Method for detecting and quantifying analytes in microfluidic devices

Abstract

A microfluidic device (1) comprising at least one fluid channel (2) with at least one inlet (3), the at least one fluid channel (2) being fluidly connected to a first sensor (4) downstream of the at least one fluid inlet (3), the first sensor (4) comprising at least one sensor cathode (5) and at least one sensor anode (6) formed on an essentially electrically isolating substrate (7), the sensor cathode (5) and the sensor anode (6) being separated by a gap (8) formed on the electrically isolating substrate (7), at least one analyte capture molecule (9) being immobilized within the gap (8) on the substrate (7), the at least one capture molecule (9) being adapted to capture at least one analyte (10) of a fluid sample introduced to the inlet (3) and transported to the first sensor (4).

Description

Technical Field

[0001]

[0001] The present invention relates to the field of microfluidic devices and methods for detecting and / or quantifying analytes in fluid samples.

Background Art

[0002]

[0002] Developing countries account for 84% of the world's population but only spend 12% of the world's healthcare costs, while simultaneously bearing 90% of the world's disease burden. As a result of this disparity, the incidence of infectious and non-infectious diseases remains extremely high in developing countries. In 2013 alone, for example, more than 8 million people died before reaching the age of 60 due to numerous diseases that are usually treatable, such as stroke, heart disease, diabetes, and influenza, hepatitis, etc. There has been no significant improvement in this situation over the past decade, and third world countries still face many healthcare challenges, including accurate diagnosis of infectious diseases and access to healthcare facilities. Due to limited access to healthcare facilities, the diagnosis of outpatients is important for identifying mild, moderate, and severe cases that require immediate attention and, in some cases, quarantine measures. Unfortunately, fully equipped functional laboratory facilities with electricity, refrigeration, and trained personnel are only available in major cities, and thus rural communities remain mostly vulnerable and unprotected.

[0003]

[0003] Also, recent experience has shown that similar challenges in the accurate diagnosis of infectious diseases also exist in developed countries during a pandemic. In these situations, due to the large number of potential infected individuals, healthcare facilities quickly become overloaded, and it has been found that the capacity of diagnostic laboratory facilities is insufficient even in countries with advanced healthcare systems.

[0004]

[0004] Lab-on-a-chip technology using microfluidic devices is known for its ability to handle complex fluids, sample processing, signal amplification, and detection, and is considered a suitable alternative for performing on-site diagnostic assays in point-of-care situations. According to WHO ASSURED criteria, the ideal point-of-care device should be affordable and easy to use, providing reliable and robust information quickly to the end user when bulky and expensive equipment is unavailable. In fact, due to the high sensitivity, specificity, and accuracy of current point-of-care testing devices, countless lives have already been saved in developing countries, for example, by detecting infectious diseases in their early stages. For example, several point-of-care devices based on molecular diagnostic assays, lateral flow assays, microfluidics, plasmons, and paper-based devices have been reported for the detection of viral infections such as HIV, Zika, Ebola, and dengue fever. The success of point-of-care device applications is also reflected in the global market value, which was US$20.15 billion in 2017 and is projected to reach US$57.85 billion by 2026, growing at a CAGR of 12.4%. [Overview of the project] [Problems that the invention aims to solve]

[0005]

[0005] The object of the present invention is to provide a means for performing inexpensive, accurate, and rapid diagnosis of infectious diseases without requiring a full-fledged experimental facility. [Means for solving the problem]

[0006]

[0006] Accordingly, the present invention relates to a microfluidic device comprising at least one fluid channel having at least one inlet. The at least one fluid channel of the microfluidic device according to the present invention is fluidly connected to a first sensor downstream of the at least one fluid inlet, the first sensor comprising at least one sensor cathode and at least one sensor anode formed on a substrate that is essentially electrically isolated. The sensor cathode and the sensor anode are separated by a gap formed on the substrate that is electrically isolated, and at least one analyte capture molecule is immobilized within the gap on the substrate. The at least one capture molecule is introduced into the inlet and adapted to capture at least one analyte of a fluid sample transported to the first sensor. The microfluidic device comprises at least one second sensor fluidly connected to the fluid channel or the first sensor, the at least one second sensor comprising at least one sensor cathode and at least one sensor anode formed on a substrate that is essentially electrically isolated. The sensor cathode and sensor anode of the second sensor are separated by a gap formed on an electrically isolated substrate, at least one capture molecule is immobilized within the gap on the substrate, and at least one capture molecule is introduced into the inlet and adapted to capture at least one analyte of the sample transported to the second sensor. The gap between the sensor cathode and sensor anode of at least one second sensor is larger or smaller in size than the gap of the first sensor, and at least one second sensor is connected to a fluid channel downstream of the first sensor. Larger or smaller means that the gap between the sensor cathode and sensor anode of at least one second sensor is larger or smaller in size compared to the gap of the first sensor. Variations in the size of the sensor gaps between the first and second sensors are not a result of variations occurring during the manufacturing process, but are intentionally selected to differ from each other in order to achieve a variety of sensitivities and measurement results. Preferably, the sensor gaps between the first and second sensors differ by about 5% to 10%. Furthermore, it is possible to use even larger differences in gap size, such as 50% or more.

[0007]

[0007] The microfluidic device according to the present invention is particularly advantageous because it allows for the determination of the presence and amount of analytes present in a fluid sample. In the course of such analysis, the analytes of the fluid sample are captured in the gap between a first sensor and a second sensor by analyte-capturing molecules. Subsequently, a second analyte-binding molecule, labeled with particles containing a metal, preferably an inert metal, and more preferably gold, is introduced into the microfluidic device, particularly the sensor. This second molecule binds to the analyte, and as a result, the analytes present in the gap are labeled. In the next step, a silver-containing solution is introduced into the microfluidic device, particularly the sensor. Subsequently, silver dendrites are formed in the gap where there are enough metal particles to bridge the non-conductive gap between the anode and cathode, thereby connecting an electrical circuit that would normally be interrupted.

[0008]

[0008] Another aspect of the present invention is a method for detecting and / or quantifying at least one analyte in one or more fluid samples, comprising: a) introducing at least one fluid sample into at least one fluid channel of a device according to the present invention, or to a first sensor, and optionally at least one second sensor; b) applying at least one analyte binding molecule to at least one fluid channel of the device, or to the first sensor, and optionally at least one second sensor; c) applying a silver agent to at least one fluid channel of the device, or to the first sensor, and optionally at least one second sensor; d) applying an electric current to at least one sensor cathode and at least one sensor anode; and e) detecting and / or determining the flow of electric current between at least one sensor cathode and at least one sensor anode. [Brief explanation of the drawing]

[0009] [Figure 1]This outlines a portable, self-powered lab-on-a-chip system (the microfluidic device of the present invention) for ultra-low detection of biomarkers, combining microfluidics, affinity-based sensors connected to a readout device, and an integrated fuel cell. [Figure 2] Figure 1 shows a cross-sectional view of a microfluidic device sensor. Figure 2a schematically illustrates a nano-based immunoassay based on an affinity-based detection principle for capturing analytes. Figure 2b shows labeling using gold nanoparticles (50 nm) modified with a secondary antibody. Figure 2c shows signal amplification using silver dendrite formation to close the gap between electrodes and provide an electrical connection between the fuel cell and the readout system. [Figure 3] (i) The operating principle of an enzyme fuel cell based on hydrogel-captured glucose oxidase and laccase, and (ii) a schematic diagram of the equivalent circuit model are shown. [Figure 4] A) Optical analysis of affinity-based assays to determine the optimal linker chemistry is shown. Absorbance values ​​for various surface modification techniques are shown in ±SD graph. B) Schematic diagram of site-specific binding orientation of primary IgG antibody to the protein A region of nanocrystalline surface protein (S layer SbpA / ZZ). C) Current-time trace of nano-gold-labeled silver dendrite formation required to form conductive crosslinks between comb-shaped electrode structures. [Figure 5] (A) Current values ​​(n=3) obtained in the presence of gradually increasing human IgG concentrations diluted in phosphate-buffered saline (0.01 M phosphate buffer concentration and 0.154 M sodium chloride concentration), and (B) electrical signals recorded in the presence of human plasma to which gradually decreasing analyte (human IgG) concentrations have been added. [Figure 6] This shows possible implementation forms of the microfluidic device according to the present invention. [Figure 7] A schematic diagram of a fuel cell usable within a microfluidic device according to the present invention is shown. [Figure 8] Figure 7 shows a schematic diagram of an alternative embodiment of the fuel cell. [Figure 9]This paper compares antibody binding on glass using various immobilization methods. [Figure 10] The bioassay used in Example 2 shows a concentration-dependent increase in absorbance. [Figure 11] This shows the crosslinking of the electrodes of the sensor of the present invention in the presence of the SARS-CoV 2 spike protein (see Example 2). [Modes for carrying out the invention]

[0010]

[0020] The microfluidic device of the present invention may be manufactured using methods known in the art. The device may be manufactured using a polymer or glass as a substrate forming the base of the device. Channels are formed on the substrate, and electrodes, an anode, and a cathode are arranged thereon. The substrate and the material used to form the channels are substantially electrically isolated. The term “substantially electrically isolated” also includes “electrically isolated.”

