CN109022552B - Single nucleotide polymorphism analysis method based on magnetic functionalized microfluidic chip - Google Patents

Abstract

The invention discloses a single nucleotide polymorphism analysis method based on a magnetic functionalized micro-fluidic chip, belonging to the technical field of micro-fluidic chips. Firstly Fe₃O₄Magnetic nano particle loaded graphene oxide surface synthesis magnetic nano composite material (GO @ Fe)₃O₄) GO @ Fe under the action of an external magnetic field₃O₄Efficiently and controllably fixed in a separation channel of a microfluidic chip to obtain GO @ Fe₃O₄And separating channels of the functionalized microfluidic chip. In addition, the invention also discloses GO @ Fe₃O₄The analysis of single nucleotide polymorphism is successfully realized in the separation channel of the functionalized microfluidic chip, and the analysis method has the advantages of simplicity, convenience, high efficiency and short time consumption.

Description

Single nucleotide polymorphism analysis method based on magnetic functionalized microfluidic chip

Technical Field

The invention relates to a single nucleotide polymorphism analysis method based on a magnetic functionalized micro-fluidic chip, belonging to the technical field of micro-fluidic chips.

Background

Single Nucleotide Polymorphisms (SNPs) are single base variations at genetic loci that are closely related to various genetic diseases in humans. It is the most common one of the heritable variations in humans. MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs of 18-25 nucleotides in length. MiRNA, an important post-transcriptional regulator, is involved in various developmental and physiological processes in animals and plants, and plays an important role in diseases. MiRNA can be used as an oncogene or tumor suppressor to regulate the expression of about one third of genes in humans. Mirnas affect cancer risk, and variation of related genes will affect the function of mirnas, and thus affect a variety of biological processes. Therefore, detection of SNPs in mirnas is essential. To date, there are many methods and techniques for identification of SNPs including specific polymerase chain reaction, microarray, pcr restriction fragment length polymorphism, etc., and most of these techniques can detect SNPs but are limited due to complicated procedures, expensive reagents, and lengthy operation time. Therefore, it is of great significance to develop simple, rapid, low-cost techniques for isolating and detecting SNPs.

The micro-fluidic chip electrophoresis is a miniaturized capillary electrophoresis technology, not only comprises the basic functions of capillary electrophoresis, but also has the characteristics of small size, low reagent consumption, high analysis speed, easy realization of multi-channel parallel analysis and integration with other operation units, is particularly suitable for microanalysis and functional research of biological samples, and is an ideal platform for separation and analysis. The common microfluidic chip is generally prepared from Polydimethylsiloxane (PDMS), and the PDMS microchip has the advantages of convenience in manufacturing, rapidness, easiness in sealing and the like, but has strong surface hydrophobicity, easiness in adsorbing a measured object and instable electroosmotic flow, so that the application of the PDMS microchip in separation is limited. By adopting a proper method to modify the microchip channel, nonspecific adsorption can be effectively inhibited, and the separation efficiency and the separation reproducibility are improved. Two-dimensional nanoplatelets have a strong ability to distinguish between single-stranded dna (ssdna) and double-stranded dna (dsdna). Graphene Oxide (GO) has unique characteristics such as a large specific surface area, easy mass availability, good dispersibility in water, high biocompatibility, and the inclusion of a series of oxygen-containing functional groups on the nanosheet surface making it easy to functionalize, which are of great interest. Magnetic nanocomposite of GO (GO @ Fe)₃O₄) With GO and Fe₃O₄Advantages of two components, high adsorption capacity of GO and Fe₃O₄The easy separation characteristics of the magnetic material are combined, so that GO @ Fe₃O₄On one hand, the material has good magnetic performance, can realize controllable fixation in a PDMS microchip channel, greatly saves the modification time, and also improves the repeated utilization rate of the microchip; GO @ Fe, on the other hand₃O₄Has good biocompatibility and hydrophilicity, and is beneficial to maintaining the biological activity of the immobilized biological molecules. However based on GO @ Fe₃O₄The single nucleotide polymorphism analysis method of the functionalized microfluidic chip has not been reported yet.

Disclosure of Invention

The invention aims to provide a single nucleotide polymorphism analysis method based on a magnetic functionalized microfluidic chip, which has the characteristics of simplicity, rapidness and high separation efficiency.

