CN110988077A - Triblock DNA probe, nucleic acid detection method and application - Google Patents

Abstract

The invention provides a triblock DNA probe, a nucleic acid detection method and application, wherein the triblock probe comprises a PolyA sequence and two probe sequences connected with the PolyA sequence; the probe sequence is complementary paired with a partial sequence of the target DNA. The invention adopts polyA to connect two segments of DNA probes to construct a triblock probe, and based on the adsorption effect of a polyA sequence and a gold electrode, the DNA probes on two sides are assembled on the surface of the gold electrode, the relative positions of the DNA probes on two sides are constant, the proportions of the DNA probes on two sides are equal, the same target molecule can be captured, different target molecules can also be captured, and the effect of detecting one or two target molecules is realized; the constructed electrochemical sensor has the advantages of obviously improved sensitivity, specificity, stability and repeatability, low detection cost, diversified functions and wide application prospect in the field of biomolecule detection.

Description

Triblock DNA probe, nucleic acid detection method and application

Technical Field

The invention belongs to the technical field of biological analysis, relates to a triblock DNA probe, a nucleic acid detection method and application, and particularly relates to a triblock DNA probe based on poly-adenine, a nucleic acid detection method and application.

Background

The biosensor as an effective analysis tool is widely applied to the fields of disease diagnosis, food safety, environmental monitoring, basic research of life science and the like, and has the advantages of real-time performance, short time consumption, low cost, high sensitivity and the like. Among them, the nucleic acid probe, which is the most important part of the biosensor, has been extensively and deeply studied, and the linearized DNA molecule has the characteristics of good physicochemical properties, easy modification, targeting to various biomolecules, etc., and thus becomes the most preferable material for constructing the probe.

In the field of biological analysis, the development of multi-block DNA probes is of great significance for realizing the detection of multiple targets and improving the sensitivity and specificity. At present, the method for constructing a multi-block DNA probe mainly comprises connecting a plurality of DNA fragments together by using organic molecules or nanomaterials, for example, Lee and the like connect a plurality of DNA fragments together by using organic molecules, so as to construct a DNA-organic molecule-DNA (dod) triblock polymer (Lee and the like, j.am.chem.soc. (2008)130, 12854-; immos et al constructed a DNA-PEG-DNA using PEG as a linker (Immos et al, J.Am.chem.Soc. (2004), 126, 10814-10815); hu et al loaded DNA probes onto the surface of gold nanoparticles (Hu et al, anal. chem. (2008), 80, 9124-. However, these methods have complicated processes and high costs, and the synthesized probes have poor biocompatibility, which greatly limits the application of multi-block DNA probes.

Therefore, it is necessary to provide a new multi-block probe, which not only has the function of targeting various molecules, but also has simple synthesis process, low cost and good biocompatibility, and has important significance in the field of bioanalysis.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides a triblock DNA probe, a nucleic acid detection method and application, wherein the probe adopts poly-adenine (polyA) to connect two DNA probes, and the triblock DNA probe is modified on the surface of a gold electrode based on the adsorption action between adenine and the gold electrode, so that the multi-target electrochemical detection is realized.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a triblock probe comprising a PolyA sequence and two probe sequences linked thereto;

the probe sequence is complementary paired with a partial sequence of the target DNA.

In the invention, the triblock probe adopts polyA to connect two DNA probes, and based on the adsorption effect of a polyA sequence and a gold electrode, the DNA probes on two sides are assembled on the surface of the gold electrode, and can capture the same target molecule and different target molecules, thereby realizing the effect of detecting one or two target molecules.

Preferably, a spacer sequence is further included between the polyA sequence and the probe sequence.

In the invention, the spacing sequence is arranged between the polyA sequence and the probe sequence, so that the triblock probe adsorbed on the gold electrode is not influenced by the gold electrode when being combined with a target molecule, and the detection sensitivity is favorably improved.

Preferably, the triblock probe comprises a probe one sequence, a spacer sequence, a PolyA sequence, a spacer sequence, and a probe two sequence.

Preferably, the PolyA sequence comprises 10-40 bases A.

In the invention, the length of the polyA sequence can be adjusted according to the actual situation so as to obtain the optimal signal-to-noise ratio.

In a second aspect, the present invention provides an electrochemical detection system, where the detection system includes a gold electrode, a capture probe is modified on a surface of the gold electrode, and the capture probe is the triblock probe according to the first aspect.

In the invention, the gold electrode is modified by taking the triblock probe as the capture probe, and the DNA probe for specifically recognizing the target molecules is assembled on the surface of the gold electrode based on the adsorption action of the polyA sequence and the gold electrode, so that the aim of detecting the same or different target molecules by using an electrochemical detection method is fulfilled.

