CN118620888A - DNA template silver nanocluster label-free fluorescent logic circuit, biosensor and construction method thereof - Google Patents

Abstract

The present invention relates to a DNA template silver nanocluster label-free fluorescent logic circuit, a biosensor and a construction method thereof, and belongs to the field of biotechnology. The present invention proposes a screened silver nanocluster DNA template sequence Sn _/m ( _CnATATACm ), _and in situ synthesizes Sn _/m -AgNC. Through complementary hybridization reaction, the G-rich base sequence is brought close to the spatial position of Sn _/m -AgNC, and its fluorescence emission signal is significantly enhanced. The probe has the advantages of high signal-to-noise ratio, fast response kinetics and excellent stability. The present invention applies the Sn _/m -AgNC probe to Boolean logic circuits, and develops high-performance basic DNA and serial-parallel advanced logic circuits (DLCs). The computational reliability of different DLCs and the processing efficiency of signal conversion are significantly improved. Therefore, the present invention constructs a biosensor based on the DLC, and realizes the distinguishable high-performance detection of two targets such as miRNA-21 and miRNA-141 at the same fluorescence emission wavelength.

Description

DNA template silver nanocluster label-free fluorescent logic circuit, biosensor and its construction method

Technical Field

The invention belongs to the field of biotechnology and relates to a DNA template silver nanocluster label-free fluorescent logic circuit, a biosensor and a construction method thereof.

Background Art

Programmable DNA logic circuits (DLCs) use DNA molecules as reaction substrates to perform data processing or signal transduction, thereby achieving the expected information processing. DLCs can not only construct molecular computing, but also realize purposeful analysis of biological systems through complex algorithmic instructions. Thanks to the solution of the Hamiltonian path problem, DLCs have been widely used to develop a variety of possible applications, such as digital circuit computing, molecular modular recognition, logical nanodevices, and cell signal regulation. At present, a variety of basic Boolean logic circuits (such as OR, AND, NAND, and XOR) have been constructed through toehold-mediated chain migration or enzyme-catalyzed reactions to meet different signal processing needs. Different basic logic gates can be used to construct multi-layer digital circuits through series or parallel stages to perform complex signal processing, such as adders/subtractors and parity checkers. However, traditional fluorescent-labeled signal probes have uncontrollable circuit leakage risks, enzyme-mediated circuit design may have nonspecific interference, and the calculation process of input conversion is cumbersome and time-consuming. These deficiencies seriously restrict the further development of DLC. Therefore, it is of great practical significance to explore enzyme-free reaction routes with low circuit leakage, screen efficient label-free signal probes, and construct high-performance DLCs.

Silver nanoclusters (DNA/AgNCs) synthesized with DNA templates of specific base sequences are label-free fluorescent signal probes with great application prospects. Due to their unique advantages such as significant photophysical properties, good biocompatibility, simple preparation and low cost, they have attracted widespread attention in many fields. The spectral behavior and fluorescence properties of DNA/AgNCs can be adjusted by specific base sequences and their secondary structures. These unique advantages of DNA/AgNCs provide great possibilities for solving the inherent deficiencies of quantum dots and fluorescent groups. Studies have shown that when a guanine (G)-rich DNA sequence is close to a non-fluorescent DNA/AgNC, its fluorescence emission can be significantly enhanced (i.e., "signal-on" type). Through DNA conformational transformation, chain migration or DNA complementary hybridization, it is easy to adjust the spatial distance between AgNC and the G-rich base sequence, customize the control of AgNC fluorescence emission "on" or "off", and achieve highly specific response signal output to specific inputs. Therefore, the flexible programmability of DNA/AgNCs has given them a wide range of application potentials in bioanalysis, transient dissipation, living cell imaging, environmental and food monitoring, etc. However, there are few reports on the construction of enzyme-free, label-free, fast-operating integrated and advanced DLC Boolean logic circuits using the fluorescence signal output mode of G-rich in situ enhanced DNA/AgNCs.

