A high-throughput and highly sensitive microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers and its preparation method and application

Abstract

The present invention belongs to the technical field of microfluidic detection, and particularly relates to a high-throughput and highly sensitive microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, a preparation method thereof, and an application. The microfluidic-photoelectrochemical sensor includes a substrate and a microfluidic multi-channel chip. In its preparation, an S-type CsPbBr3-BiOBr nanorod array is modified on four working electrodes of the microfluidic multi-channel as a sensing substrate, and then capture antibody molecules of four liver cancer markers, namely AFP, CEA, AFU, and GP73, are respectively modified. An active nanozyme Cu-Mn@MOF with superoxide dismutase-like and catalase-like activities is used as a signal amplification tag, which further promotes the separation of photogenerated electron-hole pairs and effectively improves the sensitivity of the sensor. It can be used to diagnose early liver cancer by detecting the contents of AFP, CEA, AFU, and GP73 in serum, and has the advantages of less sample / reagent consumption, rapid detection, and good stability.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic detection technology, and specifically relates to a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, and a preparation method and application thereof. Background Art

[0002] Clinical studies have shown that abnormal changes in the content of liver cancer markers are closely related to the occurrence of liver cancer. In the early stages of liver cancer, the levels of multiple molecules in the serum have changed significantly, such as: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), fucosidase (AFU), Golgi protein 73 (GP73), etc. Therefore, simultaneous quantitative detection of these molecular levels in serum can be used as an effective means of accurate early diagnosis of liver cancer.

[0003] At present, there are many methods for detecting liver cancer markers, such as enzyme-linked immunosorbent assay, radioimmunoassay, electrochemical immunoassay and chemiluminescent immunoassay, but these methods have the disadvantages of not being able to detect multiple markers simultaneously in real time, rapidly and with high throughput, consuming a lot of samples / reagents and being complicated to operate. Therefore, it is of great significance to construct a highly sensitive, high-throughput and rapid combined detection method for multiple liver cancer markers at the same time.

[0004] Microfluidics is the science and technology involved in systems that use microchannels (tens to hundreds of millimeters in size) to process or manipulate tiny fluids. Microfluidic detection technology can be used in many areas, especially in in vitro diagnosis, with the advantages of low sample consumption, high sensitivity and rapid analysis. At present, most of these technology products only detect single markers, and when detecting multiple markers, they can generally only rely on multiple products for simultaneous detection, which increases the number of operating steps, makes the process cumbersome, has a long reaction cycle, and has high detection costs. Summary of the invention

[0005] The purpose of the present invention is to provide a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, as well as a preparation method and application thereof, which has the advantages of low sample / reagent consumption, rapid detection, and good stability, and can realize high-sensitivity and high-throughput combined detection of multiple liver cancer markers at the same time, thus solving the problems existing in the prior art.

[0006] The present invention provides one of the following technical solutions:

[0007] A high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, comprising a microfluidic four-channel chip and a substrate; the microfluidic four-channel chip comprises four injection ports and microchannels arranged in parallel, each injection port and microchannel is connected to a working electrode slot, an outlet port and a microchannel are arranged on the other side of each working electrode slot away from the injection port and the microchannel, a reference-counter electrode slot is arranged between the working electrode slots, and the reference-counter electrode slot is arranged in communication with each working electrode slot; the substrate is used for screen-printing electrodes and bonding the microfluidic multi-channel chip, and working electrodes, reference electrodes and counter electrodes are respectively arranged on the substrate corresponding to each working electrode slot and reference-counter electrode slot.

[0008] Furthermore, the substrate is an ITO conductive glass screen-printed electrode substrate.

[0009] Furthermore, the working electrode slots include first, second, third and fourth working electrode slots, and each working electrode slot is arranged around the outside of the reference-counter electrode slot.

[0010] Furthermore, the reference electrode and the counter electrode are separated in the counter electrode-reference electrode groove by screen printing carbon paste and Ag / AgCI paste, respectively. The specific operation belongs to the existing operation mode, using different screen printing plates for screen printing, first using the designed screen printing plate 1 to print the carbon paste, and then using the designed screen printing plate 2 to print the Ag / AgCI paste.

[0011] Furthermore, the four liver cancer markers are AFP, CEA, AFU and GP73; the injection ports and microchannels are respectively: a first injection port and microchannel for AFP capture antibody Ab1, bovine serum albumin BSA, AFP standard solution, AFP antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through, a second injection port and microchannel for CEA capture antibody Ab1, bovine serum albumin BSA, CEA standard solution, CEA antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a third injection port and microchannel for AFU capture antibody Ab1, bovine serum albumin BSA, AFU standard solution, AFU antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a fourth injection port and microchannel for GP73 capture antibody Ab1, bovine serum albumin BSA, GP73 standard solution, GP73 antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through;

[0012] The sample outlets and microchannels are respectively: a first sample outlet and microchannel for GP73 capture antibody Ab1, bovine serum protein BSA, GP73 standard solution, GP73 antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a second sample outlet and microchannel for AFP capture antibody Ab1, bovine serum protein BSA, AFP standard solution, AFP antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a third sample outlet and microchannel for AFU capture antibody Ab1, bovine serum protein BSA, AFU standard solution, AFU antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a fourth sample outlet and microchannel for CEA capture antibody Ab1, bovine serum protein BSA, CEA standard solution, CEA antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through.

