Device and method for determining an amount of analyte in a biological sample

Abstract

The present invention relates to a method and a device for determining an amount of a specific analyte (39) in a biological sample comprising: a support substrate (11); sensing means, arranged on the support substrate and configured to produce an electric current in response to the amount of analyte in the sample; receiving means (38) configured to capture the specific analyte; and a means of absorbent material (14) that is in contact with the sensing means and configured to receive the biological sample and convey at least part of same to the sensing means.

Description

[0001] DEVICE AND METHOD FOR DETERMINING THE AMOUNT OF ANALYTE IN A BIOLOGICAL SAMPLE

[0002] DESCRIPTION

[0003] OBJECT OF THE INVENTION

[0004] The present invention relates to the technical field of biosensor devices and more specifically to the quantification of the analyte present in a biological sample based on its electrical response.

[0005] BACKGROUND OF THE INVENTION

[0006] In recent years, various integrated diagnostic kits have been developed for personal qualitative testing. These portable diagnostic devices (point-of-care testing, or POCT) provide early results on-site without requiring specialized medical personnel, enabling faster and more cost-effective implementation of medical treatments for healthcare systems.

[0007] In the state of the art, lateral flow devices are known, which rely on capture reagents fixed to a membrane (such as a nitrocellulose strip) to capture an analyte from a sample. This process involves the use of antibodies and visual detection elements such as colloidal gold. Generally, these lateral flow devices are intended for applications that do not require high sensitivity and prioritize the ability to obtain a result in situ. However, one of the major drawbacks of this technology is the transducer element they employ, usually optical, which requires analyte titration and adds complexity to the final system.

[0008] On the other hand, high-precision quantitative tests still require complex procedures that typically demand the expertise of qualified technicians in a laboratory with advanced equipment. The application of electrolyte-controlled organic field-effect transistors (EGOFETs) as biosensors is currently being investigated, with particular attention to the electrical detection of non-dilution biomolecules in an aqueous medium (electrolyte), as shown in Figure 1. However, the portability and ease of use of systems based on this type of sensor restrict their implementation to laboratory environments controlled by specialized personnel. Specifically, one of the major limitations to their portability is that sample analysis requires microfluidic chambers with an inlet, an outlet, and a pumping system for analyte / electrolyte transport.

[0009] Given the limitations in the state of the art, there is a need for highly sensitive portable solutions that allow for analytical performance in immediate diagnostic devices at the same level as laboratory tests.

[0010] DESCRIPTION OF THE INVENTION

[0011] In order to achieve the objectives and avoid the aforementioned drawbacks, the present invention describes, in a first aspect, a biosensor device for determining the quantity of a specific analyte in a biological sample. The biosensor comprises:

[0012] - a support substrate;

[0013] - sensor means, arranged on the support substrate, configured to produce an electric current in response to the amount of the specific analyte in the sample;

[0014] - receptor media configured to capture the specific analyte; and

[0015] - an absorbent material, in contact with the sensing media, configured to receive the biological sample and transport at least a portion of it to the sensing media. It is also responsible for absorbing the electrolyte (aqueous medium).

[0016] Thus advantageously, the present invention allows the analyte in the sample to be quantified in real time using an electrical reading, providing high sensitivities reflected in small variations in current intensity associated with different concentrations of the analyte, in a range that can extend from zeptomolar to nanomolar.

[0017] On the other hand, the use of an absorbent material, such as a nitrocellulose strip, to receive the sample and transport it to the sensor media, allows the device to be portable in a manner similar to those based on lateral flow strips.

[0018] According to one embodiment of the invention, the sensing means comprise an electrolyte-controlled organic field-effect transistor (EGOFET), wherein the transistor has a first electrode G, a second electrode D, a third electrode S, and an organic semiconductor layer disposed in contact with the second electrode D and the third electrode S, wherein the absorbent material medium is disposed in contact, at least, with the first electrode G and with the organic semiconductor layer. Specifically, according to one embodiment of the invention, the absorbent material medium comprises a nitrocellulose strip configured to provide an electrolytic medium to the electrolyte-controlled organic field-effect transistor when the strip receives the biological sample in an aqueous medium.Thus, advantageously, because the nitrocellulose strip handles sample transport, the need for pumps and microfluidic elements to perform this function is eliminated. Furthermore, the aqueous medium that enables biomolecule transport within the strip makes it the essential electrolyte medium for the EGOFET's electrolyte function.