[0011]

[0021] The microfluidic device of the present invention comprises a plurality of sensors fluidly connected to at least one fluid channel. Accordingly, the microfluidic device according to the present invention comprises at least one second sensor fluidly connected to the fluid channel or the first sensor, the at least one second sensor comprising at least one sensor cathode and at least one sensor anode formed on an essentially electrically isolated substrate, the sensor cathode and sensor anode being separated by a gap formed on the electrically isolated substrate, at least one capture molecule being immobilized within the gap on the substrate, and at least one capture molecule being introduced into the inlet and adapted to capture at least one analyte of a sample transported to the second sensor.

[0012]

[0022] The presence of at least one second sensor is particularly advantageous for enabling the simultaneous determination of the presence of multiple analytes in a sample when an analyte capture molecule immobilized within the gap of the at least one second sensor can bind to and capture a different analyte as the first sensor. Further, by selecting the dimension of the gap of at least one second sensor that is different from the dimension of the gap of the first sensor, a wider range of analyte concentrations can be detected by the microfluidic device according to the present invention. Quantitative or semi - quantitative determination of analytes within a sample can also be achieved by using different sensors having the same gap size.

[0013]

[0023] According to a preferred embodiment of the microfluidic device according to the present invention, the device comprises a first reservoir for containing a buffer solution, a second reservoir for containing at least one analyte - binding molecule, and a third reservoir for containing a silver agent, and the first reservoir, the second reservoir, and the third reservoir are adapted to discharge their respective contents into a fluid channel, into the first sensor, and / or into the second sensor.

[0014]

[0024] By discharging the contents of the first reservoir, the second reservoir, and the third reservoir within the fluid channel, a silver cross - bridge of the first sensor and / or at least one second sensor is formed within the gap of the sensor, thereby facilitating the conductivity between the cathode and the anode of the sensor as described above.

[0015]

[0025] According to an example that is not part of the present invention, the gap between the sensor cathode and the sensor anode of at least one second sensor is essentially the same size as the gap of the first sensor.

[0016]

[0026] According to the present invention, the gap between the sensor cathode and the sensor anode of at least one second sensor is larger or smaller than the gap of the first sensor, and the at least one second sensor is connected to a fluid channel downstream of the first sensor. With this configuration, when the gap of the first sensor is saturated by the analyte, the second sensor can be used to detect a wider range of analyte concentrations.

[0017]

[0027] Preferably, the sensor cathode and the sensor anode of the first sensor are arranged in a comb-shaped configuration. The comb-shaped configuration enhances the sensitivity of the sensor.

[0018]

[0028] According to a preferred embodiment, the microfluidic device comprises a power source that can be connected to or is connected to the first sensor and / or at least one second sensor.

[0019]

[0029] In order to apply a current to the electrodes of the device of the present invention, the electrodes can be connected to an external power source or an internal power source. The use of an internal power source is particularly preferred because it enables the use of the microfluidic device without the use of additional devices.

[0020]

[0030] According to a preferred embodiment of the present invention, the power source, preferably an internal power source, comprises a fuel cell having a fuel cell cathode and a fuel cell anode, and the fuel cell cathode and the fuel cell anode are separated by a fluid channel. This embodiment provides the advantage that no external power source is required for the operation of the microfluidic device according to the present invention.

[0021]

[0031] According to an alternative embodiment, the power source comprises an inductively coupled coil. This provides the advantage of contactless power transfer.

[0022]

[0032] The microfluidic device according to the present invention may include a power source comprising a fuel cell, which is preferably a membrane-free fuel cell comprising a fuel cell cathode and a fuel cell anode separated from the fuel cell cathode by a fuel cell gap, wherein the fuel cell cathode and fuel cell anode are arranged such that the fuel cell gap can be filled with a fluid sample transported through a fluid channel from the inlet to the fuel cell gap, and the materials of the fuel cell cathode and the fuel cell anode are selected to generate a voltage when the fuel cell gap is filled with a fluid. According to this embodiment, the operating voltage of the microfluidic device can be generated directly by using the sample to be analyzed.

[0023]

[0033] According to this embodiment, the fuel cell cathode and fuel cell anode may be arranged in a single plane, and the fluid channel passes over the fuel cell cathode and fuel cell anode.

[0024]

[0034] Alternatively, the fuel cell cathode and fuel cell anode may be located on either side of the fluid channel.

[0025]

[0035] A membrane-free fuel cell may also comprise multiple fuel cell cathodes and multiple fuel cell anodes arranged in parallel or series electrical circuits.

[0026]

[0036] The fuel cell cathode and fuel cell anode may have a right-angled triangular shape, and the fuel cell cathode and fuel cell anode may be arranged with their respective hypotenuses facing each other.

[0027]

[0037] The fuel cell cathode and fuel cell anode may also have a right-angled triangular shape, and the fuel cell cathode and fuel cell anode may be arranged with their respective hypotenuses facing each other, and the fluid channel passes between the hypotenuses of the fuel cell anode and fuel cell cathode.

[0028]

[0038] According to a preferred embodiment, the first sensor and / or at least one second sensor are connected to or connectable to their respective sensor reading devices. The sensor reading devices are preferably connected to or connectable to a power supply. Advantageously, the sensor reading devices include LEDs, and each sensor of the microfluidic device is connected to or connectable to at least one LED.

[0029]

[0039] According to an alternative embodiment, the sensor reading device includes an active RFID circuit.

[0030]

[0040] Preferably, the microfluidic device includes a fluid absorption region connected to the fluid channel at the end of the fluid channel, located opposite the fluid inlet. The fluid absorption region improves the fluid transport rate through the fluid channel. The fluid absorption region may comprise cellulose or its derivatives, or any suitable material typically used to absorb liquid in a test strip.

[0031]

[0041] In a preferred embodiment, the reservoirs are fluidly connected to a fluid channel between the fluid inlet and the first sensor, with the first reservoir connected upstream of the first sensor, the second reservoir connected upstream of the first reservoir, and the third reservoir connected upstream of the second reservoir. This configuration ensures that the fluid contained in the first reservoir reaches the first sensor before the fluid contained in the second reservoir, and the fluid contained in the second reservoir reaches the first sensor before the fluid contained in the third reservoir.

[0032]

[0042] Preferably, the first, second, and third reservoirs each include a membrane positioned between the reservoir and the fluid channel. The membrane also allows for control over the order in which the fluid is introduced into the fluid channel.

[0033]

[0043] According to one embodiment of the present invention, the membrane of the first reservoir is thinner than the membrane of the second reservoir, and the membrane of the second reservoir is thinner than the membrane of the third reservoir.

[0034]

[0044] To control the fluid flow, the membranes are preferably adapted to dissolve upon contact with the fluid sample, the membrane of the first reservoir being adapted to dissolve faster than the membrane of the second reservoir, and the membrane of the second reservoir being adapted to dissolve faster than the membrane of the third reservoir.

[0035]

[0045] The membrane preferably contains or consists of a water-soluble material such as cellulose (paper), polymer, or mineral.

[0036]

[0046] In a favorable embodiment, at least one analyte capture molecule and / or at least one analyte binding molecule is an antibody or a fragment thereof, preferably selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments. It is particularly preferable that the antibody of the fragment can bind to a viral protein, preferably a coronavirus protein, and more preferably a SARS-CoV-2 viral protein. The viral protein is preferably an envelope protein, more preferably a spike protein, more preferably a coronavirus spike protein, more preferably a SARS-CoV-2 spike protein, and more preferably a SARS-CoV-2 spike S1 protein.

