1. A probe set comprising a nucleotide sequence set forth as SEQ ID: a hairpin probe represented by NO. 1; the nucleotide sequence is shown as SEQ ID: a hairpin probe represented by NO. 2; the nucleotide sequence is shown as SEQ ID: the hairpin probe shown in NO. 3.

2. A kit comprising a set of probes according to claim 1, each probe being packaged separately.

3. A system for detecting SARS-CoV-2RNA comprising the probe set of claim 1, an ECL biosensor and a CRISPR/Cas12a system.

4. The system of claim 3, wherein the CRISPR/Cas12a system comprises a CRISPR-Cas12a protein and a nucleotide sequence set forth in SEQ ID: a gRNA shown in NO. 4.

5. The system of claim 3 or 4, wherein the ECL biosensor is prepared by a method comprising the steps of:

1) modifying GCE by Nafion through electrostatic adsorption to obtain Nafion/GCE;

2) modifying a metal-organic framework material on Nafion/GCE through electrostatic adsorption to obtain Ru-PEI/Au @ ZIF-8/Nafion/GCE;

3) and modifying the C3N4 to Ru-PEI/Au @ ZIF-8/Nafion/GCE to obtain the ECL biosensor.

6. The system of claim 5, wherein the metal-organic framework material is ZIF-8 selective and the metal-organic framework material is ZIF-8 doped with gold modified with self-enhancing ruthenium complex.

7. An ECL biosensor prepared according to the method of claim 5 or 6.

8. A method for detecting SARS-CoV-2, which comprises the step of detecting SARS-CoV-2RNA using the system for detecting SARS-CoV-2RNA according to any one of claims 3 to 7.

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

1) containing Mg²⁺Reacting the solution of the DSN and H1 hairpin probe with the solution to be tested with different concentrations at 60 +/-5 ℃ for more than 75 minutes to obtain reaction solution 1; the nucleotide sequence of the hairpin probe of H1 is shown in SEQ ID: shown as NO. 1;

2) inactivating the DSN enzyme to obtain a reaction solution 2;

3) sequentially adding equimolar H2 hairpin probe and H3 hairpin probe into the reaction solution 2; keeping the reaction solution at 4 ℃ for more than 16 hours to obtain a reaction solution 3 containing H2/H3 double chains; the nucleotide sequence of the hairpin probe of H2 is shown in SEQ ID: NO. 2; the nucleotide sequence of the hairpin probe of H3 is shown in SEQ ID: NO. 3;

4) mixing the Cas12a protein, gRNA and the reaction solution 3, and incubating at room temperature to obtain a reaction solution 4; the nucleotide sequence of gRNA is shown in SEQ ID: NO. 4;

5) adding the Fc-labeled DNA probe into the reaction solution 4, and reacting to obtain a final reaction solution; the nucleotide sequence of the DNA probe is shown as SEQ ID: NO. 9;

6) dropwise adding the final reaction solution to the surface of the ECL biosensor as described in any of claims 3-5.

10. Use of the probe set according to claim 1, the kit according to claim 2, the system according to any one of claims 3 to 7 for the detection of SARS-CoV-2 RNA.

Background

The 2019 coronavirus disease (COVID-19) caused by a novel coronavirus (Severe acute respiratory syndrome associated coronavirus 2, SARS-CoV-2) has posed a significant threat and challenge worldwide (Lukas et al, 2020). Due to its high infectivity, high morbidity and high mortality, it causes enormous harm and loss to global public health and world economic activities, cumulatively depriving more than one million people worldwide. At present, early diagnosis of this virus and development of antiviral vaccines and drugs provide the main methods for inhibiting the SARS-CoV-2 pandemic. Particularly, with the emergence of viral mutations, the need for early and rapid diagnosis of viruses is continuing and urgent. Reverse transcription polymerase chain reaction (qRT-PCR) based SARS-CoV-2 detection methods often require the extraction of RNA from viral samples by complex steps. The whole experimental process usually requires 6 hours, which can not achieve the goal of real-time diagnosis and also affects the efficiency of virus detection.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems, i.e. CRISPR/Cas, are revolutionary powerful genome editing tools, only a small segment of RNA sequence is needed, and the workload and cost of new protein synthesis are greatly reduced by anchoring the position of a new gene by utilizing the interaction between RNA and DNA. Cas proteins such as Cas9, Cas12a, Cas13 and Cas14 can non-specifically hydrolyze single-stranded DNA or RNA, which is called trans-cleavage property, and shows great potential in molecular diagnostics. When Cas12a/crRNAs complexes recognize the target dsDNA, they exhibit trans-cleavage properties similar to non-specific hydrolysis of surrounding ssDNA by single-stranded deoxyribonuclease (ssDNase). However, application in point-of-care diagnostics is limited by large and expensive optical equipment. Therefore, it has become very important and relevant to develop detection application methods in other modes that do not rely on expensive optical instruments.

