CN114720537A

CN114720537A - Polyelectrolyte hydrogel ion diode, preparation method thereof and application thereof in nucleic acid detection

Info

Publication number: CN114720537A
Application number: CN202210279670.6A
Authority: CN
Inventors: 刘一凡; 熊陈玮; 张蓉
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-07-08
Anticipated expiration: 2042-03-21
Also published as: CN114720537B

Abstract

The invention relates to the field of biochemistry, in particular to a polyelectrolyte hydrogel ion diode, a preparation method thereof and application thereof in nucleic acid detection, wherein the polyelectrolyte hydrogel ion diode comprises a micro-fluidic chip and a hydrogel heterojunction, a micro-channel is arranged on the micro-fluidic chip and comprises a working micro-channel and a reference micro-channel, the working micro-channel and the reference micro-channel are communicated through a bridge channel, and the working micro-channel and the reference micro-channel are respectively provided with electrode holes for mounting a working electrode and a reference electrode; the hydrogel heterojunction comprises a P-type hydrogel and an N-type hydrogel, the P-type hydrogel and the N-type hydrogel are both arranged in the bridge channel, the P-type hydrogel is a cationic polyelectrolyte hydrogel, and the N-type hydrogel is an anionic polyelectrolyte hydrogel. The present invention can detect nucleic acids using electrical signals.

Description

Polyelectrolyte hydrogel ion diode, preparation method thereof and application thereof in nucleic acid detection

Technical Field

The invention relates to the field of biochemistry, in particular to a polyelectrolyte hydrogel ion diode, a preparation method thereof and application thereof in nucleic acid detection.

Background

Measurement of nucleic acid species often relies on gene amplification techniques such as the use of fluorescent quantitative polymerase chain reaction (qPCR) and Multiple Displacement Amplification (MDA), but these techniques often use expensive fluorescent signal reading equipment. In order to detect nucleic acid molecules quickly, accurately and at low cost, a technology relying on an electrical signal is also an efficient and desirable means, for example, a nanocapillary diode or a graphene field effect transistor is used to detect the change of the electrical signal caused by the change of the concentration of nucleic acid, so as to complete the detection and calibration functions. However, the detection elements used in these techniques are mainly composed of materials with poor biocompatibility, such as glass and metal, and furthermore, signal transmission in most biological structures is through ion conduction, and glass and metal do not simulate such a process well. There is a need for further development to provide products and methods that are efficient, convenient and compatible with ionic conductivity, while using electrical signals to detect nucleic acid concentration and to calibrate nucleic acid amplification processes.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a polyelectrolyte hydrogel ion diode, a method for preparing the same, and a use thereof in nucleic acid detection, which are used to solve the problems of the prior art.

In order to achieve the above and other related objects, the present invention provides a polyelectrolyte hydrogel ion diode, which comprises a microfluidic chip and a hydrogel heterojunction, wherein the microfluidic chip is provided with a microchannel, the microchannel comprises a working microchannel and a reference microchannel, the working microchannel and the reference microchannel are communicated through a bridge channel, and the working microchannel and the reference microchannel are respectively provided with electrode holes for installing a working electrode and a reference electrode; the hydrogel heterojunction comprises a P-type hydrogel and an N-type hydrogel, the P-type hydrogel and the N-type hydrogel are arranged in the bridge channel, the P-type hydrogel is a cationic polyelectrolyte hydrogel, and the N-type hydrogel is an anionic polyelectrolyte hydrogel.

The invention also provides a preparation method of the polyelectrolyte hydrogel ion diode, which comprises the following steps:

1) preparing the micro-fluidic chip, and performing surface modification on a micro-channel of the micro-fluidic chip;

2) introducing a P-type hydrogel pre-solution into the microchannel, and crosslinking to form a P-type hydrogel in the bridge channel;

3) and (3) introducing the N-type hydrogel pre-solution into the microchannel, and crosslinking to form the N-type hydrogel in the bridge passage.

The invention also provides application of the polyelectrolyte hydrogel ion diode in preparation of a nucleic acid detection device.

The invention also provides a nucleic acid detection device which comprises the polyelectrolyte hydrogel ion diode, an electrochemical workstation and a computer, wherein the electrochemical workstation is in signal connection with the polyelectrolyte hydrogel ion diode and the computer.