Preferably, the detection system further comprises a signal probe, and the 3' end of the signal probe is labeled with biotin.

In a third aspect, the present invention provides an electrochemical sensor comprising a triblock probe according to the first aspect and/or a detection system according to the second aspect.

In a fourth aspect, the present invention provides an electrochemical detection kit comprising a triblock probe according to the first aspect and/or a detection system according to the second aspect.

Preferably, the kit further comprises avidin labeled HRP, TMB or H₂O₂Any one or a combination of at least two of them.

In the invention, HPR is marked on a sandwich structure of a capture probe-target DNA-signal probe through the strong affinity of biotin-avidin, and TMB or H is added₂O₂And detecting the DNA through the generated electrochemical signal.

Preferably, the triblock probe comprises a nucleic acid sequence shown as SEQ ID NO 1-10.

Preferably, the signaling probe comprises a nucleic acid sequence shown as SEQ ID NO. 11-13.

In a fifth aspect, the present invention provides an electrochemical detection method for detecting nucleic acid using the triblock probe of the first aspect, the detection system of the second aspect, the sensor of the third aspect, or the kit of the fourth aspect.

Preferably, the detection method comprises the following steps:

(1) after incubating the target DNA and the biotin-labeled signal probe together, carrying out hybridization reaction with a gold electrode modified with a capture probe to form a sandwich structure on the gold electrode;

(2) adding avidin labeled HRP into the hybridization product obtained in the step (1), and incubating;

(3) addition of TMB and H₂O₂And carrying out electrochemical detection.

Preferably, the temperature of the co-incubation in step (1) is 70-85 ℃, for example, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃ or 85 ℃.

Preferably, the hybridization reaction in step (1) is at a temperature of 20-40 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃, preferably 35-37 ℃.

Preferably, the capture probe in the step (1) comprises a nucleic acid sequence shown as SEQ ID NO 1-10;

preferably, the signaling probe in the step (1) comprises a nucleic acid sequence shown as SEQ ID NO: 11-13.

In a sixth aspect, the present invention provides a use of the tri-block probe according to the first aspect, the detection system according to the second aspect, the sensor according to the third aspect, and the kit according to the fourth aspect for preparing a biomolecule detection reagent.

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

(1) the invention adopts polyA to connect two segments of DNA probes to construct a triblock probe, and based on the adsorption effect of a polyA sequence and a gold electrode, the DNA probes on two sides are assembled on the surface of the gold electrode, the relative positions of the DNA probes on two sides are constant, the proportions of the DNA probes on two sides are equal, the same target molecule can be captured, different target molecules can also be captured, and the effect of detecting one or two target molecules is realized;

(2) the electrochemical sensor constructed by the invention has the advantages of remarkably improved stability and repeatability, detection limit of 10fM, detection range of 10 fM-1 nM, good specificity, capability of distinguishing SNP single base mismatch and suitability for biological sample detection;

(3) the biosensor has low detection cost and diversified functions, and has wide application prospect in the field of biomolecule detection.

Drawings

FIG. 1(A) is a schematic diagram of a triblock DNA probe, and FIG. 1(B) is a schematic diagram of a 16-channel electrochemical chip electrode;

FIG. 2 is a schematic diagram of electrochemical detection of bacterial genomic DNA;

FIG. 3(A) is a graph showing data of cyclic voltammetry for 1nM target DNA and blank set, and FIG. 3(B) is a graph showing data of chronoamperometry for 1nM target DNA and blank set;

FIG. 4 is a layer-by-layer assembly of the surface of an electrode characterized by an AC impedance method;

FIG. 5(A) is a graph comparing data for PAP probes with different length polyA sequences detecting 1nM target DNA and blank set, and FIG. 5(B) is a graph comparing data for PAP probes with different types detecting 1nM target DNA and blank set;

FIG. 6(A) is a graph of current time for detecting different concentrations of target DNA, wherein the curves represent the target DNA concentrations of 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, 0fM in the order from top to bottom, FIG. 6(B) is a graph of current intensity versus log of target DNA concentration, FIG. 6(C) is the detection result using PAP with a 5 'end mismatch, FIG. 6(D) is the detection result using PAP with 3' and 5 'end mismatches, and FIG. 6(E) is the detection result using PAP with a 3' end mismatch;

FIG. 7(A) is stability data based on the PAP probe detection method, and FIG. 7(B) is reproducibility data based on the PAP probe detection method;

