CN113278683A - Ratio-type ECL biosensor and preparation method and application thereof - Google Patents
Ratio-type ECL biosensor and preparation method and application thereof Download PDFInfo
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Abstract
The invention constructs a ratio electrochemical luminescence (ECL) biosensor, which is used for detecting RNA-dependent RNA polymerase (RdRp) gene of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). The entropy-driven circulation amplification reaction is participated by SARS-COV-2RdRp gene, then a piece of band DNA is output, and can be combined with another two single-stranded S1 and S2 to form double-stranded DNAwalker, and the next circulation reaction is started. After the biped DNA Walker completes the walking process, the hairpin structure at the top of the DNA Tetrahedron (TDNAs) is removed. Subsequently, using PEI-Ru @ Ti3C2@ AuNPs-S7 probe and Au-g-C3N4The surface-cleaved TDNAs hairpin portion is specifically bound by electrochemiluminescenceVibration energy transfer (ECL-RET) effects signal changes.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a ratio-type ECL biosensor and a preparation method and application thereof.
Background
The outbreak of new coronavirus disease (COVID-19) in 2019 caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has endangered the health of people worldwide. Prior to the development of an effective vaccine, COVID-19 was first diagnosed rapidly and accurately, especially in asymptomatic patients, in order to prevent the spread of new coronavirus diseases. Currently, there are two main strategies for the detection of novel coronavirus diseases in the market. One strategy is to detect SARS-COV-2RNA using Polymerase Chain Reaction (PCR) based techniques. However, the PCR operation process is complicated, different primers and temperatures need to be designed, the accuracy is low, and false results usually occur. Another strategy is based on serological and immunological detection of antibodies, but the virus is detected immediately after the patient has been infected with SARS-COV-2 by waiting for the antibodies to develop in vivo. Therefore, a rapid, accurate, simple, and reliable detection method for detecting SARS-COV-2 is needed to manage this epidemic.
Compared with other detection technologies, Electrochemiluminescence (ECL) has the characteristics of high sensitivity and simplicity in operation, and can be used for testing various analytes. Conventional ECL strategies rely on the intensity of a single signal to detect the analyte and are susceptible to environmental and substrate interference factors. However, the ratio ECL for detecting the analyte using the ratio signal ratio can effectively avoid interference of the environment and the matrix, and the detection accuracy is improved. Compared with the dual voltage comparison method, the dual wavelength ratio-emitting ECL biosensor has two advantages of multiple coreactants, narrow voltage range and the like. The former ECL biosensor relies primarily on electrochemiluminescence resonance energy transfer (ECL-RET) between the donor and the acceptor, improving the dual signal ratio by overlapping the ECL emission spectrum of the donor and the ultraviolet-visible light (UV-Vis) absorption spectrum of the acceptor. Albeit Au nanoparticle (Au NPs) decorated graphite-C3N4(Au-g-C3N4) Has good biocompatibility and excellent luminous efficiency, has attracted attention as a donor material, but a large number of suitable acceptor materials are still lacking at present. Coincidently, Ru (II) has a stronger ultraviolet-visible light absorption peak at 460-470nm, and Au-g-C3N4The ECL emission peaks of (a) overlap.
To improve the sensitivity of ECL biosensors, various cyclic amplification strategies have been introduced, including hybrid strand reaction (HCR), Rolling Circle Amplification (RCA), Catalytic Hairpin Assembly (CHA), Polymerase Chain Reaction (PCR), and the like. These cyclic amplification strategies either require complex design procedures, complex secondary structures, or require enzymes and variable temperatures to participate in the reaction. Entropy-driven reactions have been proposed in recent years as a simple, isothermal, enzyme-free, rapid detection strategy. The entropy-driven reaction is a reaction by chain displacement acting on the toes. Multiple hybridizations and separations occur during the course of cyclic amplification, followed by the formation of large amounts of target DNA. In addition, spontaneous, controllable, continuously mobile enzyme-driven orbitals of DNA Walker was introduced into biosensors as an amplification strategy. However, since the walking space of the single-foot DNA Walker is limited, the walking efficiency cannot be improved. In contrast, biped DNA Walker has a larger walking space and higher efficiency. Therefore, the sensitivity of the biosensor can be greatly improved by simultaneously introducing the entropy driving and the biped DNA Walker amplification strategies.
The electrode surface constructed by DNA Tetrahedrons (TDNAs) can reduce background signals and improve the hybridization efficiency of target DNA. As one of the four single strands of each TDNA, the longest single strand that binds to the target DNA can be designed, and the method has the advantages of easy synthesis, strong mechanical rigidity, uniform distribution of the captured probes on the surface of the electrode, and the like. The ECL signal of the DNA tetrahedral sensor is higher and the background signal of the TDNAs sensor is lower when no target is present in the system. This is because the biosensor constructed with TDNAs can effectively prevent nonspecific adsorption. Therefore, the construction of TDNAs biosensor can improve the detection sensitivity.