Electrochemiluminescence (ECL) is a combination of traditional Electrochemistry (EC) and chemiluminescence (EL) with fast reaction time and low background informationHigh sensitivity and the like, and has wide application prospect in the analysis of nucleic acid, protein and small biological molecules. Common ECL biosensing systems require an ECL reagent and a co-reactant, which together constitute the signal output of the system. However, since the ECL reagent and the co-reactant are separated from each other, an electron transfer distance is increased, which causes problems in that the reagent is consumed seriously and the ECL strength is affected. Based on the above considerations, intramolecular ECL reagents have been designed to improve electron energy loss and reagent consumption. For example, Sun et al, Ru (bpy) by constructing an intermolecular amide bond₃ ²⁺The derivative is connected with a coreactant tripropylamine to form a novel ECL emitter, and the ECL performance and the emission efficiency are improved. Furthermore, Gui et al teach a branched polymer polyethylene (BPEI) and Ru (bpy)₃ ²⁺The self-strengthening compound connected by covalent bonds improves the defects that the traditional ECL emission reagent is easy to diffuse into water and the ECL strength of intermolecular reaction is insufficient. The Polyethyleneimine (PEI) serving as an active core reagent can improve the ECL strength of Ru (bpy)32+, so that the stable ECL emission reagent and PEI intramolecular reaction system is constructed, and the reagent loss and the ECL emission efficiency are improved.

Metal Organic Framework (MOF) materials, also known as metal organic coordination polymeric materials, are composites formed from inorganic metals bound to organic ligands through covalent bonds. The structure of the material has network-shaped pores, and the material has important application in gas adsorption, material transportation, biocatalytic reaction, cell imaging and other fields. The zeolite imidazole framework-8 (ZIF-8), which is constructed of zinc ions and imidazole ligands, has been widely used in the biomedical and catalytic fields due to its simple synthesis, large specific surface area, and excellent performance in photocatalysis. It is at high concentration of H⁺Or is unstable under GSH and can be used as a drug carrier in the tumor microenvironment. In addition, it is widely used for loading of nanomaterials because it can prevent aggregation of nanomaterials and improve distribution of nanomaterials to improve optical, electrical and catalytic properties thereof.

Catalytic Hairpin Assembly (CHA), an enzyme-free signal amplification technique, has shown excellent signal amplification effects. Recently, it has been widely used for bioassays and greatly improves the sensitivity of biosensing systems. In the universal CHA reaction, the two hairpin-structured DNA strands have complementary sequences on the hairpin handles. In the presence of single-stranded nucleic acid, toe-mediated strand displacement (TMSD) is triggered, opening the two hairpin-structured DNA strands, and then rapidly forming a thermodynamically stable DNA double strand. In particular, by programming the DNA sequence, the double stranded DNA (dsDNA) formed by a first CHA reaction can trigger another CHA reaction to form new dsDNA for signal cascade amplification. micro-RNA (miRNA) is a non-coding single-stranded DNA well suited for triggering the CHA reaction and for miRNAs detection (Si et al, 2020). However, to achieve higher sensitivity, miRNAs were converted to DNA strands using double strand specific nucleases (DSNs) as trigger strands to overcome the limitations of degradation of miRNAs.

Disclosure of Invention

Therefore, to overcome the disadvantages of the prior art that the detection of SARS-CoV-2 antibody in serum can be used to diagnose coronavirus infection after the onset of symptoms, but is limited because of the sufficient amount of detectable antibody only at the late stage of infection. The invention provides a method for quickly and sensitively detecting SARS-CoV-2, a probe set, a system for detecting SARS-CoV-2RNA and an ECL biosensor.

The invention provides a probe set, which comprises a nucleotide sequence shown as SEQ ID: a hairpin probe represented by NO. 1; the nucleotide sequence is shown as SEQ ID: a hairpin probe represented by NO. 2; the nucleotide sequence is shown as SEQ ID: the hairpin probe shown in NO. 3.

The kit comprises the probe sets, and each probe is independently packaged.

A system for detecting SARS-CoV-2RNA comprising a probe set and a CRISPR/Cas12a system.

Optionally, an ECL (electrochemiluminescence) biosensor is also included.

Optionally, the CRISPR/Cas12a system includes a CRISPR-Cas12a protein and a nucleotide sequence set forth in SEQ ID: a gRNA shown in NO. 4.

Optionally, the preparation method of the ECL biosensor comprises the following steps:

1) modifying GCE by Nafion through electrostatic adsorption to obtain Nafion/GCE;

2) modifying a metal-organic framework material on Nafion/GCE through electrostatic adsorption to obtain Ru-PEI/Au @ ZIF-8/Nafion/GCE;

3) and (3) dropwise adding the C3N4 onto Ru-PEI/Au @ ZIF-8/Nafion/GCE to obtain the ECL biosensor.