FIG. 8(A) is the polyacrylamide gel electrophoresis imaging after PCR amplification of different concentrations of template, wherein lane M is DNA molecular weight, lane 1 is 200pg/μ L, lane 2 is 20pg/μ L, lane 3 is 2pg/μ L, lane 4 is 0.2pg/μ L, lane 5 is 0pg/μ L, and FIG. 8(B) is the electrochemical detection data after PCR amplification of different concentrations of template;

FIG. 9 is a diagram of the detection of bacterial genome data based on the PAP detection method.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Materials:

DNA oligonucleotides were synthesized and purified by Invitrogen tracing Co., Ltd. (Shanghai) with sequence information as shown in Table 1, bacterial genomic DNA extraction kit, 6-Mercaptohexanol (MCH) available from Sigma-Aldrich (Shanghai), color developing solution 3,3',5,5' -Tetramethylbenzidine (TMB) available from Neogen Corporation (Shanghai), avidin-labeled horseradish peroxidase (avidin-HRP) available from eBioscience Inc. (San Diego, CA), and diluent available from Fitzer industries International (Acton, MA), Escherichia coli (E.coli) Dh5 α, dNTP mix, Ex Taq DNA polymerase, 10 × Taq buffer available from Takara Biotechnology Co., Ltd. (Large Union), enterococcus faecalis (E.faecal) provided by the Industrial Center for culture of microorganisms (culture of culture)₃[Fe(CN)₆](Potassium ferricyanide) and K₄Fe(CN)₆·3H₂O (potassium ferrocyanide) purchased from China pharmaceutical group; the chemical reagents were analytically pure and the solutions were prepared using Milli-Q water (18M. omega. cm resistance).

TABLE 1 sequence information

Example 1 PAP Structure and PAP-based electrode preparation

The structural schematic diagram of a poly-adenine (polyA) -based triblock DNA probe (PAP) is shown in FIG. 1(A), and the structure schematic diagram sequentially comprises a probe I sequence, a spacer sequence, a PolyA sequence, a spacer sequence and a probe II sequence from a 5 'end to a 3' end, wherein the probe I sequence and the probe II sequence are combined with a target molecule, and the PolyA sequence, the probe I sequence and the probe II sequence are separated by the spacer sequence;

the electrode is a 16-channel electrochemical chip electrode as shown in fig. 1(B), each channel comprises a round gold working electrode, a square gold reference electrode and a ring-shaped gold counter electrode;

in this example, PAP was immobilized on the surface of a gold electrode by using the adsorption of polyA on the surface of gold, specifically, 0.1. mu.M triblock polyA probe was added to the gold electrode, incubated overnight at room temperature, and then blocked with 1mM MCH at room temperature for 30min, and then washed and placed at 4 ℃ for further use.

Example 2 electrochemical detection

The electrochemical detection principle is shown in figure 2, PAP probe modified on the surface of gold electrode is used as capture probe to generate specific complementary hybridization with target DNA, after biotin-labeled signal probe is combined with target DNA, HPR is labeled on the sandwich structure of capture probe-target DNA-signal probe by using the strong affinity of biotin-avidin, and TMB or H is added₂O₂And detecting the DNA through the generated electrochemical signal.

The method comprises the following steps: the prehybridization solution (pH 7.4, 10mM Tris-HCl, 1mM EDTA, 1M NaCl) containing 100nM of the biotinylated signaling probe and an amount (1nM) of the target DNA was heated at 80 ℃ for 5min, and cooled to room temperature for 20 min; the solution was then added to the PAP/MCH electrode prepared in example 1 and incubated at 37 ℃ for 2 h; after hybridization reaction, the electrode is placed in 5U/mLavidin-HRP, and incubated for 15min at room temperature; after washing the electrode with a washing solution (pH 7.4, 10mM Tris-HCl, 1mM EDTA, 1M NaCl), electrochemical detection was performed;

detecting by Cyclic Voltammetry (CV) and chronoamperometry by adopting a three-electrode system (Au working electrode, Ag/AgCl reference electrode and Pt counter electrode) of an electrochemical workstation, wherein the scanning rate of CV is 30mV/s, the voltage is-200 mV, when HRP redox reaction reaches a stable state for 60s, recording the electrocatalytic reduction current, and carrying out alternating-current impedanceDetection by electrochemimescence spectroscopy (EIS); the solution system of EIS contains 5mM Fe (CN)₆ ^3-/4-And 0.1M KCl in 10mM PBS (pH 7.4); the amplitude of the AC voltage is 5mV, and the voltage frequency is 0.01 Hz-100 kHz.