Disclosure of Invention
The invention provides a probe set, a reagent or a kit for quickly, accurately, simply and reliably detecting SARS COV-2 RdRp.
First, the present invention provides a probe set, comprising:
the first probe is a tetrahedral probe formed by 4 single-stranded DNA molecules, and the nucleotide sequence of the 4 single-stranded DNA molecules is shown in SEQ ID NO.3-SEQ ID NO. 6;
the second probe is obtained by complementary hybridization of 2 single-stranded DNA molecules, and the nucleotide sequences of the 2 single-stranded DNA molecules are shown as SEQ ID NO.1 and SEQ ID NO 2;
the third probe is 1 single-stranded DNA molecule, and the nucleotide sequence is shown in SEQ ID NO. 7.
Optionally, the probe set further comprises a fourth probe, which is obtained by complementary hybridization of 3 single-stranded DNA molecules, wherein the nucleotide sequence of the 3 single-stranded DNA molecules is a single-stranded DNA molecule shown as SEQ ID No.8-SEQ ID No. 10; optionally, the probe set further comprises a fifth probe, wherein the fifth probe is 1 single-stranded DNA molecule, and the nucleotide sequence is shown in SEQ ID No. 11.
Optionally, the tetrahedral probe is a tetrahedral probe modified by a hairpin structure.
A reagent or kit for detecting SARS COV-2RdRp, comprising the probe set described above.
A biosensor comprising the first probe of claim 1.
A method for preparing a biosensor, comprising the steps of:
s1: Au-g-C for glassy carbon electrode3N4Modification to obtain Au-g-C3N4/GCE；
S2: will be fixed to Au-g-C by gold-sulfur bonds at TDNAs3N4a/GCE surface; TDNAs is the first probe.
Optionally, the method also comprises the step of mixing Au-g-C3N4The step of soaking the MCH-containing solution in GCE.
A method for detecting SARS COV-2RdRp, which comprises the step of detecting SARS COV-2RdRp gene by using the probe set, the reagent or the kit, the biosensor, and the biosensor prepared by the method.
Optionally, the method specifically includes the following steps:
1) incubating the prepared biosensor in a solution to be detected; optionally, the incubation time is more than 40 min; the incubation temperature was 37 ℃;
2) adding a solution containing a second probe and an endonuclease into the mixed solution incubated in the step 1);
3) modified by PEI-Ru @ Ti3C2Adding a third probe of @ AuNPs into the mixed solution in the step 2);
optionally, the solution to be detected comprises Substrate and Fuel;
a hybridization complex formed by a single-stranded DNA molecule represented by SEQ ID NO.8 to SEQ ID NO. 10;
fuel is a single-stranded DNA molecule shown in SEQ ID NO. 11.
Optionally, the biosensor is a ratiometric ECL biosensor.
The invention reasonably designs a dual-wavelength ratio type ECL biosensor, combines entropy drive, biped DNA Walker circulation amplification strategy and DNA tetrahedron, and detects SARS-COV-2RdRp gene, and the ECL biosensor has the following advantages:
first, in the rate-type ECL biosensor, Au-g-C was designed3N4As good donors, PEI-Ru @ Ti3C2@ AuNPs as an excellent acceptor to achieve ECL-RET;
second, unlike the conventional DNA Walker, the bipedal DNA Walker walks on a walking trajectory by binding the adjacent two feet (S1 and S2 as the two feet of the bipedal DNA Walker) with the band DNA. However, the two feet which are not bound by the band DNA cannot walk on the track because they are far apart. In addition, TDNA modified with a hairpin structure was used as a walking trace of a bipedal DNA walker on the electrode surface.