[0013] Furthermore, the diameters of the working electrode slot and the counter electrode-reference electrode slot are both 1600 ~ 3600 µm; the diameter of the injection port is 400 ~ 600 µm, the diameter of the microchannel connected to the injection port is 500 ~ 700 µm, the diameter of the outlet port is 500 ~ 700 µm, and the diameter of the microchannel connected to the outlet port is 600 ~ 800 µm.

[0014] The present invention also provides the following technical solution 2:

[0015] A method for preparing a high-throughput and highly sensitive microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers comprises the following steps:

[0016] (1) Design and draw the microfluidic four-channel pattern using Auto CAD software, design the pattern and draw the mask, and use soft lithography technology to process polydimethylsiloxane (PDMS) to obtain a microfluidic four-channel chip;

[0017] (2) 4 cm × 3 cm ITO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively, dried with nitrogen, and the cleaned ITO conductive glass was etched and screen-printed in turn to obtain a substrate containing a reference electrode, a counter electrode and four working electrodes;

[0018] (3) 30 μL of 5.5 ~ 8.5 mg / mL bismuth oxybromide BiOBr solution was drop-coated on the four working electrodes of the substrate and dried at room temperature. Then 20 μL of 0.5 ~ 2.0 mmol / L CsPbBr3 quantum dot solution was drop-coated and dried at room temperature to obtain an S-type CsPbBr3-BiOBr composite material modified working electrode substrate.

[0019] (4) Add 15 μL of 0.09-0.12% (w / v) chitosan solution containing 1% acetic acid to the surface of the working electrode modified with the S-type CsPbBr3-BiOBr composite material, and then add 15 μL of 1.5-3.0% (v / v) glutaraldehyde solution to the surface of the modified working electrode, and dry it at room temperature to obtain the substrate of the amino-modified S-type CsPbBr3-BiOBr working electrode;

[0020] (5) treating the microfluidic four-channel chip prepared in step (1) and the substrate of the S-type CsPbBr3-BiOBr working electrode modified by amino in step (4) with oxygen plasma, and then bonding the microfluidic four-channel chip to the substrate, thereby completing the preparation of the microfluidic four-channel chip electrode;

[0021] (6) Using a syringe pump at 40 to 70 µL / min, 10 µg / mL of four liver cancer marker capture antibodies Ab1 were simultaneously injected onto the four working electrodes of the microfluidic chip electrode, incubated at room temperature for 10 to 25 min, and washed by injecting a buffer solution through each injection port to obtain an S-type CsPbBr3-BiOBr / Ab1 working electrode;

[0022] (7) Using a syringe pump at 40 ~ 70 µL / min, a 1.0% bovine serum albumin (BSA) solution was injected into each S-type CsPbBr3-BiOBr / Ab1 working electrode to block the unbound nonspecific active sites on the electrode surface, and a buffer solution was injected from each injection port for washing to obtain an S-type CsPbBr3-BiOBr / Ab1 / BSA working electrode;

[0023] (8) Use a syringe pump to continue injecting 10 pg / mL to 100 ng / mL of four liver cancer marker standard solutions at different concentrations into each working electrode S-type CsPbBr3-BiOBr / Ab1 / BSA at 40 to 70 µL / min, incubate at room temperature for 10 to 25 min, and inject buffer solution into each injection port for washing to obtain an S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker working electrode;

[0024] (9) Use a syringe pump to inject the liver cancer marker antibody marker solution of Cu-Mn@MOF nanozyme into the four working electrodes at 40 ~ 70 µL / min, incubate in a 4 °C refrigerator for 10 ~ 25 min, and inject buffer solution through each injection port for washing to obtain a completely modified S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker / Cu-Mn@MOF-Ab2 four-channel microfluidic-photoelectrochemical sensor.

[0025] Furthermore, under visible light irradiation, the photoelectrons in the conduction band of S-type CsPbBr3-BiOBr reduce dissolved oxygen to produce superoxide radicals, and the Cu-Mn@MOF nanozyme has superoxide dismutase and catalase-like activities; it can dismutate superoxide radicals to produce oxygen and hydrogen peroxide, and hydrogen peroxide can be catalyzed by Cu-Mn@MOF to produce oxygen. The produced oxygen is partially reduced by photoelectrons, thereby effectively promoting the separation efficiency of electron-hole pairs and realizing the sensor mode of photocathode signal amplification.