The invention also provides application of the polyelectrolyte hydrogel ion diode or the nucleic acid detection device in nucleic acid detection.

The invention also provides a nucleic acid detection method, which comprises the steps of detecting electric signals when the working micro-channel does not contain the nucleic acid to be detected and contains different nucleic acids to be detected, and comparing the difference of the electric signals to obtain the difference of the nucleic acids to be detected.

As described above, the polyelectrolyte hydrogel ion diode, the preparation method thereof and the application thereof in nucleic acid detection have the following beneficial effects:

1. the polyelectrolyte hydrogel ion diode is simple to process and low in cost, can detect nucleic acid by using an electrical signal and does not depend on detection through a fluorescence signal required by expensive equipment like a PCR instrument.

2. Compared with the traditional devices based on metal and glass materials, the polyelectrolyte hydrogel has better biocompatibility and ion conductivity, so that the signal transmission in a biological structure can be better simulated.

3. The in-situ amplification calibration can be realized in the nucleic acid amplification process.

Drawings

FIG. 1 is a schematic view of a process flow of a polyelectrolyte hydrogel ion diode, which is performed in the order of a-b-c-d-e-f;

FIG. 2 is a layout of a microchannel in a polyelectrolyte hydrogel ion diode, wherein a is a layout used in examples 1 and 2, and b is a layout used in example 3;

FIG. 3 is an electron micrograph of the pore structure inside the polyelectrolyte hydrogel, wherein a is a P-type hydrogel, and b is an N-type hydrogel;

FIG. 4 is a schematic diagram of the internal structure of a polyelectrolyte hydrogel ion diode;

FIG. 5 is a fluorescence micrograph of the region of hydrogel within the polyelectrolyte hydrogel ion diode;

FIG. 6 is a schematic view of the operating principle of polyelectrolyte hydrogel ion diode during detection, wherein a is a schematic view of the change of the hydrogel before and after the influence of DNA, and b is a schematic view of the operating environment in the hydrogel region and the micro-channel around the hydrogel region;

FIG. 7 is a current-voltage graph of a polyelectrolyte hydrogel ion diode in an initial state, under the influence of DNA, and under the influence of PLL;

FIG. 8 is a bar graph of rectification ratio change of a polyelectrolyte hydrogel ion diode under the influence of DNA and PLL in an initial state;

FIG. 9 is a histogram of the change in rectification ratio of the polyelectrolyte hydrogel ion diode in detecting DNAs of different bp lengths at the same mass concentration;

FIG. 10 is a bar graph showing the change in rectification ratio of polyelectrolyte hydrogel ion diode in detection of DNA of different bp lengths at the same molarity;

FIG. 11 is a histogram of the change in rectification ratio of polyelectrolyte hydrogel ion diodes for different PCR materials and DNA amplification products;

FIG. 12 is a graph showing the change of the rectification ratio of the polyelectrolyte hydrogel ion diode in real-time detection of DNAs with different mass concentrations;

FIG. 13 is a bar graph of rectification ratio as the number of amplification cycles increases during calibration of PCR amplification with polyelectrolyte hydrogel ion diodes;

FIG. 14 is a graph of rectification ratio change during calibration of the polyelectrolyte hydrogel ion diode for PCR amplification and variation of qPCR fluorescence values with increasing amplification cycle number;

FIG. 15 is a fluorescent representation of a polyelectrolyte hydrogel ion diode used for in situ amplification calibration using MDA;

FIG. 16 is a schematic diagram showing the structure of the polyelectrolyte hydrogel ion diode shown in FIG. 15 and a micrograph of the change in fluorescence in the corresponding region;

FIG. 17 is a graph of changes in fluorescence values in the hydrogel region and the chamber region of a polyelectrolyte hydrogel ion diode during in situ amplification calibration;

FIG. 18 is a graph of the change in DNA concentration and rectification ratio with time for polyelectrolyte hydrogel ion diodes subjected to in situ amplification calibration;

FIG. 19 is a graph of current-voltage curves for different mass concentrations of DNA detected by polyelectrolyte hydrogel ion diodes;

FIG. 20 is a graph of current-voltage curves for different amplification times of polyelectrolyte hydrogel ion diodes in situ amplification calibration;

FIG. 21 is a graph of current versus voltage for different amplification cycle numbers during the calibration of the PCR amplification with polyelectrolyte hydrogel ion diodes.