[0037]

[0047] Furthermore, at least one analyte-bound molecule may be labeled with metal nanoparticles, preferably inert metal nanoparticles, and more preferably gold nanoparticles.

[0038]

[0048] The metal nanoparticles used in the present invention may have a diameter of 10 to 100 nm, preferably 20 to 80 nm, more preferably 30 to 60 nm, even more preferably 40 to 60 nm, and even more preferably about 50 nm.

[0039]

[0049] The silver agent may be a silver salt, preferably silver nitrate, or a commercially available silver enhancement kit.

[0040]

[0050] Another aspect of the present invention is a method for detecting and / or quantifying at least one analyte in one or more fluid samples, a) The step of introducing at least one fluid sample into at least one fluid channel of the device according to the present invention, or into a first sensor, and optionally at least one second sensor, b) The step of applying at least one analyte-binding molecule to at least one fluid channel of the above device, or to a first sensor, and optionally to at least one second sensor, c) The step of applying a silver agent to at least one fluid channel of the above device, or to a first sensor, and optionally to at least one second sensor, The method comprises the steps of: d) applying current to at least one sensor cathode and at least one sensor anode; and e) detecting and / or determining the flow of current between at least one sensor cathode and at least one sensor anode.

[0041]

[0051] The present invention also relates to a membrane-free fuel cell comprising a fluid channel having an inlet for introducing a fluid sample into the fluid channel, wherein the fuel cell comprises a fuel cell cathode and a fuel cell anode separated from the fuel cell cathode by a fuel cell gap, the fuel cell cathode and fuel cell anode are arranged such that the fuel cell gap can be filled with a fluid sample transported through the fluid channel from the inlet to the fuel cell gap, and the materials of the fuel cell cathode and the fuel cell anode are selected to generate a voltage when the fuel cell gap is filled with a fluid.

[0042]

[0052] The fuel cell cathode and fuel cell anode of a membrane-free fuel cell may be located in a single plane, and the fluid channel passes over the fuel cell cathode and fuel cell anode.

[0043]

[0053] The fuel cell cathode and fuel cell anode may also be located on either side of the fluid channel.

[0044]

[0054] A membrane-free fuel cell may also comprise multiple fuel cell cathodes and multiple fuel cell anodes arranged in parallel or series electrical circuits.

[0045]

[0055] The fuel cell cathode and fuel cell anode may also have a right-angled triangular shape, and the fuel cell cathode and fuel cell anode may be arranged with their respective hypotenuses facing each other.

[0046]

[0056] The fuel cell cathode and fuel cell anode may have a right-angled triangular shape, and the fuel cell cathode and fuel cell anode may be arranged with their respective hypotenuses facing each other, and the fluid channel passes between the hypotenuses of the fuel cell anode and fuel cell cathode.

[0047]

[0057] The present invention also relates to a method for generating voltage using the above-mentioned membrane-less fuel cell, • Steps to provide bodily fluids The present invention relates to a method characterized by the step of introducing bodily fluids into the inlet of a membrane-free fuel cell. The present invention also relates to a microfluidic device comprising the above-mentioned membrane-free fuel cell.

[0048]

[0058] According to another alternative example, the microfluidic device comprises at least one fluid channel having at least one inlet, the at least one fluid channel being fluidly connected to a first sensor downstream of the at least one fluid inlet, the first sensor comprising at least one sensor cathode and at least one sensor anode formed on a substrate that is essentially electrically isolated, the sensor cathode and sensor anode being separated by a gap formed on the substrate that is electrically isolated, at least one analyte capture molecule being immobilized within the gap on the substrate, and at least one capture molecule being introduced into the inlet and adapted to capture at least one analyte of a fluid sample transported to the first sensor. According to this example, the microfluidic device also comprises a power supply that is connectable to or connected to the first sensor and / or at least one second sensor, the power supply comprising a fuel cell comprising a fuel cell cathode and a fuel cell anode, the fuel cell cathode and fuel cell anode being separated by a fluid channel.

[0049]

[0059] According to a preferred embodiment of this example, the microfluidic device comprises a fluid channel or at least one second sensor fluidly connected to the first sensor, the at least one second sensor comprising at least one sensor cathode and at least one sensor anode formed on an essentially electrically isolated substrate, the sensor cathode and sensor anode being separated by a gap formed on the electrically isolated substrate, at least one capture molecule being immobilized within the gap on the substrate, and at least one capture molecule being introduced into the inlet and adapted to capture at least one analyte of a sample transported to the second sensor.

[0050]

[0060] Furthermore, in the microfluidic device according to this example, the gap between the sensor cathode and sensor anode of at least one second sensor is preferably varies in size with respect to the gap of the first sensor, or preferably larger or smaller than the gap of the first sensor, and at least one second sensor is connected to a fluid channel downstream of the first sensor. Alternatively, the gap between the sensor cathode and sensor anode of at least one second sensor is essentially the same size as the gap of the first sensor.

[0051]

[0061] An alternative microfluidic device preferably comprises a first reservoir containing a buffer, a second reservoir containing at least one analyte-binding molecule, and a third reservoir containing a silver agent, wherein the first, second, and third reservoirs are adapted to discharge their respective contents into fluid channels to a first and / or second sensor.

[0052]

[0062] In an alternative example of a microfluidic device, the sensor cathode and sensor anode of the first sensor may be arranged in a comb-like configuration.

[0053]

[0063] The sensor cathode and sensor anode of at least one second sensor may also be arranged in a comb-like configuration.

[0054]

[0064] In an alternative example of a microfluidic device, the first sensor and / or at least one second sensor may be connected to or be connectable to their respective sensor reading devices.

[0055]

[0065] Furthermore, the reservoirs may be fluidly connected to a fluid channel between the fluid inlet and the first sensor, the first reservoir being connected upstream of the first sensor, the second reservoir being connected upstream of the first reservoir, and the third reservoir being connected upstream of the second reservoir, and the first reservoir, the second reservoir and the third reservoir (14) preferably each comprises a membrane located between the respective reservoir and the fluid channel.

[0056]

[0066] At least one analyte capture molecule and / or at least one analyte binding molecule may be an antibody or a fragment thereof, preferably selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments.

[0057]

[0067] At least one analyte-bound molecule may be labeled with metal nanoparticles, preferably inert metal nanoparticles, and more preferably gold nanoparticles.

[0058]

[0068] The silver agent may be a silver salt, preferably silver nitrate.

[0059]

[0069] Methods for detecting and / or quantifying at least one analyte in one or more fluid samples may also be performed using microfluidic devices as described in the alternative examples above. a) The step of introducing at least one fluid sample into at least one fluid channel of a microfluidic device, or into a first sensor, and optionally at least one second sensor, b) The step of applying at least one analyte-binding molecule to at least one fluid channel of the above device, or to a first sensor, and optionally to at least one second sensor, c) The step of applying a silver agent to at least one fluid channel of the above device, or to a first sensor, and optionally to at least one second sensor, d) The step of applying current to at least one sensor cathode and at least one sensor anode, e) The step of detecting and / or determining the flow of current between at least one sensor cathode and at least one sensor anode. Within the scope of this invention, the terms biofuel cell and fuel cell may be used interchangeably. Furthermore, a fuel cell is defined within the scope of this invention as a fuel cell or battery, where the electrolyte is supplied by a fluid channel.

[0060]

[0070] With reference to the drawings, preferred alternative embodiments of the microfluidic devices and methods according to the present invention are described below.

[0061]

[0071] Detailed description of the drawing Figure 1 shows a microfluidic device 1 according to the present invention in a preferred embodiment. The microfluidic device 1 comprises at least one fluid channel 2 having at least one inlet 3. The microfluidic device shown in Figure 1 comprises one fluid channel 2 and one inlet 3. The fluid channel 2 is fluidly connected to a first sensor 4 downstream of the at least one fluid inlet 3. The first sensor 4 comprises at least one sensor cathode 5 and at least one sensor anode 6 formed on a substrate 7 that is essentially electrically isolated. Cross-sections of the first sensor 4 are shown in Figures 2a, 2b, and 2c. The sensor cathode 4 and sensor anode 5 are separated by a gap 8 formed on the electrically isolated substrate 7. At least one analyte capture molecule 9 is immobilized in the gap 8 on the substrate 7 and is adapted to be introduced into the inlet 3 and capture at least one analyte 10 of a fluid sample transported to the first sensor.