[0026] Furthermore, the buffer solution is a mixture of 1 / 15 mol / L disodium hydrogen phosphate and potassium dihydrogen phosphate with a pH value of 7.4.

[0027] Further, step (3) preparation of CsPbBr3 quantum dot solution: 1.2 mmol of Cs2CO3 and 1.4 mL of oleic acid were dissolved in 14-17 mL of octadecene and heated to 100 °C for 30 minutes to obtain a Cs precursor solution; 0.76 mmol of PbBr2, 2.6 mL of oleic acid and 3 mL of oleamide were dissolved in 26-30 mL of octadecene and heated to 100 °C for 30 minutes to obtain a Pb precursor solution; 1.6 mL of the Cs precursor solution was quickly injected into the 170 °C Pb precursor solution, and reacted in an ice water bath for 20 s, then centrifuged and washed with acetone and methyl acetate, vacuum dried at 60 °C for 12 h, and dissolved in ultrapure water to obtain a CsPbBr3 quantum dot solution.

[0028] The present invention also provides the following technical solution three:

[0029] An application of a microfluidic-photoelectrochemical sensor for detecting liver cancer markers AFP, CEA, AFU and GP73, wherein the microfluidic-photoelectrochemical sensor is prepared by the aforementioned preparation method, and the detection steps are as follows:

[0030] (1) Use an electrochemical workstation to test, inject 150 ~ 350 µL, 0.1 mol / L Tris-HCl (pH = 7.4) through the injection port into each working electrode and microchannel of the microfluidic-photoelectrochemical sensor, and test under LED light;

[0031] (2) Use the time-current method to detect AFP, CEA, AFU, and GP73, set the voltage to 0 V, and run for 200 s;

[0032] (3) When the background current tends to be stable, turn on the light every 10 to 20 seconds for 10 to 20 seconds, then record the photocurrent changes and draw a working curve;

[0033] (4) Serum sample solution was used to replace the standard solution of AFP, CEA, AFU, and GP73, and the test results were obtained through the working curve.

[0034] Beneficial effects of the present invention:

[0035] (1) The microfluidic-photoelectrochemical sensor prepared in the present invention can overcome the shortcomings of traditional photoelectrochemical sensors such as low flux, poor repeatability, and low integration, and can realize the detection of multiple protein molecules at the same time, and has broad application prospects.

[0036] (2) The microfluidic-photoelectrochemical sensor prepared in the present invention integrates a three-electrode system of a multi-channel working electrode, a reference electrode and a counter electrode into a microfluidic chip. By controlling a syringe pump, automated detection can be achieved, and accurate detection results can be obtained quickly without human interference.

[0037] (3) The present invention uses S-type CsPbBr3-BiOBr as a sensing substrate and a dual nanozyme Cu-Mn@MOF having superoxide dismutase and catalase-like activities as an antibody marker. The Cu-Mn@MOF nanozyme has a dismutation effect on superoxide radicals generated by the reduction of dissolved oxygen by photoelectrons in the conduction band of S-type CsPbBr3-BiOBr, which can promote the separation efficiency of electron-hole pairs and realize the amplification of the photocathode current signal, thereby effectively improving the sensitivity of the sensor.

[0038] (4) The microfluidic-photoelectrochemical sensor prepared by the present invention has the advantages of low sample / reagent consumption, rapid detection, good stability, and a wide signal response range. It can achieve high-sensitivity and high-throughput detection of four liver cancer markers AFP, CEA, AFU, and GP73 at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0040] Figure 1 Schematic diagram of the structure of the high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor of the present invention;

[0041] Figure 2 for Figure 1 Schematic diagram of the structure of the middle substrate.

[0042] Wherein, 1 working electrode slot a; 2 working electrode slot b; 3 counter electrode-reference electrode slot; 4 working electrode slot c; 5 working electrode slot d; 6 first injection port; 7 second injection port; 8 third injection port; 9 fourth injection port; 10 first outlet port; 11 second outlet port; 12 third outlet port; 13 fourth outlet port; 14 injection microchannel; 15 outlet microchannel; 16 working electrode a; 17 working electrode b; 18 working electrode c; 19 working electrode d; 20 reference electrode; 21 counter electrode;