Optionally, the metal-organic framework material is zif-8; the metal-organic framework material is gold-doped ZIF-8 modified with self-reinforced ruthenium compound

A method for detecting SARS-CoV-2, comprising the step of detecting SARS-CoV-2 using the above-mentioned system for detecting SARS-CoV-2 RNA.

Optionally, the method specifically comprises the following steps:

1) containing Mg²⁺The solution of the DSN and H1 hairpin probe (solution A) and the SARS-CoV-2RNA solution (solution B) with different concentrations react for more than 75 minutes at the temperature of 60 plus or minus 5 ℃ to obtain reaction solution 1; the nucleotide sequence of the hairpin probe of H1 is shown in SEQ ID: shown as NO. 1;

optionally, Mg in solution A²⁺Concentration of 10mM, concentration of DSN of 0.05U μ/L, H1 hairpin probe concentration of 5 μ M;

the volume parts of the solution A and the solution B are both 6;

2) inactivating the DSN enzyme to obtain a reaction solution 2; 3) adding a solution (solution C) containing equimolar H2 hairpin probe and H3 hairpin probe into the reaction solution 2 in sequence; keeping the reaction solution at 4 ℃ for more than 16 hours to obtain a reaction solution 3 containing H2/H3 double chains; the nucleotide sequence of the hairpin probe of H2 is shown in SEQ ID: NO. 2; the nucleotide sequence of the hairpin probe of H3 is shown in SEQ ID: NO. 3; optionally, the volume part of the solution C is 3;

4) mixing the Cas12a protein, gRNA and the reaction solution 3, and incubating at room temperature to obtain a reaction solution 4; the nucleotide sequence of gRNA is shown in SEQ ID: NO. 4; specifically, 25 parts by volume of a mixture containing 50nM Cas12a, 50nM gRNA, and 10U RNase inhibitor was mixed with 25 parts by volume of reaction solution 3;

5) adding an Fc-labeled DNA probe into the reaction solution 4;

6) the reaction solution 4 was dropped onto the surface of the ECL biosensor.

Optionally, step 2) further comprises the step of inhibiting the enzymatic activity of the DSN.

The 9 th to 29 th positions of the H1 hairpin probe are circular regions, and the rest are stalk regions;

the 31 st to 51 th positions of the H2 hairpin probe are circular regions, and the rest are stalk regions;

the 16 th to 35 th positions of the H3 hairpin probe are circular regions, and the rest are stalk regions;

the application of the probe group, the kit and the system in the detection of SARS-CoV-2 RNA. The method or application is a non-disease diagnostic treatment method or application.

The technical scheme of the invention has the following advantages:

1. the present invention provides a probe set which can be used for detecting SARS-CoV-2 RNA.

2. The invention provides a system for detecting SARS-CoV-2RNA, which links self-enhanced ruthenium compound on the surface of ZIF-8, and obtains high intramolecular electron transfer efficiency under the condition of not adding co-reactant, thereby obtaining strong and stable ECL signal. The target circulation and CHA signal amplification which are participated in by the DSN not only realize the signal cascade amplification, but also convert unstable target RNA into stable dsDNA for output. The dsDNA in this strategy can activate the trans-cleavage property of Cas12a, cleaving Fc-labeled DNA probes that exhibit affinity to C3N 4. Therefore, the concentration of target RNA can determine the number of Fc-labeled DNA probes adsorbed on the surface of C3N4, thereby directly affecting ECL intensity. The SARS-CoV-2RNA detection strategy provided by the invention has good specificity and repeatability, can quickly detect the target RNA in human serum diluent, and has hope for clinical and biochemical analysis.

3. The invention synthesizes ZIF-8, then modifies the amino group on the surface of the ZIF-8, and couples the modified ZIF-8 with a self-reinforced Ru-PEI complex to obtain Ru-PEI/ZIF-8, wherein PEI is an active substance, has the characteristic of enhancing the ECL strength of Ru, and is beneficial to improving the emission intensity and efficiency of ECL. Meanwhile, in order to improve the charge transfer efficiency on the electrode and enhance the conductivity of the ECL emitter, gold nanoparticles are modified on the surface of the Ru-PEI @ ZIF-8, so that an excellent Ru-PEI/Au @ PEI emitting material is formed on the surface of the electrode and is used for biological induction.

4. The combination of the DSN enzyme and the CHA reaction greatly improves the sensitivity of miRNAs detection in biological samples and avoids reprogramming of the DNA sequence of the CHA reaction.