Detailed Description

As shown in fig. 4, the present invention provides a polyelectrolyte hydrogel ion diode, which includes a microfluidic chip and a hydrogel heterojunction, wherein the microfluidic chip is provided with a microchannel, the microchannel includes a working microchannel and a reference microchannel, the working microchannel and the reference microchannel are communicated via a bridge channel, and the working microchannel and the reference microchannel are respectively provided with electrode holes for installing a working electrode and a reference electrode; the hydrogel heterojunction comprises a P-type hydrogel and an N-type hydrogel, the P-type hydrogel and the N-type hydrogel are both arranged in the bridge channel, the P-type hydrogel is a cationic polyelectrolyte hydrogel, and the N-type hydrogel is an anionic polyelectrolyte hydrogel.

Polyelectrolyte hydrogels are a class of aqueous network polymers with positively and negatively charged groups on the backbone of the polymer. By controlling the ratio of positively and negatively charged groups in the network structure, the polymer gel exhibits characteristic polyelectrolyte properties (large number of charges) and reverse polyelectrolyte properties (in the vicinity of isoelectric point). For example, a cationic polyelectrolyte hydrogel backbone is negatively charged, and a layer of space is formed around it that is primarily occupied by a counterion (here, a cation); the anionic polyelectrolyte hydrogel backbone is positively charged and a layer of space is formed around it that is primarily occupied by counterions (here anions). Wherein, when the proportion of positive charges to negative charges is the same, the unique amphoteric structure and corresponding characteristics are closer to natural macromolecules, such as protein, nucleic acid and the like.

The microfluidic chip is selected from a PDMS-glass bonding chip, a PDMS-PDMS chip, a PDMS-hydrogel chip or a hydrogel-hydrogel chip.

And two ends of the working micro-channel are provided with electrode holes (the motor hole is the reservoir shown in figure 4) for installing working electrodes.

And two ends of the reference micro-channel are provided with electrode holes for mounting reference electrodes.

In one embodiment, the polyelectrolyte hydrogel ion diode further comprises a working electrode and a reference electrode. The working electrode and the reference electrode may employ electrodes commonly used in the art. The working electrode is typically made of an inert material. The working electrode is, for example, a gold electrode, a platinum electrode or a glassy carbon electrode. The reference electrode is an electrode used to assist in determining the potential of the working electrode. The reference electrode should have a known and stable electrochemical potential. The reference electrode commonly used in the laboratory was a Saturated Calomel Electrode (SCE) or a silver/silver chloride electrode.

The shapes of the working microchannel and the reference microchannel can be adjusted according to experimental conditions. In the embodiments shown in fig. 1, 2a, 4, the working microchannel and the reference microchannel are each "U" shaped. In the embodiment shown in fig. 2b, the remaining portions of the working microchannel excluding the electrode aperture portions are square, and the reference microchannel is "U" shaped.

In one embodiment, the interior of the microchannel is surface modified.

In one embodiment, the surface modification inside the microchannel is a hydrophilic modification. The hydrophilic modification is to crosslink the P-type hydrogel and the N-type hydrogel with the channel surface to immobilize them at the bridge channel.

In one embodiment, the interior of the microchannel is surface modified with 3- (trimethoxysilyl) propyl methacrylate (TMSMA).

The relative positions of the P-type hydrogel and the N-type hydrogel are not particularly limited. In the embodiment shown in fig. 4, the P-type hydrogel is disposed in the bridge channel on the side near the reference microchannel, and the N-type hydrogel is disposed in the bridge channel on the side near the working microchannel.

In the polyelectrolyte hydrogel ion diode, the hydrogel heterojunction consists of a P-type hydrogel and an N-type hydrogel.

The cationic polyelectrolyte hydrogel can be hydrogel formed by 3-sulfopropyl acrylate potassium salt or polystyrene sulfonate.

The anionic polyelectrolyte hydrogel may be, for example, a hydrogel formed from diallyldimethylammonium chloride or 3- (methacrylamide) propyltrimethylammonium chloride.