[0062]

[0072] The microfluidic device 1 according to the present invention comprises at least one second sensor 11 fluidly connected to the fluid channel 2 or the first sensor 4. In Figure 1, two second sensors 11 are provided. At least one second sensor 11 comprises at least one sensor cathode 5 and at least one sensor anode 6 formed on a substrate 7 that is essentially electrically isolated. Thus, the second sensor 11 has the same basic structure as the first sensor. The sensor cathode 5 and sensor anode 6 of the second sensor 11 are separated by a gap 8 formed on the electrically isolated substrate 7, at least one capture molecule 9 is immobilized in the gap 8 on the substrate 7, and at least one capture molecule 9 is introduced into the inlet 3 and adapted to capture at least one analyte 10 of the sample transported to the second sensor 11. By providing the second sensor 11, verification of the results of the first sensor 4 can be achieved. Furthermore, by implementing a second sensor 11, different analytes 10 may be detected within the same sample by selecting different capture molecules 9 within the second sensor 11 compared to the first sensor 4. Figures 2a, 2b, and 2c also serve to illustrate the second sensor 11.

[0063]

[0073] As shown in Figure 1, according to a preferred embodiment, the microfluidic device 1 comprises a first reservoir 12 containing a buffer, a second reservoir 13 containing at least one analyte-binding molecule 15, and a third reservoir 14 containing a silver agent, the first reservoir 12, the second reservoir 13, and the third reservoir 14 being adapted to discharge their respective contents to a fluid channel 2, a first sensor 4, and / or a second sensor 11. Discharge to the first sensor 4 and / or the second sensor 11 should be interpreted as the contents being discharged directly or indirectly into the gap 8 between the first sensor 4 and / or the second sensor 11. Reservoirs 12, 13, and 14 may have a fixed connection to the fluid channel 2, as shown in Figure 1, or they may be provided as separate reservoirs 12, 13, and 14 from the fluid channel 2, and may be connected to the fluid channel 2 to introduce their respective contents into the fluid channel 2, the first sensor 4, and / or the second sensor 11. According to this embodiment, reservoirs 12, 13, and 14 may be provided as, for example, syringes. In Figures 2a to 2c, reservoirs 12, 13, and 14 are shown as syringes. By introducing the buffer into the fluid channel 2, followed by the introduction of the analyte binding molecule 15, and finally the silver agent, the silver bridge 16 of the first sensor 4 and / or the second sensor 11 shown in Figure 2c is formed between the cathode 5 and anode 6 of the first sensor 4 and / or the second sensor 11, in proportion to the amount of analyte 10 captured in the sensor gap 8 by the analyte capturing molecule 9. These silver bridges 16 affect the conductivity between the sensor cathode 5 and the sensor anode 6. By measuring the conductivity, the amount of analyte 10 contained in the fluid sample can be derived. According to an example not part of the present invention, the sensor gap 8 between the sensor anode 5 and the sensor cathode 6 of at least one second sensor 11 may be selected to be essentially the same size as the gap 8 of the first sensor 4. This provides verification of the results obtained by the first sensor 4.

[0064]

[0074] According to the microfluidic device 1 of the present invention, the gap 8 between the sensor cathode 5 and sensor anode 6 of at least one second sensor 11 is larger or smaller than the gap 8 of the first sensor 4, and at least one second sensor 11 is connected to a fluid channel 2 downstream of the first sensor 4. By providing different gaps 8 between the first sensor 4 and the second sensor 11, the first sensor 4 and the second sensor 11 can be designed to exhibit different detection limits.

[0065]

[0075] Preferably, the sensor cathode 5 and sensor anode 6 of the first sensor 4 and / or the second sensor 11 are arranged in a comb-like configuration. The comb-like configuration is shown in Figure 1. The comb-like configuration increases the surface area of ​​the sensor cathode 5 and sensor anode 6 that open into the gap 8, thereby increasing the possibility of bridging the gap 8 by forming a silver bridge 16 between the sensor cathode 5 and the sensor anode 6.

[0066]

[0076] According to a preferred embodiment of the microfluidic device 1 according to the present invention, the microfluidic device 1 comprises a power supply 17 that is connectable to or connected to a first sensor 4 and / or at least one second sensor 11. In the embodiment shown in Figure 1, the power supply 17 comprises a biofuel cell comprising a fuel cell cathode 18 and a fuel cell anode 19, the fuel cell cathode 18 and the fuel cell anode 19 being separated by a fluid channel 2. The power supply shown in Figure 1 features three biofuel cells, each connected to either the first sensor 4 or one of the second sensors 11 of the microfluidic device 1. Thus, according to the embodiment of Figure 1, each sensor 4, 11 is connected to a separate biofuel cell. This embodiment offers the advantage that the operation of the microfluidic device 1 according to the present invention does not require an external power supply. Alternatively, a power supply 17 known in the prior art, such as a battery, storage battery, power grid, and solar panel, may be used as the power supply.

[0067]

[0077] According to the alternative embodiment, the power supply 17 includes an inductive coupling coil. This provides the advantage of contactless power transmission.

[0068]

[0078] Figure 1 shows a sensor reading device 20, where a first sensor 4 and / or at least one second sensor 11 are connected to or connectable to the respective sensor reading device 20. The sensor reading device 20 is further connected to a power supply 17, and the sensor reading device 20 is equipped with LEDs, where each sensor of the microfluidic device is connected to or connectable to at least one LED. In Figure 1, each LED has a different color. A green LED is connected to the first sensor 4, a blue LED is connected to the second sensor 11 downstream of the first sensor 4, and a red LED is connected to the second sensor 11 downstream of the second sensor 11 connected to the blue LED. Simple visual reading of the amount of detected analyte 10 in the fluid sample is achieved by selecting the gap 8 between the first sensor 4 and the two second sensors 11 to gradually increase downstream in the fluid channel 2.

[0069]

[0079] In a more technically sophisticated embodiment of the microfluidic device 1 according to the present invention, the sensor reading device 20 includes an active RFID circuit for wirelessly transmitting sensor readings to, for example, a computer unit such as a PC, or a portable device such as a smartphone, tablet, or laptop. As shown in Figure 1, a microfluidic device 1 according to a preferred embodiment of the present invention includes a fluid absorption region 21 located opposite the fluid inlet 3 and connected to the fluid channel 2 at the end of the fluid channel 2. The fluid absorption region 21 may comprise a fluid absorption medium such as a sponge or cotton. The fluid absorption region 21 increases the velocity of fluid transport through the microfluidic device 1.

[0070]

[0080] In a preferred embodiment, reservoirs 12, 13, and 14 are fluidly connected to a fluid channel 2 between a fluid inlet 3 and a first sensor 4, with the first reservoir 12 connected upstream of the first sensor 2, the second reservoir 13 connected upstream of the first reservoir 12, and the third reservoir 14 connected upstream of the second reservoir 13. One possible adaptation of this configuration is shown in Figure 1. Alternatively, reservoirs 12, 13, and 14 may be connected, for example, by a second fluid channel arranged linearly and joining the fluid channel 2 of the microfluidic device 1 between the fluid inlet 3 and the first sensor 4.

[0071]

[0081] Preferably, the first reservoir 12, the second reservoir 13, and the third reservoir 14 each include a membrane located between the respective reservoir and the fluid channel 2. The membrane is not shown in the figure. By including such membranes, the device 1 according to the present invention can be equipped with an automated series discharge mechanism that does not require user intervention or power supply. In a preferred embodiment of this modification, the membrane of the first reservoir 12 is thinner than the membrane of the second reservoir 13, and the membrane of the second reservoir 13 is thinner than the membrane of the third reservoir 14. According to this embodiment, the membranes are adapted to dissolve upon contact with the fluid sample, the membrane of the first reservoir 12 is adapted to dissolve faster than the membrane of the second reservoir 13, and the membrane of the second reservoir 13 is adapted to dissolve faster than the membrane of the third reservoir 14. The membranes preferably contain or consist of a water-soluble material such as cellulose (paper), polymer, or mineral.