[0019] According to one embodiment of the invention, the receptor media are fixed to the absorbent material medium in the area where the G electrode is located. This advantageously provides great versatility to the device, allowing the biosensor to be adapted to multiple biomolecules simply by replacing the absorbent material medium with another containing receptor media selected to capture another specific analyte.

[0020] The receiving means, according to one embodiment of the invention, are attached to the surface of the first electrode G of the electrolyte-controlled organic field-effect transistor.

[0021] In a specific embodiment of the present invention, the receiving means comprise an antibody selected to bind to a specific analyte.

[0022] The present invention, in one embodiment, provides a system for determining the quantity of a specific analyte in a biological sample. The system comprises: a first device according to the biosensor described in any of the preceding embodiments; and a second measuring device configured to measure the electrical current produced by the sensing means in response to the quantity of analyte in the sample; compare the measured electrical current with a prior calibration; and determine the quantity of analyte based on the comparison between the measured electrical current and the prior calibration. Specifically, in one embodiment of the invention, the second measuring device comprises a potentiostat.

[0023] A second aspect of the invention relates to a method for determining a quantity of a specific analyte in a biological sample comprising the following steps:

[0024] - receive the biological sample by means of an absorbent material;

[0025] - transporting, by means of absorbent material, at least a part of the sample to sensing means arranged in contact with or within the absorbent material;

[0026] - to capture, by means of receptors, the specific analyte present in the biological sample;

[0027] - to produce, by means of sensors, an electric current in response to the amount of specific analyte present in the sample; and

[0028] - determine the amount of the specific analyte present in the sample based on the electrical current produced.

[0029] The process begins by establishing a relationship between different quantities of the specific analyte and the corresponding electrical currents produced by the sensing media. Specifically, in one embodiment, establishing this relationship involves associating known quantities of analyte with the characteristic transfer curve of the sensing media. This advantageously calibrates the device for the subsequent identification of analyte quantities.

[0030] In one embodiment of the invention, determining the amount of the specific analyte present in the sample comprises: electrically connecting the sensing means to a measuring device; measuring, by the measuring device, the electric current produced by the sensing means; and determining the amount of specific analyte based on the relationship established between different amounts of the specific analyte and the corresponding electric currents produced by the sensing means.

[0031] Additionally, one embodiment of the invention contemplates establishing wireless communication between the measuring device and an electronic display device; and representing on the electronic display device the amount of specific analyte determined in the biological sample.

[0032] BRIEF DESCRIPTION OF THE FIGURES To complete the description of the invention and to aid in a better understanding of its characteristics, according to a preferred embodiment thereof, a set of drawings is included in which, for illustrative and non-limiting purposes, the following figures have been represented:

[0033] - Figure 1 (state of the art) schematically represents a sensor based on an EGOFET requiring microfluidics.

[0034] - Figures 2A and 2B schematically represent an embodiment of the present invention in which an EGOFET-based sensor is combined with a nitrocellulose strip.

[0035] - Figure 3 schematically represents two alternative embodiments of the biosensor device of the present invention, which differ in the fixation of the receiving means in the absorbent material or in the gate electrode G.

[0036] - Figure 4 schematically represents the process of fixing and preparing the receptor media to the nitrocellulose strip, including blocking with BSA (bovine serum albumin), according to a first embodiment of the invention in which the receptor media are fixed to the nitrocellulose strip.

[0037] - Figure 5 schematically represents the process of attaching and preparing the receiving media to an electrode connected to the gate terminal of the electrolyte-controlled organic field-effect transistor, of the EGOFET type, according to a second embodiment of the invention in which the receiving media are attached to the G (gate) electrode of the transistor.

[0038] - Figure 6 illustrates the detection of biomolecules after functionalization of the device, according to any of the embodiments described in Figures 4 and 5.

[0039] - Figure 7 shows a graph representing a characteristic transfer curve of the EGOFET transistor used for calibration of the biosensor results.

