CN108918620B - Photoelectrochemical DNA detection method based on single-double-stranded DNA adsorption difference of cobalt phosphide nanowire - Google Patents

Photoelectrochemical DNA detection method based on single-double-stranded DNA adsorption difference of cobalt phosphide nanowire Download PDF

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CN108918620B
CN108918620B CN201810360572.9A CN201810360572A CN108918620B CN 108918620 B CN108918620 B CN 108918620B CN 201810360572 A CN201810360572 A CN 201810360572A CN 108918620 B CN108918620 B CN 108918620B
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dna
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cobalt phosphide
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CN108918620A (en
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梁汝萍
邱伟斌
邱建丁
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Nanchang University
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

The invention discloses a photoelectrochemical DNA detection method based on adsorption difference of cobalt phosphide nanowires on single-stranded DNA, and belongs to the technical field of photoelectric biosensing detection. Firstly, preparing a cobalt phosphide nanowire, wherein the cobalt phosphide nanowire adsorbs single-stranded DNA marked by fluorescein, and the fluorescein enhances photoelectric induced electron transfer so as to enhance a photocurrent signal of the cobalt phosphide nanowire; when the target DNA exists, the target DNA and the fluorescein-labeled single-stranded DNA are subjected to complementary hybridization to generate double-stranded DNA and cannot be adsorbed to the surface of the cobalt phosphide nanowire, so that the fluorescein is far away from the cobalt phosphide nanowire, the photocurrent signal of the cobalt phosphide nanowire is reduced, the photocurrent signal intensity is related to the target DNA concentration, and the sensitive detection of the DNA is realized. The method has the advantages of high sensitivity, low detection limit and simple required instrument.

Description

Photoelectrochemical DNA detection method based on single-double-stranded DNA adsorption difference of cobalt phosphide nanowire
Technical Field
The invention discloses a photoelectrochemical DNA detection method based on the adsorption difference of cobalt phosphide nanowires on single-stranded DNA, and belongs to the technical field of photoelectric biosensing detection.
Background
DNA has important connection with pathogenic genes and genetic diseases, gene expression regulation, cell proliferation and apoptosis and the occurrence and development of cancers. Biological tissues, bacteria and viruses all have unique DNA sequences, and the detection of the specific sequences plays an important role in the fields of gene analysis, disease diagnosis, food pollution, forensic identification, environmental monitoring and the like. In the past decades, the detection method of specific DNA sequence has the technical defects of long useful time, complex operation, time and labor waste and the like. The research of new DNA detection technology has great significance for promoting biological function research, disease diagnosis and related gene drug development.
The photoelectrochemical analysis method is a novel analysis method which is developed after an optical method, a photochemical method and an electrochemical method and utilizes a photoelectrochemical process to realize the purposes of energy conversion, energy utilization, analysis and detection and the like. The device utilizes illumination as an excitation signal of the whole system, and uses the generated electric signal (current or voltage) as a detection signal to realize photoelectrochemical detection. Because the excitation signal and the detection signal of the photoelectrochemistry analysis method are different energy forms, the photoelectrochemistry analysis method has the advantages of low background signal, high sensitivity, simple device, easy miniaturization, reasonable and effective utilization of solar energy and the like. At present, the application of the photoelectric analysis method is mainly to construct a photoelectrochemical biosensing mode. Photoelectrochemical biosensing is a new sensing technology developed by combining a photoelectrochemical reaction process and a biomolecule recognition process, and can be developed into a DNA detection method with high sensitivity, low background current, simple equipment and great potential.
Research shows that the cobalt phosphide can selectively adsorb single-stranded DNA but not double-stranded DNA, and the fluorescein can enhance the photoelectric induction effect when being close to the surface of the cobalt phosphide, so that the photocurrent intensity of the cobalt phosphide is enhanced. Based on the principle, the photoelectrochemical DNA detection method which is high in sensitivity, low in detection limit and simple in instrument can be researched and developed as a new design idea by utilizing the corresponding relation between the photocurrent intensity of the cobalt phosphide and the concentration of fluorescein adsorbed on the surface.
Disclosure of Invention
The invention aims to provide a photoelectrochemical DNA detection method based on the adsorption difference of cobalt phosphide nanowires on single-stranded and double-stranded DNA, which is constructed by utilizing the adsorption difference of the cobalt phosphide nanowires (CoP NWs) on the single-stranded DNA and the double-stranded DNA and the enhancement effect of fluorescein on a photoelectric current signal of the CoP NWs.