[0072]

[0082] According to a preferred embodiment, at least one analyte capture molecule 9 and / or at least one analyte binding molecule 15 is an antibody or fragment thereof, preferably selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments.

[0073]

[0083] Furthermore, according to a preferred embodiment, at least one analyte-binding molecule 15 is labeled with metal nanoparticles, preferably inert metal nanoparticles, and more preferably gold nanoparticles.

[0074]

[0084] Preferably, the silver agent is a silver salt, preferably silver nitrate, or a commercially available silver enhancement kit.

[0075]

[0085] Figure 6 shows an alternative embodiment of the microfluidic device 1 according to the present invention, in which the fluid channel 2 comprises a first channel 22 and a second channel 23 downstream of the inlet 3. In Figure 3, the inlet 3 is shown with a droplet of sample fluid introduced into the fluid channel. The first channel 22 connects the inlet 3 to reservoirs 12 and 13, which are fluidly connected to the first channel 22 of the fluid channel 2 between the fluid inlet 3 and the first sensor 4. One or more additional reservoirs may be provided at essentially the locations of reservoirs 12 and 13, such as reservoir 14 shown in Figure 1. One or more second sensors 11 may be provided at essentially the location and downstream of sensor 4, as disclosed with respect to the embodiment of Figure 1. Downstream of sensors 4 and 11, a fluid absorption region 21 is provided, which may comprise a fluid absorption medium such as a sponge or cotton.

[0076]

[0086] The second channel 23 connects the inlet 3 to the aforementioned fuel cell, preferably a membrane-free fuel cell, which serves as a power source 17 for sensors 4 and 11. The fuel cell comprises multiple fuel cell cathodes 18 and multiple fuel cell anodes 19 arranged in series. Alternatively, the fuel cell cathodes 18 and fuel cell anodes 19 may also be arranged in parallel. As shown in Figure 6, the fluid channel 2 introduces a sample into the fuel cell gap 25, thereby supplying an electrolyte to generate electricity. The fuel cell is connected to sensors 4 and 11 to supply electricity. The aforementioned sensor readout device 20 is also provided, electrically connected between the fuel cell and sensors 4 and 11. A conjugate pad 24 may also be provided, fluidly connected to the fluid channel 2 downstream of the inlet 3 and upstream of the fuel cell. The conjugate pad 24 serves to supply additional electrolyte to the fuel cell.

[0077]

[0087] Figures 7 and 8 schematically illustrate different embodiments of the fuel cell according to the present invention. The power source 17 in the form of the fuel cell is designed as described above, and the fuel cell gap 25 between the fuel cell cathode 18 and the fuel cell anode 19 is connected to the inlet 3 via a fluid channel 2. The fuel cell cathode 18 and the fuel cell anode 19 are electrically connected to a sensor readout device 20. In the embodiment shown in Figure 8, the fuel cell includes an additional pad 26. [Examples]

[0078]

[0088] [Examples]

[0079]

[0089] material and method

[0080]

[0090] Fabrication of microfluidic biochips using rapid prototyping

[0081]

[0091] Using AutoCAD, we designed the microfluidic channel layout and shadow mask required for metal deposition to create electrical leads and electrodes for biosensor arrays and biofuel cells. Overall, the microdevice shown in Figure 1 consisted of (1) a glass bottom substrate housing microelectrodes, microsensor arrays, and LEDs, (2) a PDMS microfluidic layer, and (3) a glass top cover housing perforated inlet and outlet ports. The microfluidic channel network and shadow mask were fabricated by cutting 250 μm thick PDMS (polydimethylsiloxane) foil (MVQ Silicones, SIP40HT6240GK0,25) using a Roland Cutter (CAMM-1, GS-24). Electrodes were fabricated by first placing a PDMS shadow mask on a cleaned glass substrate (sonicated in the presence of a 2% Helminex (Hellme Analytics) solution, followed by isopropanol washing and a final rinse in deionized water (DI)), and then depositing a 50 nm titanium bonding layer and an 80 nm gold layer using a Von Ardenne LSxxx sputtering system. After washing with Harrick Plasma (PDC-002-CE) for 2 minutes, the electrodes were bonded. Two biofuel cell electrodes (2.5 mm × 5 mm) were placed in the center of each circular hydrogel chamber (r=3 mm) separated by a 1 mm wide microfluidic channel.

[0082]

[0092] Characterization of redox-mediated enzyme reactions using cyclic voltammetry

[0083]

[0093] The electron transfer reaction from enzyme to electrode was characterized using potassium hexacyanoferrate(III / II) redox pairs (Alfa Aesar, 033357; Fluka, 60279) in a CV assay. Potentiostat VMP3 (Bio-logic SA) was used to determine the potential required for maximum electron transfer between the redox pairs and the enzymes glucose oxidase and laccase. A 40 μL aliquot obtained from a stock solution consisting of potassium hexacyanoferrate(III / II) in a 1:2 ratio and 0.5 mg / mL of glucose oxidase (Sigma Aldrich, G7141) was mixed with 500 μL and 100 mM D-glucose (Sigma Aldrich, G5400) and immediately placed on a gold electrode. For three electrodes (Au WE, Pt-CE, and Ag / AgCl Ref) including an electrochemical cell, a scanning speed of 100 mV / s was applied using Potentiostat VMP3, and the resulting peak values ​​were recorded using EC-lab V9.98.

[0084]

[0094] Enzyme and mediator capture in hydrogels, and characterization of biofuel cells.

[0085]

[0095] Both the enzyme and the mediator were mixed in a fibrin hydrogel and polymerized to obtain a stiffness of 1 kPa. Here, 15 μL of fibrin diluted in CaCl2 and 50 μL of 2 U / mL thrombin were mixed with 20 μL of potassium hexacyanoferrate(III / II) solution (60 mM in PBS (Roche)) containing either (a) 15 μL of glucose oxidase (Sigma Aldrich, G7141; 2.5 mg / mL in PBS) for the anode side or (b) 15 μL of laccase (Sigma Aldrich, 40452) at a concentration of 2.5 mg / mL in PBS (Roche) for the cathode side. Each enzyme-mediator-fibrin hydrogel mixture was packed into its respective chamber, sealed with PCR foil, and left in RG for 30 minutes to complete hydrogel polymerization. The performance of the microfluidic enzyme biofuel cells was evaluated in the presence of the escalating glucose solution using cyclic voltammetry at a scanning speed of 10 mV / s (Potentiostat VMP3).

[0086]

[0096] Biofunctionalization of capture regions using antibodies with EDC, ascorbic acid, and the S layer

[0087]

[0097] A) Anti-human IgG (Sigma Aldrich, I9135-2ML) antibody was diluted to a concentration of 1 mg / mL in reaction buffer PBS (pH 7.5), centrifuged using an Amicon Ultra-0.5 centrifuge, and filtered for buffer exchange. Crosslinking of Ab to a glass surface was achieved using standard EDC / sulfo-NHS linker chemistry. In summary, 1.1 mg of sulfo-NHS (Thermo Scientific, 24510) was added to 1 mL of antibody solution, incubated at RT for 1 hour, and centrifuged to remove excess sulfo-NHS. Next, 1 mL of MES buffer (pH 6.0) was added to the Ab solution, centrifuged for buffer exchange, and then 0.4 mg of EDC (Thermo Scientific, 22980) and 1.1 mg of sulfo-NHS were added sequentially. The reaction mixture was incubated at RT for 15 minutes, centrifuged to remove excess EDC and Sulfo-NHS, and reconstituted in PBS to obtain a final Ab concentration of 1 μg / mL. Prior to biofunctionalization of the glass bottom substrate (VWR, 631-1577), the glass was rinsed with isopropanol and DI water, plasma-treated for 2 minutes using Harrick Plasma (PDC-002-CE), and incubated for 30 minutes with 3% APTES (3-aminopropyltriethoxysilane) (Sigma Aldrich, 440140-100ML) in anhydrous ethanol. In the final step, the Ab solution was added, incubated for 2 hours, and then thoroughly rinsed with DI water.

[0088]

[0098] B) The ascorbic acid linker protocol is known in the art. In summary, a washed and APTES-modified glass substrate (see protocol above) was incubated with a 0.5 M ascorbic acid (sigma Aldrich, A7506-100G) solution (methanol) at RT for 30 minutes and then thoroughly rinsed with DI. The modified glass substrate was then coated with an anti-human IgG solution (see above) and incubated at RT for 16 hours.