Drawings

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(A) is a TEM image of ZIF-8 in example 1;

FIG. 1(B) is a TEM image of Ru-PEI/Au @ ZIF-8 in example 1;

FIG. 1(C) is an element map of Ru-PEI/Au @ ZIF-8 (Ru, Zn, N, and Au,)

FIG. 2(A) is an XPS spectrum of Ru-PEI/Au @ ZIF-8 of example 1 with the inset showing gold 4f magnified in FIG. 2A;

FIG. 2(B) is the EDS spectrum of Ru-PEI/Au @ ZIF-8 of example 1;

FIG. 3(A) EIS spectra in example 2, which characterize the ECL biosensor assembly process and SARS-CoV-2RNA detection; impedance (Ω) in real part of abscissa, impedance (Ω) in imaginary part of ordinate; the inset in FIG. 3A is the analog circuit of a modified GCE (ECL biosensor);

in fig. 3 (a):

(a)Nafion/bare GCE,

(b)Ru-PEI/[email protected]/Nafion/bareGCE,

(c)C₃N₄/Ru-PEI/[email protected]/Nafion/bare GCE,

(d) fc labeled DNA probe/Ru-PEI/Au @ ZIF-8/Nafion/bare GCE;

(e) target (100fM) reaction solution/Ru-PEI/Au @ ZIF-8/Nafion/bare GCE,

(f) target (1pM) reaction solution/Ru-PEI/Au @ ZIF-8/Nafion/bare GCE;

FIG. 3(B) ECL reaction characterization of biosensor assembly process and SARS-CoV-2RNA detection; ECL intensity denotes ECL intensity; in fig. 3 (B):

(a)Nafion/bare GCE,

(b)Ru-PEI/[email protected]/Nafion/bareGCE,

(c)C3N4/Ru-PEI/[email protected]/Nafion/bare GCE,

(d) fc labeled DNA probe/Ru-PEI/Au @ ZIF-8/Nafion/bare GCE;

(e) target (100fM) reaction solution/Ru-PEI/Au @ ZIF-8/Nafion/bare GCE,

(f) target (1pM) reaction solution/Ru-PEI/Au @ ZIF-8/Nafion/bare GCE;

the inset in FIG. 3B is an ECL-potential curve corresponding to the ECL-time curve in FIG. 3B; potential represents a potential;

FIG. 4(A) ECL-time curves for SARS-CoV-2RNA detection at a range of concentrations (1fM,10fM,20fM,100fM,200fM,1pM,2pM,10pM,20pM, and 100pM, from a to j) in example 3; ECL intensity denotes ECL intensity;

FIG. 4(B) is a mathematical relationship between the intensity of ECL and the log value of SARS-CoV-2RNA concentration in example 3; ECL intensity denotes ECL intensity;

FIG. 4B is an inset of a linear relationship between ECL intensity and the logarithmic value of the boxed target (SARS-CoV-2 RNA concentration) in FIG. 4B;

figure 5 ECL performance of the ECL biosensor of example 4 under the following conditions:

(A) detecting target RNA (1pM) or other non-specific RNA (100pM) for verifying the specificity of ECL biosensors; ECL intensity denotes ECL intensity;

(B) replacing the gRNA in example 2 with grnas with mutation sites (gRNA1, gRNA2, and gRNA3) for verification of the specificity of the ECL biosensor; ECL intensity denotes ECL intensity;

(C) detecting 1pM of target RNA in a 25 cycle constant potential scan to characterize the stability of the ECL biosensor; ECL intensity denotes ECL intensity;

FIG. 6 is a schematic of the detection of SARS-CoV-2RNA using CRISPR-Cas12a trans-cleavage properties and an ECL biosensor.

Detailed Description

Materials and reagents

Related nucleic acids for detection of SARS-CoV-2RNA were purified by polyacrylamide gel electrophoresis (PAGE), both from Genscript Biotechnology Inc. (Nanjing, China), and the sequences are listed in Table 1.

Analytical grade chemical Zinc acetate dehydrating agent (Zn (AC)₂-2H₂O), 2-methylimidazole, ethylenediaminetetraacetic acid (EDTA), 3-aminopropyltriethoxysilane, N- (3- (dimethylamine) propyl) -N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium borohydride (HAuCl)₄) And organic solvents such as methanol, N-Dimethylformamide (DMF) were purchased from Aladdin Biotechnology Ltd. Double-stranded specific nucleases (DSN) and C3N4 were from EVROGEN (beijing, china) and XFNANO (south beijing, china), respectively. CRISPR-Cas12a protein and tris (4,4 '-dicarboxylic acid-2, 2' -bipyridine) ruthenium dichloride (Ru (dcbpy)₃ ²⁺) From the new england biological laboratory (NEB, shanghai, China) and the Suna Tech Inc (Suzhou, China), the triborate-edta (tbe) buffer and the Phosphate Buffered Saline (PBS), respectively, were purchased from Sangon biotechnology limited. The water in all experiments was purified by a Milli-Q purification system (Branstead) and maintained at a resistance of 5M Ω.

TABLE 1 sequences used in the following examples.

TABLE 2

miRNA 133a	agc ugg uaa aau gga acc aaa u(SEQ ID：NO.10)
		miRNA 499	uua aga cuu gca gug aug uuu(SEQ ID：NO.11)
miRNA 208b	aag cuu uuu gcu cga auu aug u(SEQ ID：NO.12)
		miRNA 328	cug gcc cuc ucu gcc cuu ccg u(SEQ ID：NO.13)

Instrument and test method

Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) were used to illustrate the electrochemical activity describing the energy and mass transfer during electrode modification, and these measurements were performed by CHI 660E (shanghai chenhua instruments ltd).

