Automated Enzyme-Labeled Catalytic Substrate Amplified Hybrid DNA Signal Electrochemical Microfluidic Biochip and Its Application

Abstract

The present invention discloses an electrochemical microfluidic biochip for automatically amplifying hybrid DNA signals by enzyme-labeled catalysts and its applications. The biochip includes a reagent supply unit, a microfluidic device, and a waste liquid collection device. The reagent supply unit is connected to the inlet of the microfluidic device, and the waste liquid collection device is connected to the outlet of the microfluidic device. By modifying nucleic acid probes on the surface of the sensing electrode, the nucleic acid sample to be tested hybridizes with the nucleic acid probes, and then the streptavidinylated nucleic acid capture probe 2 hybridizes with the nucleic acid sample to be tested and binds to the electrode surface. Finally, through the biological affinity between biotin and streptavidin, or by labeling the sample to be tested, the biotinylated electrochemically active enzyme is bound to the electrode surface to catalyze the substrate into an electrochemically active product, thereby amplifying the signal for detection on the electrode. This chip greatly improves the detection sensitivity, reduces the sample consumption, lowers the detection cost, expands the user population and geographical area, and can be made into an intelligent device with good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical detection, and particularly to an electrochemical microfluidic biochip for automatically amplifying hybrid DNA signals by enzyme-labeled catalytic substrates, and also relates to a method for detecting nucleic acids using the biochip and its applications. Background Art

[0002] One of the key steps in constructing an electrochemical immunosensor is to select a suitable probe immobilization method. The most widely used method is the microsphere-based immobilization technique when the probe is physically adsorbed or covalently bound to the surface of polystyrene microspheres with magnetic cores. Although this method has high sensitivity, it cannot provide relevant immunoreagents with controllable spatial resolution, thus limiting its application in biochips. Another alternative method is to use electro-polymerized conducting polymers as the matrix for immobilizing immunoreagents. After the pioneering work of Foulose, the immobilization of biomolecules such as enzymes, DNA, antibodies, and even whole cells in conducting polymers has been widely applied in the fabrication of biosensors, including immunosensors. To improve the detection sensitivity, antibodies in enzyme-linked immunosorbent assays can be labeled with DNA-functionalized nanostructures, and the DNA-labeled functional materials can achieve signal amplification through polymerase chain reaction, hybridization chain reaction, or rolling circle amplification technology. Through this technology, the sensitivity of immunoreaction can be improved by several orders of magnitude compared with traditional enzyme-linked immunosorbent assays. In the enzyme-linked immunosorbent assay based on DNA signal amplification, the amplification degree of the catalytic hairpin DNA probe self-assembly reaction and hybridization chain reaction is limited; although the polymerase chain reaction has a high signal amplification degree, its reaction process requires strict heating and cooling processes, which limits its wide application. In comparison, the rolling circle amplification technology has unique advantages. It uses isothermal amplification and can achieve signal molecule amplification by 10 5 to 10 9 or even exponential order of magnitude. However, it also requires temperature control equipment, thus limiting its use in different regions and environments.

[0003] Therefore, there is an urgent need for a product with high sensitivity, no additional equipment required, low storage conditions requirements, and can be used in various environments. Summary of the Invention

[0004] In view of this, one of the purposes of the present invention is to provide an electrochemical microfluidic biochip for automatically amplifying hybrid DNA signals by enzyme-labeled catalytic substrates; the second purpose of the present invention is to provide the application of the microfluidic biochip in the manufacture of an automatic detection device.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] An electrochemical microfluidic biochip for automating the enzymatic labeling of catalytic substrates to amplify hybrid DNA signals. The electrochemical microfluidic biochip includes a reagent supply unit, a microfluidic device, and a waste liquid collection device. The reagent supply unit is connected to the inlet of the microfluidic device, and the waste liquid collection device is connected to the outlet of the microfluidic device;

[0007] The main body of the microfluidic device is a microfluidic channel. The microfluidic channel is provided with at least one sensing electrode for detecting electrochemical signals. The sensing electrode is modified with a nucleic acid probe that specifically recognizes the nucleic acid to be detected;

[0008] The reagent supply unit includes a cleaning solution reservoir, a nucleic acid to be detected reservoir, a substrate reservoir, a streptavidinylated nucleic acid probe reservoir, a biotinylated electrochemically active enzyme reservoir, and a waste liquid reservoir;