[0089]

[0099] C) From Rothbauer, M., et al. (2017) ACS applied materials & interfaces, 9(39), 34423-34434, we adapt the establishment of a nanobiointerface using an engineered surface protein layer rSbpA31-1068 / ZZ. Here, 2 mg of lyophilized rSbpA31-1068 / ZZ protein was dissolved in 1 ml of 5 M GHCL (guanidine hydrochloride) pH 7.2 for 20 minutes at RT, transferred to a 6 mm diameter dialysis tube (Biomol, 08390.30), placed in a flask filled with water, stirred at 4°C for 30 minutes, and centrifuged at 13000 rpm for 15 minutes. The final protein concentration was determined by UV measurement at 280 nm, and 100 μL aliquots were stored at 4°C. Prior to application, the rSbpA31-1068 / ZZ protein solution was diluted 1:10 with TRIS buffer (10 ml CaCl2 in 0.5 mM tris(hydroxymethyl)-aminomethane, pH-9), pipettered onto a plasma-treated glass substrate, and incubated overnight at 40C to allow recrystallization of rSbpA31-1068 / ZZ. After several rinsing steps with DI water, the anti-human IgG solution (see above) was added, and Ab was bound to the protein A region (ZZ) of the S layer over 2-4 hours at RT (room temperature). To ensure covalent binding of the antibody (ab) to the S layer, in the final step, a 10-fold excess of DMP linker (thermo scientific, 21666) in TRIS buffer solution was added over 30-60 minutes at room temperature.

[0090] [000100] Absorbance spectroscopy was used to compare the quality of selected biofunctionalization strategies using the Enspire plate reader. Absorbance spectroscopy was performed with wavelengths from 320 to 700 nm and a step size of 1 nm.

[0091] [000101] Affinity-based nanogold-labeled sandwich immunoassay for detecting human IgG using silver-enhanced chemistry

[0092] [000102] The sandwich bioassay involved first binding human IgG (Sigma Aldrich, I4506) to a biofunctionalized surface containing anti-human IgG Ab for 10 minutes at RT, followed by labeling with a solution of secondary anti-human IgG conjugate gold nanoparticles (BBI solutions, BA.GAH40 / X) (diluted 1:2 with DI water) for a further 10 minutes at RT. After a rinsing step to remove unbound secondary nano-gold-labeled Ab, signal enhancement with a silver-enhancing mixture was added to the surface-bound gold nanoparticles to form silver dendrites that electrically connect the suspended sensor leads. Silver reduction on the gold surface was achieved by mixing two reagents A and B from a commercially available silver enhancer kit (Sigma Aldrich, SE100) in a 1:2 ratio. Reduction of silver nitrate to elemental silver was carried out within 5–10 minutes at RT, followed by removal of the access silver solution using DI or PBS.

[0093] [000103] Characterization of silver dendrite formation using conventional current-detection atomic force microscopy.

[0094] [000104] To visualize and verify the formation of conductive bridges between interrupted electrode leads, time-resolved AFM was performed in tapping mode using a Nanoscope(viii) multimode scanning probe microscope (Brucker, California). Single-crystal silicone cantilevers (NCH, Nanosensors) were used for tapping mode, and boron-doped diamond-coated silicone cantilevers (Piontprobe CONT, NanoWorld, Switzerland) were used for cAFM.

[0095] [000105] Results and Discussion

[0096] [000106] Design concept and characterization of a self-powered lab-on-a-chip system

[0097] [000107] Current self-powered lab-on-a-chip designs combine biofuel cells, microsensors, and display units on a common microfluidic platform for remote sample handling, sensing, and readout. Figure 1 shows a diagram of a biochip featuring a sample inlet port, three reservoirs containing silver reagent, nano-gold-labeled secondary antibody, and buffer, and a comb-type electrode structure that acts as a sensor, a one-way connector switch, and an enzyme biofuel cell. The basic principle of the microfluidic biofuel cell and the underlying circuit equivalence are shown in Figure 3, and Figure 2 reveals implemented bioassay strategies. A key design criterion for self-powered lab-on-a-chips is the ability to identify biomarkers and pathogens at ultra-low detection limits. To achieve this objective, two strategies have been investigated, including (1) optimization of surface biofunctionalization to increase antibody-antigen binding events (Figure 2a) and (2) signal amplification using silver-enhanced chemistry (Figure 2c).

[0098] [000108] A key step in any affinity-based detection method is optimizing the biointerface required to capture, retain, and detect the analyte. For a total of three antibodies, we first evaluated immobilization strategies, including two crosslinking agent chemistrys and S-layer technology, using absorbance spectroscopy. Figure 4A shows the results of a comparative study in which anti-IgG antibodies were immobilized on nine coverslips (n=3) using EDC-NHS and ascorbic acid (ASA) linker chemistry as well as engineered S-layer SbpA / ZZ. The S-layer specific protein A binding sequence to IgG yielded the highest absorbance value of 0.07, followed by EDC-NHS at 0.06 and ASA at 0.05 (e.g., control 0.01). Although all three immobilization protocols yielded similar results, the recombinant S-layer rSbpA / ZZ was selected for subsequent experiments due to its favorable orientation of the antibody at the binding site (see also Figure 4B), the inherent antifouling properties of the recrystallized rSbpA / ZZ protein layer, and its high porosity, which allows for efficient electron transfer on the electrode surface. This also means that a single immobilization procedure allows for the simultaneous deposition of Ab onto two different surface materials, such as a gold electrode and a separation glass substrate. AFM and fluorescence assays were performed in subsequent experiments to verify effective S-layer deposition on the gold surface and Ab binding within the ZZ region. The results shown in Figure 4C demonstrate successful protein recrystallization on the gold surface and complete coating across the entire electrode region. In the final step, cyclic voltammetry was used to functionally confirm the electrode in the presence of a biprotein surface layer, such as rSbpA / ZZ-anti-human IgG.

[0099] [000109] Following the optimization of antibody immobilization, the ability of silver-enhanced chemistry to form conductive crosslinks across a 5 μm wide gap was investigated in detail. In particular, subsequent experiments evaluated the silver concentration and time required to electrically connect the two individual fingers of the comb-shaped electrode structure. The sensing region (5 μm × 5 μm) was covered with an S layer, the S layer was incubated overnight with 1 μg / mL anti-human IgG, followed by the addition of 1 μg / mL human IgG over 30 minutes at RT, and then anti-human IgG conjugated gold nanoparticles. The PDMS microwells were thoroughly rinsed with PBS between each step. As a final step, a 10 μL aliquot of silver reagent was pipetted onto the top of the sensor, and current readings were obtained at 0, 1, 3, 5, 7, and 10 minutes using Potentiostat set to 0.3 V. In the absence of human IgG (negative control), no silver dendrite formation or current flow was observed. It was found that a minimum of 3 minutes was required to obtain a measurable current that significantly increased with reaction time in the presence of gold nanoparticles. Removal of the silver reagent by extensive rinsing of the sensor area with DI water stopped the formation of silver dendrites at each time point. Microscopic images taken after 10 minutes clearly showed the formation of a thick silver layer on the top of the sensing area, and reaction times exceeding 30 minutes resulted in the nonspecific and spontaneous formation of elemental silver aggregates. To eliminate the undesirable formation of silver aggregates within a 10-minute reaction time, the silver reagent was diluted with DI to reduce the concentration of reactive silver nitrate.