FIG. 3(A) shows two typical redox peak pairs (redox peaks pairs), illustrating the electron transfer between TMB and gold electrodes; after the electrode is hybridized with 1nM target DNA, the reduction peak at 50mV is obviously increased, which indicates HRP catalytic electrochemical reaction; fig. 3(B) shows a current signal in a steady state.

The EIS is adopted to carry out layer-by-layer assembly characterization on the structure of the electrochemical sensor, as shown in FIG. 4, A is a bare gold electrode and basically has no charge transfer resistance (charge transfer resistance), which indicates that the surface of the electrode is very clean; b is a gold electrode modified with PAP, and it can be seen that EIS signal is significantly improved when PAP is fixed on the electrode due to the increase of negative charge density on the surface of the electrode, which in turn leads to [ Fe (CN) ]₆]^3-/4-The electrostatic repulsion of the ions increases; c, after MCH is added, EIS signals are further improved; d is after adding target DNA and signal probe, the electrode surface negative charge density further increases, EIS also improves correspondingly.

Example 3 Condition optimization

(1) Optimization of polyA length

Construction of PAP probes PA containing polyA (10, 20, 30 and 40nt) of varying lengths based on the target DNA (SEQ ID NO:14)₁₀P-1、PA₂₀P-1、PA₃₀P-1 and PA₄₀P-1, the signal probe is SP-1, and the target DNA with the concentration of 1nM is detected. As shown in fig. 5(a), when the length of polyA is increased from 10nt to 30nt, the current signal is increased and the background signal is decreased, and when the length of polyA is 30nt, there is the highest signal-to-noise ratio (S/N) 22.1; when the length of polyA is 40nt, the current signal is greatly reduced, probably because polyA40 probe occupies the surface area of gold electrode, affecting the amount of capture probe assembly.

(2) Optimization of PAP design

The inventors designed different PAP probe PA based on the sequence of the target DNA₃₀P-1、PA₃₀P-2 and PA₃₀P-3 when the PAP probe is PA₃₀P-1, the signal probe SP-1 is bound to the 5' end of the target DNA, and when the PAP probe is PA₃₀P-2, the signal probe SP-2 is combined in the middle part of the target DNA; as shown in FIG. 5(B), when the PAP probe is PA₃₀The signal value was significantly reduced for P-2, probably because the DNA hybridization sites were not easily accessible, when PAP was PA₃₀In P-3, the biotin label of the signal probe SP-3 is close to the electrode, the combination of avidin-HRP is influenced by steric hindrance, and PA₃₀P-1 has the highest signal-to-noise ratio and is PA₃₀1.8 times of P-2, is PA₃₀6.3 times of P-3.

Example 4 sensitivity and specificity experiments

After the target DNA is diluted in a gradient manner, the sensitivity detection is carried out. The I-t analysis curve is shown in FIG. 6(A), and as the concentration of the target DNA increases, the signal value also increases; the signal values at 60s were used to construct a standard curve as in fig. 6(B), and the non-linear fit formula was y 270.7-196.7exp (-x/3.36), y being the signal value, x being the DNA concentration, and the limit of detection (LOD) reached 10 fM.

PAP probes containing different numbers of mismatched bases at different positions were synthesized for target DNA detection. As shown in FIG. 6(C), when there is a mismatch of 1, 3 or 5 bases at the 5 'end of PAP (5 MS-1, 5MS-3 and 5MS-5 for the probes used at the 5' end, respectively), the signal values decreased by 27.2%, 51.5% and 69.5%, respectively; as shown in FIG. 6(E), when the mismatched base is located at the 3 'end of PAP (the probes used at the 3' end are 3MS-1, 3MS-3 and 3MS-5, respectively), similar results are obtained; when the mismatched bases are located at the 5 'end and the 3' end, the signal value is greatly reduced, indicating that the PAP probe of the invention has better specificity.

Example 5 stability and reproducibility experiments

And (3) respectively placing the gold electrode modified with PAP in an environment of 4-8 ℃ for 2 days, 3 days and 30 days for electrochemical detection. As shown in FIG. 7(A), after 30 days, the performance is only reduced by 5%, which shows that the sensor constructed by the invention has good stability.

In the first detection, 1nM target DNA is detected by using a gold electrode modified with PAP, after urea treatment, double-stranded DNA is completely melted, a signal probe is eluted, and the background value is very low, as shown in FIG. 7(B), the sensor constructed by the invention can be reused for at least 5 times.