In one embodiment, the microfluidic chip is selected from a PDMS-glass bonded chip. The micro-fluidic chip can be prepared by adopting the existing micro-processing technology, and is different from the prior art that the mask plate is customized in a third party company, and the pattern on the customized mask plate is the shape of the micro-channel on the polyelectrolyte hydrogel ion diode.

In one embodiment, the step of preparing the PDMS-glass bonded chip described in step 1) is as follows:

1a) pre-baking the silicon wafer coated with the photoresist;

1b) placing a mask on the pre-baked silicon wafer, and exposing under an ultraviolet lamp; then post-baking the exposed silicon wafer;

1c) putting the silicon wafer after being dried into a developing solution for soaking, cleaning and drying, and then putting the silicon wafer into a culture dish;

1d) and adding the PDMS pre-solution mixture into the culture dish for crosslinking, and bonding and fixing the crosslinked PDMS and the glass sheet to form the PDMS-glass bonded chip.

In one embodiment, step 1) comprises hydrophilic surface modification of the interior of the microchannel.

In one embodiment, the hydrophilic surface modification is by 3- (trimethoxysilyl) propyl methacrylate (TMSMA).

In one embodiment, the surface modification method in step 1) is to sequentially introduce a methanol solution, a TMSMA solution and a methanol solution into the microchannel.

In one embodiment, the solution for surface modification in the microchannel is blown dry and then the P-type hydrogel pre-solution or the N-type hydrogel pre-solution is introduced.

In one embodiment, the P-type hydrogel pre-solution is a 3-sulfopropyl acrylate potassium salt solution.

In one embodiment, the N-type hydrogel pre-solution is a solution of diallyldimethylammonium chloride.

The solvent of the P-type hydrogel pre-solution or the N-type hydrogel pre-solution is water, and the mass concentration of the solute is 1-40%. The mass concentration of the solute in the P-type hydrogel pre-solution or the N-type hydrogel pre-solution is selected from any one of the following: 1% -30%, 1% -25%, 3% -18%, 5% -15%, 7% -13% and 8% -12%.

In one embodiment, the method of crosslinking in steps 2) and 3) is crosslinking under uv light.

In one embodiment, the method for preparing the polyelectrolyte hydrogel ion diode further comprises the step of introducing a buffer solution into the microchannel after the P-type hydrogel or the N-type hydrogel is formed so as to remove the P-type hydrogel pre-solution or the N-type hydrogel pre-solution.

And the computer is provided with electrochemical workstation software.

The nucleic acid detection is a nucleic acid concentration detection. The nucleic acid is selected from DNA or RNA. The nucleic acid concentration detection is semi-quantitative detection. The nucleic acids may be homogeneous nucleic acid fragments or heterogeneous nucleic acid fragments. In one embodiment, the nucleic acid can be detected by extracting isolated nucleic acid, PCR amplification products of different cycle cycles, and in situ detection of products of multiple displacement amplification at different amplification times.

The invention also provides a method for detecting nucleic acid, which comprises the steps of detecting electric signals when the working micro-channel does not contain the nucleic acid to be detected and contains different nucleic acids to be detected, and comparing the difference of the electric signals to obtain the difference of the nucleic acids to be detected.

In one embodiment, the nucleic acid detection is a nucleic acid concentration detection.

The nucleic acid is selected from DNA or RNA.

In one embodiment, the difference between the test nucleic acids obtained by comparing the difference between the electric signals is the difference between the concentrations of the test nucleic acids.

When the polyelectrolyte hydrogel ion diode shown in FIG. 2a is used, the method for detecting nucleic acid comprises the following steps:

1) inserting a working electrode into an electrode hole of the working microchannel, inserting a reference electrode into an electrode hole of the reference microchannel, and connecting the electrochemical workstation with a computer;

2) introducing a buffer solution into the working microchannel and the reference microchannel, and measuring an initial electric signal;

3) and (3) introducing the nucleic acid to be detected with different concentrations into the working microchannel, detecting to obtain electric signals corresponding to the nucleic acid to be detected with each concentration, and comparing the difference between the electric signals and the initial electric signal to obtain the relative concentration of the nucleic acid to be detected.

The ion concentration of the buffer solution in the step 2) is kept consistent with that of the solvent of the nucleic acid to be detected. For example, when PCR amplification products are detected over different cycle periods, the ion concentration of the buffer in step 2) is kept consistent with the ion concentration in the PCR buffer solution.

