CN116203093B - Electrochemical microfluidic biochip for automated enzyme-labeled substrate-catalyzed amplification of hybrid DNA signals and its application - Google Patents
Electrochemical microfluidic biochip for automated enzyme-labeled substrate-catalyzed amplification of hybrid DNA signals and its application Download PDFInfo
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Abstract
The invention discloses an electrochemical microfluidic biochip for amplifying hybridization DNA signals by an automatic enzyme-labeled catalytic substrate and application thereof, wherein the biochip comprises a reagent supply unit, a microfluidic device and a waste liquid collecting device, the reagent supply unit is connected with an inlet of the microfluidic device, the waste liquid collecting device is connected with an outlet of the microfluidic device, a nucleic acid probe is modified on the surface of a sensing electrode, a nucleic acid sample to be detected is hybridized with the nucleic acid probe, then a streptavidin nucleic acid capture probe 2 is hybridized with the nucleic acid sample to be detected and combined with the surface of the electrode, finally, the biotin-labeled electrochemical active enzyme is combined with the surface of the electrode by the biological affinity between biotin and streptavidin, or the sample to be detected is marked, and the catalytic substrate is changed into an electrochemical active product, so that signals are amplified and detected on the electrode.
Description
Technical Field
The invention relates to the field of electrochemical detection, in particular to an electrochemical microfluidic biochip for amplifying hybridization DNA signals by using an automatic enzyme-labeled catalytic substrate, a method for detecting nucleic acid by using the biochip and application thereof.
Background
One of the key steps in constructing an electrochemical immunosensor is the selection of an appropriate probe immobilization method. The most widely used method is microsphere-based immobilization techniques when probes are physically adsorbed or covalently bound to the surface of polystyrene microspheres with magnetic cores. Although this approach is highly sensitive, it does not provide the relevant immunoreagent with a controllable spatial resolution, thereby limiting its use in biochips. Another alternative is to use electropolymerized conductor polymers as the matrix for the immobilized immunoreagent. Following the pioneering work of fossa, immobilization of biomolecules such as enzymes, DNA, antibodies and even whole cells in conductive polymers has been widely used to fabricate biosensors, including immunosensors. In order to improve the detection sensitivity, antibodies in the enzyme-linked immunosorbent assay can be marked by a DNA functionalized nanostructure, and the DNA marked functionalized material can realize signal amplification by polymerase chain reaction, hybridization chain reaction or rolling circle amplification technology, and by the technology, the sensitivity of the immune reaction can be improved by a plurality of orders of magnitude compared with that of the traditional enzyme-linked immunosorbent assay. In the ELISA method based on DNA signal amplification, the amplification degree of the catalytic hairpin DNA probe self-assembly reaction and the hybridization chain reaction is limited, and the polymerase chain reaction has high signal amplification degree, but the reaction process requires strict heating and cooling processes, so that the expansion application of the polymerase chain reaction is limited. In comparison, the rolling circle amplification technique has unique advantages in that isothermal amplification is employed to achieve amplification of signal molecules 10 5 to 10 9, even on the order of an exponential. But also require temperature control equipment and thus limit use to the territory and environment.
Therefore, there is a need for a product that has high sensitivity, requires no additional equipment, requires low storage conditions, and can be used in a variety of environments.
Disclosure of Invention
In view of the above, one of the purposes of the present invention is to provide an electrochemical microfluidic biochip for amplifying hybridization DNA signals by using an automated enzyme-labeled catalytic substrate, and the other purpose of the present invention is to provide an application of the microfluidic biochip in manufacturing an automated detection device.