Thirdly, two amplification strategies of entropy driving and biped DNA Walker are combined, and ultra-sensitive detection is realized. The detection range of the biosensor can be from 10aM to 10pM, and the detection limit is as low as 7.8 aM. The dual wavelength ratio ECL biosensor shows excellent recovery rate when used in human serum, and can be used for clinical early diagnosis.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a ratiometric ECL biosensor based on entropy-driven and bipedal DNA Walker amplification strategy, DNA tetrahedron and for detecting the SARS-COV-2RdRp gene;
FIG. 2(A) PEI-Ru @ Ti3C2Ultraviolet-visible absorption spectra (Gray curves) of @ AuNPs and Au-g-C3N4ECL spectra (red curve) of (1), obtained through a series of filters, at a spectrum containing 0.1M S2O8 2-In 0.1M PBS (pH 7.4);
(B)Au-g-C3N4(curve a) and PEI-Ru @ Ti3C2ECL spectra of @ AuNPs/GCE (curve b), scanned at 0V to-1.5V;
(C) is PEI-Ru @ Ti3C2The ECL intensity change curve before and after modification of the @ AuNPs-S7 probe; curve a is that the electrode has not passed through PEI-Ru @ Ti3C2@ AuNPs-S7 probe modification; curve b is the electrode passing PEI-Ru @ Ti3C2@ AuNPs-S7 probe modification;
(D) ratiometric ECL biosensors using SARS COV-2RdRp at different concentrations (0aM, 10aM, 10fM and 10pM) were tested using a series of filters spaced 20nm apart in a sample containing 0.1M S2O8 2-In 0.1M PBS (pH 7.4);
FIG. 3(A) Au-g-C3N4TEM characterization of (1), inset is g-C3N4；
(B)Au-g-C3N4EDX spectrum of (1), inset is g-C3N4The EDX of (1);
(C)g-C3N4(Curve a) and Au-g-C3N4(curve b) uv-vis absorption spectrum;
(D)g-C3N4(Curve a) and Au-g-C3N4(curve b) ECL Strength Curve, g-C3N4(Curve a') and Au-g-C3N4(curve b') in a cyclic voltammogram containing 0.1M S2O8 2-Modification in the GCE test of (1);
FIG. 4(A) Ti3C2TEM characterization of (4); (B) PEI-Ru @ Ti3C2The TEM characterization of @ AuNPs; (C) the corresponding size distribution profile of the nanoparticles of (B); (D) PEI-Ru @ Ti3C2The EDX spectrum of @ AuNPs;
FIG. 5 gel electrophoresis analysis
(A) TDNAs construction process; lane 1: s3, lane 2: S3-S4, lane 3: S3-S4-S5, lane 4: S3-S4-S5-S6;
(B) entropy-driven cycle amplification reaction, lane 1: Scaffold, lane 2: blocker, run 3: band, lane 4: substrate, lane 5Substrate + SARS-COV-2RdRp, lane 6 Substrate + SARS-COV-2RdRp + Fuel;
(C) biped DNA Walker, lane 1, S1 probe, lane 2, S2 probe, lane 3, S1+ S2, lane 4, S1+ S2+ band;
FIG. 6 features of the stepwise construction Process of the biosensor
(A) Characterization of biosensor construction by Cyclic Voltammetry (CV)
(B) Characterizing the construction process of the biosensor by electrochemical alternating current impedance spectroscopy (EIS);
(C) ECL response of electrodes modified with different substances;
(A) in (C): (a) naked GCE; (b) GCE/Au-g-C3N4；(c)GCE/Au-g-C3N4/TDNAs；(d)GCE/Au-g-C3N4/TDNAs/MCH；(e)GCE/Au-g-C3N4TDNAs/MCH with SARS COV-2RdRp and Nb.BbvCI treatment; (f) with PEI-Ru @ Ti3C2@ AuNPs-S7 further incubation;
FIG. 7A (a) correlation between ECL signal intensity at 460nm and the logarithm of the concentration of SARS-COV-2RdRp (10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10 pM); (b) relationship between ECL Signal Strength at 620nm and the logarithm of the concentration of SARS-COV-2RdRp (10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM)
(B) A calibration curve for SARS-COV-2 RdRp;
(C) stability of ECL biosensors;
16 consecutive measurements were made at a concentration of 10pMSARS-COV-2RdRp at 460nm (left) and 620nm (right), respectively;
(D) selectivity of ECL biosensor
(a)1pM of SARS-COV-2RdRp, (b)100pM of SARS-COV RdRp (single-stranded DNA molecule shown by SEQ ID NO. 14), (c)100pM of random DNA, (d) ratio of blank solution;
ECL Signal measurement Using a series of filters spaced 20nm apart, in a volume of 0.1M S2O8 2-In 0.1M PBS (pH 7.4).
Detailed Description
Chemicals and materials
Gold chloride trihydrate (HAuCl4-3H2O), sodium citrate, potassium persulfate (K)2S2O8) And sodium borohydride (NaBH)4) All from Aladdin Biotechnology Ltd. Both the Ti3C2 dispersion and the g-C3N4 were from Jiangsu New agricultural materials science and technology, Inc. Tris (4,4'-dicarboxylic acid-2,2' -bipyridyl) ruthenaum (II) dicblock (Ru (dcbpy)3Cl 2); from suzhou, china. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 6-Mercaptohexanol (MCH) were obtained from Sigma-Aldrich (St Louis, Mo., USA). BbvCI and 10 XNEB buffers were obtained from New England Biolabs (USA). The DNA sequence was synthesized by Genscript Bio-technology Co.Ltd
Transmission Electron Microscope (TEM) images were obtained by JEM-2100F of JEOL (Japan). Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were measured by an electrochemical workstation (Shanghai Chenghua instruments, Inc., China) at 5.0mM [ Fe (CN)6]3-/4-In solution. ECL signals were collected using ECL-6B from the national emphasis laboratory of analytical chemistry, Life sciences, university of Nanjing. Ultraviolet-visible absorption spectroscopy was measured using a microplate reader (Spectra Max M5e) and had a wavelength range of 250nm to 700nm (Molecular Devices co. ltd., USA). Atomic Force Microscope (AFM) images were obtained from Dimension ICON (Bruker). Gel images were taken by Bio-Rad ChemDoc XRS.