[0009] During detection, the nucleic acid fragment to be detected is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe 1 on the sensing electrode; cleaning solution is introduced for washing, leaving the paired substances; then it is combined with the electrochemically enzyme-labeled nucleic acid probe 2, and cleaning solution is introduced for washing; the bottom solution is introduced to enable the labeled electrochemically active enzyme to catalyze the substrate to be converted into electrochemically active substances, and electrochemical detection is carried out; the electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time;

[0010] Or during detection, first the nucleic acid fragment to be detected is electrochemically enzyme-labeled, and then it is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe 1 on the sensing electrode; cleaning solution is introduced to wash away the unpaired substances and the unreacted enzyme labels, leaving only the paired substances; the bottom solution is introduced to enable the labeled electrochemically active enzyme to catalyze the substrate to be converted into electrochemically active substances, and electrochemical detection is carried out; the electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time.

[0011] Preferably, in the present invention, the microfluidic channel is composed of one or more injection channels. The injection channel is provided with at least one shunt channel, and the sensing electrode modified with the nucleic acid probe is arranged on the shunt channel.

[0012] Preferably, in the present invention, a microfluidic cleaning unit is further connected between the reservoir and the outlet of the microfluidic channel. One end of the microfluidic cleaning unit is connected to the outlet of the microfluidic channel, and the other end is connected to the waste liquid reservoir. A pipeline communicating with the substrate reservoir is also provided for substrate recovery and recycling.

[0013] Preferably, in the present invention, the biochip is an addressable microfluidic device.

[0014] Preferably, in the present invention, the cleaning solution is but not limited to PBS solution; the electrochemically active enzyme is but not limited to alkaline phosphatase; the substrate is but not limited to PBS solution containing aminophenyl phosphate.

[0015] Preferably, the material of the sensing electrode is, but not limited to, metal, metal oxide, metal carbide, conductive plastic, conductive polymer, carbon material, or a composition or mixture thereof.

[0016] Preferably, the nucleic acid probe is, but not limited to, a nucleic acid sequence for detecting cancer, chronic diseases, or pathogenic microorganisms.

[0017] Preferably, the reaction solution collected by the substrate reservoir can be recycled by an electrochemical reverse reaction and used continuously.

[0018] Preferably, the microfluidic device is prepared by printing, 3-D printing, microfabrication, electrodeposition, or vacuum deposition.

[0019] Preferably, the control system of the microfluidic device uses an ARM architecture STM32 microprocessor as the core chip to build a circuit.

[0020] In the present invention, the electrochemical enzyme can also be first modified on the nucleic acid or fragment to be detected, and then complementary paired and combined with the nucleic acid probe on the sensing electrode; a cleaning solution is introduced for cleaning, leaving the paired substances; then a substrate is introduced so that the electrochemically active enzyme labeled on the nucleic acid or nucleic acid fragment captured by the nucleic acid probe can catalyze the substrate to be converted into an electrochemically active substance, which can be detected by an electrochemical method; the electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time.

[0021] 2. Application of the electrochemical microfluidic biochip in making a portable point-of-care medical device.

[0022] The beneficial effects of the present invention are as follows: The present invention discloses a sensor for amplifying the hybrid DNA signal by enzyme-labeled catalytic substrate. The sensor modifies the nucleic acid probe on the electrode surface, then hybridizes with the nucleic acid sample to be detected, and then hybridizes with the streptavidinylated nucleic acid capture probe 2. Finally, through the biological affinity between biotin and streptavidin, the biotinylated electrochemically active enzyme is combined with the electrode surface by methods such as labeling the sample, catalyzing the non-electrochemically active substrate to become an electrochemically active product, so that a signal can be detected by the electrode. Based on this principle, a highly sensitive electrochemical biochip that uses substrate enzyme-catalyzed labeling to amplify the hybrid DNA signal can also be made for simultaneously detecting multiple major diseases or chronic diseases based on nucleic acid probes. Compared with the traditional method of immobilizing biomolecules, increasing the substrate concentration or prolonging the detection time will greatly improve the detection sensitivity, reduce the sample consumption, and greatly reduce the detection cost. And it can be used by people in different regions, has low requirements for the product storage conditions, can be used in a variety of environments, and is basically not affected by temperature and humidity.