In order to achieve the above purpose, the present invention provides the following technical solutions:
an electrochemical microfluidic biochip for amplifying hybridization DNA signals by an automatic enzyme-labeled catalytic substrate, wherein the electrochemical microfluidic biochip comprises a reagent supply unit, a microfluidic device and a waste liquid collecting device, the reagent supply unit is connected with an inlet of the microfluidic device, and the waste liquid collecting device is connected with an outlet of the microfluidic device;
The microfluidic device body is a microfluidic channel, the microfluidic channel is provided with at least one sensing electrode for detecting electrochemical signals, and a nucleic acid probe for specifically identifying nucleic acid to be detected is modified on the sensing electrode;
The reagent supply unit comprises a cleaning solution reservoir, a nucleic acid reservoir to be tested, a substrate reservoir, a streptavidin nucleic acid probe reservoir, a biotinylated electrochemical active enzyme reservoir and a waste liquid reservoir;
During detection, the nucleic acid fragment to be detected is sent to the sensing electrode through a microfluidic channel to be complementarily matched and combined with the nucleic acid probe 1 on the sensing electrode, a cleaning solution is introduced to clean and leave a matched substance, then the nucleic acid fragment is combined with the electrochemical enzyme-labeled nucleic acid probe 2, the cleaning solution is introduced to clean, a base solution is introduced to convert a labeled electrochemical active enzyme catalytic substrate into an electrochemical active substance, and the electrochemical active substance is detected by an electrochemical method;
Or during detection, firstly, the nucleic acid fragment to be detected is marked by electrochemical enzyme, then is sent to a sensing electrode through a microfluidic channel to be complementarily paired and combined with a nucleic acid probe 1 on the sensing electrode, a cleaning solution is introduced to clean the unpaired substances and unreacted enzyme labels, only the paired substances are left, a base solution is introduced to convert the marked electrochemical active enzyme catalytic substrate into the electrochemical active substance, the electrochemical active substance is detected by an electrochemical method, and an electrochemical signal can be amplified by adjusting the concentration of the base solution or increasing the reaction time.
Preferably, the microfluidic channel is composed of one or more sample injection channels, the sample injection channels are provided with at least one sub-channel, and the sensing electrode of the modified nucleic acid probe is arranged on the sub-channel.
In the invention, preferably, a microfluidic cleaning unit is also connected between the reservoir and the outlet of the microfluidic channel, one end of the microfluidic cleaning unit is connected with the outlet of the microfluidic channel, and the other end is connected with the waste liquid reservoir.
Preferably, the biochip is a microfluidic device for address management.
Preferably, the cleaning solution is not limited to PBS solution, the electrochemically active enzyme is not limited to alkaline phosphatase, and the substrate is not limited to PBS solution containing aminophenyl phosphate.
Preferably, the material of the sensing electrode is, but not limited to, metal oxide, metal carbide, conductive plastic, conductive polymer, carbon material or a combination or mixture thereof.
Preferably, the nucleic acid probes of the present invention are, but are not limited to, nucleic acid sequences that detect cancer, chronic disease or pathogenic microorganisms.
In the invention, preferably, the reaction liquid collected by the substrate storage can be recycled by electrochemical reverse reaction and then used continuously.
Preferably, the microfluidic device is prepared by printing, 3-D printing, micromachining, electrodeposition or vacuum deposition.
In the invention, preferably, the control system of the microfluidic device adopts an ARM architecture STM32 microprocessor as a core chip building circuit.
In the invention, electrochemical enzyme can be modified on the nucleic acid or fragment to be detected and complementarily paired with the nucleic acid probe on the sensing electrode, a cleaning solution is introduced to clean the nucleic acid or fragment to be detected and the substrate is introduced to enable the electrochemical active enzyme mark marked on the nucleic acid or fragment to be detected and captured by the nucleic acid probe to catalyze the substrate to be converted into electrochemical active substance, the electrochemical detection can be carried out by an electrochemical method, and the electrochemical signal can be amplified by adjusting the concentration of the primer or increasing the reaction time.