[0100] [000110] Verification of a one-way switch sensing strategy using standard current-sensing atomic force microscopy (AFM)

[0101] [000111] To verify that the results obtained above are indeed based on signal generation caused by the formation of silver dendrites in gold nanoparticles, time-resolved AFM, XPS, and conductive AFM tests were performed to identify (a) the number of gold nanoparticles required to form a conductive bridge, (b) the material and layer thickness, and (c) the current flow through the submicron-thick silver bridge. The first AFM image revealed the presence of nano-gold-labeled secondary Ab located on the electrode surface and in the gap region between the finger electrodes. Next, an AFM image obtained after silver deposition (5 min) using 1 μg / mL human IgG and 1 μg / mL nano-gold labeling, with a layer thickness of 100 μm on the electrode in the presence of a fully saturated nano-gold surface. Additional XPS analysis revealed the presence of an upper silver layer, followed by gold and glass substrates, thus confirming the selective deposition of elemental silver after a short reaction time of 5 minutes at RT. Next, time-resolved tapping-mode AFM was performed to track the initial formation and growth of silver dendrites from individual gold nanoparticles to create a solid bridge between the two electrode fingers. Figure 5 shows a series of AFM images capturing the initiation, growth, and formation of silver bridges. Results from this initial AFM test revealed that at least 2–3 gold nanoparticles were required to reduce silver nitrate sufficiently to bridge the 5 μm gap between the two electrode fingers during a 5-minute reaction period. In other words, within a 5-minute reaction time, the formation of silver dendrites significantly increased the size of the gold nanoparticles by 30–50 times. To further verify whether the formation of individual silver bridges actually conducts electricity, current-sensing AFM was used to measure the flow of current between the tip and the surface of the material. From cAFM images in and without silver bridges, the ability of silver-enhanced chemistry to electrically connect the two separate electrode leads and thus act as a one-way switch in the lab-on-a-chip configuration of the present invention was confirmed.

[0102] [000112] Performance evaluation of a self-powered lab-on-a-chip system

[0103] [000113] The main premise of applying silver-enhanced chemistry was to obtain an ultra-low detection limit. To initially estimate the detection limit of the microfluidic unidirectional sensing switch, we investigated reducing the concentration of IgG in the buffer and human plasma samples from 15 pg / mL to 2 pg / mL. For example, Figure 5A shows the current readings obtained from a comb-type electrode structure arranged with 6 mm and 400 μm microfluidic channels connected to an external readout. The average current value over a 4-minute measurement period already yielded 172 ± 0.9 μA in the presence of an IgG concentration of 2 pg / mL, but nonlinear signal behavior was observed in the presence of gradually increasing IgG concentrations. This means that the LOD of our current bioassay is 1 / 48th of that generally achieved by commercially available ELISA kits of approximately 100 μg / mL. To further evaluate the bioassay performance over a wider range of IgG concentrations, IgG was added to human plasma samples. Figure 5B shows current readings in the presence of 2, 5, 10, 50, and 100 pg / mL IgG, yielding 11.9±0.2, 79±12, 168±2, 220±5, 326±1.6, and 433±0.7 μA, respectively. Interestingly, 2 pg / mL IgG was readily detected by the lab-on-chip despite a 14-fold decrease in current from 172 μA to 12 μA in the presence of human plasma. Interestingly, a linear signal increase up to 20 pg / mL was observed, followed by the establishment of a current plateau after 100 pg / mL IgG, indicating saturation in the presence of sufficient conductive crosslinking. In the final experiment, an integrated self-powered lab-on-chip consisting of a microfluidic biofuel cell connected to a one-way sensing switch and an external readout (Ohmmeter) was validated with IgG-added plasma and whole blood samples. While whole blood samples were unable to generate a measurable current, 10 μL aliquots of human plasma samples supplemented with 10, 20, and 30 pg / mL yielded 12.5 μA, 18.3 μA, and 29.4 μA, respectively.

[0104] [000114] [Examples]

[0105] [000115] Materials and methods

[0106] [000116] Fabricated well array

[0107] [000117] The microscope slides were first washed by immersing them in a 2% Hellmanex® III solution and placing them in an ultrasonic bath for 5 minutes. The solution was then discarded and replaced with isopropanol and then water, each for 5 minutes in the ultrasonic bath. The slides were first dried with pressurized air and then dried in an oven at 80°C for 1 hour.

[0108] [000118] For the well array, three polydimethylsiloxane (PDMS) layers were bonded onto glass. To do this, a Roland DG cutting plotter CAMM-1 GS-24 was used to cut 3 mm diameter wells into the PDMS sheet. The notches were removed using tweezers, and the layers were bonded to each other using a Harrick Plasma Plasma Cleaner PDC-002-CE. The first PDMS layer was bonded onto a clean microscope slide, and the subsequent layers were bonded onto the PDMS. The bond was strengthened by placing the chip in an oven for 15 minutes.

[0109] [000119] Immobilization method

[0110] [000120] Adsorption

[0111] [000121] No further surface modification was performed on the washed microscope slides using adsorption as the antibody immobilization method. Alexa Fluor 488 goat anti-rat IgG antibody was diluted in PBS to obtain three different concentrations (1, 10, and 100 μg / ml), and 5 μl was added to each well. The antibodies were incubated for 1 hour and then discarded. The wells were washed with washing buffer (0.05% Tween® 20 in PBS) and then imaged.

[0112] [000122]Covalent bond

[0113] [000123] For covalent binding of the antibody to the glass surface, (3-mercaptopropyl)-trimethoxysilane (3-MPS) was diluted with anhydrous ethanol to a final concentration of 0.56 mg / ml. Washed microscope slides (prepared as described in 1) were activated by plasma treatment along with the wells. Then, 10 μl of 3-MPS solution was added to each well and incubated for 30 minutes. Meanwhile, N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) was dissolved in DMSO to obtain a 100 mg / ml stock solution, which was further diluted with ethanol to a final concentration of 0.56 mg / ml.

[0114] [000124] After 3-MPS incubation, the wells were washed with ethanol. 10 μl of the prepared GMBS solution was added and incubated for a further 35 minutes. The wells were then washed with ethanol and PBS, and finally, the antibody solution could be incubated under absorption as described above.

[0115] [000125] Protein G

[0116] [000126] The preparation of the wells was the same as described above (covalent bonding), but instead of covalently immobilizing the antibody, protein G was added at a concentration of 1 mg / ml over 1.5 hours. Then, the wells were washed with washing buffer (0.05% Tween® 20 in PBS), and the different antibody concentrations described above (absorption) were added.

[0117] [000127] Sandwich ELISA using a glass substrate

[0118] [000128] Using the above (covalent) method, capture antibodies against the SARS-CoV-2 spike S1 protein were immobilized. After immobilization, 5 μl of spike S1 protein was incubated for 30 minutes, and then the wells were washed. To complete the sandwich, 5 μl of biotinylated detection antibody against spike S1 protein was incubated for 30 minutes. The wells were washed with wash buffer, and 10 μl of horseradish peroxide (HRP) conjugated with streptavidin was added. Finally, after 30 minutes of incubation and washing of the wells, 10 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and incubated for a further 15 minutes. The enzymatic reaction was stopped with 0.18 M H2SO4, and the wells were analyzed using a plate reader.

[0119] [000129] Sandwich ELISA using electrodes

[0120] [000130] The first step up to the detection antibody incubation is the same as above (sandwich ELISA using a glass substrate), but it is performed using an electrode and a shorter incubation time of 15 minutes. Next, streptavidin-labeled gold nanoparticles were used instead of HRP / streptavidin. In the final step, the size of the gold nanoparticles was enhanced by adding a silver-enhancing solution over 10 minutes.

[0121] [000131]A commercially available electrode with a 5 μm gap was used.

[0122] [000132] Results and Discussion

[0123] [000133] Since the commercially available electrodes used in this example use glass as the substrate, different antibody immobilization methods for glass substrates were tested. The results (Figure 9) show that orientation immobilization using protein G yielded the best results. However, covalent bonding also showed an increase in signal at even higher antibody concentrations. As a result, these methods should be suitable for immobilizing antibodies on glass.

[0124] [000134] The bioassay in this lab-on-a-chip platform is similar to a sandwich ELISA. However, instead of enzymatic readout, the detection antibody is labeled with gold nanoparticles. To demonstrate that the sandwich assembly works on a glass substrate, a sandwich ELISA was performed using a well array. Figure 10 shows the concentration-dependent increase in absorbance. Thus, the sandwich assay works and can be used in assays using electrodes.

[0125] [000135] Instead of enzymatic readout, streptavidin-labeled gold nanoparticles were used. Commercial silver enhancement was added to enhance these gold nanoparticles. After enhancement, spots were visible under a microscope, indicating crosslinking of the electrodes in the presence of spike proteins (Figure 11).

