CN109738503B - Positive feedback amplification electrochemical sensor based on exonuclease III - Google Patents

Abstract

The invention discloses a positive feedback amplification electrochemical sensor based on exonuclease III, which comprises a gold electrode and Hybrid DNA fixed on the gold electrode, wherein the Hybrid DNA sequentially comprises a sulfur-hydrogen bond, a double-stranded DNA structure and a single-stranded DNA structure from a 5 'end to a 3' end, the double-stranded DNA structure is prepared by hybridizing C-DNA, G4-DNA and A-DNA, and the alkali sequence of the single-stranded DNA structure is complementary with the base sequence of the T-DNA; the Hybrid DNA was immobilized on the gold electrode via S-Au bonds. The positive feedback amplification strategy combining homogeneous reaction and heterogeneous reaction is realized through the skillfully designed Hairpin DNA and Hybrid DNA and the target object cyclic amplification based on Exo III. The strategy shows ultra-high sensitivity, detection limit is as low as 0.12aM T-DNA, and high selectivity to T-DNA and other base mismatch nucleic acids.

Description

Positive feedback amplification electrochemical sensor based on exonuclease III

Technical Field

The invention belongs to the technical field of electrochemical sensors, and particularly relates to a positive feedback amplification electrochemical sensor based on exonuclease III, and a preparation method and application thereof.

Background

Acquired immune syndrome, also known as AIDS, is an acquired infectious disease. In 1983, Human Immunodeficiency Virus (HIV) was first discovered in the united states, and it was accompanied by acquired immunodeficiency syndrome (aids), one of the most fatal diseases in the world. The genes of the HIV group are two identical RNA strands that, upon reverse transcription, can be transcribed into DNA for further expression of the genes in the host cell. It attacks the host immune system and causes destruction of T4 lymphocytes, leading to a breakdown of the human immune system, rendering the body unable to defend against many diseases and leading to death. Therefore, the detection of HIV biomarkers or genes is very important for the early diagnosis, clinical treatment and prevention of virus transmission of AIDS. At present, the most common method for the early diagnosis of AIDS is to detect antibodies in the HIV host. However, it takes several weeks to months from infection to the production of antibodies to HIV, a window period known as HIV. During this time, aids cannot be detected because there is not enough antibody production to complete. To achieve early and rapid diagnosis of human AIDS, HIV-associated DNA in host cells can be detected, thereby overcoming the window phase problem.

In the early stages of the disease, the levels of biomarkers are usually low and ultrasensitive detection of trace nucleic acid biomarkers is crucial for early diagnosis. To meet the requirements for highly sensitive detection, researchers have implemented signal amplification using various DNA-based biological reactions, such as Polymerase Chain Reaction (PCR), enzyme assisted target cycling (EATR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), Hybrid Chain Reaction (HCR), and Catalytic Hairpin Amplification (CHA). Among them, EATR has attracted extensive research interest because nucleases, such as nickases and exonucleases, can achieve rapid and efficient amplification at mild temperatures. However, nicking enzymes have special recognition sites, which place higher demands on DNA sequences, resulting in increased design complexity. Moreover, it usually requires recycling to be achieved with the aid of a polymerase, which increases costs.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention aims to provide a positive feedback amplification electrochemical sensor based on exonuclease III and a preparation method and application thereof. The positive feedback amplification strategy combining homogeneous reaction and heterogeneous reaction is realized through the skillfully designed Hairpin DNA and Hybrid DNA and the target object cyclic amplification based on Exo III. The strategy shows ultra-high sensitivity, detection limit is as low as 0.12aM T-DNA, and high selectivity to T-DNA and other base mismatch nucleic acids.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a positive feedback amplification electrochemical sensor based on exonuclease III comprises a gold electrode and Hybrid DNA fixed on the gold electrode, wherein the Hybrid DNA sequentially comprises a sulfur-hydrogen bond, a double-stranded DNA structure and a single-stranded DNA structure from a 5 'end to a 3' end, the double-stranded DNA structure is prepared by hybridizing C-DNA, G4-DNA and A-DNA, and the base sequence of the single-stranded DNA structure is complementary with the base sequence of the T-DNA; the Hybrid DNA was immobilized on the gold electrode via S-Au bonds.