2. The electrochemical microfluidic biochip is applied to the preparation of portable timely diagnostic medical devices.
The invention has the beneficial effects that the invention discloses a sensor for amplifying hybridization DNA signals by enzyme-labeled catalytic substrates, the sensor adopts a nucleic acid probe modified on the surface of an electrode, then the sensor is hybridized with a nucleic acid sample to be detected, then the sensor is hybridized with a streptavidin-type nucleic acid capture probe 2, finally the biotin-type electrochemical active enzyme is combined on the surface of the electrode through the bioaffinity effect between biotin and streptavidin by a method of labeling the sample, and the catalytic substrate without electrochemical activity is changed into an electrochemical active product, so that the signals are detected through the electrode. Based on such principle, a highly sensitive electrochemical biochip employing substrate enzyme catalytic labelling to amplify hybridized DNA signals can also be fabricated for simultaneous detection of a variety of major diseases or chronic conditions based on nucleic acid probes. Compared with the traditional method for fixing the biological molecules, the method has the advantages that the substrate concentration is increased or the detection time is prolonged, the detection sensitivity is greatly improved, the sample consumption is reduced, and the detection cost is greatly reduced. And the product can be used by people and regions, has low requirement on storage conditions, can be used in various environments, and is basically not influenced by temperature and humidity.
The sensor and the biochip detection device thereof of the invention are composed of a microfluidic sample acquisition unit, a sensing array unit and a detection unit. The invention has high sensitivity and small amount of required samples, and expands the detection means.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a schematic diagram of a sensor construction (arrows indicate reagent flow direction);
FIG. 2 is a block diagram of a microfluidic sample acquisition unit;
FIG. 3 is a schematic diagram of the detection;
FIG. 4 is a schematic illustration of alkaline phosphatase-catalyzed hydrolysis of aminophenyl phosphate to para-aminophenol;
FIG. 5 is the oxidation of para-aminophenol to para-benzoquinone imine;
FIG. 6 shows differential impulse response curves of different concentrations of nucleic acid samples to be tested;
FIG. 7 is a stability test chart of an electrochemical sensor;
FIG. 8 is a diagram showing a selective test of an electrochemical sensor, wherein (a) is a fully complementary target DNA, (b) is a single base mismatched target DNA, (c) is a multi-base mismatched target DNA, and (d) is a non-complementary target DNA.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
Example 1, enzyme-labeled catalytic substrate amplification of hybridized DNA Signal electrochemical microfluidic biochip
The structure of the electrochemical microfluidic biochip for amplifying hybridization DNA signals by using enzyme-labeled catalytic substrates is shown in figure 1. As shown in the figure, the sensor comprises a reagent supply unit 1, a microfluidic device 2 and a waste liquid collection device 3, wherein the reagent supply unit 1 is connected with an inlet of the microfluidic device 2, and the waste liquid collection device 3 is connected with an outlet of the microfluidic device 4.
The main body of the microfluidic device 2 is a microfluidic channel 5 for conveying a nucleic acid sample to be detected, a substrate, a streptavidin nucleic acid capture probe 2, biotinylated electrochemical enzyme and a cleaning solution;
The microfluidic device further comprises a processor, an ARM architecture STM32 microprocessor is used as a core chip to build a circuit to control the injection pump, the circuit is connected through a signal output line, dynamic adjustment of samples and other additives is achieved, the performance is high, the cost is low, the power consumption is low, and the operation software uses an intelligent algorithm to deeply excavate detection data. The control unit is shown in a system block diagram in fig. 2, wherein the injection pump and the processor are powered by a power supply, the injection pump is controlled by the processor through a stepping motor driver, and detection signals are finally transmitted to the man-machine interface through the processor. The automatic control unit circuit of the microfluidic device 2 is shown in fig. 3.
The microfluidic channel is provided with at least one sensing electrode 6 and a counter electrode 7 for detecting electrochemical signals, and nucleic acid probes for specifically identifying nucleic acids to be detected are modified on the sensing electrode. Preferably, the microfluidic channel is composed of one or more sample injection channels, the sample injection channels are provided with at least one sub-channel, sensing electrodes for modifying the nucleic acid probes are arranged on the sub-channels, the plurality of sample injection channels and the plurality of sub-channels are arranged to form an array structure, and the sensing electrodes at different positions modify different nucleic acid probes so as to realize high-flux detection and realize the microfluidic device through address management.