EIS experiments were conducted by setting parameters in a medium containing 5mM [ Fe (CN)₆]^3-/4-In PBS solution (0.1M, pH 7.4), electrodes of different modification states were scanned with an amplitude of 5mV at a frequency of 0.1 to 10 kHz.

The entire ECL test used a three electrode system provided by the national focus laboratory of life science analytical chemistry (university of tokyo) with the working electrode being a (glassy carbon) electrode (GCE), the reference electrode being an Ag/AgCl electrode and the counter electrode being a platinum electrode. The characterization of a Transmission Electron Microscope (TEM) was performed with 1400PLUS (Japanese Electron).

UV-Vis Spectroscopy was used to describe the synthesis of Ru-PEI/Au @ ZIF-8 using Spectra Max M5e (Molecular Devices Co. Ltd.).

X-ray photoelectron spectroscopy (XPS) characterization was obtained from Thermo Fisher K-Alpha.

Example 1

1.1 Synthesis of Ru-PEI/Au @ ZIF-8

1.1.1 Synthesis of ZIF-8

1) Dehydrating agent of zinc acetate (Zn (AC)₂-2H₂O) heating to 55 ℃ and holding for 45 minutes to remove water from the crystals to obtain Zn (AC)₂Powder;

2) reduction of Zn (AC)₂The powder (200mg) and 2-methylimidazole (295mg) were dissolved in methanol (22 ml). The mixture was then diluted with water under continuous stirring to give Zn (AC) in final concentrations of 1mmol each₂And 3.3mmol of 2-methylimidazole. When the mixture became milky white, it was aged at room temperature for one day with stirring.

3) The mixture was centrifuged, washed repeatedly with methanol and water, and then dried in a thermostat to give a white powder, ZIF-8, for further modification.

Amino modification of ZIF-8 surface, comprising the steps of:

1) sonicate 1.5mg of ZIF-8 into 20 ml of N, N-Dimethylformamide (DMF) to obtain a uniformly dispersed suspension;

2) to the suspension was added 150 μ L of 3-Aminopropyltriethoxysilane (APTES) with continuous stirring and heating was maintained at 125 ℃ for 20 minutes to form amino-modified ZIF-8;

3) the suspension was centrifuged at 12000rpm and washed three times with DMF to give NH₂-ZIF-8。

1.12 Synthesis of self-reinforced Ru-PEI complexes the following procedure was followed:

1) 10mg of Ru (dcbpy)₃ ²⁺Dissolved in 2mL of ultrapure water to give 5mg/mL of Ru (dcbpy)₃ ²⁺An aqueous solution.

2) In Ru (dcbpy)₃ ²⁺The aqueous solution was added to EDC and NHS at final concentrations of 15mg/mL and 3mg/mL, respectively, to activate Ru (dcbpy)₃ ²⁺A carboxyl group of (2).

3) Ru (dbbpy) activated 30 minutes after activation₃ ²⁺And reacting with 2mL of PEI solution with the mass fraction of 1% for 2.5 hours to form a self-reinforced Ru-PEI complex solution.

1.1.3 synthetic method of Ru-PEI/Au @ ZIF-8 is as follows:

1) the self-reinforced Ru-PEI complex solution formed in 4.5mL L1.1.2, 30mg EDC and 5mg NHS were added to 10mL of prepared NH₂ZIF-8(30mM), and stirred at room temperature for 4 hours, and then added to the mixture a final concentration of 0.01% (mass percent) of uric chloride acid and sodium citrate (equimolar to uric chloride acid).

2) To the mixture of step 1), 100mM HAuCl was added with stirring₄To reduce Au³⁺And reacted for 4 hours with stirring to ensure intercalation and adsorption of Au on Ru-PEI @ ZIF-8.

3) The product (Ru-PEI/Au @ ZIF-8) was centrifuged, washed with ultrapure water and dispersed in 10mL of ultrapure water for further use.

1.2 characterization of Ru-PEI/Au @ ZIF-8

First, the synthesis of Ru-PEI/Au @ ZIF-8 is illustrated by TEM. FIG. 1A is a TEM image of ZIF-8, and it can be seen that ZIF-8 exhibits dodecahedral particles with a uniform particle distribution of about 200nm in size. With the modification of amino groups on the surface of ZIF-8, loading Ru-PEI and Au nanoparticles, as shown in FIG. 1B, it was found that the surface of the regular geometric particles became blurred, which was attributed to the adsorption of Ru-PEI on the surface and Au³⁺And (4) reducing the product. To further verify this conclusion, this example discusses the elements on the surface of the nanoparticles, and the elemental map (FIG. 1C) shows that the ZIF-8 with Zn as the core element constitutes the adsorption carrier for Ru-PEI with Ru as the basic element and Au nanoparticles with Au as the basic element, which also concludes that Ru-PEI/Au @ ZIF-8 was synthesized according to the synthetic route designed in this example.