[0040] - Figure 8 represents a calibration graph that relates different quantities of analyte to the variations in electrical current produced in the biosensor.

[0041] - Figure 9 schematically represents an embodiment of the system of the present invention comprising a measuring device for reading and interpreting the electrical output signals of the biosensor device.

[0042] - Figure 10 shows a specific transfer curve of the biosensor device that fixes antibodies on a nitrocellulose strip, used in calibration for the detection of hepatitis B and C antigens. - Figure 11 shows a calibration graph for hepatitis B that relates different concentrations of analyte dissolved in buffer medium and saliva matrix with the variations in electrical current produced in the biosensor.

[0043] - Figure 12 shows a calibration graph for hepatitis C that relates different concentrations of analyte dissolved in buffer medium and saliva matrix with the variations in electrical current produced in the biosensor.

[0044] List of references used in the figures

[0045] 1. Electrolyte-controlled organic field-effect transistor (EGOFET)

[0046] 2. Aqueous medium (electrolyte)

[0047] 3. Microfluidic chamber entrance

[0048] 4. Microfluidic chamber outlet

[0049] 11. Substrate

[0050] 12. Conducting terminals S and D.

[0051] 13. Organic Semiconductor Layer (OSC)

[0052] 14. Absorbent material medium

[0053] 15. Terminal Gate

[0054] 21. Sample pad

[0055] 22. Absorbent pad

[0056] 31. Flexible substrate layer

[0057] 33. Drain terminal

[0058] 35. Fuel dispenser terminal

[0059] 36. Nitrocellulose strip

[0060] 37. Production with receiving media fixed to the strip

[0061] 38. Receiving media

[0062] 39. Specific Analyte

[0063] 40. Implementation with receiving means fixed to the gate terminal of the transistor

[0064] 41. Human anti-IgG antibodies

[0065] 42. Human IgG analyte

[0066] 43. Bovine serum albumin (BSA)

[0067] 50. Cleaning procedure

[0068] 51. Self-assembled monolayer (SAM)

[0069] 52. Covalent bonding to the SAM of biological recognition components

[0070] 53. Anchoring of capture antibodies 54. Blocking stage.

[0071] 60. Incubation time

[0072] 61. Cleaning the strip with water

[0073] 70. Transfer curve associated with an EGOFET sensor in the absence of IgG analyte. 71-78. Transfer curves associated with samples with IgG ligand concentrations between 10' 17 M and 10- 10 M.

[0074] 90. Measuring device

[0075] 91. Wireless communication

[0076] 92. Electronic display device

[0077] DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

[0078] The present invention discloses a device, a system, and a method for determining the quantity of a specific analyte in a biological sample, combining technologies from lateral flow assays and electrolyte-controlled organic field-effect transistors (EGOFETs) as an electrical transducer for the development of a portable biosensor. Thus, the present invention provides an alternative method for the electrical detection of biomolecules in aqueous media, using strips of absorbent material (e.g., nitrocellulose) as the electrolyte medium, where the receptors can also be attached, enabling the development of a point-of-care (POC) device.

[0079] Figures 2A and 2B illustrate an electrolyte-controlled field-effect transistor (EGOFET) that functions as an electrical transducer and comprises: a substrate 11; two conducting terminals 12, comprising a drain terminal 33 and a source terminal 35, for example, made of gold and manufactured by photolithography followed by thermal evaporation of the metal; an organic semiconductor (OSC) layer 13, covering the area between the drain and source terminals; an electrolyte covering the organic semiconductor layer 13; and a third conducting contact (gate) 15, where the gate is in contact with the electrolyte. Additionally, the device of the present invention includes an absorbent material 14 that acts as the electrolyte. The absorbent material 14 is physically positioned on top of the semiconductor layer 13 and the third contact, the gate 15.In one of the preferred embodiments, along with the absorbent material medium 14, the following are also provided: a sample pad 21, made of glass fiber, for depositing the sample and allowing it to flow along the absorbent material medium 14; and an absorbent pad 22, made of glass fiber, which is the component that drives the flow to move along the absorbent material medium 14.