The invention is realized by the following technical scheme:

the single nucleotide polymorphism analysis method based on the magnetic functionalized microfluidic chip comprises the following steps:

(1) preparation of GO @ Fe₃O₄Nanocomposite, using GO @ Fe₃O₄Good magnetic property, GO @ Fe under the action of an external magnet₃O₄Fixed in a PDMS micro-fluidic chip separation channel to prepare GO @ Fe₃O₄Separating channels of the functionalized PDMS microfluidic chip;

(2) designing a single-stranded DNA marked by methylene blue, enabling the single-stranded DNA to be completely complementary with a microRNA-21 sequence and also partially complementary with a microRNA-21 single-base mismatch sequence sm-microRNA-21, hybridizing the single-stranded DNA with the microRNA-21 and the microRNA-21 single-base mismatch sequence sm-microRNA-21 respectively to form a completely complementary stable DNA/microRNA-21 heteroduplex and a DNA/sm-microRNA-21 heteroduplex with a single-base mismatch loose structure, and then mixing the DNA/microRNA-21 and the DNA/sm-microRNA-21 to prepare a mixed sample;

(3) applying a 1000V separation voltage to two ends of a separation channel of the PDMS microfluidic chip by using a high-voltage power supply as a fluid driving device, and washing the channel for 10min by using an operation buffer solution; adding a mixed sample into a sample cell of a PDMS micro-fluidic chip, applying 800V sample injection voltage between the sample cell and a waste liquid cell by a high-voltage power supply for 5s to drive the mixed sample to a cross part of a sample channel and a separation channel of the PDMS micro-fluidic chip, then switching the high-voltage power supply, applying 1000V separation voltage at two ends of the separation channel to drive the sample to flow through the separation channel of the PDMS micro-fluidic chip and GO @ Fe₃O₄After the PDMS microfluidic chip is functionalized to separate channels, DNA/microRNA-21 and DNA/sm-microRNA-21 in a mixed sample are separated, and then carbon fiber is used as a working electrode, a platinum wire is used as a counter electrode, and Ag/AgCl is used as a referenceThe three-electrode system constructed by the specific electrode detects the electrochemical signal of methylene blue marked on DNA, judges the separation degree of DNA/microRNA-21 and DNA/sm-microRNA-21 according to the retention time of the electrochemical signal of the methylene blue, and can judge the concentration of the microRNA-21 and sm-microRNA-21 according to the intensity of the electrochemical signal of the methylene blue to realize GO @ Fe @ Si₃O₄And (3) separating and detecting single nucleotide polymorphism by the functionalized PDMS microfluidic chip.

In the above method, the hybridization in the step (2) is carried out in a hybridization buffer solution consisting of a 20mM Tris-hydrochloride buffer solution having a pH of 7.4 and containing 500mM NaCl and 100mM MgCl₂(ii) a The running buffer solution described in step (3) was a phosphate buffer solution at a concentration of 20mM and pH 7.17.

In the present invention, GO @ Fe₃O₄The functional PDMS microfluidic chip separation channel is prepared by the following method:

(1) preparing graphene oxide: 0.5g of graphite powder and 0.5g of NaNO were mixed₃Add to 23mL of concentrated H₂SO₄In the middle, slowly adding 3g KMnO under ice bath condition₄Stirring thoroughly to mix well, transferring the solution into 35 deg.C water bath, stirring for 1H to form a grey brown paste, adding 40mL ultrapure water, stirring at room temperature for 30min, adding ultrapure water to dilute to 140mL, and dropwise adding 3mL 30% H₂O₂Changing the solution from dark brown to bright yellow, filtering the obtained product while the solution is hot, centrifuging the product for 2min at 8000r/min by using ultrapure water, and then cleaning the centrifuged product until supernatant is neutral to obtain graphene oxide GO;

(2) preparation of GO @ Fe₃O₄Nano composite material: dissolving 40mg of GO prepared in the step (1) into 20mL of ultrapure water, ultrasonically dispersing for 3h, and then introducing N₂Heating to 70 deg.C for 30min, adding 21mg FeCl₃·6H₂O and 72mgFeCl₂·4H₂O, stirring for 40 min; then 1.0ml NH was added dropwise₃·H₂O and stirring vigorously for 40 min; after the reaction solution was cooled to room temperature, the product was separated with a magnet and washed with ultrapure water to obtainProduct GO @ Fe₃O₄Dispersing the mixture into ultrapure water to prepare 1mg/mL GO @ Fe₃O₄A solution;