Au-g-C3N4The preparation of (1):
50 μ L of HAuCl4(0.01M) solution and 4mL of g-C3N4(0.15mg mL-1) Mix together, stir ultrasonically for 10 minutes, then stir for 1 hour. Then, 100. mu.L of fresh NaBH was injected into the mixed solution4(0.01M) solution, treated in ice bath for 20 minutes. To the above mixed solution was continuously added 50. mu.L of a 1% by mass sodium citrate solution, and the mixed solution was continuously stirred at 25 ℃ for 20 minutes. Finally, to remove excess AuNPs, sodium citrate and NaBH4Centrifuging the mixed solution at 12000rpm, washing with ultrapure water, and ultrasonically treating for 3 times to obtain pure Au-g-C3N4Redispersed in 1mL H2O, and stored at 4 ℃ for use in the next experiment.
PEI-Ru@Ti3C2Synthesis of the @ AuNPs-S7 probe according to the following procedure:
1. 10mg of tris (4,4'-dicarboxylic acid-2,2' -bipyridine) ruthenium (II) dichloride [ Ru (dcbpy) ]3 2+]EDC and NHS were dissolved in 5mL PBS (0.1M, pH 7.4) and stirred for 2h to obtain a solution containing Ru (dcbpy)3 2+The final concentration of EDC in the mixture is 200mM, and the final concentration of NHS in the mixture is 50 mM;
2. 5ml of 5mg/ml Ti3C2Uniformly mixing the suspension and PEI to form a mixed solution A, wherein the mass fraction of PEI in the mixed solution is 1%, and adding the mixed solution A into the mixed solution A containing Ru (dcbpy)3 2+For 1 hour. Subsequently, 600. mu.L of HAuCl was added to the above solution4(0.01M) and 600. mu.L of fresh NaBH4(0.01M) and stirred for 1 hour, and the prepared PEI-Ru @ Ti was added to remove excess impurities3C2@ AuNPs was centrifuged, washed 3 times, and redispersed in 1mL PBS (0.1M, pH 7.4).
3. To prepare PEI-Ru @ Ti3C2@ AuNPs-S7 Probe to 1mL Ru @ Ti3C2200 μ L S7(10 μ M, S7 is labeled with the 3' -thiol group of the single-stranded DNA molecule shown in SEQ ID NO. 12) was added to the @ AuNPs, and the mixture was stored overnight in a refrigerator at 4 ℃. Successfully prepare PEI-Ru @ Ti3C2@AuNPAn S-S7 probe.
Preparation of TDNAs
Dissolving 10 μ L of 4 single-stranded DNAs (S3, S4, S5 and S6; S3 is obtained by labeling the 5 '-end thiol group of the single-stranded DNA molecule shown in SEQ ID NO.3, S4 is obtained by labeling the 5' -end thiol group of the single-stranded DNA molecule shown in SEQ ID NO.4, S5 is obtained by labeling the 5 '-end thiol group of the single-stranded DNA molecule shown in SEQ ID NO.5, and S6 is obtained by labeling the 5' -end thiol group of the single-stranded DNA molecule shown in SEQ ID NO. 6) in a solution containing 10mM TCEP and 50mM MgCl2In PBS buffer (pH 7.4). Then, the four single-stranded DNAs were mixed together, left at 95 ℃ for 5 minutes, cooled to room temperature, and stored for 3 hours in this order. Finally, robust TDNAs were obtained.
TABLE 1 different sequences of oligonucleotides
EXAMPLE 1 construction of a ratiometric ECL biosensor and use for detecting the SARS COV-2RdRp Gene
A method for constructing a ratio-type ECL biosensor comprises the following steps:
1. the Glassy Carbon Electrode (GCE) was cleaned. 10 μ L of Au-g-C3N4Immobilization on GCE to obtain Au-g-C3N4(ii)/GCE, and drying at room temperature. Subsequently, the obtained Au-g-C3N4the/GCE was soaked in 100. mu.L of TDNAs (1. mu.M) solution containing 10mM TCEP for 8 hours to reduce the S-S bonds. Subsequently, the modified GCE was soaked in 60. mu.L MCH (100. mu.M) solution for 1 hour to prevent non-specific adsorption. In each of the above steps, it is necessary to wash away the unlinked material with a PBS solution. The well-constructed electrode (MCH/TDNAS/Au-g-C)3N4the/GCE) was stored in a refrigerator at 4 ℃ for the next experiment.