The invention realizes the aim through the following technical scheme:
the photoelectrochemical DNA detection method based on the single-double-stranded DNA adsorption difference of the cobalt phosphide nanowire comprises the following steps:
polishing glassy carbon electrode with alumina suspension of 1 μm, 0.3 μm and 0.05 μm, cleaning with nitric acid, anhydrous ethanol and ultrapure water, blowing the electrode with nitrogen, dropping CoPNWs suspension on the surface of the electrode, naturally drying, cleaning with ultrapure water, soaking the electrode in 50nM fluorescein-labeled single-stranded DNA solution, reacting at 37 ℃ for 30min, placing the electrode in hybridization solution containing target DNA with different concentrations, reacting at 37 ℃ for 30min, cleaning the surface of the electrode with 10mM phosphate buffer solution with pH of 7.4 to serve as a working electrode, taking Ag/AgCl as a reference electrode and platinum wire as a counter electrode, a phosphate buffer solution with the pH value of 0.5M and the pH value of 7.4 and containing 5 percent of triethanolamine by volume ratio is taken as an electrolyte solution to self-prepare a photoelectrochemical device, the photocurrent intensity is measured under the condition that the illumination wavelength is 420nm, and judging the concentration of the target DNA according to the linear relation between the photocurrent signal intensity of the CoPNWs and the concentration of the target DNA.
Further preferably, the DNA hybridization solution contains 50mM NaCl and 10mM MgCl220mM pH 7.4 buffer solution of tris hydrochloride.
Further preferably, the photoelectrochemical DNA detection method has a good linear relationship with the target DNA in the concentration range of 0.1-20nM, and the detection limit is 28.4 pM.
Further preferably, the CoP NWs suspension is prepared by the following process:
0.56g of CoSO4·7H2Dissolving cobalt sulfate O in 40mL of solution containing 8mL of glycerol and 6mg of polyvinylpyrrolidone, uniformly stirring, adding 0.1g of urea, stirring to obtain a uniform and transparent solution, pouring the solution into a 50mL high-pressure reaction kettle, putting the solution into a forced air drying oven, reacting for 24 hours at 170 ℃, cooling to room temperature, respectively centrifugally washing the obtained solid with ethanol and ultrapure water for 3 times, and performing vacuum drying at 60 ℃ to prepare a nanowire precursor; respectively putting the nanowire precursor and sodium hypophosphite into upstream and downstream crucibles of a tube furnace, under the protection of argon atmosphere,heating to 300 ℃ at the heating rate of 1 ℃ per minute and keeping for 2 hours, cooling to room temperature to prepare CoP NWs, weighing 5mg of cobalt phosphide nanowire and dispersing in 1mL of ultrapure water to prepare cobalt phosphide nanowire suspension.
The principle of the invention is as follows: the photoelectrochemical DNA detection method based on the adsorption difference of cobalt phosphide nano-wires on single-double-stranded DNA comprises the steps of firstly preparing CoP NWs, dripping the CoP NWs on the surface of a glassy carbon electrode to prepare a CoP NWs modified electrode, adsorbing the single-stranded DNA marked by fluorescein to the surface of the electrode by utilizing the specific adsorption effect of the CoP NWs on the single-stranded DNA marked by the fluorescein, wherein the fluorescein can enhance photoelectric induced electron transfer so as to enhance the photocurrent signal intensity of the CoP NWs; when the target DNA exists, the target DNA and the single-stranded DNA marked by the fluorescein are subjected to complementary hybridization to generate double-stranded DNA, the double-stranded DNA cannot be adsorbed to the surface of the CoP NWs, so that the photocurrent signal of the CoP NWs is reduced, the photocurrent signal intensity of the CoP NWs is related to the concentration of the target DNA, and therefore the DNA photoelectrochemical detection method based on the adsorption difference of the CoP NWs on the single-stranded DNA and the double-stranded DNA and the enhancement effect of the fluorescein on the photocurrent signal of the CoP NWs is established, and the concentration of the target DNA is judged according to the photocurrent signal intensity of the CoP NWs.
Compared with the prior art, the photoelectrochemistry DNA detection method has the advantages of being high in sensitivity, low in detection limit, simple in instrument and the like.