(3) preparation of GO @ Fe₃O₄Functional PDMS micro-fluidic chip separation channel: washing the PDMS microfluidic separation channel with ultrapure water for 5min, placing a permanent magnet with the length of 2cm above and below the chip respectively, and using a vacuum pump to remove GO @ Fe prepared in the step (2)₃O₄The solution is pumped into a separation channel, GO @ Fe₃O₄Fixed at the position of a magnet in a PDMS microfluidic separation channel, placing the modified microfluidic chip for 1h, continuously washing the separation channel with running buffer solution for 5min to obtain GO @ Fe₃O₄Functional PDMS micro-fluidic chip separation channel.

The principle of the invention is as follows:

GO @ Fe in the mixture of DNA/microRNA-21 and DNA/sm-microRNA-21₃O₄The adsorption effect on the DNA/sm-microRNA-21 heteroduplex with a single base mismatch loose structure is strong, and the adsorption effect on the completely complementary stable DNA/microRNA-21 heteroduplex is weak, so that the DNA/sm-microRNA-21 is adsorbed at GO @ Fe₃O₄The retention time in the separation channel of the functionalized PDMS microfluidic chip is long, and the DNA/microRNA-21 is at GO @ Fe₃O₄The retention time in the separation channel of the functionalized PDMS microfluidic chip is short, and the difference of the retention time enables DNA/microRNA-21 and DNA/sm-microRNA-21 to be at GO @ Fe₃O₄Separating the functional PDMS microfluidic chip in a separation channel, and judging the separation degree of the DNA/microRNA-21 and the DNA/sm-microRNA-21 according to the retention time of the electrochemical signal of methylene blue marked on the DNA; in addition, the intensity of the electrochemical signal of methylene blue is enhanced along with the increase of the concentrations of the DNA/microRNA-21 and the DNA/sm-microRNA-21, and the content of the microRNA-21 and the sm-microRNA-21 can be judged according to the intensity of the electrochemical signal of the methylene blue.

Compared with the prior art, the invention has the beneficial effects that:

1. based on the difference of the adsorption capacity of graphene oxide to a complete complementary strand double strand and a mismatched double strand, the molecular weight distribution of graphene oxide is GO @ Fe₃O₄The functional PDMS microfluidic chip realizes the good separation and detection of single nucleotide polymorphism in the separation channel;

2. GO @ Fe prepared by method₃O₄Nanocomposite having both GO and Fe₃O₄The method has the advantages of simple synthesis method, good biocompatibility and hydrophilicity, contribution to keeping the activity of biomolecules and realization of simple, quick and efficient separation and detection of single nucleotide polymorphism.

Drawings

FIG. 1 is (A) GO and (B) GO @ Fe₃O₄Transmission electron micrograph (D). (C) Is GO @ Fe₃O₄Photographs in the absence of (1) and the presence of (2) an applied magnetic field.

FIG. 2 shows (a) GO and (b) Fe₃O₄And (c) GO @ Fe₃O₄The Fourier transform infrared spectrogram of (1).

FIG. 3 shows (A) bare chip, (B) GO and (C) GO @ Fe₃O₄Contact angle characterization graph of functionalized PDMS chip.

FIG. 4 is a running buffer pH (4.83-9.18) versus (a) PDMS microchip and (b) GO @ Fe₃O₄Functionalized PDMS microchip EOF effect.

FIG. 5 is a diagram of (a) electrophoretic separation of P1T1+ P1M1 on a PDMS microchip; (b) P1T1, (c) P1M1 and (d) P1T1+ P1M1 at GO @ Fe₃O₄Electrophoresis separation on functionalized PDMS microchip.

FIG. 6 is GO @ Fe₃O₄Functionalized PDMS microchips were used to analyze miR-21 and sm-miR-21.