The complementary strand DNA (C-DNA) was designed to consist of three fragments, complementary to the quadruplex-forming oligonucleotide (G4-DNA), the helper strand (A-DNA) and the T-DNA bases, respectively. First, a Hybrid DNA probe (Hybrid DNA) was prepared by hybridizing C-DNA with G4-DNA and A-DNA. Then, the Hybrid DNA was immobilized on the gold electrode by forming a strong S-Au bond between the 3' end of G4-DNA and the electrode surface. The electrochemical sensor was completed after blocking with 6-mercapto-1-hexanol (MCH). When T-DNA is present, Hybrid DNA hybridizes with it and forms a Hybrid DNA/T-DNA double-stranded structure having blunt 3' -ends, and T-DNA, A-DNA and G4-DNA are subsequently released from the Hybrid DNA/T-DNA double-stranded structure by Exo III hydrolysis. The released T-DNA is recognized by other Hybrid DNA, initiating the next cleavage process, constituting cycle I in the schematic. On the other hand, A-DNA released from cycle I hybridizes with hairpin DNA (hairpin DNA) and cycle II is triggered with the aid of Exo III, thereby producing T' -DNA. Since the T' -DNA sequence is designed to be similar to T-DNA, it can also be recognized by Hybrid DNA and trigger cycle III with the aid of Exo III. And the A-DNA released from the cycle III enters a cycle II to induce and generate more T' -DNA, so that positive feedback signal amplification is realized. Finally, G4-DNA released from cycles I and III is in K⁺And the presence of hemin further forms a G-quadruplex/hemin complex, thereby generating an electrochemical signal. In contrast, when T-DNA is not present, it cannot be hydrolyzed by Exo III digestion due to the protruding 3' end in the specially designed Hybrid DNA. Meanwhile, G4-DNA is prevented from forming an active conformation to which hemin is bound because G4-DNA hybridizes with C-DNA. Therefore, no electrochemical signal is generated.

Unlike nickases, exonuclease III (Exo III) has no stringent requirements for DNA sequence. For double-stranded DNA having a flush or a depressed 3' end, it can catalyze stepwise hydrolysis of the 3' end of the double-stranded DNA and exhibits limited activity on double-stranded DNA or single-stranded DNA having a protruding 3' end.

The Exo III-assisted cascade amplification strategy is characterized by cyclic amplification of T-DNA, with the production of large amounts of target analogs (T' -DNA). Since these newly generated T' -DNAs have a base sequence similar to that of T-DNA, they can trigger new cycles and produce significant signal amplification.

Preferably, the C-DNA has a base sequence of 5'-ATCCCGCCCAACCCAGTCCAAGTGTCGTTCGTCAAAATCTCTAGCAGT-3';

the base sequence of the G4-DNA is as follows: 5'-GACTGGGTTGGGCGGGATGGGTTTTTT- (CH2) 6-SH-3';

the base sequence of the A-DNA is as follows: 5'-GACGAACGACACTTG CAGCAT-3' are provided.

The preparation method of the electrochemical sensor comprises the following steps:

1) pretreating a gold electrode;

2) preparing Hybrid DNA, namely mixing G4-DNA, C-DNA and A-DNA in a Tris-HCl buffer solution, heating for a set time, cooling, and standing for a set time to obtain a Hybrid DNA solution;

3) dropwise adding the Hybrid DNA solution prepared in the step 2) onto the pretreated gold electrode, standing at a set temperature for a set time, and drying to obtain the electrochemical sensor.

Preferably, in step 1), the gold electrode is pretreated by polishing the gold electrode on a flannelette containing alumina slurry, and then washing the gold electrode with ultrapure water and ethanol in sequence to remove residual alumina powder.

Further preferably, the gold electrode polished with alumina is placed in a dilute sulfuric acid solution at-0.2V to 1.6V at 100 mV. s^-1The gold electrode is subjected to electrochemical pretreatment until a stable cyclic voltammogram is obtained, and the gold electrode is cleaned by ultrapure water.