The reagent supply unit comprises a cleaning solution reservoir 8, a nucleic acid reservoir 9 to be tested, a substrate reservoir 10, a nucleic acid capture probe 2 reservoir 11 with streptavidin and an electrochemical enzyme reservoir 12 with biotinylation, wherein the cleaning solution, the nucleic acid to be tested, the substrate, the nucleic acid capture probe 2 with streptavidin and the electrochemical enzyme with biotinylation are respectively stored, and DNA in the nucleic acid capture probe 2 reservoir with streptavidin can be complementarily paired with partial gene sequences of the nucleic acid to be tested. Preferably, the cleaning solution is a PBS solution, the electrochemical enzyme is alkaline phosphatase, and the substrate is an aminophenyl phosphate-containing PBS solution.
Further, a microfluidic cleaning unit 12 is further connected between the waste liquid collecting device 3 and the outlet of the microfluidic device 2, one end of the microfluidic cleaning unit is connected with the outlet of the microfluidic device 2, and the other end is connected with the waste liquid collecting device 3, and a pipeline communicated with the substrate storage 10 is further arranged for recycling and reusing the substrate. The oxidation reaction product benzoquinone imine (PQI) is reduced under the action of an electrode in the reaction liquid, so that an electroactive product p-aminophenol (PAP) is generated and conveyed back to a substrate storage, the detection sensitivity is improved, and the sample consumption is reduced.
The material of the sensing electrode in the embodiment can be metal, metal oxide, metal carbide, conductive plastic, conductive polymer, carbon material or their composition or mixture, printing, 3-D printing, micromachining, electrodeposition or vacuum deposition preparation
In this embodiment, the nucleic acid probe designs a specific recognition sequence according to a specific detected cancer, chronic disease or pathogenic microorganism.
During detection, the nucleic acid fragment to be detected is sent to the sensing electrode through a microfluidic channel to be complementarily matched and combined with the nucleic acid probe 1 on the sensing electrode, a cleaning solution is introduced to clean and leave a matched substance, then the nucleic acid fragment is combined with the electrochemical enzyme-labeled nucleic acid probe 2, the cleaning solution is introduced to clean, a base solution is introduced to convert a labeled electrochemical active enzyme catalytic substrate into an electrochemical active substance, and the electrochemical active substance is detected by an electrochemical method;
Or during detection, firstly, the nucleic acid fragment to be detected is marked by electrochemical enzyme, then is sent to a sensing electrode through a microfluidic channel to be complementarily paired and combined with a nucleic acid probe 1 on the sensing electrode, a cleaning solution is introduced to clean the unpaired substances and unreacted enzyme labels, only the paired substances are left, a base solution is introduced to convert the marked electrochemical active enzyme catalytic substrate into the electrochemical active substance, the electrochemical active substance is detected by an electrochemical method, and an electrochemical signal can be amplified by adjusting the concentration of the base solution or increasing the reaction time.
Example 2 methods for detecting nucleic acid markers Using the biochip
The principle of the method for detecting the nucleic acid marker by using the biochip is shown in fig. 3, and the specific steps are as follows:
a method for detecting nucleic acid based on electrochemical signals, comprising the steps of:
1) The method comprises the specific steps of modifying a nucleic acid probe for specifically recognizing nucleic acid to be detected on a sensing electrode, namely dripping a PBS solution of the nucleic acid probe with the concentration of 1 mug/mL on the surface of the sensing electrode, incubating for 2 hours at room temperature, and then washing the surface of the electrode by using the PBS solution to remove the nucleic acid probe which is not modified on the surface of the electrode, wherein the sequence of the nucleic acid probe is 5'-ttttttttttttttt TCCGTCCCACCTCATGTGT-3'.