Detailed Description

The invention will be further elucidated with reference to the drawings and the embodiments without being limited thereto;

example 1

(1) Preparing graphene oxide: 0.5g of graphite powder and 0.5g of NaNO were mixed₃Add to 23mL of concentrated H₂SO₄In the middle, slowly adding 3g KMnO under ice bath condition₄Stirring thoroughly to mix well, transferring the solution into 35 deg.C water bath, stirring for 1 hr to form a grey brown paste, adding 40mL ultrapure waterWater, stirring at room temperature for 30min, diluting with ultrapure water to 140mL, and adding dropwise 3mL of 30% H₂O₂And changing the solution from dark brown to bright yellow, filtering the obtained product while the solution is hot, centrifugally cleaning the product by using ultrapure water until the supernatant is neutral, and centrifuging at 8000r/min for 2min to obtain the Graphene Oxide (GO).

(2) Preparation of GO @ Fe₃O₄Nano composite material: dissolving 40mg GO into 20mL of ultra-pure water, ultrasonically dispersing for 3h, and then introducing N₂Heating to 70 deg.C for 30min, adding 21mg FeCl₃·6H₂O and 72mg FeCl₂·4H₂O, stirring for 40 min; then 1.0ml NH was added dropwise₃·H₂O and stirring vigorously for 40 min; cooling the reaction solution to room temperature, separating the product by using a magnet, and cleaning the product by using ultrapure water to obtain a product GO @ Fe₃O₄GO @ Fe₃O₄Dispersing in ultrapure water to prepare 1mg/mL GO @ Fe₃O₄And (3) solution.

GO and GO @ Fe produced₃O₄Respectively scanning the nano composite material by an electron microscope as shown in figure 1A, B to obtain GO @ Fe₃O₄The results of the nanocomposite with and without an applied magnetic field are shown in FIG. 1C. As can be seen in fig. 1A, GO is a thin, transparent, nano-platelet structure. Carry Fe₃O₄The GO surface after the nano particles has a large number of nano particles with uniform particle size, the average particle size is 10nm, and no obvious agglomeration phenomenon is seen (figure 1B). As can be seen from FIG. 1C, GO @ Fe₃O₄The suspension of (1) was uniformly dispersed and brownish black, and when a magnet was placed near the suspension, GO @ Fe₃O₄Rapidly moves towards the direction of the magnet to form a brownish black GO @ Fe near the magnet₃O₄Spots, and the solution was clear and transparent (bottle 2). The experimental results show that GO @ Fe prepared by the method₃O₄Has good aqueous solution dispersibility and Fe supported on GO surface₃O₄The nano particles still keep good magnetic performance, so that GO @ Fe can be well realized under the action of an external magnetic field only₃O₄In PDMS microFixation in the chip channel.

Mixing GO and Fe₃O₄And GO @ Fe₃O₄The structure characterization was performed by fourier transform infrared spectroscopy, respectively, and the results are shown in fig. 2. As can be seen from FIG. 2, Fe₃O₄NPs at 571cm^-1The characteristic absorption peak of the Fe-O function (curve a) appears. GO is at 3438cm^-1Has a stretching vibration absorption peak of-OH, 1735cm^-1Has a stretching vibration absorption peak of C ═ O in carbonyl and carboxyl, 1618cm^-1Where C is C and 1229cm^-1There is a C-OH stretching vibration peak (curve b). At GO @ Fe₃O₄In infrared spectrum, GO is 1735cm^-1The stretching vibration peak of the position C ═ O is moved to 1585cm^-1Fe-O at 571cm^-1The absorption peak at (A) moved to 588cm^-1Showing that Fe₃O₄Interacts with carboxyl on GO to convert Fe₃O₄Nanoparticles were supported on the GO surface (curve c).

Example 2

Preparation of GO @ Fe₃O₄Functional PDMS micro-fluidic chip separation channel: firstly, a PDMS microfluidic separation channel is washed for 5min by ultrapure water, then permanent magnets with the length of 2cm are respectively arranged above and below a chip, and GO @ Fe prepared in example 1 is pumped by a vacuum pump₃O₄The solution is pumped into a separation channel, GO @ Fe₃O₄Fixed in a PDMS microfluidic separation channel, placing the modified microfluidic chip for 1h, continuously washing the separation channel with running buffer solution for 5min to obtain GO @ Fe₃O₄Functional PDMS micro-fluidic chip separation channel.