Claims

1. A microfluidic device (1) comprising at least one fluid channel (2) having at least one inlet (3), The at least one fluid channel (2) is fluidly connected to a first sensor (4) downstream of the at least one fluid inlet (3), The first sensor (4) comprises at least one sensor cathode (5) and at least one sensor anode (6) formed on a substrate (7) that is essentially electrically isolated, The sensor cathode (5) and the sensor anode (6) are separated by a gap (8) formed on the electrically isolated substrate (7). At least one analyte-capturing molecule (9) is immobilized within the gap (8) on the substrate (7), The at least one capture molecule (9) is introduced into the inlet (3) and adapted to capture at least one analyte (10) of the fluid sample transported to the first sensor (4), The microfluidic device (1) comprises at least one second sensor (11) fluidly connected to the fluid channel (2) or the first sensor (4), The at least one second sensor (11) comprises at least one sensor cathode (5) and at least one sensor anode (6) formed on a substrate (7) that is essentially electrically isolated, The sensor cathode (5) and the sensor anode (6) are separated by a gap (8) formed on the electrically isolated substrate (7). At least one capture molecule (9) is immobilized within the gap (8) on the substrate (7), At least one capture molecule (9) is introduced into the inlet (3) and adapted to capture at least one analyte (10) of the sample transported to the second sensor (11), The size of the gap (8) between the sensor cathode (5) and the sensor anode (6) of at least one second sensor (11) is different from the size of the gap (8) of the first sensor (4). A microfluidic device (1) in which at least one second sensor (11) is connected to the fluid channel (2) downstream of the first sensor (4).

2. A membrane-free fuel cell comprising a fluid channel (2) having an inlet (3) for introducing a fluid sample into the fluid channel (2), The aforementioned fuel cell, Fuel cell cathode (18), The fuel cell comprises a fuel cell anode (19) separated from the fuel cell cathode (18) by a fuel cell gap (25), The fuel cell cathode (18) and the fuel cell anode (1) are arranged such that the fuel cell gap (25) can be filled with the fluid sample transported from the inlet (3) to the fuel cell gap (25) via the fluid channel (2), The material of the fuel cell cathode (18) and the material of the fuel cell anode (1) are selected to generate a voltage when the fuel cell gap (25) is filled with bodily fluids. The fuel cell cathode (18) and the fuel cell anode (1) are arranged in a single plane. A membrane-free fuel cell in which the fluid channel (2) passes over the fuel cell cathode (18) and the fuel cell anode (19).

3. The aforementioned device (1) A first reservoir (12) containing a buffer solution, A second reservoir (13) containing at least one analyte-binding molecule (15), It comprises a third reservoir (14) for containing the silver agent, The microfluidic device (1) according to claim 1, wherein the first reservoir (12), the second reservoir (13), and the third reservoir (14) are configured to discharge their respective contents to the fluid channel (2), the first sensor (4), and / or the second sensor (11).

4. The microfluidic device (1) according to any one of claims 1 or 3, wherein the sensor cathode (5) and sensor anode (6) of the first sensor (4) are arranged in a comb-like configuration.

5. The microfluidic device (1) according to any one of claims 1 or 3 to 4, wherein the sensor cathode (5) and sensor anode (6) of at least one second sensor (11) are arranged in a comb-like configuration.

6. A microfluidic device (1) according to any one of claims 1 or 3 to 5, comprising a power supply (17) that is connectable to or connected to the first sensor (4) and / or the at least one second sensor (11).

7. The microfluidic device (1) according to claim 6, wherein the power supply (17) comprises an inductively coupled coil.

8. The microfluidic device (1) according to any one of claims 1 or 3 to 7, wherein the first sensor (4) and / or the at least one second sensor (11) are connected to or connectable to the respective sensor reading devices (20).

9. The reservoirs (12, 13, 14) are fluidly connected to the fluid channel (2) between the fluid inlet (3) and the first sensor (4). The first reservoir (12) is connected upstream of the first sensor (4), The second reservoir (13) is connected upstream of the first reservoir (12), The microfluidic device (1) according to claim 3, wherein the third reservoir (14) is connected upstream of the second reservoir (13).

10. The microfluidic device (1) according to any one of claims 1 or 3 to 9, wherein at least one analyte-capturing molecule (9) and / or at least one analyte-binding molecule (15) is an antibody or a fragment thereof.

11. The microfluidic device (1) according to claim 3, wherein the at least one analyte-binding molecule (15) is labeled with metal nanoparticles.

12. The microfluidic device (1) according to claim 3, wherein the silver agent is a silver salt.

13. The power source (17) includes a fuel cell, The fuel cell comprises a fuel cell cathode (18) and a fuel cell anode (19) separated from the fuel cell cathode (18) by a fuel cell gap (25). The fuel cell cathode (18) and the fuel cell anode (19) are arranged such that the fuel cell gap (25) can be filled with the fluid sample transported from the inlet (3) to the fuel cell gap (25) via the fluid channel (2), The microfluidic device (1) according to claim 7, wherein the material of the fuel cell cathode (18) and the material of the fuel cell anode (19) are selected to generate a voltage when the fuel cell gap (25) is filled with a bodily fluid.

14. The fuel cell cathode (18) and the fuel cell anode (19) are arranged in a single plane. The microfluidic device (1) according to claim 13, wherein the fluid channel (2) passes over the fuel cell cathode (18) and the fuel cell anode (19).

15. The microfluidic device (1) according to claim 13, wherein the fuel cell cathode (18) and the fuel cell anode (19) are arranged on both sides of the fluid channel (2).

16. The microfluidic device (1) according to any one of claims 13 to 15, wherein the fuel cell comprises a plurality of fuel cell cathodes (18) and a plurality of fuel cell anodes (19) arranged in parallel or in series electrical circuits.

17. The fuel cell cathode (18) and the fuel cell anode (19) are triangular in shape with right angles, The microfluidic device (1) according to claim 14, wherein the fuel cell cathode (18) and the fuel cell anode (1) are arranged so that their respective hypotenuses face each other.

18. The fuel cell cathode (18) and the fuel cell anode (1) are triangular in shape with right angles, The fuel cell cathode (18) and the fuel cell anode (19) are arranged so that their respective hypotenuses face each other. The microfluidic device (1) according to claim 13, wherein the fluid channel (2) passes between the hypotenuses of the fuel cell anode (18) and the fuel cell cathode (19).

19. A method for detecting and / or quantifying at least one analyte (10) in one or more fluid samples, a) The step of introducing at least one fluid sample into at least one fluid channel (2) of the device according to any one of claims 1 to 17, or into the first sensor (4), b) The step of applying at least one analyte-binding molecule (15) to at least one fluid channel (2) of the device or to the first sensor (4), c) The step of applying a silver agent to at least one fluid channel (2) of the device, or to the first sensor (4), d) The step of applying current to at least one sensor cathode (5) and at least one sensor anode (6), e) A method comprising the steps of detecting and / or determining the flow of current between the at least one sensor cathode (5) and the at least one sensor anode (6).

20. A membrane-free fuel cell according to claim 2, comprising a plurality of fuel cell cathodes (18) and a plurality of fuel cell anodes (19) arranged in parallel or series electrical circuits.

21. The fuel cell cathode (18) and the fuel cell anode (19) are triangular in shape with right angles, The membrane-free fuel cell according to claim 2, wherein the fuel cell cathode (18) and the fuel cell anode (19) are arranged so that their respective hypotenuses face each other.

22. A method for generating voltage using a membrane-free fuel cell according to any one of claims 2, 20, and 21, - Steps to provide bodily fluids A method characterized by the step of introducing the bodily fluid into the inlet (3) of the membrane-less fuel cell.

23. A microfluidic device comprising a membrane-free fuel cell according to any one of claims 2 and 20 to 21.

24. The microfluidic device according to claim 9, wherein the first reservoir (12), the second reservoir (13), and the third reservoir (14) each include a membrane located between the respective reservoirs (12, 13, 14) and the fluid channel (2).

25. The microfluidic device according to claim 10, wherein the antibody or fragment thereof is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments.

26. The microfluidic device according to claim 11, wherein the metal nanoparticles are inert metal nanoparticles or gold nanoparticles.

27. The microfluidic device according to claim 12, wherein the silver salt is silver nitrate.

28. The microfluidic device according to claim 13, wherein the fuel cell is a membrane-free fuel cell.

29. Step a) includes introducing at least one fluid sample into the at least one second sensor (11) of the microfluidic device according to any one of claims 1 to 17, Step b) above includes applying at least one analyte-binding molecule (15) to the at least one second sensor (11) of the device, The method according to claim 19, wherein step c) comprises applying a silver agent to the at least one second sensor (11) of the device.

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