2. Mix MCH/TDNAS/Au-g-C3N4Entropy-driven reaction of/GCE at 37 deg.CIncubation in the mixture for 40min, entropy-driven reaction mixtures including different concentrations of SARS-COV-2RdRp gene, Substrate and Fuel (single-stranded DNA molecule shown in SEQ ID NO.11 at a concentration of 1. mu.M); the Substrate is formed by hybridization of Scaffold, Blocker and Bandage (Scaffold, Blocker and Bandage are single-stranded DNA molecules shown by SEQ ID NO.8-SEQ ID NO.10 in sequence, three strands (the molar ratio is 1: 1: 1) are incubated at 95 ℃ for five minutes, and then cooled to room temperature to obtain a stable hybrid complex), and the concentration of the Substrate is 1 mu M. And two single-stranded S1(SEQ ID NO.1), S2(SEQ ID NO.2) and Nb.BbvCI endonuclease (10U) were simultaneously added to the mixed solution, the final concentration of S1 was 60nM, and the final concentration of S2 was 60nM, followed by completion of the DNA Walker walking process on the electrode surface. Finally, the synthesized PEI-Ru @ Ti with the concentration of 1.67 μ M3C2Dropping the @ AuNPs-S7 probe solution on the surface of the modified electrode to obtain PEI-Ru @ Ti3C2@AuNPs-S7/MCH/TDNAs/Au-g-C3N4(ii)/GCE (ratiometric ECL biosensor).
Verification of successful construction of ratio-type ECL biosensor
1) Based on Au-g-C3N4With PEI-Ru @ Ti3C2The ECL-RET principle between @ AuNPs, and a ratio-type ECL biosensor is designed. As shown in FIG. 2A, Au-g-C3N4ECL emission spectrum of (E) and PEI-Ru @ Ti3C2The UV-visible absorption spectra of @ AuNPs match well. Description of Au-g-C3N4Donor and PEI-Ru @ Ti3C2ECL-RET can be achieved between the @ AuNPs receptors. In addition, when the scanning voltage is-1.5-0V, S exists in the system2O8 2-In the presence of Au-g-C3N4There was an ECL emission peak on the/GCE (FIG. 2B). However, PEI-Ru @ Ti3C2No ECL emission peak appears at @ AuNPs/GCE, which indicates that PEI-Ru @ Ti3C2@ AuNPs receptor does not interfere with Au-g-C at cathode voltage3N4ECL emission of donor.
FIG. 2C is PEI-Ru @ Ti3C2Curve of ECL intensity before and after modification of the @ AuNPs-S7 probe. When the electrode does not pass through PEI-Ru @ Ti3C2@ AuNPs-S7 Probe repairWhen decorated, a strong ECL signal appears at 460nm and a weak ECL signal appears at 620 nm. In contrast, when PEI-Ru @ Ti is used3C2When the @ AuNPs-S7 probe is used for modifying the electrode, a weak ECL signal appears at 460nm, and a strong ECL signal appears at 620 nm. It was further shown that ECL-RET between the Au-g-C3N4 donor and the PEI-Ru @ Ti3C2@ AuNPs acceptor is reasonable.
As shown in FIG. 2D, a series of filters were used to investigate the change in ECL intensity with increasing concentrations of SARS-COV-2RdRp gene. When SARS-COV-2RdRp is absent from the system, only the ECL emission peak appears at 460 nm. However, as the concentration increased, the ECL signal intensity at 460nm gradually decreased, and correspondingly, the ECL emission peak at 620nm continued to increase. These results successfully demonstrate that the rate-based ECL biosensor of ECL-RET can efficiently detect the SARS-COV-2RdRp gene.
2) Characterization of Au-g-C3N4
As can be seen from FIG. 3A, 8. + -.1 nm AuNPs were uniformly dispersed in Au-g-C3N4Of (2) is provided. Fig. 3B is an energy dispersive X-ray (EDX) analysis. The results show that Au-g-C3N4Has an element peak of C, N, Au, and g-C3N4No elemental peak of Au was observed in the EDX spectrum of (fig. 3B inset).
Using UV-visible absorption spectrum for Au-g-C3N4Characterization was performed as shown in fig. 3C. Wherein g-C3N4The AuNPs have a unique peak at 360nm and a characteristic peak at 520 nm. Prepared Au-g-C3N4Contains two characteristics, prove Au-g-C3N4The synthesis is successful.
FIG. 3D verifies Au-g-C3N4ECL characteristics of (A), Au-g-C3N4Has an ECL signal intensity (curve b) greater than g-C3N4(curve a), which benefits from the excellent conductivity of the Au NPs. The CV curve also verifies Au-g-C3N4(Curve b') with a monocomponent g-C3N4(curve a') has excellent conductivity.
3)PEI-Ru@Ti3C2Characteristics of @ AuNPsAnalysis of
FIG. 4A is a single layer of Ti3C2TEM characterization of (d). As can be seen from FIG. 4B, AuNPs are uniformly dispersed in Ti3C2No aggregation appeared, indicating that Ti was present3C2As an excellent carrier, the AuNPs can be prevented from aggregating. As can be seen from FIG. 4C, the AuNPs have a diameter of 4.5. + -.1 nm. PEI-Ru @ Ti characterized by EDX3C2The @ AuNPs nanocomposite shows element peaks of C, N, O, Ti, Au and Ru (FIG. 4D), which indicates that PEI-Ru @ Ti3C2The synthesis of the @ AuNPs nano composite material is successful.