Summary of the invention

In view of this, the object of the present invention is to provide a S _5/4 -AgNC fluorescent signal probe synthesized by a new template sequence Sn _{/m ( CnATATACm} ) of silver nanoclusters. The probe exhibits advantages such as high signal-to-noise ratio, fast kinetics and high stability. After combining Boolean logic circuits with S _5/4 -AgNC, we developed a high-performance basic DLC. Even if the computational complexity is increased by series or parallel connection, their computational reliability and signal processing efficiency are significantly improved. Therefore, we have proposed a biosensor based on the DLC, which realizes the detection of dual targets at only one fluorescence emission wavelength.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a silver nanocluster fluorescent signal probe, which is formed by incubating a template signal chain of a silver nanocluster with AgNO ₃ and NaBH ₄ , wherein the silver nanocluster template sequence in the template signal chain is C _n ATATAC _m , n/m=3/4, 4/4, 4/3, 3/3, 2/4, 4/2, 4/5 or 5/4, and as a preferred embodiment, n/m=5/4.

In a further embodiment, the template signal chain of the silver nanocluster contains a silver nanocluster template sequence and a hybridization sequence.

The preparation method of the silver nanocluster fluorescent signal probe is as follows: incubate the prepared AgNO ₃ and the template signal chain of the silver nanocluster in a dark environment at 4°C. Quickly add the newly prepared NaBH ₄ solution to the incubated mixture and shake it quickly. Then place it in a dark environment to generate Sn _/m -AgNC.

The present invention also provides an application of the silver nanocluster fluorescent signal probe in constructing a programmable DNA logic circuit.

The present invention also provides a programmable DNA logic circuit constructed based on a silver nanocluster fluorescent signal probe. The logic circuit comprises a silver nanocluster fluorescent signal probe and a fluorescent signal enhancement chain;

The logic circuit uses whether the fluorescence signal enhancement chain can be complementary paired with the template signal chain of the silver nanocluster to perform logic operations and signal output.

If the fluorescence signal enhancement chain in the logic circuit can complementally pair with the template signal chain of the silver nanocluster and the fluorescence enhancement is higher than the threshold, the output signal is true, otherwise it is false.

In a further embodiment, the template signal chain of the silver nanocluster contains a silver nanocluster template sequence and a hybridization sequence one connected in sequence by 5'-3', and the fluorescence signal enhancement chain contains a hybridization sequence two and a guanine-rich sequence (G-rich sequence) connected in sequence by 5'-3', and the hybridization sequence two is complementary to the hybridization sequence one.

In a further embodiment, the logic circuit is an OR logic circuit, an AND logic circuit, an AND and INH logic circuit, or an AND-INH/AND logic circuit, etc.

The method for constructing the above logic circuit is: adding the fluorescent signal enhancement chain to the above synthesized Sn _/m -AgNC solution and incubating at 37° C. The basic components of the logic circuit adopt the existing technology.

The present invention further provides an application of a silver nanocluster fluorescent signal probe in constructing a biosensor, and a biosensor constructed based on the silver nanocluster fluorescent signal probe. Specifically, the biosensor is constructed using the above logic circuit, and the biosensor can be used to detect dual-target miRNAs. The logic circuit is an AND-INH/AND logic circuit, etc.

In a further optimized scheme, the biosensor contains silver nanocluster fluorescent signal probe 1, fluorescent signal enhancement chain 1, silver nanocluster fluorescent signal probe 2, fluorescent signal enhancement chain 2, and target miRNA 1 (such as miR-21) and target miRNA 2 (such as miR-141);

The silver nanocluster fluorescent signal probe 1 includes a silver nanocluster template sequence (C-rich sequence) and a hybridization sequence A connected in sequence from 5' to 3', and the fluorescent signal enhancement chain 1 includes a hybridization sequence B, a hybridization sequence C and a G-rich sequence connected in sequence from 5' to 3', the hybridization sequence A is complementary to the 5' end of miRNA1, and the hybridization sequence C is complementary to the 3' end of miRNA1;

The silver nanocluster fluorescent signal probe 2 includes a C-rich sequence, a hybridization sequence D, a hybridization sequence E and a hybridization sequence F connected in sequence from 5' to 3', and the fluorescent signal enhancement chain 2 includes a hybridization sequence G and a G-rich sequence connected in sequence from 5' to 3'. The hybridization sequence D is complementary to the 5' end portion of miRNA2, the hybridization sequence G is complementary to the 3' end portion of miRNA2, the hybridization sequence B is complementary to the hybridization sequence F, and the hybridization sequence C is complementary to the hybridization sequence E.