This example also performed elemental characterization of Ru-PEI/Au @ ZIF-8 by X-ray photoelectron spectroscopy (XPS) (see FIG. 2A), in which characteristic peaks such as O1s (531.1eV), N1s (399.6eV), C1s (284.6eV), Ru3p, Au4f (4f5/2 is 87.1eV, 4f7/2 is 83.5eV), Zn2p (2p1/2 is 1044.9eV, 2p3/2 is 1021.7eV), indicating the successful synthesis of Ru-PEI/Au @ ZIF-8. To further characterize the formation of Ru-PEI/Au @ ZIF-8, this example also performed EDS spectral characterization of the elemental composition of Ru-PEI/Au @ ZIF-8, as shown in FIG. 2B, which indicates that the nanocomposite contains all of the elements expected to provide an ideal nanocomposite emitter.

Example 2

2.1 preparation of an ECL biosensor comprising the steps of:

s1: the GCE needs to be cleaned before the sensor is built. GCE 3mm in diameter was immersed in a piranha solution (98% (v/v) of H₂SO₄And 30% (v/v) of H₂O₂At a molar ratio of 4 to 1) to remove non-specifically adsorbed species from the electrode surface, followed by repeated polishing with 0.05 μm alumina powder and ultrasonic cleaning in ethanol and water until a bright, clean interface appears. The electrodes were then activated in sulfuric acid at a concentration of 0.1M, at a voltage of-0.8-0.8V, and at a scan rate of 0.1V/s, until a continuous stable CV peak appeared. After rinsing with ultrapure water, the electrodes are in dry N₂Drying is carried out, and the product is used for the next construction of the biosensor.

S2: 0.5 percent (mass percent) of Nafion (N169478-5ml, alatin) and 8 mu L of Ru-PEI/Au @ ZIF-8 are dripped on the spot-free surface of GCE, and Nafion/GCE and Ru-PEI/Au @ ZIF-8/Nafion/GCE are sequentially obtained. Subsequently, 8. mu. L C3N4 was added dropwise to Ru-PEI/Au @ ZIF-8/Nafion/GCE in the absence of light to give C3N4/Ru-PEI/Au @ ZIF-8/Nafion/GCE. Then, the C3N4/Ru-PEI/Au @ ZIF-8/Nafion/GC is washed by PBS solution, and the ECL biosensor for detecting SARS-COV-2RNA is successfully constructed.

2.2 detection of SARS-CoV-2RNA Using CRISPR-Cas12a Trans-cleavage Properties and ECL biosensor

Relates to a DSN target cycle and a CHA amplification technical program, and specifically comprises the following steps:

1) all hairpin DNA (H1 hairpin probe, H2 hairpin probe and H3 hairpin probe in Table 1) was heated to 95 ℃ and cooled to 25 ℃ in 10 min.

2) mu.L of a solution containing 10mM Mg²⁺The solution of DSN (0.05U. mu.l) and H1 hairpin probe (5. mu.M) was reacted with 6. mu.l of SARS-CoV-2RNA solution of different concentrations at 60. + -. 5 ℃ for 75 minutes to give reaction mixture 1.

3) To the reaction solution 1, 15. mu.L of EDTA solution (10mM) was added and the reaction was continued at 50 ℃ for 30 minutes to inhibit the enzymatic activity of DSN. Next, the mixture was rapidly heated to 90 ℃ for 10 minutes and then cooled to 4 ℃ to completely lose the activity of the DSN enzyme, thereby obtaining a reaction solution 2.

4) Mu. L H2 and H3 hairpin probe concentrations were 10. mu.M solution were added to reaction solution 2 and held at 4 ℃ for 16 hours to obtain H2/H3 hybridized dsDNA, and reaction solution 3 was obtained.

Subsequently, H2/H3 was involved in Cas12 a-directed trans cleavage. Specifically, the method comprises the following steps:

s1: a mixture (25. mu.L) containing 50nM Cas12a, 50nM gRNA, and 10U RNase inhibitor was mixed with 25. mu.L of reaction solution 3 and incubated at room temperature for 20 minutes to obtain reaction solution 4.

S2, reacting the reaction solution 4 with a ferrocene (Fc) modified DNA probe (the final concentration of the Fc modified DNA probe is 100nM) at 25 ℃ for 2 hours to obtain a final reaction solution.

S3 mu.L of the final reaction solution was dropped onto the surface of ECL biosensor for SARS-COV-2RNA detection at 4 ℃ to react for 1 hour, and washed with PBS solution (0.1M, pH 7.4) to obtain Fc labeled probe/C₃N₄/Ru-PEI/[email protected]/Nifion/GCE。

S4: the electrode was washed with PBS solution (0.1M, pH 7.4) to remove non-specifically adsorbed material.