4) Characterization of TDNAs
Polyacrylamide gel electrophoresis (PAGE) was used to demonstrate the construction of TDNAs, as shown in figure 5A. Lanes 1 to 4 show the construction of single stranded S1 to four stranded TDNAs. As can be seen from the gel, the corresponding bands are further back with increasing number of DNA strands, which is consistent with the expected results. The TDNA was characterized by AFM, and the height of TDNAs was 5. + -. 0.2nm, which was almost the same as the theoretical value of 5.27 nm. This further demonstrates the successful construction of TDNAs.
5) Entropy-driven and biped DNA Walker characterization
PAGE was used to verify entropy-driven reactions, and bands of Scaffold, Blocker and Bandrege DNA are shown in lanes 1, 2 and 3, respectively, as shown in FIG. 5B.
SARS-COV-2RdRp was introduced in the Substrate system and with the aid of Scaffold the Substrate (Substrate) was converted to Intermediate 2 and Bandage DNA, two new bands being visible in lane 5.
Waste was finally generated by introducing into the system Substrate, SARS-COV-2 (a single-stranded DNA molecule having a nucleotide sequence shown in SEQ ID NO.13) RdRp and Fuel, and Banpage and Blocker were observed in lane 6, but SARS-COV-2RdRp did not participate in the reaction.
FIG. 5C reflects the structure of the biped DNA Walker. When no band DNA was present in the system, S1 and S2 could not bind to each other (lane 3), and the position of the band coincided with that of a single S1 (lane 1) or S2 (lane 2). However, when the DNA forming both feet is present in the system as Banpage, S1 and S2, the molecular weight is large and the resulting complex band is very slow (lane 4). Therefore, it was confirmed that by introducing the band DNA, the biped DNA Walker was successfully constructed.
6) Characterization of step-and-assemble ratio-based ECL biosensors
In fig. 6A, CV was used to characterize the construction process of the ratiometric ECL biosensor. The bare electrode (curve a) maintains the maximum current peak. When Au-g-C3N4(curve b) the current peak decreases as it deposits on the electrode surface. Then, TDNAs (curve c) and MCH (curve d) are continuously modified on the electrode, and the current peak is continuously reduced to show that the conductivity of the adsorbed substances on the surface of the electrode is poor. When SARS-COV-2RdRp and Nb. BbvCI were introduced (curve e), the electrode surface completed the DNA Walker walking process. Thus, TDNAs is cut off and peak current increases. Finally, to PEI-Ru @ Ti3C2Specific binding of the @ AuNPs-S7 probe (curve f) was modified and the peak current increased with the modified electrode.
The EIS shown in fig. 6B characterizes the construction process of the ratiometric ECL biosensor. The resistance value of the bare electrode (curve a) is minimal. Continuous modification of Au-g-C3N4(curve b), TDNAs (curve c), MCH (curve d), the impedance value gradually increases. When SARS-COV-2RdRp and Nb. BbvCI (curve e) appear, the TDNA of the surface is clipped and the impedance value is slightly reduced. Finally, PEI-Ru @ Ti with good conductivity is added3C2The @ AuNPs-S7 probe (curve f) was modified on the electrode to give a decrease in impedance.
Figure 6C shows ECL responses characterizing electrodes modified with different substances. Soaking the electrode in solution containing 0.1M S2O8 2-In PBS solution (b). The bare electrode (curve a) has no ECL response. Fixing Au-g-C on the surface of the electrode3N4(curve b), has good luminescence properties, and exhibits a higher ECL signal. When TDNA (curve c) and MCH (curve d) with poor conductivity were modified on the electrode surface, the ECL signal gradually decreased. Subsequently, SARS-COV-2RdRp was introduced (curve e) and the ECL signal slightly recovered. Finally, PEI-Ru @ Ti3C2@AuNPThe S-S7 probe (curve f) was linked to TDNAs and the ECL signal decreased.
The success of the construction of the ratiometric ECL biosensor can be confirmed by the above three methods.
7) Optimizing analysis conditions
In order to obtain the best performance of the rate-based ECL biosensor, several experimental parameters were optimized, including the concentration of DNA Walker S1 and S2, the amount of nb.bbvci used, the digestion time of nb.bbvci, and the incubation time of the entropy-driven reaction. The results show that as the concentration of S1 and S2 increased, the ECL signal gradually decreased and tended to stabilize at 60 nM. The ECL curve tends to stabilize when the nb. The optimal digestion time for nb. The optimal time for the entropy-driven reaction was 40min (compare MCH/TDNAS/Au-g-C)3N4The optimal time for the incubation of the/GCE in the entropy-driven reaction mixture at 37 ℃ was 40 min). Thus, the concentrations of S1 and S2 of DNA walker were chosen to be 60nM, 10U and 60min were the optimum amount of Nb. BbvCI and the optimum digestion time, and the optimum entropy-driven reaction time was 40 min.