Preferably, in the step 2), the molar ratio of the G4-DNA, the C-DNA and the A-DNA is 1:8-12: 8-12. In order to bind as much G4-DNA as possible to C-DNA and A-DNA and to reduce the amount of G4-DNA that is not hybridized on the electrodes and thus to reduce the background signal of the sensor, low concentrations of G4-DNA and high concentrations of C-DNA and A-DNA are selected.

Preferably, in step 2), the Tris-HCl buffer has a pH value of 8. The pH of the reaction system has an influence on the stability of the DNA structure, and the peracid and the overbase both destroy the structure, so that the DNA structure is stable at a pH value of about 8.

Preferably, in the step 2), the heating temperature is 85-95 ℃, and the heating time is 15-25 min.

Preferably, in step 2), after cooling, the time of standing at 25-35 ℃ is more than 12 hours. The time includes two times, the first time is that after heating in the step 2), the DNA is placed at room temperature for more than 2 hours, so that the DNA forms a stable double-helix structure. Thereafter, in step 3), in order to immobilize a sufficient amount of Hybrid DNA on the electrode, the electrode was left for 12 hours or more.

Preferably, the step 3) further comprises the step of immersing the dried gold electrode in a 1mM MCH solution for a set time to reduce non-specific adsorption. Preparation method of 6-mercapto-1-hexanol (MCH) solution: 1mM mercaptohexanol solution in Tris-HCl buffer.

The application of the electrochemical sensor in the detection of T-DNA.

The method for detecting the T-DNA by using the electrochemical sensor comprises the following steps:

adding a Tris-HCl solution containing Exo III and Hairpin DNA into a solution containing T-DNA, then dropwise adding the solution onto the surface of an electrochemical sensor, incubating for 50-70min at 30-40 ℃, after washing, dropwise adding HEPES buffer containing hemin onto the surface of the electrochemical sensor, incubating for a set time to induce the folding of the released G4-DNA to form a G-quadruplex/hemin complex, and detecting the generated electrochemical signal.

The invention has the beneficial effects that:

the invention realizes the positive feedback amplification strategy combining homogeneous reaction and heterogeneous reaction by smartly designed Hairpin DNA and Hybrid DNA and target object cyclic amplification based on Exo III. The strategy shows ultra-high sensitivity, detection limit is as low as 0.12aM T-DNA, and high selectivity to T-DNA and other base mismatch nucleic acids.

The entire detection procedure can be carried out in several steps and demonstrates the great potential for quantitative analysis of DNA in complex biological samples. Furthermore, by coupling different aptamer probe sequences, this strategy can also be readily used to develop new methods to detect various types of target molecules (proteins, small molecules, metal ions, etc.).

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.

FIG. 1 is a schematic diagram of a positive feedback amplification electrochemical sensing strategy based on exonuclease III for ultrasensitive detection of nucleic acids according to the present invention;

FIG. 2 is a 12% polyacrylamide electrophoretogram; a band M, a DNA Marker (20,40,60,80,100,120,140,160,180,200,300,400,500 bp); band 1, C-DNA; band 2, G4-DNA; band 3, A-DNA; band 4, hybrid dna; band 5, T-DNA; band 6, Hybrid DNA + T-DNA; band 7, Hybrid DNA + T-DNA + Exo III; band 8, Hairpin DNA; band 9, Hairpin DNA + A-DNA; band 10, Hairpin DNA + A-DNA + Exo III; a band 11, Hybrid DNA + T-DNA + Hairpin DNA; a band 12, Hybrid DNA + T-DNA + Hairpin DNA + Exo III;

FIG. 3 is an electrochemical impedance spectrum and corresponding signals of DPV; (A) electrochemical impedance spectroscopy (Nyquist plots). A bare gold electrode (a); immobilizing Hybrid DNA (b); MCH closure (c); incubating in the presence of T-DNA, Exo III and Hairpin DNA (d); forming a G-quadruplex/hemin complex (e). The inserted diagram represents the relevant equivalent circuit diagram. (B) A DPV response signal. T-DNA (a); (b) no T-DNA; comparative experiments were performed without T-DNA and Exo III (c) and without Hairpin DNA (d);