S5: ECL signals were collected in PBS (0.1M, pH 7.4) solution at a potential ranging from 0 to 1.3V.

2.3 principles of using CRISPR-Cas12a trans-cleavage Properties and ECL biosensor SARS-CoV-2RNA detection

In this example, an ECL biosensor for detecting SARS-CoV-2RNA was constructed using CRISPR-Cas12a trans-cleavage properties. According to the invention, ZIF-8 is synthesized, as shown in FIG. 6A, the amino group on the surface of the ZIF-8 is modified and coupled with a self-reinforced Ru-PEI complex to obtain Ru-PEI/ZIF-8, wherein PEI is an active substance, has the characteristic of enhancing the intensity of Ru ECL, and is beneficial to improving the emission intensity and efficiency of the ECL. Meanwhile, in order to improve the charge transfer efficiency on the electrode and enhance the conductivity of the ECL emitter, gold nanoparticles are modified on the surface of the Ru-PEI @ ZIF-8, so that an excellent Ru-PEI/Au @ PEI emitting material is formed on the surface of the electrode and is used for biological induction.

Fig. 6B illustrates a DSN assisted target recovery and CHA signal amplification method. In a target recovery process involving DSN, target RNA first binds to hairpin H1, opening the neck loop structure of H1. Due to the unique property of DSN enzymes to cleave DNA in double stranded DNA or DNA/RNA, the target RNA is constantly cycled, creating a residual fragment of H1 to open hairpin H2. Likewise, the residual segment of H1 was combined with H2 and the neck ring structure of H2 was opened. Since the opened H2 has a nucleic acid fragment capable of opening hairpin H3 and H2 binds H3 more strongly than the residual fragment of H1, the residual fragment of H1 is released, cycling open H2, and ultimately creating a large number of H2/H3 double strands. These duplexes then in turn activate the trans-cleavage properties of Cas12a/gRNA, constantly cleaving Fc-labeled DNA probe molecules.

FIG. 6C depicts Ru-PEI/Au @ ZIF-8 modified onto the GCE surface, and then C3N4 was drop-coated onto the electrode to stabilize the ECL signal. The Fc-labeled DNA probe can adsorb strongly to the C3N4 surface and quench the ECL signal greatly. However, the Fc-labeled DNA probe is hydrolyzed in the presence of the target RNA, so the intensity of ECL is stronger than in the absence of the target RNA. Thus, the target concentration can be inferred from the ECL intensity.

2.4 validation of ECL biosensor construction and validation of SARS-CoV-2RNA detection strategy

To illustrate the step-wise assembly and detection process of the proposed sensor, EIS characterization was performed and the results are shown in fig. 3 (a). First, the electron transfer conditions of the bare GCE modified with Nafion (curve a) were tested and exhibited a relatively low electron transfer resistance. The inset of fig. 3(a) shows the analog circuit during electrode modification, where the resistance to charge transfer (Rct) is mainly shown by the high-frequency half circle of the Nyquist plot. With the modification of Ru-PEI/Au @ ZIF-8 (curve b), the charge transfer efficiency is improved due to the good conductivity of Au on ZIF-8, resulting in a decrease in Rct. When C is present₃N₄When dropped on the modified GCE (curve c), the modification due to the non-conductive material increases the resistance to charge transfer, resulting in an increase in Rct. When Fc-labeled DNA probes were adsorbed on C3N4(curve d), Rct increases because it results in a greater resistance to charge transfer. With the appearance and change in concentration of the target RNA (curves e) and (curve f), partial hydrolysis of the Fc-labeled DNA probe eventually resulted, resulting in a decrease in Rct.

This example also investigated ECL reactions during modification and detection at each step. As shown in FIG. 3(B), the Nafion-modified GCE (curve a) has no ECL signal. Whereas the ECL signal showed a strong reaction when Ru-PEI/Au @ ZIF-8 was dropped onto the modified GCE (curve b). With the modification of C3N4 (curve C), ECL signal decreased slightly, since C3N4 masks the emitter. With Fc labeled DNA Probe at C₃N₄The ECL signal is significantly reduced by the strong quenching of ferrocene (curve d). However, with the presence of target DNA, due to the trans-lytic nature of Cas12a, the Fc-labeled DNA probe was partially hydrolyzed, resulting in a significant increase in ECL signal (curves e and f) compared to incubating the Fc-labeled DNA probe alone. Both EIS characterization and ECL reaction demonstrated the successful manufacture of ECL biosensors and the establishment of the proposed SARS-CoV-2RNA detection strategy.