[0043] Among them, the first injection port 6 is the injection port for AFP capture antibody Ab1, bovine serum albumin BSA, AFP standard solution, AFP antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0044] The second injection port 7 is the injection port for CEA capture antibody Ab1, bovine serum albumin BSA, CEA standard solution, CEA antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0045] The third injection port 8 is the injection port and microchannel for AFU capture antibody Ab1, bovine serum albumin BSA, AFU standard solution, AFU antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0046] The fourth injection port 9 is the injection port for GP73 capture antibody Ab1, bovine serum albumin BSA, GP73 standard solution, GP73 antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0047] The first sample outlet 10 is the sample outlet for GP73 capture antibody Ab1, bovine serum albumin BSA, GP73 standard solution, GP73 antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0048] The second sample outlet 11 is the sample outlet for AFP capture antibody Ab1, bovine serum albumin BSA, AFP standard solution, AFP antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0049] The third sample outlet 12 is the sample outlet for AFU capture antibody Ab1, bovine serum albumin BSA, AFU standard solution, AFU antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0050] The fourth sample outlet 13 is the sample outlet for CEA capture antibody Ab1, bovine serum albumin BSA, CEA standard solution, CEA antibody marker of Cu-Mn@MOF nanozyme and cleaning solution. DETAILED DESCRIPTION

[0051] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings. The scope of the present invention is not limited to the following embodiments. Professionals in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0052] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art. The parts, percentages, etc. involved in the following embodiments, unless otherwise specified, are all weight units.

[0053] Example 1

[0054] A method for preparing a high-throughput and highly sensitive microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, the steps are as follows:

[0055] (1) Design and draw the microfluidic four-channel graphics using AutoCAD software, design the graphics and draw the mask, and use soft lithography technology to process polydimethylsiloxane (PDMS) to obtain a microfluidic four-channel chip;

[0056] (2) 4 cm × 3 cm ITO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively, dried with nitrogen, and the cleaned ITO conductive glass was etched and screen-printed in sequence to obtain a substrate containing four working electrodes, a single reference electrode and a single counter electrode;

[0057] (3) 30 μL of 5.5 mg / mL bismuth oxybromide BiOBr solution was drop-coated on the four working electrodes of the electrode substrate and dried at room temperature. Then 20 μL of 0.5 mmol / L CsPbBr3 quantum dot solution was drop-coated and dried at room temperature to obtain an S-type CsPbBr3-BiOBr composite material modified working electrode substrate.

[0058] (4) Add 15 μL of 0.09% (w / v) chitosan solution containing 1% acetic acid to the surface of the S-type CsPbBr3-BiOBr modified working electrode, and continue to add 15 μL of 1.5% (v / v) glutaraldehyde solution to the modified working electrode surface, dry at room temperature, and rinse with ultrapure water; obtain an amino-modified S-type CsPbBr3-BiOBr working electrode substrate;

[0059] (5) The PDMS microfluidic four-channel prepared in the above step (1) and the substrate containing the amino-modified S-type CsPbBr3-BiOBr working electrode, the reference electrode, and the counter electrode in step (4) are treated with oxygen plasma, and then the microfluidic channel is bonded to the electrode substrate, thereby completing the preparation of the microfluidic four-channel chip electrode;

[0060] (6) Using a syringe pump at 40 µL / min, 10 µg / mL of four liver cancer marker capture antibodies Ab1 were simultaneously injected onto the four working electrodes in the microfluidic chip, incubated at room temperature for 10 min, and washed by injecting a buffer solution through the injection port to obtain S-type CsPbBr3-BiOBr / Ab1;

[0061] (7) Using a syringe pump to inject a 1.0% bovine serum albumin (BSA) solution at a rate of 40 µL / min into the working electrode S-type CsPbBr3-BiOBr / Ab1 to block the unbound nonspecific active sites on the electrode surface, and inject a buffer solution from the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA;

[0062] (8) Using a syringe pump at 40 µL / min, four liver cancer marker standard solutions of different concentrations ranging from 10 pg / mL to 100 ng / mL were injected into four working electrodes S-type CsPbBr3-BiOBr / Ab1 / BSA, incubated at room temperature for 10 min, and a buffer solution was injected into the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer markers;

[0063] (9) The liver cancer marker antibody labeling solution of Cu-Mn@MOF nanozyme was injected into the working electrode at 40 µL / min using a syringe pump, incubated at room temperature for 10 min, and washed by injecting a buffer solution through the injection port to obtain a fully modified S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker / Cu-Mn@MOF-Ab2 four-channel microfluidic-photoelectrochemical sensor; that is, a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for the simultaneous detection of four liver cancer markers.

[0064] Example 2

[0065] A preparation method and application of a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, the preparation steps are as follows:

[0066] (1) Design and draw the microfluidic four-channel pattern using Auto CAD software, design the pattern and draw the mask, and use soft lithography technology to process polydimethylsiloxane (PDMS) to obtain a microfluidic four-channel chip;

[0067] (2) 4 cm × 3 cm ITO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively, dried with nitrogen, and the cleaned ITO conductive glass was etched and screen-printed in turn to obtain a substrate containing four working electrodes, a single reference electrode, and a single counter electrode;

[0068] (3) 30 μL of 6.5 mg / mL bismuth oxybromide BiOBr solution was drop-coated on the four working electrodes of the electrode substrate and dried at room temperature. Then 20 μL of 1.0 mmol / L CsPbBr3 quantum dot solution was drop-coated and dried at room temperature to obtain an S-type CsPbBr3-BiOBr composite modified working electrode substrate.