FIG. 4 shows the effect of Hybrid DNA concentration (A), reaction time (B), Exo III concentration (C) on the DPV signal difference;

FIG. 5 (A) shows DPV curves for detection of T-DNA at different concentrations. Concentrations (from a to l) were 0, 0.05, 0.1,0.5,1,5,10, 50, 100, 500, 1000 and 10000 aM. (B) The difference in DPV response varies with target concentration. Positive feedback amplification experiment (point a) and control experiment (point b). Error bars represent standard deviations of three parallel experiments.

FIG. 6(A) detects the difference in DPV response (. DELTA.I) of T-DNA, Sm-DNA, Tm-DNA and N-DNA having the same concentration (100 aM); (B) stability test chart. Error bars represent standard deviations of three parallel experiments.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As shown in fig. 1, a novel DNA electrochemical sensor based on Exo III positive feedback amplification strategy was designed in this application for detecting DNA sequences associated with HIV. As shown in the schematic diagram, the complementary strand DNA (C-DNA) was designed to be composed of three fragments, which were base-complementary to the quadruplex-forming oligonucleotide (G4-DNA), the helper strand (A-DNA) and the T-DNA, respectively. First, a Hybrid DNA probe (Hybrid DNA) was prepared by hybridizing C-DNA with G4-DNA and A-DNA. Then, the Hybrid DNA was immobilized on the gold electrode by forming a strong S-Au bond between the 3' end of G4-DNA and the electrode surface. The electrochemical sensor was completed after blocking with 6-mercapto-1-hexanol (MCH). When T-DNA is present, Hybrid DNA hybridizes with it and forms a Hybrid DNA/T-DNA double-stranded structure having blunt 3' -ends, and T-DNA, A-DNA and G4-DNA are subsequently released from the Hybrid DNA/T-DNA double-stranded structure by Exo III hydrolysis. The released T-DNA is recognized by other Hybrid DNA, initiating the next cleavage process, constituting cycle I in the schematic. On the other hand, A-DNA released from cycle I hybridizes with hairpin DNA (HairpinDNA) and triggers cycle II with the aid of Exo III, thereby producing T' -DNA. Because the T' -DNA sequence is designed to beSimilar to T-DNA, it can also be recognized by Hybrid DNA and trigger cycle III with the aid of Exo III. And the A-DNA released from the cycle III enters a cycle II to induce and generate more T' -DNA, so that positive feedback signal amplification is realized. Finally, G4-DNA released from cycles I and III is in K⁺And the presence of hemin further forms a G-quadruplex/hemin complex, thereby generating an electrochemical signal. In contrast, when T-DNA is not present, it cannot be hydrolyzed by Exo III digestion due to the protruding 3' end in the specially designed Hybrid DNA. Meanwhile, G4-DNA is prevented from forming an active conformation to which hemin is bound because G4-DNA hybridizes with C-DNA. Therefore, no electrochemical signal is generated.

Experimental part

Material reagent

The oligonucleotide sequences for HPLC purification were purchased from Sangon biotech limited (shanghai, china) and are listed in table 1:

TABLE 1 oligonucleotide sequences used herein^a

^aThe underlined sequences of the Hairpin DNA are complementary and the bold letters represent mismatched bases.

4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), NaCl, MgCl₂KCl, [ tris (hydroxymethyl-1) aminomethane](Tris), Tris-HCl, boric acid from Sangon Biotech, Inc. (Shanghai, China). SYBR Gold was purchased from Invitrogen Biotech co, Ltd (shanghai, china). Exonuclease III is purchased from Takara Biotechnology co, Ltd (chinese continental). Hemin was purchased from Sigma-Aldrich Inc (st. louis, MO). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 6-mercapto-1-hexanol (MCH) were purchased from adadin Reagents (shanghai, china). Ultrapure water (18.25M omega cm) was obtained and used in a Upu water purification systemIn all experimental procedures.