Example 3 detection of SARS-CoV-2RNA Using CRISPR-Cas12a Trans-cleavage Properties and ECL biosensor

In order to demonstrate the reverse cleavage property using CRISPR-Cas12a and the detection level of SARS-CoV-2RNA by ECL biosensor in example 2, this example analyzed the relationship between ECL intensity and target RNA concentration, and obtained the mathematical relationship between them, thereby realizing the possibility of inferring the target concentration from signal intensity. As shown in FIG. 4(A), ECL intensity becomes progressively stronger as the concentration of SARS-CoV-2RNA (SEQ ID: NO.5) in the sample increases, further demonstrating that the target RNA can drive the DSN enzyme to cycle with the CHA reaction process, activating the activity of the CRISPR-Cas12a system, effecting hydrolysis of the Fc-labeled DNA probe. This example also explores a linear relationship between ECL intensity and target RNA concentration, as shown in fig. 4B. The linear relationship obtained is Y1013.3 +322.2lgCtarget (R)²0.9998), where Y represents ECL intensity and lgCtarget represents the log of SARS-CoV-2RNA concentration. The limit of detection (LOD) of the sensor calculated by the 3 σ method was 6.7fM, which was previously reported for RNThe A detection is at the leading level in the common method, which also shows the advancement and sensitivity of the method of the embodiment.

Example 4 specificity and stability of CRISPR 12 a-based strategy

To illustrate the CRISPR-Cas12a trans-cleavage properties and specificity of ECL biosensors for SARS-CoV-2RNA detection in example 2, several common non-specific RNAs were selected in this example, including miRNA-133a, miRNA-499, miRNA-208, and miRNA-328 (see Table 2 for sequence), and SARS-CoV-2RNA in example 2 was replaced with miRNA-133a, miRNA-499, miRNA-208, or miRNA-328, and Blank sample was used as a control. To more fully illustrate the selectivity of the sensor, the concentration of non-specific RNA (100fM) was 10-fold higher than SARS-CoV-2RNA (10fM), and other conditions were as described in example 2. The results shown in FIG. 5A indicate that the ECL response for non-specific RNA is about the same as the response for the blank, but much lower than the ECL intensity for the target RNA, indicating that the system has superior specificity.

Replacing the gRNA in example 2 with a gRNA with a mutation site (gRNA1, gRNA2, or gRNA3) was used to verify the specificity of the sensor, other conditions are seen in example 2. Activation of Cas12a was shown to be under the control of a specific gRNA, as depicted in fig. 5 (B). Under constant SARS-CoV-2RNA conditions, grnas with mutation sites did not significantly increase ECL intensity, indicating that the activity of CRISPR-Cas12 cannot be activated by the mutated grnas.

This example also discusses the stability of detecting SARS-CoV-2RNA at a concentration of 1pM using CRISPR-Cas12a trans-cleavage properties and an ECL biosensor, and ECL signal was scanned 25 consecutive times at a cyclic voltage (as shown in FIG. 5C), and the Relative Standard Deviation (RSD) of the signal was calculated to be 2.9%, indicating that the stability of the sensor was excellent.

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.

Sequence listing

<110> atomic medical institute of Jiangsu province

<120> probe set for detecting SARS-CoV-2RNA, ECL biosensor, preparation method and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 57

<212> DNA

<213> Artificial sequence

<400> 1

ccaaacgtaa tgcggggtgc atttcgctga cgtttggcct cgaaagtgcg tagtagc 57

<210> 2

<211> 76

<212> DNA

<213> Artificial sequence

<400> 2

gaaatggccg aagatgcgaa agtgcgtagt agccgctcgc taaaacacgc tactacgcac 60

tttcgaggcc aaacgt 76

<210> 3

<211> 58

<212> DNA

<213> Artificial sequence

<400> 3

cgctcgctaa acacggcctc gaaagtgcgt agtagcgtgt ttagcgagcg gctactac 58

<210> 4

<211> 40

<212> RNA

<213> Artificial sequence

<400> 4

uaauuucuac ucuuguagau gcgagcggcu acuacgcacu 40

<210> 5

<211> 40

<212> RNA

<213> Artificial sequence

<400> 5

gaccccaaaa ucagcgaaau gcaccccgca uuacguuugg 40

<210> 6

<211> 40

<212> RNA

<213> Artificial sequence

<400> 6

uaauuucuac ucuuguagau gcgagccccu acuacgcacu 40

<210> 7

<211> 40

<212> RNA

<213> Artificial sequence

<400> 7

uaauuucuac ucuuguagau gcgagacccu acuaugcacu 40

<210> 8

<211> 40

<212> RNA

<213> Artificial sequence

<400> 8

uaauuucuac ucuuguagau gggagaccau acuaugcacu 40

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<400> 9

atttccttgg attttaggac gt 22

<210> 10

<211> 22

<212> RNA

<213> Artificial sequence

<400> 10

agcugguaaa auggaaccaa au 22

<210> 11

<211> 21

<212> RNA

<213> Artificial sequence

<400> 11

uuaagacuug cagugauguu u 21

<210> 12

<211> 22

<212> RNA

<213> Artificial sequence

<400> 12

aagcuuuuug cucgaauuau gu 22

<210> 13

<211> 22

<212> RNA

<213> Artificial sequence

<400> 13

cuggcccucu cugcccuucc gu 22