2) During detection, the nucleic acid or fragment to be detected is sent to the sensing electrode through the microfluidic channel to be complementarily paired and combined with the nucleic acid probe on the sensing electrode, a cleaning solution is introduced to clean and leave paired substances, then the streptavidin nucleic acid probe 2 is introduced, the streptavidin nucleic acid probe 2 is combined with the nucleic acid or fragment to be detected to clean and remove unbound molecules, and electrochemical enzyme is fixed on the sensing electrode in combination with biotinylated electrochemical enzyme, so that the electrochemical active enzyme marked on the nucleic acid or nucleic acid fragment to be detected captured by the nucleic acid probe can catalyze the substrate to be converted into electrochemical active substances, the electrochemical active substances can be detected by an electrochemical method, and the electrochemical signal can be amplified by adjusting the concentration of a base solution or increasing the reaction time.
2) In this example, a sample containing a nucleic acid to be tested (5'-tggtggcgtctctaacacatgaggtgggacgga-3') is introduced into a microfluidic device modified with a nucleic acid probe, after incubation for 30min at room temperature, the nucleic acid sample not bound to the nucleic acid probe is washed with PBS, then 1. Mu.g/mL of streptavidin-coated nucleic acid capture probe 2 (5'-tttttttttttttttttttttACCACCGCAGAGAT-3') is introduced into the microfluidic device and incubated for 30min, after washing with PBS to remove unbound streptavidin-coated nucleic acid capture probe 2, and then 1. Mu.g/mL of biotinylated alkaline phosphatase is introduced into the microfluidic device and incubated for 30min at room temperature, and the alkaline phosphatase not bound to the electrode surface is washed off with PBS. Then, a substrate is input for enzyme-catalyzed reaction, wherein the substrate is PBS solution containing aminophenyl phosphate (PAPP). In the detection, alkaline phosphatase (ALP) was enzymatically converted to p-aminophenyl phosphate (PAPP), as shown in FIG. 4, which is an electrochemically active substance, and voltage was applied to the working electrode based on the reference electrode, and PAP was oxidized to p-benzoquinone imine (PQI), to generate electrons, as shown in FIG. 5, and the presence of ALP was detected by the current value in the electrochemical measurement by two reactions, i.e., the enzymatic reaction and the redox reaction.
The nucleic acid samples to be tested were tested at different concentrations, respectively 0M and 10 -18 M、10-16 M、10-14 M、10-12 M、10-10 M、10-8 M、10-6 M, according to the same procedure as described above, and the results are shown in FIG. 6. The results show that the method for detecting nucleic acid has the advantage of high sensitivity.
The sensors constructed were left for 5 days, 10 days and 15 days, respectively, and then were tested, and their response signals were reduced by only 4.35%, 8.79% and 11.74% from the initial state, and the results are shown in fig. 7. The results show that the stability of the constructed sensor is very good.
The constructed sensor was used to detect full-complement target DNA, single base mismatched target DNA (mismatch at position 10 of target sequence), multiple base mismatched target DNA (mismatch at positions 5, 10, 15, and 20 of target sequence), non-complementary target DNA (using random sequences), respectively, to evaluate the selectivity of the gene sensor, and the results are shown in fig. 8. The results show that the constructed sensor can distinguish non-complementary target DNA, multi-base mismatched target DNA and single-base mismatched target DNA.
In this embodiment, the sensing electrode may be a blank electrode, and the nucleic acid probe is first immobilized and then detected during detection.
Example 3 construction of automated inspection device
The biochip prepared in example 1 is combined with a control device to construct an automatic detection device, and the detection control device comprises a sensing electrode signal acquisition module, a microfluidic sample injection and flow control module, an electric signal amplification module, an analog-to-digital conversion processing module, a wireless signal transmission module and a data processing module. The STM32 microprocessor adopting the ARM architecture is used as a core chip to build a circuit, has high performance, low cost and low power consumption, and is used for collecting electrochemical signals, amplifying electric signals, carrying out analog-to-digital conversion processing, transmitting wireless signals and carrying out data processing, and transmitting the signals into a mobile phone terminal to realize automatic electrochemical rapid detection.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
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| CN1499195A (en) * | 2002-10-31 | 2004-05-26 | 惠普开发有限公司 | Microflow system for analyzing nucleic acid |
| JP2010156605A (en) * | 2008-12-26 | 2010-07-15 | Mitsubishi Chemical Medience Corp | Electrical analysis method |
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