[0080] The physical operation of the transistor involves applying a potential between its gate and source (S) terminals. This causes the electrolyte to form an electrical double layer at two interfaces: gate / electrolyte and electrolyte / oscillator. In this scenario, the cations and anions of the electrolyte migrate, on the one hand, towards the gate surface, creating an electrical layer at the interface with the electrolyte, and, on the other hand, towards the oscillator / electrolyte interface, creating the second electrical layer. This allows for the accumulation of charges in the oscillator layer. Finally, when a potential is applied between the drain (D) and source (S) terminals, a flow of mobile charges appears along the organic semiconductor. The output signal of an EGOFET transistor, called the transfer characteristic curve, is obtained by measuring the current between the drain and source terminals (IDS) against a sweep of the gate potential (VG) with a fixed potential between the drain and source (VDS).

[0081] The application of EGOFET transistors for biomolecule detection according to the present invention comprises attaching receptor media as a recognition element (e.g., an antibody or an aptamer) to one of the gate / electrolyte or semiconductor / electrolyte interfaces. Upon recognition of the biomolecule of interest, the electrical double layer created at the gate / electrolyte or semiconductor / electrolyte interface, respectively, undergoes modifications that produce changes in the electrical properties of the EGOFET. These changes are reflected in the transistor's transfer characteristic curve, for example, in a change in the drain current (IDS). In other words, a highly sensitive electrical output is provided by functionalizing the device interfaces with the appropriate bioreceptor molecules to capture a specific analyte.In the device described herein, the receiving media are fixed at the gate / electrolyte interface.

[0082] Figure 3 illustrates two embodiments of the invention that differ in the location of the receptor media 38, but both comprise the same components: an electrolyte-controlled organic field-effect transistor (EGOFET) 1, with a flexible substrate layer 31, a gate terminal (G) 15, a drain terminal (D) 33, and a source terminal (S) 35, having an organic semiconductor layer 13 between the drain (D) and source (S) terminals; and a nitrocellulose strip 36, as the absorbent material, disposed on the transistor 1 and in contact with the G, D, and S terminals. In a first embodiment 37, antibodies are disposed directly on the nitrocellulose strip as receptor media 38 selected to bind to a specific analyte 39 present in a biological sample. In a second embodiment 40, the antibodies are attached directly to the gate terminal (G) 15 of the transistor 1.

[0083] Figure 4 illustrates the process of fixing and preparing the receptor media on the nitrocellulose strip 36, including blocking with BSA 43 (bovine serum albumin), according to an embodiment of the invention in which the nitrocellulose strip 36 is functionalized. The anchoring of biomolecules to the nitrocellulose strip 36 is possible thanks to the presence of nitro functional groups (-NO2), which interact with groups present in the biomolecules primarily through electrostatic, hydrophobic, and hydrogen bonding interactions. The steps to follow for the functionalization protocol of the nitrocellulose strip 36 are as follows:

[0084] - a solution of the receptor (in this case, human anti-IgG antibodies 41) is dispensed onto a nitrocellulose strip 36 and left to dry at 37 °C overnight;

[0085] - the next day, the nitrocellulose strip 36 is blocked with bovine serum albumin protein 43 (BSA) at 2% by mass for 20 minutes, washed twice with PBS (aqueous saline solution, pH=7.4) and dried for 4 hours at 37 °C;

[0086] - Sample pad 21 is soaked with buffer (0.01 M PBS and 0.5% BSA) and left to dry at 37°C overnight; and

[0087] - Once all components are completely dry, sample pad 21 and absorbent pad 22 are carefully attached to nitrocellulose strip 36 with a 2 mm overlap between them.

[0088] Figure 5 illustrates an embodiment of the invention in which the gate G of the transistor is functionalized. The process of chemically attaching the receiving media to the electrode connected to the Gate 15 terminal of the electrolyte-controlled organic field-effect transistor, type EGOFET 1, can be carried out using various known techniques. One of these is described below as an example:

[0089] - prior to the (biofunctionalization of Gate 15, Gate 15 is subjected to a cleaning procedure 50 in which the gold surface is cleaned in a basic 5:1 :1 piranha bath (H2O / NH4OH / H2O2) for 30 minutes at 60°C and subsequently rinsed with copious Milli-Q water and isopropanol, drying it with a stream of N2; - to fix the receptor media to the surface, a self-assembled monolayer 51 (SAM) is used with 3-mercaptopropionic acid (3-MPA) as a reagent; - the biological recognition components are covalently linked 52 to the SAM using the well-known EDC / NHS chemistry, activating the carboxylic groups of the SAM so that capture antibodies 41 can be anchored 53 via their amine groups;

[0090] - After antibody binding, a blocking step 54 is performed in which BSA 43 is added to the electrode to complete biofunctionalization and prevent non-specific absorption.