[0023] The sensor and its biochip detection device of the present invention are composed of a microfluidic sample collection unit, a sensing array unit, and a detection unit. The present invention has high sensitivity and requires a small amount of sample, expanding the detection means. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the following drawings are provided for the description of the present invention:

[0025] Figure 1 Schematic diagram for constructing the sensor (the arrow indicates the reagent flow direction);

[0026] Figure 2 Block diagram of the microfluidic sample collection unit;

[0027] Figure 3 Principle diagram of detection;

[0028] Figure 4 Alkaline phosphatase catalyzes the hydrolysis of aminophenyl phosphate to p-aminophenol;

[0029] Figure 5 p-aminophenol is oxidized to p-benzoquinone imine;

[0030] Figure 6 Differential pulse response curves of nucleic acid samples to be detected at different concentrations;

[0031] Figure 7 Stability test diagram of the electrochemical sensor;

[0032] Figure 8 Selectivity test diagram of the electrochemical sensor; where (a) is fully complementary target DNA, (b) single-base mismatched target DNA, (c) multi-base mismatched target DNA, and (d) non-complementary target DNA. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The following further describes the present invention in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0034] Embodiment 1: Electrochemical microfluidic biochip with enzyme-labeled catalytic substrate for amplifying hybrid DNA signal

[0035] The electrochemical microfluidic biochip with enzyme-labeled catalytic substrate for amplifying hybrid DNA signal has a structure as Figure 1 shown. As shown in the figure, the sensor includes a reagent supply unit 1, a microfluidic device 2, and a waste liquid collection device 3. The reagent supply unit 1 is connected to the inlet of the microfluidic device 2, and the waste liquid collection device 3 is connected to the outlet of the microfluidic device 4.

[0036] The main body of the microfluidic device 2 is a microfluidic channel 5, which is used to transport the nucleic acid sample to be detected, substrates, streptavidinylated nucleic acid capture probes 2, biotinylated electrochemical enzymes, and cleaning solutions;

[0037] The microfluidic device also includes a processor. An ARM architecture STM32 microprocessor is used as the core chip to build a circuit to control the injection pump, which is connected by signal output lines to achieve dynamic adjustment of samples and other additives, with high performance, low cost, and low power consumption; the operating software uses intelligent algorithms to deeply mine detection data. The system block diagram of the control unit is as Figure 2 shown. Among them, the injection pump and the processor are powered by a power supply. The injection pump is controlled by the processor through a stepper motor driver, and the detection signal is finally transmitted to the human-machine interaction interface through the processor. The automatic control unit circuit of the microfluidic device 2 is as Figure 3 shown.

[0038] The microfluidic channel is provided with at least one sensing electrode 6 and a counter electrode 7 for detecting electrochemical signals. The sensing electrode is modified with a nucleic acid probe that specifically recognizes the nucleic acid to be detected. Preferably, the microfluidic channel is composed of one or more sampling channels. The sampling channel is provided with at least one shunt channel. The sensing electrode modified with the nucleic acid probe is arranged on the shunt channel. Multiple sampling channels and multiple shunt channels are arranged to form an array structure. The sensing electrodes at different positions are modified with different nucleic acid probes to achieve high-throughput detection and through an addressable microfluidic device.

[0039] The reagent supply unit includes a cleaning solution reservoir 8, a nucleic acid reservoir 9 to be detected, a substrate reservoir 10, a streptavidinylated nucleic acid capture probe 2 reservoir 11, and a biotinylated electrochemical enzyme reservoir 12; they store cleaning solutions, nucleic acids to be detected, substrates, streptavidinylated nucleic acid capture probes 2, and biotinylated electrochemical enzymes respectively; the DNA in the streptavidinylated nucleic acid capture probe 2 reservoir can be complementary paired with a partial gene sequence of the nucleic acid to be detected. Preferably, the cleaning solution is a PBS solution; the electrochemical enzyme is alkaline phosphatase; the substrate is a PBS solution containing aminophenyl phosphate.

[0040] Furthermore, a microfluidic cleaning unit 12 is also connected between the waste liquid collection device 3 and the outlet of the microfluidic device 2. One end of the microfluidic cleaning unit is connected to the outlet of the microfluidic device 2, and the other end is connected to the waste liquid collection device 3. A pipeline communicating with the substrate reservoir 10 is also provided for substrate recovery and recycling. The oxidation reaction product benzoquinone imine (PQI) in the reaction solution is reduced under the action of the electrode to generate the electroactive product p-aminophenol (PAP) and transported back to the substrate reservoir, improving the detection sensitivity and reducing the sample consumption.

[0041] In this embodiment, the material of the sensing electrode can be metal, metal oxide, metal carbide, conductive plastic, conductive polymer, carbon material or their combination or mixture; prepared by printing, 3-D printing, microfabrication, electrodeposition or vacuum deposition

[0042] In this embodiment, the nucleic acid probe is designed with a specific recognition sequence according to the specific cancer, chronic disease or pathogenic microorganism to be detected.