The prepared GO @ Fe₃O₄The functionalized PDMS microfluidic chip was subjected to contact angle characterization, and compared with a PDMS bare chip and a GO-modified PDMS chip, the result is shown in fig. 3. As can be seen from fig. 3A, the contact angle of the PDMS microchip is 106 °, and the PDMS microchip is clearly hydrophobic; the contact angle of the GO modified PDMS microchip was reduced to 11.7 ° (fig. 3B); GO @ Fe₃O₄The contact angle of the functionalized PDMS microchip surface was 29 ° (fig. 3C). GO @ Fe compared to PDMS microchips₃O₄ModifiedThe hydrophilicity of the PDMS microchip was greatly improved.

Observation of the produced GO @ Fe₃O₄The functional PDMS microfluidic chip and the PDMS bare chip showed the variation of electroosmotic flow (EOF) in the running buffer solutions with different pH, and the results are shown in fig. 4. As can be seen from fig. 4, EOF showed strong pH dependence due to the strong hydrophobicity of the surface of the PDMS microchip, and poor stability, not favorable for electrophoretic separation (fig. 4 a). At GO @ Fe₃O₄On the functionalized PDMS microchip, the EOF has little change with the increase of pH (FIG. 4b) and good reproducibility. At GO @ Fe₃O₄The relative standard deviation of EOF measured at pH 7.17 on functionalized PDMS microchips was 0.51% (n ═ 5), approximately one-seventh of that of the PDMS chips, further indicating GO @ Fe₃O₄The stability and reproducibility of the surface of the functionalized PDMS microchip are good, and the application of biological analysis is facilitated.

Example 3

GO@Fe₃O₄Analysis of single nucleotide polymorphism by functionalized PDMS microfluidic chip

A methylene blue labeled probe DNA (P1, 5 '-methyl blue-TCAACATCAGTCTGATAAGCTA-3') which is completely complementary to the mircoRNA-21(T1, 5'-UAGCUUAUCAGACUGAUGUUGA-3') is designed, the P1 and the T1 are hybridized to form a completely complementary double strand (P1T1), and the P1 and the single base mismatch strand sm-mircoRNA-21(M1, 5'-UAGCUUAUAAGACUGAUGUUGA-3') of the T1 are hybridized to form a single base mismatch double strand (P1M 1). T1, 50. mu.L of 2.5. mu.M P1 and 20. mu.L of hybridization buffer (20mM Tris-HCl buffer, pH 7.4) were mixed at different concentrations, and ultrapure water was added to give a final volume of 200. mu.L, followed by hybridization at 37 ℃ for 2.5 hours. The hybridization reaction of M1 with P1 was carried out in a similar manner as described above. Hybridization reactions of different concentrations of T1 and different concentrations of M1 with 0.5. mu. M P1 were performed as described above.

Applying 1000V voltage to two ends of a separation channel of a PDMS micro-fluidic chip by using a high-voltage power supply as a fluid driving device, flushing the channel for 10min by using an operating buffer solution, adding a mixture of P1T1 and P1M1 into a sample cell of the PDMS micro-fluidic chip, applying 800V sampling voltage between the sample cell and a waste liquid cell by using the high-voltage power supply for 5s, and driving the sample to PAnd switching a high-voltage power supply at the cross part of the sample channel and the separation channel of the DMS microfluidic chip, applying 1000V separation voltage at two ends of the separation channel, and driving the sample to flow through the separation channel of the PDMS microfluidic chip. A three-electrode system constructed by using a carbon fiber working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode records an i-T curve under a detection potential of +0.6V in an ampere detection mode, detects an electrochemical signal of methylene blue marked on DNA, judges the separation degree of P1T1 and P1M1 according to the retention time of the electrochemical signal of the methylene blue, in addition, the strength of the electrochemical signal of the methylene blue is enhanced along with the increase of the concentrations of P1T1 and P1M1, judges the contents of microRNA-21 and sm-microRNA-21 according to the strength of the electrochemical signal of the methylene blue, and realizes the purpose of GO @ Fe @ pair₃O₄And (3) separating and detecting single nucleotide polymorphism by the functionalized PDMS microfluidic chip.