Drawings
FIG. 1 is a schematic diagram of a photoelectrochemical DNA detection method based on differences in adsorption of CoP NWs to single/double stranded DNA.
FIG. 2 shows (A) SEM images, (B) TEM images, (C) TEM images and (D) X-ray diffraction patterns of CoP NWs.
FIG. 3 is an AC impedance profile of various modified electrodes: (a) bare glassy carbon electrode, (b) CoP NWs, (c) CoP NWs + FAM-PHIV+T1，(d)CoP NWs+FAM-PHIV。
FIG. 4 is a graph of (A) fluorescence spectrum: (a) FAM-PHIV，(b)FAM-PHIV+T1+CoP NWs，(c)FAM-PHIV+ CoPNWs. (B) Photo-amperometry: (a) FAM-PHIV+CoP NWs，(b)FAM-PHIV+T1+CoP NWs，(c)CoP NWs。
FIG. 5 is (A) graphs of photocurrent responses detected at different concentrations of target DNA (a-i)0, 0.1, 1, 5, 10, 15, 20, 100 and 200 nM. (B) Photocurrent intensity versus target DNA concentration was plotted, with the inset being a standard curve.
FIG. 6 is a graph showing selectivity of detection of target DNA.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific embodiments, it should be noted that the present invention is not limited thereto;
example 1
Preparation of CoP NWs: 0.56g of CoSO4·7H2Dissolving O in 40mL of solution containing 8mL of glycerol and 6mg of polyvinylpyrrolidone, stirring uniformly, adding 0.1g of urea, stirring to obtain a uniform and transparent solution, pouring the solution into a 50mL high-pressure reaction kettle, placing the high-pressure reaction kettle into a forced air drying oven, reacting at 170 ℃ for 24h, cooling to room temperature, respectively centrifuging and washing the obtained solid with ethanol and ultrapure water for 3 times, and drying in vacuum at 60 ℃ to obtain Co (CO) (CO is prepared)3)0.5(OH)·0.11H2An O nanowire precursor; respectively putting the nanowire precursor and sodium hypophosphite into upstream and downstream crucibles of a tube furnace, heating to 300 ℃ at the heating rate of 1 ℃ per minute under the protection of argon atmosphere, keeping for 2 hours, cooling to room temperature to prepare CoP NWs, and dispersing 5mg of CoP NWs in 1mL of ultrapure water to prepare CoP NWs suspension.
Scanning electron microscopy and transmission electron microscopy are adopted to represent the structure and the morphology of the synthesized CoPNWs, and the result is shown in figure 2.
As can be seen from FIGS. 2A and 2B, the CoPNWs synthesized by the method of the present invention are nanowires with a diameter of 20-30nm, and the surface thereof is porous and rough. The size of the crystal lattice is 0.283nm, which is seen by high-power transmission electron microscopy and corresponds to the (011) crystal plane of cobalt phosphide (FIG. 2C). As can be seen in FIG. 2D, the diffraction peaks on the XRD pattern of the CoP NWs correspond to those of standard card JCPDS No. 29-0497.
The results show that the method successfully synthesizes the CoP NWs.
Example 2
FIG. 1 is a diagram of a CoP-based NWs pairSchematic diagram of photoelectrochemical DNA detection method of single/double-stranded DNA adsorption difference. Polishing glassy carbon electrode with alumina suspension of 1 μm, 0.3 μm and 0.05 μm in sequence, cleaning with nitric acid, anhydrous ethanol and ultrapure water in sequence, blow-drying the electrode with nitrogen gas, dropping the suspension CoPNWs prepared in example 1 on the surface of the electrode, naturally drying, cleaning with ultrapure water, and soaking the electrode in 50nM fluorescein-labeled single-stranded DNA (FAM-P)HIVThe sequence is 5 '-FAM-AGTCAG TGT GGA AAA TCT CTA GC-3', PHIVIs single-stranded DNA complementary-paired with target DNA of AIDS virus-related oligonucleotide sequence, and is reacted at 37 deg.C for 30min, and the electrodes are placed in the target DNA (T) containing different concentrations15'-GCT AGA GAT TTT CCA CAC TGA CT-3'), washing the surface of the electrode with 10mMpH 7.4 phosphate buffer solution, using the prepared electrode as a working electrode, Ag/AgCl as a reference electrode, a platinum wire as a counter electrode, 0.5M phosphate buffer solution with pH 7.4 and triethanolamine of 5% by volume as electrolyte solution, preparing a self-made photoelectrochemical device, and measuring the photocurrent intensity under the condition that the illumination wavelength is 420 nm.