Mechanism of three, ratio ECL biosensor
Based on entropy-driven and DNA walker's cyclic amplification strategy, this example synthesizes a ratiometric ECL biosensor for detecting the SARS-COV-2RdRp gene. The principle of the entropy-driven and DNA walker amplification strategy is shown in FIG. 1. The SARS-COV-2RdRp gene acts as a trigger to initiate the entropy-driven reaction in the upper left corner. SARS-COV-2RdRp can bind to Scaffold by means of a substrate (consisting of three strands of Scaffold, Blocker, Bandage) to form Intermediate 1. At this time, the binding force between the band DNA and the Scaffold is weak, the band DNA is released, and the Intermediate 1 is converted into the Intermediate 2. The exposed single-stranded "toe" -like part of Intermediate 2 (called toehold) binds to Fuel, generating double-stranded Waste, Blocker and SARS-COV-2RdRp genes as catalysts, stimulating the next cycle.
After many cycles, a large amount of band DNA is output. Subsequently, the S1 strand, the S2 strand and the band were combined into a bipedal DNA Walker to initiate the DNA Walker reaction. Initially, at Au-g-C3N4TDN modification on surface of/GCEA, to obtain a strong ECL signal at 460 nm. Bipedal DNA Walker can spontaneously walk on hairpins at the top of TDNAs with the energy of nb. Thus, a large amount of the hairpin at the top of TDNAs was removed and reacted with PEI-Ru @ Ti3C2The @ AuNPs-S7 probe binds specifically. By ECL-RET, the ECL signal at 460nm is quenched, producing an ECL signal at 620 nm. The SARS COV-2RdRp gene can be quantitatively detected according to the reduction degree of ECL signal at 460nm and the increase degree of ECL signal at 620 nm. The ratio-based ECL biosensor effectively detects the SARS-COV-2RdRp gene by combining two signal amplification strategies.
EXAMPLE 2 detection of SARS-COV-2RdRp Using a ratiometric ECL biosensor
1. The SARS-COV-2RdRp gene was detected using the rate-type ECL biosensor obtained in example 1 under the optimum conditions found in example 1 (see the first in example 1 for the detection method). FIG. 7A depicts that as the concentration of SARS-COV-2RdRp increases from 10aM to 10pM, the ECL signal intensity decreases at 460nm and increases at 620 nm. Fig. 7A also depicts that the change in ECL signal intensity at 460nm has a very good linear relationship, i.e., y-502.82 lgc (rdrp) -986.17, R20.9944. The change in ECL signal intensity at 620nm also shows a good linear relationship of y-805.79 lgc (rdrp) +14704.28, R20.9950. To obtain higher reliability, the log values of ECL (460nm)/ECL (620nm) with increasing concentration of SARS-COV-2RdRp were evaluated, as shown in FIG. 7B. From the detection Limit (LOD) of 3 σ/k, the LOD was calculated to be 7.8 aM.
2. Selectivity and stability of a ratiometric ECL biosensor (of example 1)
To evaluate the selectivity of the ratiometric biosensors, some interference was introduced. When the entropy-driven reaction mixture detected by the ratio-type ECL biosensor contains 1pM of SARS-COV-2RdRp, 100pM of SARS-COV RdRp, 100pM of random DNA (single-stranded DNA molecule having a nucleotide sequence shown in SEQ ID NO. 7) and a blank (the detection method is described in the first in example 1), respectively. As can be seen from FIG. 7D, the ratio-based ECL biosensor treated with SARS-COV-2RdRp showed a clear difference compared to the ratio-based ECL biosensor treated with other interferents. Therefore, the constructed ratio-type ECL biosensor has excellent selectivity. To evaluate the stability of the ratiometric ECL biosensor, the concentration of 10pM SARS-COV-2RdRp (SEQ ID NO.13) contained in PBS was measured using the ratiometric ECL biosensor and scanned for 16 cycles in succession. As can be seen from FIG. 7C, the ECL signal is still stable at 460nm with a Relative Standard Deviation (RSD) of 2.34%, and the ECL signal is stable at 620nm with an RSD of 3.61%.
3. Applicability of ratiometric ECL biosensors in real sample analysis
Biosensors are necessary for early clinical detection. Recovery experiments were performed in 50-fold dilutions of human serum as shown in table 2. The ratio type ECL biosensor detects serum containing SARS-COV-2RdRp at different concentrations (the detection method is shown as the first in example 1), and the recovery value is 92.00-103.56%, and the RSD is 1.51-4.30%. The results indicate that the ratio-type ECL biosensor can be used for detection of actual samples.
TABLE 2 detection recovery of SARS-COV-2RdRp in 50 fold diluted human serum.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
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Claims (9)
1. A probe set, wherein the probe set comprises:
the first probe is a tetrahedral probe formed by 4 single-stranded DNA molecules, and the nucleotide sequence of the 4 single-stranded DNA molecules is shown in SEQ ID NO.3-SEQ ID NO. 6;
the second probe is obtained by complementary hybridization of 2 single-stranded DNA molecules, and the nucleotide sequences of the 2 single-stranded DNA molecules are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the third probe is 1 single-stranded DNA molecule, and the nucleotide sequence is shown in SEQ ID NO. 7.