Electrode pretreatment

The gold electrode was polished on a fleece containing a slurry of alumina trioxide (diameter 50nm), and then washed with ultrapure water and ethanol in order to remove the remaining alumina powder. Then, in order to further clean and activate the gold electrode surface, the surface is cleaned by adding H between-0.2V and 1.6V₂SO₄At a value of 100mV · s in (0.5M)^-1The gold electrode is electrochemically pretreated until a stable cyclic voltammogram is obtained. The electrodes are then thoroughly rinsed with ultrapure water for further use.

Preparation of the sensor

For the preparation of Hybrid DNA, G4-DNA, C-DNA and A-DNA in a ratio of 1:10:10 in 10mM Tris-HCl buffer (50mM NaCl, 10mM MgCl)₂pH 8.0) and heated to 90 ℃ for 20 minutes. The mixture was then cooled to room temperature and continued for at least 2 hours before use. Before fixation, Hybrid DNA was incubated with 10mM TCEP for 1 hour in the dark. Subsequently, 5. mu.L of the incubated Hybrid DNA solution was dropped onto the clean gold electrode and left at 4 ℃ for more than 12 hours. Then, the gold electrode was thoroughly washed with Tris-HCl buffer and dried under a nitrogen stream, and then immersed in a 1mM MCH solution for 1 hour to reduce nonspecific adsorption. The finished electrochemical sensor was placed at 4 ℃ for further use.

T-DNA detection based on Exo III positive feedback signal amplification strategy

T-DNA containing different concentrations was added to 5. mu.L of 10mM Tris-HCl solution (pH 8.0,50mM NaCl, 10mM MgCl) containing 1U/. mu.L of Exo III and 0.2. mu.M of Hairpin DNA₂) Then added dropwise to the electrochemical sensor surface, followed by incubation at 37 ℃ for 60 minutes. After washing, 5 μ L of 10mM HEPES buffer (pH 8.0,50mM KCl, 1% DMSO) containing 0.2mM hemin was added dropwise to the resulting sensor surface and incubated for 30 minutes to induce folding of the released G4-DNA to form a G-quadruplex/hemin complex.

Electrochemical test and apparatus

Electrochemical measurements were performed on CHI 660E electrochemical workstation (Shanghai Chenghua, China), in which gold was presentElectrode (area 0.07 cm)²) An Ag/AgCl electrode (in saturated KCl) and a platinum wire were used as the working, reference and counter electrodes, respectively. Differential Pulse Voltammograms (DPV) were recorded in 10mM HEPES buffer (pH 8.0,50mM KCl) with potential windows from 0V to-0.6V. Prior to the measurement, the electrolyte solution was purged with high purity nitrogen for about 20 minutes to avoid interference with oxygen reduction. In the presence of 5mM [ Fe (CN) ] containing 0.1MKCl₆]^3-/4-Electrochemical Impedance Spectroscopy (EIS) was measured in solution at frequencies ranging from 0.01Hz to 100 kHz.

12% Polyacrylamide gel electrophoresis test

To verify the implementation of the positive feedback amplification strategy, 12% polyacrylamide gel electrophoresis (PAGE) analysis of DNA was performed in 1 × TBE (90mM Tris, 90mM boronic acid, 2mM EDTA) at 10 ℃ for 80 minutes at a constant current of 30 mA. Then, the obtained Gel was stained in SYBR Gold solution for 40 minutes and Gel Doc XR was used⁺Imaging systems (Bio-Rad, Hercules, Calif., USA).