[0069] (4) Add 15 μL of 0.1% (w / v) chitosan solution containing 1% acetic acid to the surface of the S-type CsPbBr3-BiOBr modified working electrode, and continue to add 15 μL of 2.0% (v / v) glutaraldehyde solution to the modified working electrode surface, dry at room temperature, and rinse with ultrapure water; obtain an amino-modified S-type CsPbBr3-BiOBr working electrode substrate;

[0070] (5) The PDMS microfluidic four-channel prepared in the above step (1) and the substrate containing the amino-modified S-type CsPbBr3-BiOBr working electrode, the reference electrode, and the counter electrode in step (4) are treated with oxygen plasma, and then the microfluidic channel is bonded to the electrode substrate, thereby completing the preparation of the microfluidic four-channel chip electrode;

[0071] (6) Using a syringe pump at 50 µL / min, 10 µg / mL of four liver cancer marker capture antibodies Ab1 were simultaneously injected onto the four working electrodes in the microfluidic chip, incubated at room temperature for 15 min, and washed by injecting a buffer solution through the injection port to obtain S-type CsPbBr3-BiOBr / Ab1;

[0072] (7) Using a syringe pump to inject a 1.0% bovine serum albumin (BSA) solution into the working electrode S-type CsPbBr3-BiOBr / Ab1 at a rate of 50 µL / min to block the unbound nonspecific active sites on the electrode surface, and inject a buffer solution from the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA;

[0073] (8) Using a syringe pump at 50 µL / min, four liver cancer marker standard solutions of different concentrations ranging from 10 pg / mL to 100 ng / mL were injected into four working electrodes S-type CsPbBr3-BiOBr / Ab1 / BSA, incubated at room temperature for 15 min, and a buffer solution was injected into the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer markers;

[0074] (9) The liver cancer marker antibody labeling solution of Cu-Mn@MOF nanozyme was injected into the working electrode at a rate of 50 µL / min using a syringe pump, incubated at room temperature for 15 min, and washed by injecting a buffer solution through the injection port to obtain a fully modified S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker / Cu-Mn@MOF-Ab2 four-channel microfluidic-photoelectrochemical sensor; that is, a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for the simultaneous detection of four liver cancer markers.

[0075] Example 3

[0076] A preparation method and application of a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, the preparation steps are as follows:

[0077] (1) Design and draw the microfluidic four-channel pattern using Auto CAD software, design the pattern and draw the mask, and use soft lithography technology to process polydimethylsiloxane (PDMS) to obtain a microfluidic four-channel chip;

[0078] (2) 4 cm × 3 cm ITO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively, dried with nitrogen, and the cleaned ITO conductive glass was etched and screen-printed in turn to obtain a substrate containing four working electrodes, a single reference electrode, and a single counter electrode;

[0079] (3) 30 μL of 7.5 mg / mL bismuth oxybromide BiOBr solution was drop-coated on the four working electrodes of the electrode substrate and dried at room temperature. Then 20 μL of 1.5 mmol / L CsPbBr3 quantum dot solution was drop-coated and dried at room temperature to obtain an S-type CsPbBr3-BiOBr composite material modified working electrode substrate.

[0080] (4) Add 15 μL of 0.11% (w / v) chitosan solution containing 1% acetic acid to the surface of the S-type CsPbBr3-BiOBr modified working electrode, and continue to add 15 μL of 2.5% (v / v) glutaraldehyde solution to the modified working electrode surface, dry at room temperature, and rinse with ultrapure water; obtain an amino-modified S-type CsPbBr3-BiOBr working electrode substrate;

[0081] (5) The PDMS microfluidic four-channel prepared in the above step (1) and the substrate containing the amino-modified S-type CsPbBr3-BiOBr working electrode, the reference electrode, and the counter electrode in step (4) are treated with oxygen plasma, and then the microfluidic channel is bonded to the electrode substrate, thereby completing the preparation of the microfluidic four-channel chip electrode;

[0082] (6) Using a syringe pump at 40-70 µL / min, 10 µg / mL of four liver cancer marker capture antibodies Ab1 were simultaneously injected onto the four working electrodes in the microfluidic chip, incubated at room temperature for 20 min, and washed by injecting a buffer solution through the injection port to obtain S-type CsPbBr3-BiOBr / Ab1;

[0083] (7) Using a syringe pump to inject a 1.0% bovine serum albumin (BSA) solution at a rate of 60 µL / min into the working electrode S-type CsPbBr3-BiOBr / Ab1 to block the unbound nonspecific active sites on the electrode surface, and inject a buffer solution from the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA;

[0084] (8) Using a syringe pump at 60 µL / min, four liver cancer marker standard solutions of different concentrations ranging from 10 pg / mL to 100 ng / mL were injected into four working electrodes S-type CsPbBr3-BiOBr / Ab1 / BSA, incubated at room temperature for 20 min, and a buffer solution was injected into the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer markers;

[0085] (9) The liver cancer marker antibody labeling solution of Cu-Mn@MOF nanozyme was injected into the working electrode at 60 µL / min using a syringe pump, incubated at room temperature for 20 min, and washed by injecting a buffer solution through the injection port to obtain a fully modified S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker / Cu-Mn@MOF-Ab2 four-channel microfluidic-photoelectrochemical sensor; that is, a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for the simultaneous detection of four liver cancer markers.