2. The probe set of claim 1, further comprising a fourth probe, wherein the fourth probe is a single-stranded DNA molecule obtained by complementary hybridization of 3 single-stranded DNA molecules, and the nucleotide sequence of the 3 single-stranded DNA molecules is represented by SEQ ID No.8-SEQ ID No. 10; optionally, the probe set further comprises a fifth probe, wherein the fifth probe is 1 single-stranded DNA molecule, and the nucleotide sequence is shown in SEQ ID No. 11.
3. A reagent or kit for detecting SARS COV-2RdRp comprising a probe set according to claim 1.
4. A biosensor comprising the first probe of claim 1.
5. A method for preparing a biosensor, comprising the steps of:
s1: modifying the glassy carbon electrode with Au-g-C3N4 to obtain Au-g-C3N 4/GCE;
s2: TDNAs is fixed on the surface of Au-g-C3N4/GCE through a gold-sulfur bond; the TDNAs is the first probe of claim 1.
6. The method of claim 6, further comprising the step of soaking Au-g-C3N4/GCE in the MCH-containing solution.
7. A method for detecting SARS COV-2RdRp, which comprises the step of detecting the SARS COV-2RdRp gene using the probe set, the reagent or the kit, the biosensor, and the biosensor prepared by the above method.
8. The method according to claim 7, characterized in that it comprises in particular the steps of:
1) incubating the biosensor prepared according to claim 5 or 6 in a test solution; optionally, the incubation time is more than 40 min; the incubation temperature was 37 ℃;
2) adding a solution containing a second probe and an endonuclease into the mixed solution incubated in the step 1); optionally, the concentration of the 2 single-stranded DNA molecules in the second probe is 60-100 nM; the endonuclease is Nb.BbvCI, and the dosage of the endonuclease is 10-100U; the digestion time of the endonuclease is 60min-100 min;
3) adding a third probe modified with a PEI-Ru @ Ti3C2@ AuNPs material into the mixed solution obtained in the step 2);
optionally, the solution to be detected comprises Substrate and Fuel;
a hybridization complex formed by a single-stranded DNA molecule represented by SEQ ID NO.8 to SEQ ID NO. 10;
fuel is a single-stranded DNA molecule shown in SEQ ID NO. 11.
9. The method of manufacturing of claim 5, or the method of claim 7 or 8, wherein the biosensor is a ratiometric ECL biosensor.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263725A (en) * | 2014-09-19 | 2015-01-07 | 上海市计量测试技术研究院 | Spire tetrahedral DNA nano-structural probe and telomerase electrochemical detection |
| CN107254550A (en) * | 2017-05-25 | 2017-10-17 | 重庆医科大学 | A kind of spr sensor of detection HIV related genes and its preparation and application |
| CN111505284A (en) * | 2020-04-23 | 2020-08-07 | 中国科学院重庆绿色智能技术研究院 | Test paper strip and sensor for detecting novel coronavirus SARS-CoV-2, preparation and application thereof |
| CN112649479A (en) * | 2020-11-25 | 2021-04-13 | 重庆医科大学 | Multi-clamp-string cascade assembly construction universal electrochemical biosensor ultrasensitive detection target based on assistance of tetrahedral tripod |
-
2021
- 2021-05-13 CN CN202110524577.2A patent/CN113278683B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263725A (en) * | 2014-09-19 | 2015-01-07 | 上海市计量测试技术研究院 | Spire tetrahedral DNA nano-structural probe and telomerase electrochemical detection |
| CN107254550A (en) * | 2017-05-25 | 2017-10-17 | 重庆医科大学 | A kind of spr sensor of detection HIV related genes and its preparation and application |
| CN111505284A (en) * | 2020-04-23 | 2020-08-07 | 中国科学院重庆绿色智能技术研究院 | Test paper strip and sensor for detecting novel coronavirus SARS-CoV-2, preparation and application thereof |
| CN112649479A (en) * | 2020-11-25 | 2021-04-13 | 重庆医科大学 | Multi-clamp-string cascade assembly construction universal electrochemical biosensor ultrasensitive detection target based on assistance of tetrahedral tripod |
Non-Patent Citations (2)
| Title |
|---|
| ZHENQIANG FAN等: "Entropy-driven amplified electrochemiluminescence biosensor for RdRp gene of SARS-CoV-2 detection with self-assembled DNA tetrahedron scaffolds", BIOSENSORS ANDBIOELECTRONICS, vol. 178, pages 113015 * |
| ZHENQIANG FAN等: "Rational engineering the DNA tetrahedrons of dual wavelength ratiometric electrochemiluminescence biosensor for high efficient detection of SARS-CoV-2 RdRp gene by using entropy-driven and bipedal DNA walker amplification strategy", CHEMICAL ENGINEERINGJOURNAL, vol. 427, pages 131686 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114965638A (en) * | 2022-04-29 | 2022-08-30 | 南京邮电大学 | Ratio type biosensor with internal reference signal and preparation method |
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