Results and discussion

Protocol characterization

12% polyacrylamide gel electrophoresis: the proposed strategy was first validated by 12% polyacrylamide gel electrophoresis (PAGE) experiments. As shown in FIG. 2, lanes 1 to 5 show bands of C-DNA, G4-DNA, A-DNA, Hybrid DNA and T-DNA, respectively. After incubation of Hybrid DNA with T-DNA, a band slower than that of lane 4 was observed (lane 6), indicating that Hybrid DNA recognizes T-DNA; while the band of the Hybrid DNA/T-DNA double-stranded structure disappeared in the presence of Exo III, and the bands associated with G4-DNA, A-DNA and T-DNA appeared again (lane 7), indicating that C-DNA was successfully digested and hydrolyzed. Exo III released the intact G4-DNA, A-DNA and T-DNA from the Hybrid DNA/T-DNA duplex. The band in lane 8 corresponds to Hairpin DNA. After incubation of the Hairpin DNA with A-DNA, a new band with a slower migration rate was observed (lane 9), confirming the hybridization of the Hairpin DNA and A-DNA. In the presence of Exo III, the Hairpin DNA/A-DNA double strand band disappeared, resulting in a new band of about 20bp related to T' -DNA (lane 10). Lane 11 shows a band of a mixture of Hybrid DNA, T-DNA and Hairpin DNA, while lane 12 shows a band of the mixture after addition of Exo III. The band of the hydrolysate in lane 12 is identical to the bands in lanes 7 and 10, confirming that the entire detection strategy is fully compliant with the proposed principles. PAGE experiments provide evidence for this designed strategy.

Electrochemical impedance spectroscopy: to further characterize the feasibility of the proposed strategy, the sensor fabrication steps and sensing process were confirmed by Electrochemical Impedance Spectroscopy (EIS) measurements. As shown in fig. 3A, the bare gold electrode obtained a very small half-circle domain (Rct ═ 113.7 Ω, curve a), indicating that the charge transfer process was very fast. After the Hybrid DNA was immobilized on the gold electrode, since Hybrid DNA and [ Fe (CN)₆]^3-/4-Effective repulsion between the negatively charged phosphate backbones of the anions results in a larger half-circle domain (Rct ═ 211.8 Ω, curve b). Subsequently, the electrode was closed with MCH and a further increase in Rct was obtained (Rct 699.6 Ω, curve c). After further incubation of the sensor with T-DNA in the presence of Exo III and Hairpin DNA, the radius diameter dropped sharply, with an Rct value of about 382 Ω (curve d), which is attributed to the fact that considerable Hybrid DNA converts it to single-stranded G4-DNA by digestion hydrolysis of Exo III and the implementation of a positive feedback signal amplification strategy. In addition, when K is used⁺And hemin treated electrodes, an increased Rct value of about 1036 Ω was obtained (curve d). This can be explained by the following reasons: the G-quadruplex/hemin complex formed is more orderly and densely packed on the electrode surface, more strongly repelling negatively charged [ Fe (CN)₆]^3-/4-A redox species.

Differential pulse voltammogram: in addition, the designed Exo III-assisted positive feedback amplification strategy is characterized by Differential Pulse Voltammograms (DPVs). As shown in FIG. 3B, a well-defined DPV peak around-0.35V was observed in the presence of T-DNA (curve a). This peak may contribute to the electrochemical reduction of hemin incorporated in the G-quadruplex. However, in the absence of T-DNA, only a small DPV peak was observed (curve b), and a similar response was observed in the control experiment without T-DNA and Exo III (curve c), which is attributable to the possible formation of G-quadruplex/heme complexes and non-specific adsorption of hemin by small amounts of unhybridised G4-DNA on the electrode surface. These results preliminarily verify the feasibility of the designed strategy. To further demonstrate the signal amplification effect, a control experiment without Hairpin DNA was performed. The DPV obtained showed a much smaller peak (curve c) than in the design case where there was Hairpin DNA (curve a), since in the absence of Hairpin DNA only cycle I as shown in the schematic occurred and the formation of the G-quadruplex/heme complex was greatly reduced. In contrast, when Hairpin DNA is present, both homogeneous amplification Cycle ii and heterogeneous amplification Cycle iii can be triggered, thereby achieving positive feedback signal amplification and significantly increasing the electrochemical signal. This strongly demonstrates the successful implementation of the designed positive feedback signal amplification strategy with the help of Exo III and Hairpin DNA.

To achieve ultrasensitive detection of T-DNA, parameters that may affect the detection sensitivity, such as the hybrid DNA concentration, Exo III concentration and reaction time, were optimized.