[0086] Example 4

[0087] A preparation method and application of a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, the preparation steps are as follows:

[0088] (1) Design and draw the microfluidic four-channel pattern using Auto CAD software, design the pattern and draw the mask, and use soft lithography technology to process polydimethylsiloxane (PDMS) to obtain a microfluidic four-channel chip;

[0089] (2) 4 cm × 3 cm ITO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively, dried with nitrogen, and the cleaned ITO conductive glass was etched and screen-printed in turn to obtain a substrate containing four working electrodes, a single reference electrode, and a single counter electrode;

[0090] (3) 30 μL of 8.5 mg / mL bismuth oxybromide BiOBr solution was drop-coated on the four working electrodes of the electrode substrate and dried at room temperature. Then 20 μL of 2.0 mmol / L CsPbBr3 quantum dot solution was drop-coated and dried at room temperature to obtain an S-type CsPbBr3-BiOBr composite modified working electrode substrate.

[0091] (4) Add 15 μL of 0.12% (w / v) chitosan solution containing 1% acetic acid to the surface of the S-type CsPbBr3-BiOBr modified working electrode, and continue to add 15 μL of 3.0% (v / v) glutaraldehyde solution to the modified working electrode surface, dry at room temperature, and rinse with ultrapure water; obtain an amino-modified S-type CsPbBr3-BiOBr working electrode substrate;

[0092] (5) The PDMS microfluidic four-channel prepared in the above step (1) and the substrate containing the amino-modified S-type CsPbBr3-BiOBr working electrode, the reference electrode, and the counter electrode in step (4) are treated with oxygen plasma, and then the microfluidic channel is bonded to the electrode substrate, thereby completing the preparation of the microfluidic four-channel chip electrode;

[0093] (6) Using a syringe pump at 70 µL / min, 10 µg / mL of four liver cancer marker capture antibodies Ab1 were simultaneously injected onto the four working electrodes in the microfluidic chip, incubated at room temperature for 25 min, and washed by injecting a buffer solution through the injection port to obtain S-type CsPbBr3-BiOBr / Ab1;

[0094] (7) Using a syringe pump to inject a 1.0% bovine serum albumin (BSA) solution into the working electrode S-type CsPbBr3-BiOBr / Ab1 at a rate of 70 µL / min to block the unbound nonspecific active sites on the electrode surface, and inject a buffer solution from the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA;

[0095] (8) Using a syringe pump at 70 µL / min, four liver cancer marker standard solutions of different concentrations ranging from 10 pg / mL to 100 ng / mL were injected into four working electrodes S-type CsPbBr3-BiOBr / Ab1 / BSA, incubated at room temperature for 25 min, and a buffer solution was injected into the injection port for washing to obtain S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer markers;

[0096] (9) The liver cancer marker antibody labeling solution of Cu-Mn@MOF nanozyme was injected into the working electrode at 70 µL / min using a syringe pump, incubated at room temperature for 25 min, and washed by injecting a buffer solution through the injection port to obtain a fully modified S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker / Cu-Mn@MOF-Ab2 four-channel microfluidic-photoelectrochemical sensor; that is, a high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor for the simultaneous detection of four liver cancer markers.

[0097] Example 5

[0098] See also Figure 1-2 , is the structure of a high-throughput, high-sensitivity microfluidic-photoelectrochemical sensor prepared by the aforementioned embodiments 1-4. It includes a microfluidic four-channel chip and a substrate; the microfluidic four-channel chip includes four injection ports and microchannels arranged in parallel, each injection port and microchannel is connected to a working electrode slot, a sample outlet and a microchannel are arranged on the other side of each working electrode slot away from the injection port and the microchannel, a counter electrode-reference electrode slot is arranged between the working electrode slots, and the counter electrode-reference electrode slot is arranged in communication with each working electrode slot; the substrate is used to make screen-printed electrodes and bond the microfluidic multi-channel chip, and a working electrode, a reference electrode 20 and a counter electrode 21 are arranged on the substrate corresponding to each working electrode slot and the counter electrode-reference electrode slot, respectively, and the working electrode includes Figure 2 16-19 show working electrode a, working electrode b, working electrode c and working electrode d.

[0099] The working electrode slots include working electrode slot a, working electrode slot b, working electrode slot c, and working electrode slot d, which are indicated by 1, 2, 4, and 5 in the figure respectively, and each working electrode slot is arranged around the outer side of the counter electrode-reference electrode slot 3.