[0091] All functionalization steps are performed by immersing the electrode in a vial containing the aforementioned solutions. The incubation time for SAM 51 formation is 24 hours, for covalent bonding 52 using EDC / NHS 5 hours, for antibody anchoring 53 18 hours, and for blocking 54 with BSA 1 hour.

[0092] Figure 6 illustrates the detection of biomolecules after functionalization of the device, according to any of the described embodiments. Thus, for the electrical detection of human IgG ligands 42 (the specific analyte 39 in this example), IgG solutions in PBS (pH = 7.4) are deposited onto the sample pad 21. By capillary action, the aqueous medium containing the human IgG ligands 42 circulates along the nitrocellulose strip 36 until it encounters the recognition element (i.e., the previously anchored anti-human IgG antibodies 41), finally reaching the absorbent pad 22. A 15-minute incubation period 60 is then allowed between the analyte and receptor. Afterward, the nitrocellulose strip 61 is cleaned with water to remove any excess analyte that has not interacted with the receptor, and electrical characterization is performed using the transfer curve.

[0093] Figure 7 shows several transfer characteristic curves used for calibrating the biosensor results. The transfer characteristic curve relates the source-drain current (IDS) to the gate voltage (VGS); therefore, it is obtained by measuring the current between the drain and source terminals (IDS) against a sweep of the gate potential (VGS) with a fixed potential between the drain and source (VDS). In this case, several (nine) transfer characteristic curves are shown, corresponding to different known concentrations of IgG. These include a first curve 70 associated with a sample without IgG analyte (which serves as a reference for recording the electrical variations of the other concentrations) and a group of eight curves 71-78 associated with samples with different concentrations of IgG analyte between 10 -17 M and 10' 10M. As seen in Figure 8, the device is calibrated based on the information obtained from the transfer characteristic curves represented in Figure 7, so that it allows relating different concentrations of IgG (represented on the horizontal axis between 10 -17 M and 1O' 10 M) with the maximum electrical currents (IDS) produced in the biosensor. Specifically, the vertical axis reflects the current variation caused by the analyte concentration by the ratio of the difference between the drain currents (IDS), with and without IgG, and the current without IgG (IDS, bare). In this way, the device is calibrated for the detection of a specific analyte (human IgG in this case) with a direct relationship between the transistor output currents and analyte concentrations.

[0094] Figure 9 illustrates a measuring device 90 electrically connected to the Gate 15, Drain 33, and Spout 35 terminals of the biosensor device of the present invention, wherein the measuring device 90 is a potentiostat configured to read and interpret the electrical output signals of the biosensor device. The measuring device 90 can be connected to a computer or establish a wireless connection 91, via Bluetooth or WiFi, for example, with other electronic display devices 92, such as a tablet or a mobile phone.

[0095] Figure 10 illustrates a specific embodiment of the invention, where several transfer curves relate the source-drain current (SDC) to the gate voltage (GV). Each curve corresponds to different known concentrations of hepatitis B antigens (HBsAg), including a first curve corresponding to a sample without analyte, which serves as a reference for recording the electrical variations of the remaining concentrations.

[0096] Figure 11 shows a calibration curve relating different concentrations of analyte dissolved in buffer medium (dot plot) and saliva matrix (triangle plot) to the variations in electrical current produced in the biosensor. Thus, in this specific embodiment for the detection of hepatitis B antigens, the device is calibrated based on the information obtained from the transfer curves, which allows relating the different concentrations of HBsAg (Hepatitis B surface antigen), ranging from 10' 17 and 10' 10 M, with the maximum electrical currents (IDS) produced in the biosensor. Specifically, the vertical axis reflects the current variation caused by the analyte concentration by the ratio of the difference between drain currents (IDS), with and without antibodies, and the current without antibodies (IDS, bare).