To investigate the effect of Hybrid DNA concentration on sensor performance, different concentrations of Hybrid DNA (0.1,0.5,1,5,10nM) were immobilized on gold electrodes to prepare sensors. The obtained relationship between the difference in DPV response (Δ I) in the presence of T-DNA and blank control and the Hybrid DNA concentration is shown in fig. 4A, and it can be found that the difference in DPV response increases with the increase in the Hybrid DNA concentration and reaches the highest value at 1nM of the Hybrid DNA concentration and then decreases when the Hybrid DNA concentration is higher than 1 nM. High fixation concentration of Hybrid DNA may increase the assembly density on the electrode surface, but it may limit the hybridization efficiency of Hybrid DNA and T-DNA due to steric hindrance effects. Furthermore, the high density of Hybrid DNA probes is not conducive to efficient folding of G4-DNA. These two factors may result in a relatively low electrochemical response of high concentrations of Hybrid DNA-modified sensors. Thus, a 1nM Hybrid DNA concentration was used for sensor preparation.

For reaction time, the DPV response difference increased with increasing reaction time and reached a maximum at 60 minutes and then remained stable beyond 60 minutes (fig. 4B). Whereas for Exo III concentration, the DPV response difference increased with increasing Exo III concentration and reached saturation at a concentration of 1U/. mu.l of ExoIII (fig. 4C). These results show that with increasing reaction time and Exo III concentration, more and more G4-DNA was released and further G-quadruplex/heme complex was formed, while G4-DNA was completely released at 60 min reaction time and 1U/. mu.L Exo III. Therefore, a reaction time of 60 minutes and 1U/. mu.L of Exo III was chosen for further experiments.

Establishment of titration curves

The sensitivity of the proposed strategy was studied by incubating different concentrations of T-DNA with 1U/. mu.L of Exo III and 0.2. mu.M of Airpin DNA for 1 hour under optimized experimental conditions. As shown in FIG. 5A, the DPV peak current increases as the T-DNA concentration increases from 0 to 10fM, confirming the working principle of the proposed strategy. The corresponding DPV peak current difference in the presence of T-DNA versus blank as a function of the logarithm of T-DNA concentration is shown in fig. 5B, which shows a good linear correlation (a) over the range of 0.5aM-500aM T-DNA, resulting in a linear regression equation with Δ I (μ a) of 0.1037log (C)_T-DNA(aM))+0.1455(R²0.9944), limit of detection was 0.12aM T-DNA (based on 3 σ rule), whereas control experiments without HairpinDNA yielded Δ I (μ a) 0.0471log (C)_T-DNA(aM))-0.0299(R²0.9790) calculated to have a detection limit of 54.35aM T-dna (b). Clearly, the proposed strategy shows excellent detection sensitivity compared to the control experiment, demonstrating the successful implementation of the designed positive feedback signal amplification strategy. Furthermore, the proposed strategy shows lower detection limits compared to previously reported methods using nucleases, thus providing a viable and efficient method for ultrasensitive electrochemical detection of DNA.

Investigation of specificity

The specificity of the designed detection method was examined by testing four DNA sequences, including T-DNA, single base mismatch DNA (Sm-DNA), three base mismatch DNA (Tm-DNA) and complete non-complementary DNA (N-DNA) (concentration 100 aM). As shown in FIG. 6A, the difference in the DPV peak currents of Sm-DNA and Tm-DNA was only about 32% and 10.4% of the difference in the DPV peak currents of T-DNA, respectively. The response of N-DNA is almost negligible. These results clearly show that the designed strategy has good specificity for T-DNA detection.

Determination of stability

The stability of the sensor was investigated. After the sensor was prepared, it was stored in a refrigerator at 4 ℃ for 1 day, 2 days, 3 days, 7 days and 14 days, respectively (fig. 6B). Three independent experiments showed that the sensor retained about 94% of its initial response to T-DNA after 2 weeks storage in the refrigerator, showing relatively good stability.

Actual sample detection

The application of this design strategy to the analysis of real biological samples was investigated (table 2). T-DNA was prepared at different concentrations (1aM, 2aM, 10aM, 100aM) using a 10-fold dilution of human serum sample solutions obtained from healthy individuals. The recovery rates of added T-DNA were 103% ± 2.0%, 98.7% ± 1.6%, 101% ± 2.6% and 101.4% ± 3.9% (n ═ 4), respectively, indicating acceptable accuracy for quantitative detection of T-DNA in complex biological samples.