[0043] During detection, the nucleic acid fragment to be detected is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe 1 on the sensing electrode; a cleaning solution is introduced for washing to leave the paired substances; then it is bound to the nucleic acid probe 2 labeled with an electrochemical enzyme, and a cleaning solution is introduced for washing; a bottom solution is introduced to enable the labeled electrochemically active enzyme to catalyze the substrate to be converted into an electrochemically active substance, and electrochemical detection is carried out; the electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time;

[0044] Or during detection, first the nucleic acid fragment to be detected is labeled with an electrochemical enzyme, and then it is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe 1 on the sensing electrode; a cleaning solution is introduced to wash away the unpaired substances and the unreacted enzyme label, leaving only the paired substances; a bottom solution is introduced to enable the labeled electrochemically active enzyme to catalyze the substrate to be converted into an electrochemically active substance, and electrochemical detection is carried out; the electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time.

[0045] Example 2. Method for detecting nucleic acid markers using the biochip

[0046] The method for detecting nucleic acid markers using the biochip, the principle of which is as Figure 3 shown, and the specific steps are as follows:

[0047] The method for detecting nucleic acid based on electrochemical signals includes the following steps:

[0048] 1) Modify a nucleic acid probe on the sensing electrode that specifically recognizes the nucleic acid to be detected. The specific steps are as follows: Drop a PBS solution of the nucleic acid probe with a concentration of 1 μg / mL on the surface of the sensing electrode, incubate at room temperature for 2 h, and then wash the electrode surface with a PBS solution to remove the nucleic acid probe not modified on the electrode surface. The sequence of the nucleic acid probe is: 5’-tttttttttttttttTCCGTCCCACCTCATGTGT-3’.

[0049] 2) During detection, the nucleic acid or fragment to be detected is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe on the sensing electrode; a cleaning solution is introduced for washing, leaving the paired substances; then streptavidinylated nucleic acid probe 2 is introduced, and streptavidinylated nucleic acid probe 2 binds to the nucleic acid or fragment to be detected. Unbound molecules are removed by washing, and then it is combined with biotinylated electrochemical enzyme to immobilize the electrochemical enzyme on the sensing electrode, enabling the electrochemical active enzyme label marked on the nucleic acid or nucleic acid fragment captured by the nucleic acid probe to catalyze the substrate into an electrochemical active substance, which can be detected by electrochemical methods; the electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time.

[0050] 2) In this embodiment, the nucleic acid tumor marker of breast cancer is used as the nucleic acid to be detected. A sample containing the nucleic acid to be detected (5’-tggtggcgtctctaacacatgaggtgggacgga-3’) is introduced into the microfluidic device modified with the nucleic acid probe. After incubating at room temperature for 30 min, the nucleic acid sample not bound to the nucleic acid probe is washed with PBS. Then, 1 μg / mL of streptavidinylated nucleic acid capture probe 2 (5’-tttttttttttttttttttttACCACCGCAGAGAT-3’) is introduced into the microfluidic device and incubated for 30 min, and then the unbound streptavidinylated nucleic acid capture probe 2 is removed by washing with PBS. Subsequently, 1 μg / mL of biotinylated alkaline phosphatase is introduced and incubated at room temperature for 30 min, and the alkaline phosphatase not bound to the electrode surface is washed away with PBS. Then, the substrate is input for the enzymatic reaction, where the substrate is a PBS solution containing aminophenyl phosphate (PAPP). During the detection, alkaline phosphatase (ALP) enzymatically converts aminophenyl phosphate (PAPP) to p-aminophenol (PAP), and the reaction is as Figure 4 , PAP is an electrochemical active substance. By applying a voltage to the working electrode with the reference electrode as the reference, PAP is oxidized to p-benzoquinone imine (PQI), generating electrons, and the reaction is as Figure 5 ; through these two reactions, the enzymatic reaction and the redox reaction, the presence of ALP is detected by the current value in the electrochemical measurement.

[0051] According to the same method as above, nucleic acid samples to be detected with different concentrations are detected, and their concentrations are 0 M, 10 -18 M, 10 -16 M, 10 -14 M, 10 -12 M, 10 -10 M, 10 -8 M, 10 -6 M, and the results are as Figure 6 shown. The results show that the method for detecting nucleic acid using the present invention has the advantage of high sensitivity.

[0052] The fabricated sensors were tested after being placed for 5 days, 10 days, and 15 days respectively. The response signals decreased by only 4.35%, 8.79%, and 11.74% compared to the initial state. The results are as Figure 7 shown. The results indicate that the fabricated sensors have good stability.