In one embodiment, after the buffer solution or the nucleic acid to be detected is introduced into the microchannel, the electric signal is detected after the diffusion of the introduced ion or nucleic acid is stabilized.

The electrical signal includes an I-V curve.

The polyelectrolyte hydrogel ion diode shown in FIG. 2a can be used to detect nucleic acids that have been prepared, for example, by PCR amplification products over different cycles, and extraction of isolated nucleic acids. The prepared nucleic acid is nucleic acid which does not need to be amplified in the polyelectrolyte hydrogel ion diode.

In detecting the product concentration of multiple displacement amplification at different times using a polyelectrolyte hydrogel ion diode as shown in FIG. 2b, the method for nucleic acid detection comprises the steps of:

2) introducing raw materials required by nucleic acid amplification into a working micro-channel, introducing a buffer solution into a reference micro-channel, detecting at different time points to obtain electric signals corresponding to the nucleic acid at the time points, and comparing the difference between the electric signals to obtain the relative concentration of the amplified nucleic acid at different time points.

In one embodiment, for example, a 10 μ l system, the raw materials for the multiple displacement amplification include: 3. mu.l of deionized water, 1. mu.l of phi29 buffer solution, 0.5. mu.l of random primer, 1. mu.l of template, 4. mu.l of dNTP, and 0.5. mu.l of polymerase.

In the invention, a Polyelectrolyte Hydrogel Ion Diode (PHID) is formed by using a material with good biocompatibility and ion conductivity, namely a polyelectrolyte hydrogel heterojunction, as a detection element to complete the detection of nucleic acid. In PHID, after DNA molecules diffuse into the three-dimensional network structure inside P-type hydrogel and N-type hydrogel, the charge density of the surface is influenced by negatively charged DNA molecules, thereby changing the rectification characteristic of PHID. By recording and comparing the change of the electric signal, the detection of the DNA molecule and the calibration of the concentration change can be completed.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The sources of materials used in the examples are as follows:

p-type hydrogel monomer: 3-sulfopropyl acrylate potassium salt;

n-type hydrogel monomer: diallyldimethylammonium chloride;

surface modifier: 3- (trimethoxysilyl) propyl methacrylate;

a crosslinking agent: n, N' -methylenebisacrylamide;

photoinitiator (2): lithium phenyl (2,4, 6-trimethylbenzoyl) phosphate, purchased from Sigma-Aldrich.

PCR kit (Phanta Max Super-Fidelity DNA Polymerase), Phi29 DNA Polymerase, Phi29 DNA Polymerase buffer, dNTP (10mM) was purchased from Nanjing Novozam Biotech GmbH.

Oligonucleotide primers were produced by jingzhi.

EVA Green (20X) was purchased from Biotium (USA).

Random primers (EXO-RESISTANT RANDOM PRIMER), PCR fluorescent primer sequences, and ribozyme-free water (NUCLEASE-FREE WATER 10X 50ML) were purchased from Saimer Feishell science and technology (China) Ltd.

Example 1 preparation of polyelectrolyte hydrogel ion diodes

The preparation process is shown in figure 1 and comprises the following steps:

1) setting the rotation speed of a spin coater, placing a piece of clean silicon wafer on the spin coater, and then pouring a layer of SU8-3025 photoresist on the silicon wafer. After the spin coater was operated at 500rpm for 10s and 2200rpm for 30s, the wafer was placed on a hot plate at 95 ℃ and pre-baked for 20 min.

2) A reticle having the pattern shown in FIG. 1a was placed on the pre-baked silicon wafer and exposed for 4min under a 365nm wavelength ultraviolet lamp (Thorlabs COP1-A UV light). The exposed silicon wafer was then placed on a hot plate at 95 ℃ and post-baked for 10 min.

3) And (3) soaking and cleaning the post-baked silicon wafer in a developing solution for 5min, and then sequentially cleaning with an isopropanol solution and an ethanol solution. And drying the cleaned silicon wafer by using a nitrogen gun and placing the silicon wafer into a culture dish.