Mixing the prepared mixture of P1T1, P1M1, P1T1 and P1M1 at GO @ Fe₃O₄Electrophoresis experiments were performed on functionalized PDMS microchips, and the results of P1T1 and P1M1 electrophoresis on PDMS microchips were used as controls, and are shown in FIG. 5. As can be seen from fig. 5, the P1T1 and P1M1 mixtures failed to achieve baseline separation on the PDMS chip and the peak current was low due to severe non-specific adsorption of biomolecules due to the strong hydrophobicity of the surface of the PDMS chip (curve a). From P1T1 (curve b) and P1M1 (curve c) at GO @ Fe₃O₄The peak current on the functionalized PDMS micro-fluidic chip is strong and the peak output time is different, which is caused by GO @ Fe₃O₄Improving the hydrophobicity of the PDMS microchip surface, inhibiting the nonspecific adsorption of biomolecules, and furthermore, GO @ Fe₃O₄The difference in the forces acting on P1T1 and P1M1 caused differences in their migration times. GO @ Fe₃O₄The adsorption effect on the P1M1 with a single base mismatch loose structure is strong, and the adsorption effect on the completely complementary stable P1T1 is weak, so that the P1M1 is at GO @ Fe₃O₄The retention time in the separation channel of the functionalized PDMS microfluidic chip is long, and the retention time of P1T1 in GO @ Fe₃O₄The retention time in the separation channel of the functionalized PDMS microfluidic chip is short, and the difference of the retention time enables P1T1 and P1M1 to be at GO @ Fe₃O₄And separating the functionalized PDMS microfluidic chip in the separation channel. Thus, mixtures of P1T1 and P1M1 at GO @ Fe₃O₄The separation effect on the functionalized PDMS microchip is remarkably improved, good baseline separation can be obtained within 1min, the separation degree is 2.01 (curve d), the peak currents of P1T1 and P1M1 are high, the separation efficiency of the P1T1 and the P1M1 is greatly improved, and nonspecific adsorption is effectively inhibited. The above results show that GO @ Fe prepared by the invention₃O₄The functional PDMS micro-fluidic chip separation channel can be used for analyzing single nucleotide polymorphism.

(3)GO@Fe₃O₄Functionalized PDMS microchips for the analysis of miR-21 and sm-miR-21

Detection of single nucleotide polymorphisms is important for medical diagnosis and disease prevention, and in order to verify the performance of the method of the present invention in single nucleotide polymorphism analysis, different ratios of M1/(M1+ T1) (0, 10%, 20%, 40%, 60%, 80% and 100%) at GO @ Fe were studied₃O₄The analysis effect in the separation channel of the functionalized PDMS microfluidic chip is shown in fig. 6. As the content of M1 increased, the peak current of P1T1 gradually decreased (left peak), while the peak current intensity of P1M1 gradually increased (right peak), and the relative standard deviations of six repetitions were 2.9% and 2.3%, respectively. The result shows that the method can be successfully applied to the identification and detection of the single nucleotide polymorphism, and the identification and detection can be still realized even if the content of M1 is as low as 10 percent.

Claims (6)

1. The single nucleotide polymorphism analysis method based on the magnetic functionalized microfluidic chip is characterized by comprising the following steps of:

(1) preparation of GO @ Fe₃O₄Nano composite material prepared by adding GO @ Fe under the action of external magnet₃O₄Fixed in a PDMS micro-fluidic chip separation channel to prepare GO @ Fe₃O₄Separating channels of the functionalized PDMS microfluidic chip;

(2) designing a single-stranded DNA marked by methylene blue, enabling the single-stranded DNA to be completely complementary with a microRNA-21 sequence and also partially complementary with a microRNA-21 single-base mismatch sequence sm-microRNA-21, hybridizing the single-stranded DNA with the microRNA-21 and sm-microRNA-21 respectively to form a completely complementary stable DNA/microRNA-21 heteroduplex and a single-base mismatched loose-structure DNA/sm-microRNA-21 heteroduplex, and then mixing the DNA/microRNA-21 and the DNA/sm-microRNA-21 to prepare a mixed sample;

(3) applying a 1000V separation voltage to two ends of a separation channel of the PDMS microfluidic chip by using a high-voltage power supply as a fluid driving device, and washing the channel for 10min by using an operation buffer solution; adding the mixed sample into a sample pool of a PDMS microfluidic chip, driving the sample to be injected and flow through a PDMS microfluidic chip separation channel, and flow through GO @ Fe₃O₄After a PDMS micro-fluidic chip is functionalized to separate a channel, DNA/microRNA-21 and DNA/sm-microRNA-21 in a mixed sample are separated, a three-electrode system is combined to detect an electrochemical signal of methylene blue marked on the DNA, the separation degree of the DNA/microRNA-21 and the DNA/sm-microRNA-21 is judged according to the retention time of the electrochemical signal of the methylene blue, in addition, the concentration of the microRNA-21 and the concentration of the sm-microRNA-21 can be judged according to the intensity of the electrochemical signal of the methylene blue, and GO @ Fe is realized₃O₄And (3) separating and detecting single nucleotide polymorphism by the functionalized PDMS microfluidic chip.