[0100] The injection ports include 4, namely: a first injection port 6 for AFP capture antibody Ab1, bovine serum albumin BSA, AFP standard solution, AFP antibody marker of Cu-Mn@MOF nanozyme and cleaning solution; a second injection port 7 for CEA capture antibody Ab1, bovine serum albumin BSA, CEA standard solution, CEA antibody marker of Cu-Mn@MOF nanozyme and cleaning solution; a third injection port 8 for AFU capture antibody Ab1, bovine serum albumin BSA, AFU standard solution, AFU antibody marker of Cu-Mn@MOF nanozyme and cleaning solution; a fourth injection port 9 for GP73 capture antibody Ab1, bovine serum albumin BSA, GP73 standard solution, GP73 antibody marker of Cu-Mn@MOF nanozyme and cleaning solution;

[0101] The sample outlets are respectively: a first sample outlet 10 for GP73 capture antibody Ab1, bovine serum albumin BSA, GP73 standard solution, GP73 antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a second sample outlet 11 for AFP capture antibody Ab1, bovine serum albumin BSA, AFP standard solution, AFP antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; a third sample outlet 12 for AFU capture antibody Ab1, bovine serum albumin BSA, AFU standard solution, AFU antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through; and a fourth sample outlet 13 for CEA capture antibody Ab1, bovine serum albumin BSA, CEA standard solution, CEA antibody marker of Cu-Mn@MOF nanozyme and cleaning solution to pass through.

[0102] The above-mentioned sample inlets and sample outlets are connected to the sample inlet microchannel 14 and the sample outlet microchannel 15 respectively.

[0103] The diameters of the working electrode slot and the counter electrode-reference electrode slot are both 1600-3600 µm; the diameter of the injection port is 400-600 µm, the diameter of the microchannel connected to the injection port is 500-700 µm, the diameter of the outlet port is 500-700 µm, and the diameter of the microchannel connected to the outlet port is 600-800 µm.

[0104] Example 6

[0105] The high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor prepared in the above embodiment is used for auxiliary detection of liver cancer, and can simultaneously detect four liver cancer markers AFP, CEA, AFU, and GP73. The steps are as follows:

[0106] (1) Use an electrochemical workstation to test, inject 250 µL of 0.1 mol / L Tris-HCl (pH=7.4) into the microelectrode and channel through the injection port, and test under LED light;

[0107] (2) Use the time-current method to detect AFP, CEA, AFU, and GP73, set the voltage to 0 V, and run for 200 s;

[0108] (3) When the background current tends to be stable, turn on the light every 15 seconds for 15 seconds, then record the photocurrent changes and draw a working curve;

[0109] (4) The serum sample solution was used instead of the standard solution of AFP, CEA, AFU, and GP73, and the test results were obtained using the working curve.

[0110] Example 7

[0111] The high-throughput and high-sensitivity microfluidic-photoelectrochemical sensor prepared in the above embodiment is used for auxiliary detection of liver cancer, and the steps are as follows:

[0112] (1) Use an electrochemical workstation to test, inject 350 µL of 0.1 mol / L Tris-HCl (pH=7.4) into the microelectrode and channel through the injection port, and test under LED light;

[0113] (2) Use the time-current method to detect AFP, CEA, AFU, and GP73, set the voltage to 0 V, and run for 200 s;

[0114] (3) When the background current tends to be stable, turn on the light every 20 seconds for 20 seconds, then record the photocurrent changes and draw a working curve;

[0115] (4) The serum sample solution was used instead of the standard solution of AFP, CEA, AFU, and GP73, and the test results were obtained using the working curve.

[0116] In the above detection process, the detection range of AFP, CEA, AFU, and GP73 can reach 10 pg / mL ~ 100 ng / mL, realizing simple, rapid, highly sensitive and high-throughput detection.