4) The PDMS pre-solution mixture was poured into the petri dish, which was then placed in an oven at 60 ℃ to crosslink for 8 h. The crosslinked PDMS was then cut out and placed in a plasma cleaner with a 2cm x 2cm size glass slide for 80 s. Bonding and fixing the processed PDMS cover on a glass sheet to form a PDMS-glass chip microfluidic device (as shown in figure 1c), and placing on a heating plate for 2h for use after the state is stable.

5) Methanol solution (99.9%), 3- (trimethoxysilyl) propyl methacrylate (TMSMA) solution (10% TMSMA, 90% methanol) and methanol solution (99.9%) were sequentially introduced into the channel of the PDMS-glass chip for 2min each, thereby completing surface modification inside the microchannel.

6) And blowing the microchannel into the P-type hydrogel pre-solution (the mass concentration of the monomer is 10%) after the microchannel is dried. Covering a mask chromium plate on a corresponding P-type hydrogel area of the device, and exposing for 10s under an ultraviolet lamp, so that the P-type hydrogel is bonded with the surface of the microchannel while being cross-linked and molded, and the P-type hydrogel can be fixed at one end of the bridge channel.

7) And (3) flushing the channel for 2 times by using an N-type hydrogel pre-solution (the mass concentration of the monomer is 10%), standing for 5min, and then flushing for 2 times, so that the N-type hydrogel pre-solution can completely enter the other end in the bridge channel. Covering a mask chromium plate on a corresponding N-type hydrogel area of the device, and exposing for 10s under an ultraviolet lamp, so that the N-type hydrogel is bonded with the surface of the microchannel while being cross-linked and molded, and the N-type hydrogel can be fixed at the other end of the bridge channel.

8) The buffer solution was passed into the microchannel to drain the neat hydrogel pre-solution. The washing is repeated 3 times and the mixture is left standing for 2 hours until the hydrogel ion diode reaches a stable state in the buffer solution environment, as shown in fig. 1 f.

Example 2 polyelectrolyte hydrogel ion diode real-time detection of DNA at different concentrations

And inserting the working electrode and the reference electrode into the electrode holes corresponding to the working microchannel and the reference microchannel, and connecting the electrode holes with an electrochemical workstation and a computer to form a whole set of detection system. The specific detection method comprises the following steps:

1) in the initial state, the micro-channel of the polyelectrolyte hydrogel ion diode device is filled with a buffer solution (50mmol/L KCl, 1.5mmol/L MgCl) with a certain salt ion concentration₂) (as shown in fig. 1 f), the hydrogel remains in equilibrium in this environment. And opening the software of the electrochemical workstation, scanning and measuring an I-V curve of the hydrogel ion diode in the initial state and obtaining a rectification ratio (the rectification ratio is the ratio of on-state current to off-state current).

2) And introducing a solution containing the DNA to be detected into the working micro-channel. When the process of the DNA molecule diffusing into the N-type hydrogel is stable (i.e. the rectification ratio value tends to be stable), the I-V curve in the state is measured again, and a rectification ratio larger than that in the initial state is obtained. The detection of DNA is accomplished by this change in rectification ratio.

3) Repeating the step 2) and sequentially introducing the solution containing DNA with larger mass concentration. When the mass concentration of DNA is higher, the rectification ratio is lower, and the change of the rectification ratio from that in the initial state is larger. As shown in FIGS. 12 and 19, taking the two states of 5 ng/. mu.l and 40 ng/. mu.l as an example, for the measured electrical signals, the off-state current at 40 ng/. mu.l is higher than that at 5 ng/. mu.l in the I-V graph, and the calculated rectification ratio at 40 ng/. mu.l is smaller than that at 5 ng/. mu.l. Throughout the whole testing process, the concentration value of the DNA introduced each time is continuously increased, and the measured rectification ratio value is continuously reduced (namely, the change value of the rectification ratio value is continuously increased).

Example 3 semi-quantitative calibration of PCR amplification products Using polyelectrolyte hydrogel ion diodes

The specific calibration method comprises the following steps:

1) the procedure was the same as in step 1 and step 2 of example 2 by first passing a solution containing only primers or polymerase in the working microchannel to exclude the influence of molecules other than DNA on the detection (the change in rectification ratio caused by these molecules of raw materials required for amplification of DNA is much smaller than that in the detection of DNA). And measuring an I-V curve in an initial state in a state where a solution containing only a primer or a polymerase is introduced into the working microchannel.