The preparation process of the modified electrode is characterized by adopting an electrochemical alternating-current impedance method and a cyclic voltammetry method, and the result is shown in fig. 3, wherein a curve a is a bare glassy carbon electrode, a curve b is CoP NWs, and a curve c is CoP NWs + FAM-PHIV+T1The curve d is CoP NWs + FAM-PHIV。
As can be seen from fig. 3, the electron transfer resistance (Ret) of the bare glassy carbon electrode is small (curve a); the Ret value increased after the CoPNWs was modified to the electrode surface (curve b); FAM-P by CoP NWsHIVAdsorption of FAM-PHIVAfter assembly to the electrode surface, Ret increases further (curve d); when the target DNA is present, the target DNA is complementarily hybridized with the fluorescein-labeled single-stranded DNA to form a double-stranded DNA (FAM-P)HIV/T1) The double stranded DNA cannot adsorb on the CoP NWs surface, resulting in a decrease in Ret (curve c).
The above results indicate that the modified electrode was successfully assembled and that there was an impedance difference between single/double stranded DNA.
FIG. 4 is a drawing showingFluorescence spectrum and photocurrent diagrams under different conditions, wherein in FIG. 4A, curve a is FAM-PHIVCurve b is FAM-PHIV+T1+ CoP NWs, curve c FAM-PHIV+ CoP NWs. In FIG. 4B, curve a is FAM-PHIV+ CoP NWs, curve b FAM-PHIV+T1+ CoP NWs, curve c is CoP NWs.
As can be seen in FIG. 4A, FAM-PHIVThe strong fluorescence emission peak of fluorescein FAM is at 525nm (curve a); when FAM-P is activatedHIVFAM-P when mixed with CoP NWsHIVAdsorption to the surface of CoP NWs, resulting in a sharp drop in the fluorescence emission intensity of FAM (curve c); when there is 200nM T1Then, FAM-PHIVAnd T1Complementary hybridization to form double-stranded FAM-PHIV/T1The complex cannot adsorb to the surface of the CoP NWs, and the fluorescence emission intensity of FAM slightly decreases (curve b). As can be seen from fig. 4B, the CoP NWs has a certain photocurrent signal (curve c); when FAM-P is activatedHIVUpon adsorption to the surface of CoP NWs, the photocurrent signal of CoP NWs rapidly increased (curve a); when there is 200nM T1Then, FAM-PHIVAnd T1Complementary hybridization produced double-stranded DNA and failed to adsorb to the CoP NWs surface, with slightly enhanced photocurrent signal of CoPNWs (curve b). The results of the fluorescence spectrogram and the photoelectromogram are consistent, and the results show that the FAM-PHIVCan be adsorbed on the surface of the CoP NWs, so that the fluorescence of the FAM is reduced and the photocurrent signal of the CoP NWs is enhanced, and when the target DNA exists, the target DNA and the FAM-PHIVDouble-stranded DNA generated by complementary hybridization cannot be adsorbed to the surface of CoP NWs, so that the fluorescence of FAM is not reduced and the photocurrent signal of CoPNWs is not increased. Therefore, based on the difference of the CoP NWs in single/double-stranded DNA adsorption, a photoelectrochemical method can be constructed for detecting the target DNA.
Example 3
The CoP NWs can specifically adsorb fluorescein-labeled single-stranded DNA, the fluorescein labeled on the single-stranded DNA can enhance photoelectric-induced electron transfer and further enhance the photocurrent signal of the CoP NWs, and when the target DNA exists, the target DNA and the fluorescein-labeled single-stranded DNA are subjected to complementary hybridization to form double-stranded DNA (FAM-P)HIV/T1) Keeping fluorescein away from CoP NWsAnd reducing the photocurrent signal of CoPNWs, wherein the photocurrent signal intensity of CoP NWs is related to the concentration of the target DNA, and judging the concentration of the target DNA according to the photocurrent signal intensity of the CoP NWs. FIG. 5 is a diagram showing the photocurrent response of the method of the present invention to target DNA detection at different concentrations. As the concentration of the target DNA increases (a-i: 0, 0.1, 1, 5, 10, 15, 20, 100 and 200nM), the photocurrent response of the CoP NWs gradually decreases (FIG. 5A), the photocurrent response of the CoP NWs has a good linear relationship with the target DNA concentration in the range of 0.1-20nM (FIG. 5B and the interpolation), the detection limit of the target DNA is 28.4pM, and the sensitive detection of the target DNA is realized.