[0053] The fabricated sensors were used to detect fully complementary target DNA, single-base mismatched target DNA (mismatch at the 10th position of the target sequence), multi-base mismatched target DNA (mismatches at the 5th, 10th, 15th, and 20th positions of the target sequence), and non-complementary target DNA (using a random sequence) to evaluate the selectivity of the gene sensor. The results are as Figure 8 shown. The results show that the fabricated sensors can distinguish non-complementary target DNA, multi-base mismatched target DNA, and single-base mismatched target DNA.

[0054] In this embodiment, a blank electrode can also be used as the sensing electrode. During detection, the nucleic acid probe is first introduced and fixed, and then the detection is carried out.

[0055] Example 3: Construction of an automated detection device

[0056] The biochip prepared in Example 1 was combined with a control device to construct an automated detection device. The detection control device includes a module for collecting sensing electrode signals, a module for controlling microfluidic sample injection and flow rate, a module for amplifying electrical signals, an analog-to-digital conversion processing module, a wireless signal transmission module, and a data processing module. A circuit was built using an STM32 microprocessor with an ARM architecture as the core chip, which is high-performance, low-cost, and low-power consumption, and is used for collecting electrochemical signals, amplifying electrical signals, analog-to-digital conversion processing, wireless signal transmission, and data processing. The signals are transmitted to the mobile phone side to achieve automated electrochemical rapid detection.

[0057] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. Application of an electrochemical microfluidic biochip in fabricating a portable point-of-care medical device, characterized in that: The electrochemical microfluidic biochip includes a reagent supply unit, a microfluidic device, and a waste liquid collection device. The reagent supply unit is connected to the inlet of the microfluidic device, and the waste liquid collection device is connected to the outlet of the microfluidic device; The main body of the microfluidic device is a microfluidic channel. The microfluidic channel is provided with at least one sensing electrode for detecting electrochemical signals, and a nucleic acid probe specifically recognizing the nucleic acid to be detected is modified on the sensing electrode; The microfluidic device is prepared by printing, 3D printing, microfabrication, electrodeposition or vacuum deposition, and the control system of the microfluidic device uses an ARM architecture STM32 microprocessor as the core chip to build a circuit; The nucleic acid probe is a nucleic acid sequence for detecting cancer, chronic diseases or pathogenic microorganisms; The reagent supply unit includes a cleaning solution reservoir, a nucleic acid to be detected reservoir, a substrate reservoir, a streptavidinylated nucleic acid probe reservoir, a biotinylated electrochemically active enzyme reservoir, and a waste liquid reservoir; The biotinylated electrochemically active enzyme reservoir contains alkaline phosphatase solution; The substrate reservoir contains a PBS solution containing aminophenyl phosphate; A microfluidic cleaning unit is further connected between the waste liquid reservoir and the outlet of the microfluidic channel. One end of the microfluidic cleaning unit is connected to the outlet of the microfluidic channel, and the other end is connected to the waste liquid reservoir. A pipeline communicating with the substrate reservoir is also provided for substrate recovery and recycling; The biochip is an addressable microfluidic device; The cleaning solution is PBS solution, and the electrochemically active enzyme is alkaline phosphatase; The substrate is a PBS solution containing aminophenyl phosphate; The reaction solution collected by the cleaning unit and recovered to the substrate reservoir is reused after being recovered by electrochemical reverse reaction; During detection, the nucleic acid fragment to be detected is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe 1 on the sensing electrode; The cleaning solution is introduced for washing, and the paired substances are left; Then it binds to the nucleic acid probe 2 labeled with an electrochemically active enzyme, and the cleaning solution is introduced for washing; The bottom solution is introduced to enable the labeled electrochemically active enzyme to catalyze the substrate to be converted into an electrochemically active substance, and electrochemical detection is performed; The electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time; Or during detection, first the nucleic acid fragment to be detected is labeled with an electrochemically active enzyme, and then it is sent to the sensing electrode through the microfluidic channel and complementarily paired and bound with the nucleic acid probe 1 on the sensing electrode; The cleaning solution is introduced to wash away the unpaired substances and the unreacted enzyme label, and only the paired substances are left; The bottom solution is introduced to enable the labeled electrochemically active enzyme to catalyze the substrate to be converted into an electrochemically active substance, and electrochemical detection is performed; The electrochemical signal can be amplified by increasing the concentration of the bottom solution or prolonging the reaction time; The material of the sensing electrode is metal, metal oxide, metal carbide, conductive plastic, conductive polymer, carbon material or a composition or mixture thereof.

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