2) The PCR instrument is used to obtain 6 kinds of DNA amplification products with different mass concentrations under 5, 10, 15, 20, 25 and 30 cycle periods.

3) After the solutions with the DNA amplification products were sequentially introduced into the working channels in the order of cycle number from small to large, and I-V curves were measured, it was found that the magnitude of the rectification ratio decreased with the increase of the cycle number of DNA amplification, and the increasing trend of the change in the rectification ratio was also consistent with the change in fluorescence value measured by qPCR, as shown in FIG. 11. As shown in FIGS. 13, 14 and 21, the concentration of DNA was increased with the increase of the reaction time during the PCR. Taking the states of two time points of 0cycles and 10cycles as an example, after 10cycles, the DNA concentration value represented by the fluorescence value measured by the qPCR instrument is increased; for the measured electrical signal, the off-state current at 10cycles is higher than the off-state current at 0cycles in the I-V plot, and the calculated rectification ratio at 10cycles is less than the rectification ratio at 0 cycles. Throughout the whole reaction process, the DNA concentration value represented by the fluorescence value measured by the qPCR instrument is continuously increased, the rectification ratio value measured by the device is continuously reduced (namely the change value of the rectification ratio value is continuously increased), and the increase trend of the rectification ratio change value in the whole reaction process is basically consistent with the increase trend of the DNA concentration value, so that the calibration of nucleic acid amplification is completed.

Example 4 in situ calibration of MDA Using polyelectrolyte hydrogel ion diodes

The specific calibration method comprises the following steps:

1) the micro-channels were redesigned before performing the in situ amplification calibration experiments. The working microchannel is changed into a large chamber so as to ensure that the DNA can continuously carry out the amplification reaction in the working microchannel. As shown in fig. 2 b.

2) And (3) introducing the solution with the raw materials required by the MDA into the working micro-channel, and recording the DNA concentration change represented by the change of the fluorescence value in the working micro-channel and the N-type hydrogel by using a fluorescence microscope every other hour. The current-voltage curve was measured every half hour and the rectification ratio was calculated. As shown in fig. 16 and 17. Meanwhile, a solution with raw materials required by MDA is added into an empty Ep tube as a control, and the DNA concentration value in the tube is measured by using the Qubit every half hour.

3) The change trend of the rectification ratio is compared with the change trend of the DNA concentration value measured by the Qubit, and the results are consistent, as shown in FIGS. 18 and 20, in the MDA process, the concentration of the DNA is continuously increased along with the continuous increase of the reaction time. Taking the states of two time points of 0h and 1h as an example, after 1 hour, the DNA concentration value measured by the Qubit is increased; for the measured electrical signal, in the I-V graph, the off-state current at 1h is higher than the off-state current at 0h, and the calculated rectification ratio at 1h is smaller than the rectification ratio at 0 h. Throughout the whole reaction process, the concentration value of DNA measured by the Qubit is continuously increased, the rectification ratio value measured by the device is continuously reduced (namely the variation value of the rectification ratio is continuously increased), and the increasing trend of the variation value of the rectification ratio in the whole reaction process is basically consistent with the increasing trend of the concentration value of the DNA, so that the calibration of nucleic acid amplification is completed.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. The polyelectrolyte hydrogel ion diode is characterized by comprising a micro-fluidic chip and a hydrogel heterojunction, wherein the micro-fluidic chip is provided with a micro-channel, the micro-channel comprises a working micro-channel and a reference micro-channel, the working micro-channel and the reference micro-channel are communicated through a bridge channel, and the working micro-channel and the reference micro-channel are respectively provided with an electrode hole for mounting a working electrode and a reference electrode; the hydrogel heterojunction comprises a P-type hydrogel and an N-type hydrogel, the P-type hydrogel and the N-type hydrogel are both arranged in the bridge channel, the P-type hydrogel is a cationic polyelectrolyte hydrogel, and the N-type hydrogel is an anionic polyelectrolyte hydrogel.

2. The polyelectrolyte hydrogel ion diode according to claim 1, wherein the microfluidic chip is selected from a PDMS-glass bonding chip, a PDMS-PDMS chip, a PDMS-hydrogel chip or a hydrogel-hydrogel chip.

