitive biosensor, which may be close to the sensitivity of PCR based tests, albeit with a quick response time.

In one embodiment, the system and the method for detecting analytes from biofluids using biosensor may provide a point of care system for diagnosis.

The embodiments, examples and alternatives of the preceding paragraphs or the description and drawings, including any of their various aspects or respective individual features, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.

Although implementations for detecting analytes from biofluids using biosensor have been described in language specific to structural features and/or methods, it is to be understood that the appended claims are not necessarily limited to the specific features or methods described. Rather, the specific features and methods are disclosed as examples of implementations for detecting analytes from biofluids using biosensor.
,CLAIMS:WE CLAIM:
1. A system (100) for detecting analytes from biofluids using biosensor, characterized in that, the system comprises:
a Field Effect Transistor (FET) (105), wherein the gate of the FET (105) is extended as an encapsulated electrode,
wherein the encapsulated electrode (102) and a reference electrode (104) are exposed to body fluid placed in a buffer solution and electronically communicated with the FET (105),
wherein the encapsulated electrode (105) is made up of pyrolytic graphite sheet and configured to immobilize recognition elements (103) on top of pyrolytic graphite sheet,
wherein the FET (105) is configured to detect voltage across the encapsulated electrode (102) and results in change in current of the FET (105);
a trans-impedance amplifier (106) configured to receive current from the FET and convert into a voltage signal and amplify the voltage signal;
a signal conditioning circuit (109) configured to receive the voltage signal from the trans-impedance amplifier in order to increase signal to noise ratio; and
a data acquisition system (110) configured to convert the voltage signal from the signal conditioning circuit into the digital data;
a processing and control circuit (114) configured to process the digital data and to generate the output data in a user format.

2. The system (100) as claimed in claim 1, wherein the pyrolytic graphite sheet is having thickness of at least 100 µm.

3. The system (100) as claimed in claim 1, wherein the basal plane or edge plane of the pyrolytic graphite sheet is used.
4. The system (100) as claimed in claim 1, wherein the pyrolytic graphite sheet is electrochemically oxidized with at least one of H2SO4, Potassium permanganate, sodium nitrite or a combination thereof.

5. The system (100) as claimed in claim 1, wherein recognition elements (103) are selected from a group of peptides, proteins, nucleic acids, aptamers, antibodies, antibiotics, chemical moieties or any other molecules which bind to the analyte of interest.

6. The system (100) as claimed in claim 1, wherein the reference electrode (104) is made up of platinum or gold or carbon or Ag/AgCl or non-polarizable material or combination thereof.

7. The system (100) as claimed in claim 6, wherein a pulsed gate voltage is applied to the reference electrode (104), with the source drain of FET (105) being probed with a constant DC bias.

8. A method for detecting analytes from biofluids using biosensor, characterized in that, the method comprises:
detecting, via a Field Effect Transistor (FET) (105), voltage across an encapsulated electrode (102) which results in change in current of the FET (105), wherein the gate of the FET is extended as the encapsulated electrode,
wherein the encapsulated electrode (102) and a reference electrode (104) are exposed to body fluid placed in a buffer solution and electronically communicated with the FET (105),
wherein the encapsulated electrode (102) is made up of pyrolytic graphite sheet and configured to immobilize recognition elements (103) on top of pyrolytic graphite sheet,
receiving, via a trans-impedance amplifier (106), current from the FET and convert into a voltage signal and amplify the voltage signal;
receiving, via a signal conditioning circuit (109), the voltage signal from the trans-impedance amplifier in order to increase signal to noise ratio; and
converting, via a data acquisition system (110), the voltage signal from the signal conditioning circuit into the digital data;
processing, via a processing and control circuit (114), digital data and to generate the output data in a user format.

9. The method as claimed in claim 10, wherein the pyrolytic graphite sheet is having thickness of at least 100 µm.

10. The method as claimed in claim 10, wherein the basal plane or edge plane of pyrolytic graphite sheet is used.

11. The method as claimed in claim 10, wherein recognition elements (103) are selected from a group of proteins, nucleic acid, aptamers, antibodies, antibiotics, chemical moieties or any other such similar molecules which bind to the analyte of interest.
Dated this 30th Day of November 2021

Priyank Gupta
Agent for the Applicant
IN/PA-1454