2. The method for analyzing single nucleotide polymorphism according to claim 1, wherein the hybridization in step (2) is performed in a hybridization buffer solution consisting of 20mM Tris-HCl buffer solution at pH 7.4 and containing 500mM NaCl and 100mM MgCl₂。

3. The method for analyzing single nucleotide polymorphism based on magnetically functionalized microfluidic chip according to claim 1, wherein the running buffer solution in step (3) is phosphate buffer solution with concentration of 20mM and pH of 7.17.

4. The method for analyzing single nucleotide polymorphism based on magnetically functionalized microfluidic chips of claim 1, wherein the step (3) of injecting and flowing the driving sample through the separation channel of the PDMS microfluidic chip is performed by applying a sample injection voltage of 800V for 5s between the sample cell and the waste liquid cell through a high voltage power supply to drive the sample to the cross-shaped intersection between the sample channel and the separation channel of the PDMS microfluidic chip, and then switching the high voltage power supply to apply a separation voltage of 1000V at two ends of the separation channel to drive the sample to flow through the separation channel of the PDMS microfluidic chip.

5. The method for analyzing the single nucleotide polymorphism based on the magnetically functionalized microfluidic chip according to claim 1, wherein the three-electrode system in the step (3) is a three-electrode system constructed by using carbon fibers as a working electrode, platinum wires as a counter electrode and Ag/AgCl as a reference electrode.

6. The method for analyzing single nucleotide polymorphism based on magnetically functionalized microfluidic chip according to claim 1, wherein GO @ Fe in step (1)₃O₄The preparation method of the functional PDMS microfluidic chip separation channel comprises the following steps:

(1) preparing graphene oxide: 0.5g of graphite powder and 0.5g of NaNO were mixed₃Add to 23mL of concentrated H₂SO₄In the middle, slowly adding 3g KMnO under ice bath condition₄Stirring thoroughly to mix well, transferring the solution into 35 deg.C water bath, stirring for 1H to form a grey brown paste, adding 40mL ultrapure water, stirring at room temperature for 30min, adding ultrapure water to dilute to 140mL, and dropwise adding 3mL 30% H₂O₂Changing the solution from dark brown to bright yellow, filtering the obtained product while the solution is hot, centrifuging the product for 2min at 8000r/min by using ultrapure water, and then cleaning the centrifuged product until supernatant is neutral to obtain graphene oxide GO;

(2) preparation of GO @ Fe₃O₄Nano composite material: dissolving 40mg of GO prepared in the step (1) into 20mL of ultrapure water, ultrasonically dispersing for 3h, and then introducing N₂Heating to 70 deg.C for 30min, adding 21mg FeCl₃·6H₂O and 72mgFeCl₂·4H₂O, stirring for 40 min; then 1.0ml NH was added dropwise₃·H₂O and stirring vigorously for 40 min; cooling the reaction solution to room temperature, separating the product by using a magnet, cleaning the product by using ultrapure water, and obtaining a product GO @ Fe₃O₄Dispersing the mixture into ultrapure water to prepare 1mg/mL GO @ Fe₃O₄A solution;

(3) preparation of GO @ Fe₃O₄Functional PDMS micro-fluidic chip separation channel: washing the PDMS microfluidic separation channel with ultrapure water for 5min, placing a permanent magnet with the length of 2cm above and below the chip respectively, and using a vacuum pump to remove GO @ Fe prepared in the step (2)₃O₄The solution is pumped into a separation channel, GO @ Fe₃O₄Fixed at the position of a magnet in a PDMS microfluidic separation channel, placing the modified microfluidic chip for 1h, continuously washing the separation channel with running buffer solution for 5min to obtain GO @ Fe₃O₄Functional PDMS micro-fluidic chip separation channel.

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