3. The polyelectrolyte hydrogel ion diode according to claim 1, wherein the working microchannel is provided with electrode holes at two ends for installing working electrodes; and/or two ends of the reference micro-channel are provided with electrode holes for installing reference electrodes.

4. The polyelectrolyte hydrogel ion diode of claim 1, wherein the polyelectrolyte hydrogel ion diode further comprises a working electrode and a reference electrode.

5. The polyelectrolyte hydrogel ion diode according to claim 1, wherein the working microchannel is "U" shaped or square shaped, and/or the reference microchannel is "U" shaped.

6. The polyelectrolyte hydrogel ion diode according to claim 1, wherein the interior of the microchannel is surface-modified; preferably, the surface of the microchannel is subjected to hydrophilic modification; more preferably, the surface of the microchannel is modified by 3- (trimethoxysilyl) propyl methacrylate.

7. The polyelectrolyte hydrogel ion diode according to claim 1, wherein the cationic polyelectrolyte hydrogel is a hydrogel formed from 3-sulfopropyl acrylate potassium salt, and/or the anionic polyelectrolyte hydrogel is a hydrogel formed from diallyl dimethyl ammonium chloride.

8. The method for preparing the polyelectrolyte hydrogel ion diode as claimed in any one of claims 1 to 7, wherein the preparation method comprises the following steps:

9. The method of claim 8, wherein the interior of the microchannel is subjected to hydrophilic surface modification in step 1); preferably, in step 1), hydrophilic surface modification is performed by introducing 3- (trimethoxysilyl) propyl methacrylate into the microchannel.

10. The preparation method according to claim 8, wherein the P-type hydrogel pre-solution is a 3-sulfopropyl acrylate potassium salt solution, and/or the N-type hydrogel pre-solution is a diallyl dimethyl ammonium chloride solution.

11. The preparation method according to claim 8, wherein the solvent of the P-type hydrogel pre-solution or the N-type hydrogel pre-solution is water, and the mass concentration of the solute is 1-20%.

12. The method of claim 8, wherein the crosslinking in steps 2) and 3) is performed under UV light.

13. Use of the polyelectrolyte hydrogel ion diode according to any one of claims 1 to 7 in the preparation of a nucleic acid detection device.

14. A nucleic acid detection device comprising the polyelectrolyte hydrogel ion diode according to any one of claims 1 to 7, an electrochemical workstation in signal communication with the polyelectrolyte hydrogel ion diode and a computer, and a computer.

15. Use of the polyelectrolyte hydrogel ion diode according to any one of claims 1 to 7 or the nucleic acid detection device according to claim 14 in nucleic acid detection.

16. Use according to claim 15, wherein the nucleic acid detection is a nucleic acid concentration detection and/or the nucleic acid is selected from DNA or RNA.

17. A method for detecting nucleic acid is characterized in that the method comprises the steps of detecting electric signals when no nucleic acid to be detected exists in a working micro-channel of a polyelectrolyte hydrogel ion diode and when different nucleic acids to be detected exist in the working micro-channel, and comparing the difference of the electric signals to obtain the difference of the nucleic acids to be detected.

18. The method for detecting nucleic acid according to claim 17, comprising the steps of:

3) and (3) introducing the nucleic acid to be detected with different concentrations into the working microchannel, detecting to obtain electric signals corresponding to the nucleic acid to be detected with each concentration, and comparing the differences between the electric signals and the initial electric signals to obtain the differences of the nucleic acid to be detected.

19. The method for detecting nucleic acid according to claim 18, wherein the nucleic acid to be detected is selected from the group consisting of isolated nucleic acid and nucleic acid amplification product.

20. The method for detecting nucleic acid according to claim 17, wherein the method is a method for in situ calibration of amplification products during nucleic acid amplification, and the method for detecting nucleic acid comprises the steps of:

2) introducing raw materials required by nucleic acid amplification into a working micro-channel, introducing a buffer solution into a reference micro-channel, detecting at different time points to obtain electric signals corresponding to the nucleic acid at the time points, and comparing differences among the electric signals to obtain differences of the amplified nucleic acid at different time points.

21. The method for detecting nucleic acid according to claim 20, wherein the nucleic acid to be detected is a product of multiple displacement amplification at different times.