Example 6 detection of bacterial genomes

Inoculating Escherichia coli (E.coli) Dh5 α and enterococcus faecalis (E.faecalis) in LB culture medium, culturing at 37 deg.C overnight, centrifuging the bacterial liquid at 6000rpm for 5min the next day, and extracting the genome DNA of the obtained thallus with bacterial genome DNA extraction kit;

PCR amplification is carried out by taking the extracted bacterial genome DNA as a template and adopting asymmetric primers 1(SEQ ID NO:16) and 2(SEQ ID NO:17) according to the proportion of 10:1 under the condition of 95 ℃ for 2 min; 30s at 95 ℃, 30s at 61 ℃, 30s at 72 ℃ and 50 cycles; 10min at 72 ℃; storing at 4 deg.C;

the amplification product was diluted and subjected to gel electrophoresis analysis, and the result is shown in FIG. 8 (A); and the sensor constructed by the invention is used for detection, and the result is shown in figure 8(B), the sensor of the invention can detect unpurified PCR products from a 10 mu L system, and the sensitivity is at least 3 orders of magnitude higher than that of gel electrophoresis.

In this example, the bacterial genome was directly detected by using a sensor, and the results are shown in FIG. 9, where the 10.0pM Escherichia coli (E.coli) genome was used as the target, and the signal value was-283 nA; the genome of enterococcus faecalis (E.faecalis) is used as a negative control, and the obtained signal value is equivalent to that of a blank control, which indicates that the sensor provided by the invention can be used for detecting biological samples.

In conclusion, the invention adopts polyA to connect two DNA probes to construct a triblock probe, and based on the adsorption effect of a polyA sequence and a gold electrode, the DNA probes on two sides are assembled on the surface of the gold electrode, the relative positions of the DNA probes on two sides are constant, the proportions of the DNA probes on two sides are equal, the same target molecule can be captured, different target molecules can also be captured, and the effect of detecting one or two target molecules is realized; the electrochemical sensor constructed by the method has the advantages of obviously improved sensitivity, specificity, stability and repeatability, and wide application prospect.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> research institute of metrological testing technology in Shanghai city

<120> triblock DNA probe, nucleic acid detection method and application

<130>20191125

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Claims (10)

1. A triblock probe is characterized by comprising a PolyA sequence and two probe sequences connected with the PolyA sequence;

the probe sequence is complementary paired with a partial sequence of the target DNA.

2. The triblock probe of claim 1, further comprising a spacer sequence between the polyA sequence and the probe sequence;

preferably, the triblock probe comprises a probe one sequence, a spacer sequence, a PolyA sequence, a spacer sequence, and a probe two sequence;

preferably, the PolyA sequence comprises 10-40 bases A.

3. An electrochemical detection system, characterized in that the detection system comprises a gold electrode, the surface of the gold electrode is modified with a capture probe, and the capture probe is the triblock probe of claim 1 or 2;

preferably, the detection system further comprises a signal probe, and the 3' end of the signal probe is labeled with biotin.

4. An electrochemical sensor comprising a triblock probe according to claim 1 or 2 and/or a detection system according to claim 3.

5. An electrochemical detection kit comprising a triblock probe according to claim 1 or 2 and/or a detection system according to claim 3;

preferably, the kit further comprises avidin labeled HRP, TMB or H₂O₂Any one or a combination of at least two of them.

6. The kit according to claim 5, wherein the triblock probe comprises a nucleic acid sequence as shown in SEQ ID NO 1-10;

preferably, the signaling probe comprises a nucleic acid sequence shown as SEQ ID NO. 11-13.

7. An electrochemical detection method, wherein the detection method employs the triblock probe of claim 1 or 2, the detection system of claim 3, the sensor of claim 4, or the kit of claim 5 or 6 for nucleic acid detection.

8. The detection method according to claim 7, characterized in that it comprises the steps of:

(1) after incubating the target DNA and the biotin-labeled signal probe together, carrying out hybridization reaction with a gold electrode modified with a capture probe to form a sandwich structure on the gold electrode;

(2) adding avidin labeled HRP into the hybridization product obtained in the step (1), and incubating;

(3) addition of TMB and H₂O₂And carrying out electrochemical detection.

9. The detection method according to claim 7 or 8, wherein the temperature of the co-incubation in the step (1) is 70-85 ℃;

preferably, the temperature of the hybridization reaction in the step (1) is 20-40 ℃, and preferably 35-37 ℃;

preferably, the capture probe in the step (1) comprises a nucleic acid sequence shown as SEQ ID NO 1-10;

preferably, the signaling probe in the step (1) comprises a nucleic acid sequence shown as SEQ ID NO: 11-13.

10. Use of a triblock probe according to claim 1 or 2, a detection system according to claim 3, a sensor according to claim 4, a kit according to claim 5 or 6 for the preparation of a reagent for the detection of biomolecules.

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