To assess the specificity of the method of the invention for detection of target DNA, fluorescein-labeled single base mismatched DNA (T) was used25'-GCT AGA GAT TGT CCA CAC TGACT-3' sequence), fluorescein labeled perfectly non-complementary paired DNA (T)3Sequence 5'-TTT TTT TTT TTT TTT TTT TTT TT-3') was used as a negative control, and the sample containing no DNA was used as a blank control, and the results are shown in FIG. 6.
As can be seen in FIG. 6, T3When the CoP NWs exists, the photocurrent intensity difference of the CoP NWs is very small and is close to that of a blank control; t is2The photocurrent difference of CoPNWs in the presence of the catalyst was slightly enhanced; however, when T is1When present, the photocurrent difference of CoP NWs was significantly enhanced. The results show that the method has good specificity for detecting the target DNA. In addition, the method can also distinguish completely complementary DNA from non-complementary DNA, even if only one base difference can be identified, and the method has good application prospect.

Claims (3)

1. The photoelectrochemical DNA detection method based on the single-double-stranded DNA adsorption difference of the cobalt phosphide nanowire is characterized by comprising the following steps of:
polishing a glassy carbon electrode by using alumina suspension of 1 mu m, 0.3 mu m and 0.05 mu m in sequence, cleaning the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and ultrapure water in sequence, blow-drying the electrode by using nitrogen, dropwise coating the cobalt phosphide nanowire suspension on the surface of the electrode, naturally drying the electrode, cleaning the electrode by using the ultrapure water, soaking the electrode into a 50nM fluorescein-labeled single-stranded DNA solution, reacting for 30min at 37 ℃, and then, carrying out the reactionPlacing an electrode in hybridization solution containing target DNA with different concentrations for reacting for 30min at 37 ℃, washing the surface of the electrode by using 10mM phosphate buffer solution with pH 7.4 as a working electrode, using Ag/AgCl as a reference electrode, a platinum wire as a counter electrode, and 0.5M phosphate buffer solution with pH 7.4 and containing 5% by volume of triethanolamine as an electrolyte solution, self-preparing a photoelectrochemical device, measuring the photocurrent intensity under the condition that the illumination wavelength is 420nm, and judging the concentration of the target DNA according to the linear relation between the photocurrent signal intensity of the cobalt phosphide nanowire and the concentration of the target DNA; wherein the hybridization solution of the DNA is 50mM NaCl and 10mM MgCl2Is 20mM Tris hydrochloride buffer solution at pH 7.4.
2. The photoelectrochemical DNA detection method based on the differential adsorption of single-stranded DNA by cobalt phosphide nanowires according to claim 1, wherein the photoelectrochemical DNA detection method has a good linear relationship with a detection limit of 28.4pM for target DNA in a concentration range of 0.1-20 nM.
3. The photoelectrochemical DNA detection method based on the differential adsorption of cobalt phosphide nanowires to single-double-stranded DNA according to claim 1, wherein the CoP NWs suspension is prepared by the following steps:
0.56g of CoSO4·7H2Dissolving O in 40mL of solution containing 8mL of glycerol and 6mg of polyvinylpyrrolidone, uniformly stirring, adding 0.1g of urea, stirring to obtain a uniform and transparent solution, pouring the solution into a 50mL high-pressure reaction kettle, putting the solution into a forced air drying oven, reacting for 24 hours at 170 ℃, cooling to room temperature, respectively centrifugally washing the obtained solid for 3 times by using ethanol and ultrapure water, and performing vacuum drying at 60 ℃ to prepare a nanowire precursor; respectively placing the nanowire precursor and sodium hypophosphite into upstream and downstream crucibles of a tubular furnace, heating to 300 ℃ at a heating rate of 1 ℃ per minute under the protection of argon atmosphere, keeping for 2 hours, cooling to room temperature to prepare cobalt phosphide nanowires, weighing 5mg of cobalt phosphide nanowires, dispersing in 1mL of ultrapure water to prepare the cobalt phosphide nanowire suspensionAnd (4) floating liquid.
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