Table 2 positive feedback amplification strategy added T-DNA (n-4) was detected in normal human serum.

Conclusion

In view of the above, we have proposed an ultra-sensitive and simple electrochemical strategy for detecting nucleic acids based on Exo III-assisted positive feedback amplification. The positive feedback amplification strategy combining homogeneous reaction and heterogeneous reaction is realized through the skillfully designed Hairpin DNA and Hybrid DNA and the target object cyclic amplification based on Exo III. The strategy shows ultra-high sensitivity, detection limit is as low as 0.12aM T-DNA, and high selectivity to T-DNA and other base mismatch nucleic acids. Furthermore, the entire detection procedure can be carried out in several steps and demonstrates the great potential for quantitative analysis of DNA in complex biological samples. Furthermore, by coupling different aptamer probe sequences, this strategy can also be readily used to develop new methods to detect various types of target molecules (proteins, small molecules, metal ions, etc.).

Claims (2)

1. A positive feedback amplification electrochemical sensor based on exonuclease III is characterized in that: the gold electrode structure comprises a gold electrode and Hybrid DNA fixed on the gold electrode, wherein the Hybrid DNA sequentially comprises a sulfur-hydrogen bond, a double-stranded DNA structure and a single-stranded DNA structure from a 5 'end to a 3' end, the double-stranded DNA structure is prepared by hybridizing C-DNA, G4-DNA and A-DNA, and the base sequence of the single-stranded DNA structure is complementary with the base sequence of the T-DNA; the Hybrid DNA is fixed on the gold electrode through an S-Au bond;

the base sequence of the C-DNA is

5'-ATCCCGCCCAACCCAGTCCAAGTGTCGTTCGTCAAAATCTCTAGCAGT-3'；

The base sequence of the G4-DNA is as follows: 5'-GACTGGGTTGGGCGGGAT GGGTTTTTT- (CH2) 6-SH-3';

the base sequence of the A-DNA is as follows: 5'-GACGAACGACACTTG CAGCAT-3' are provided.

2. The method of manufacturing an electrochemical sensor according to claim 1, wherein: the method comprises the following steps:

1) pretreating a gold electrode;

2) preparing Hybrid DNA, namely mixing G4-DNA, C-DNA and A-DNA in a Tris-HCl buffer solution, heating for a set time, cooling, and standing for a set time to obtain a Hybrid DNA solution;

3) dropwise adding the Hybrid DNA solution prepared in the step 2) onto the pretreated gold electrode, standing at a set temperature for a set time, and drying to prepare the electrochemical sensor;

in the step 1), the pretreatment method of the gold electrode comprises the steps of polishing the gold electrode on a flannelette containing alumina slurry, and then washing the gold electrode with ultrapure water and ethanol in sequence to remove residual alumina powder; then the gold electrode polished by the alumina is put into a dilute sulphuric acid solution at the voltage of-0.2V to 1.6V and the voltage of 100 mV.s^-1Carrying out cyclic scanning at the scanning rate, carrying out electrochemical pretreatment on the gold electrode until a stable cyclic voltammogram is obtained, and cleaning with ultrapure water;

in the step 2), the molar ratio of the G4-DNA, the C-DNA and the A-DNA is 1:8-12: 8-12; the pH value of the Tris-HCl buffer solution is 8; heating at 85-95 deg.C for 15-25 min; cooling to room temperature, and standing at 25-35 deg.C for more than 2 hr;

in step 3), before dropwise adding the Hybrid DNA to the gold electrode, incubating the Hybrid DNA with 10mM tris (2-carboxyethyl) phosphine hydrochloride for 1 hour in the dark;

in the step 3), the gold electrode dropwise added with the Hybrid DNA solution is placed at 4 ℃ for more than 12 hours;

in step 3), the dried gold electrode is immersed in a 1mM MCH solution for 1 hour to reduce non-specific adsorption, and the prepared electrochemical sensor is placed at 4 ℃ for further use.

Images