[0117] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers, characterized in that: The steps are as follows: (1) Design and draw the microfluidic four-channel pattern using Auto CAD software, design the pattern and draw the mask, and use soft lithography technology to process polydimethylsiloxane (PDMS) to obtain a microfluidic four-channel chip; (2) 4 cm × 3 cm ITO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively, dried with nitrogen, and the cleaned ITO conductive glass was etched and screen-printed in sequence to obtain a substrate containing a reference electrode, a counter electrode and four working electrodes; (3) 30 μL of 5.5 ~ 8.5 mg / mL bismuth oxybromide BiOBr solution was drop-coated on the four working electrodes of the substrate and dried at room temperature. Then 20 μL of 0.5 ~ 2.0 mmol / L CsPbBr3 quantum dot solution was drop-coated and dried at room temperature to obtain an S-type CsPbBr3-BiOBr composite material modified working electrode substrate. (4) Add 15 μL of 0.09-0.12% mass / volume chitosan solution to the surface of the working electrode modified with the S-type CsPbBr3-BiOBr composite material, and continue to add 15 μL of 1.5-3.0% volume / volume glutaraldehyde solution to the modified working electrode surface, and dry at room temperature to obtain the substrate of the amino-modified S-type CsPbBr3-BiOBr working electrode; (5) treating the microfluidic four-channel chip prepared in step (1) and the substrate of the S-type CsPbBr3-BiOBr working electrode modified by amino in step (4) with oxygen plasma, and then bonding the microfluidic four-channel chip to the substrate, thereby completing the preparation of the microfluidic four-channel chip electrode; (6) Using a syringe pump at 40 to 70 µL / min, 10 µg / mL of four liver cancer marker capture antibodies Ab1 were simultaneously injected onto the four working electrodes of the microfluidic chip electrode, incubated at room temperature for 10 to 25 min, and washed by injecting a buffer solution through each injection port to obtain an S-type CsPbBr3-BiOBr / Ab1 working electrode; (7) Using a syringe pump at 40-70 µL / min, a 1.0% bovine serum albumin (BSA) solution was injected into each S-type CsPbBr3-BiOBr / Ab1 working electrode to block the unbound nonspecific active sites on the electrode surface, and a buffer solution was injected from each injection port for washing to obtain an S-type CsPbBr3-BiOBr / Ab1 / BSA working electrode; (8) Use a syringe pump to continue injecting 10 pg / mL to 100 ng / mL of four liver cancer marker standard solutions of different concentrations onto each S-type CsPbBr3-BiOBr / Ab1 / BSA working electrode at 40 to 70 µL / min, incubate at room temperature for 10 to 25 min, and inject a buffer solution into each injection port for washing to obtain an S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker working electrode; (9) Use a syringe pump to inject the liver cancer marker antibody Ab2 marker solution of Cu-Mn@MOF nanozyme into the four working electrodes at 40 ~ 70 µL / min, incubate in a 4 °C refrigerator for 10 ~ 25 min, and inject a buffer solution through each injection port for washing to obtain a fully modified S-type CsPbBr3-BiOBr / Ab1 / BSA / liver cancer marker / Cu-Mn@MOF-Ab2 four-channel microfluidic-photoelectrochemical sensor; The four liver cancer markers are AFP, CEA, AFU and GP73 respectively.

2. The method for preparing a microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers according to claim 1, characterized in that: The buffer solution is a mixed solution of disodium hydrogen phosphate and potassium dihydrogen phosphate.

3. The method for preparing a microfluidic-photoelectrochemical sensor for simultaneously detecting four liver cancer markers according to claim 1, characterized in that: Step (3) CsPbBr3 quantum dot solution is prepared as follows: 1.2 mmol of Cs2CO3 and 1.4 mL of oleic acid are dissolved in 14-17 mL of octadecene and heated to 100 °C for 30 minutes to obtain a Cs precursor solution; 0.76 mmol of PbBr2, 2.6 mL of oleic acid and 3 mL of oleamide are dissolved in 26-30 mL of octadecene and heated to 100 °C for 30 minutes to obtain a Pb precursor solution; 1.6 mL of Cs precursor solution is rapidly injected into the 170 °C Pb precursor solution, and reacted in an ice water bath for 20 s, then centrifuged and washed with acetone and methyl acetate, vacuum dried at 60 °C for 12 h, and dissolved in ultrapure water to obtain a CsPbBr3 quantum dot solution.

4. The microfluidic-photoelectrochemical sensor prepared by the preparation method according to any one of claims 1 to 3, characterized in that: It comprises a microfluidic four-channel chip and a substrate; the microfluidic four-channel chip comprises four injection ports and microchannels arranged in parallel, each injection port and microchannel is connected to a working electrode slot, a sample outlet and a microchannel are arranged on the other side of each working electrode slot away from the injection port and the microchannel, a counter electrode-reference electrode slot is arranged between the working electrode slots, and the counter electrode-reference electrode slot is arranged in communication with each working electrode slot; the substrate is used for bonding the microfluidic four-channel chip, and a working electrode, a reference electrode and a counter electrode are respectively arranged on the substrate corresponding to each working electrode slot and the counter electrode-reference electrode slot.

5. The microfluidic-photoelectrochemical sensor according to claim 4, characterized in that: The working electrode slots include first, second, third and fourth working electrode slots, and each working electrode slot is arranged around the outside of the counter electrode-reference electrode slot.

6. The microfluidic-photoelectrochemical sensor according to claim 4, characterized in that: The diameters of the working electrode slot and the counter electrode-reference electrode slot are both 1600-3600 µm; the diameter of the injection port is 400-600 µm, the diameter of the microchannel connected to the injection port is 500-700 µm, the diameter of the outlet port is 500-700 µm, and the diameter of the microchannel connected to the outlet port is 600-800 µm.

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