[0097] Figure 12 shows a calibration curve relating different concentrations of analyte dissolved in buffer (dot plot) and saliva matrix (triangle plot) to the variations in electrical current produced in the biosensor. Thus, in this specific implementation for the detection of hepatitis C antigens, the device is calibrated based on the information obtained from the transfer curves, which allows relating the different concentrations of HCcAg (Hepatitis C core antigen), ranging from 10' 17 and 10' 10 M, with the maximum electrical currents (IDS) produced in the biosensor. Specifically, the vertical axis reflects the current variation caused by the analyte concentration by the ratio of the difference between drain currents (IDS), with and without antibodies, and the current without antibodies (IDS, bare).

[0098] The present invention is not limited to the embodiment described herein. Although implementations of the present invention as an immunosensor have been described in detail, other types of analytes and receptors may be employed. Other configurations may be implemented by those skilled in the art in light of this description. Accordingly, the scope of the invention is defined by the following claims.

Claims

CLAIMS 1. A device for determining a quantity of a specific analyte (39) in a biological sample comprising: - a support substrate (11); - sensor means, arranged on the support substrate, configured to produce an electric current in response to the amount of analyte in the sample; characterized in that it further comprises: - receptor media (38) configured to capture the specific analyte (39); and - an absorbent material medium (14), in contact with the sensing means, configured to receive the biological sample and transport, at least a part, to the sensing means, where the receiving means (38) are fixed to the absorbent material medium (14).

2. Device according to claim 1 wherein the sensing means comprise an electrolyte-controlled organic field-effect transistor, of the EGOFET type, (1) wherein the transistor has a first electrode G (15), a second electrode D (33), a third electrode S (35) and an organic semiconductor layer (13) disposed in contact with the second electrode D (33) and the third electrode S (35), wherein the absorbent material medium (14) is disposed in contact, at least, with the first electrode G (15) and with the organic semiconductor layer (13).

3. Device according to claim 2, wherein the absorbent material medium comprises a nitrocellulose strip (36) configured to provide an electrolyte medium to the electrolyte-controlled organic field-effect transistor when the strip receives the biological sample in an aqueous medium.

4. Device according to any of the preceding claims wherein the receiving means (38) comprise an antibody selected to bind to a specific analyte (39).

5. A system for determining the quantity of a specific analyte in a biological sample, wherein the system comprises: - a first device according to any of claims 1-4; and - a second measuring device (90) configured to measure electric current produced by the sensor media in response to the amount of analyte in the sample; compare the measured electrical current with a previous calibration; and determine the amount of analyte based on the comparison between the measured electrical current and the previous calibration.

6. System according to claim 5 wherein the second measuring device comprises a potentiostat.

7. Method for determining a quantity of a specific analyte (39) in a biological sample comprising the following steps: - receive, by means of an absorbent material (14), the biological sample; - transport, by means of absorbent material (14), at least a part of the sample to sensing means arranged in contact with the absorbent material (14); - to capture, by means of receptors fixed to the medium of absorbent material (14), the specific analyte (39) present in the biological sample; - to produce, by means of sensors, an electric current in response to the amount of specific analyte (39) present in the sample; and - determine the amount of the specific analyte present in the sample based on the electrical current produced.

8. Method according to claim 7 further comprising a prior step of establishing a relationship between different quantities of the specific analyte and the corresponding electric currents produced by the sensing means.

9. Method according to claim 8 wherein establishing the relationship between the amount of analyte and the electric current produced by the sensor comprises associating known quantities of analyte with the transfer characteristic curve of the sensing media.

10. Method according to claim 9 wherein determining the amount of the specific analyte present in the sample comprises: - electrically connect the sensor means to a measuring device (90); - to measure, by the measuring device (90), the electric current produced by the sensing means; and - Determine the amount of specific analyte based on the relationship established between different quantities of the specific analyte and the corresponding electrical currents produced by the sensing media.

11. Method according to claim 10 further comprising establishing wireless communication (91) between the measuring device (90) and an electronic display device (92); and displaying on the electronic display device (92) the amount of specific analyte determined in the biological sample.

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