The silver nanocluster fluorescent signal probe 1 is: CCCCCATATACCCCCTGATAAGCTA; C-rich sequence: CCCCCATATACCCC, hybridization sequence A: CTGATAAGCTA;

Fluorescence signal enhancement chain 1: CAAGAGTCTCAACATCAGTGGGTGGGGTGGGGTGGGG; G-rich sequence: GGGTGGGGTGGGGTGGGG, hybridization sequence B: CAAGAGTC, hybridization sequence C: TCAACATCAGT.

Silver nanocluster fluorescent signal probe 2:

CCCCCATATACCCCAGACAGTGTTAACTGATGTTGAGACTCTTG; C-rich sequence: CCCCCATATACCCC, hybridization sequence D: AGACAGTGTTA, hybridization sequence E: ACTGATGTTGA, hybridization sequence F: GACTCTTG;

Fluorescence signal enhancement chain 2: CCATCTTTACCGGGTGGGGTGGGGTGGGG, G-rich sequence: GGGTGGGGTGGGGTGGGG, hybridization sequence G: CCATCTTTACC.

miRNA1: UAGCUUAUCAGACUGAUGUUGA;

miRNA2:UAACACUGUCUGGUAAAGAUGG.

The construction method of the biosensor is as follows: silver nanocluster fluorescent signal probe 1 is mixed with fluorescent signal enhancement chain 1. Then, miRNA 1 and miRNA 2 are introduced into the mixture for reaction. Then, silver nanocluster fluorescent signal probe 2 and fluorescent signal enhancement chain 2 are added to the above solution and incubated.

The beneficial effects of the present invention are:

The present invention is the first to prepare Sn _/m -AgNCs with multiple excellent properties and cleverly integrate them into DLC to achieve an example of low circuit leakage and efficient signal processing.

The present invention also uses the DLC-based biosensor for detecting different biomolecules, such as dual-target detection of miR-21 and miR-141, thereby improving the detection efficiency.

Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

Figure 1. Schematic diagram of the hybridization reaction between _Sn/m -AgNC fluorescent signal probe and GS.

Figure 2. (AH) Fluorescence curves of S _3/4 , S _4/4 , S _4/3 , S _3/3 , S _2/4 , S _4/2 , S _4/5 , and S _5/4 before and after the introduction of GS; (I) Fluorescence intensity diagram before and after the introduction of GS; (J) Corresponding signal-to-noise ratio bar graph.

Figure 3. (A) Schematic diagram of the OR logic gate of S _5/4 -AgNC; (B) Corresponding logic circuit and truth table. (C) PAGE images of IN2, IN3, S _5/4 , IN2/S _5/4 , IN3/S _5/4 , IN2/IN3/S _5/4 and DNA marker; (D) Fluorescence emission spectrum, (E) Corresponding normalized fluorescence intensity, (F) Real-time fluorescence of OR logic gate; (G) TIRFM image.

Figure 4. (A) Schematic diagram of S _5/4 -AgNC AND-INH logic circuit; (B) corresponding logic circuit symbols and truth table; (C) PAGE images of IN4, S _5/4 , IN10, IN9, IN4/IN10, S _5/4 /IN10, IN4/S _5/4 /IN10, IN4/IN9/S _5/4 /IN10 and DNA marker; (D) fluorescence emission spectrum, (E) corresponding normalized fluorescence intensity and (F) corresponding real-time fluorescence.

Figure 5. (A) Schematic diagram of the miRNA-specific dual-response AND-AND-INH gate; (B) the corresponding logic circuit symbol, reaction route and circuit diagram of the modular switch for fluorescence switching (red and gray represent the bright fluorescence or dim fluorescence of S _5/4 -AgNC, respectively).

Figure 6. Design of AND-INH/AND logic circuit. (A) Dual-path logic gate based on S _5/4 -AgNC signal probe; (B) Possible situations of AND-INH/AND logic circuit; (C) Truth table; AND-INH/AND is used to analyze (D) miR-21 and (E) miR-141 fluorescence kinetic data; AND-INH/AND is used to analyze (F) miR-21 and (G) miR-141 fluorescence emission spectra and (H) corresponding normalized fluorescence intensities.

DETAILED DESCRIPTION

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Drugs and reagents used in this example: silver nitrate (AgNO ₃ , 99.98%), sodium borohydride (NaBH ₄ , 98%), disodium phosphate (Na ₂ HPO ₄ , 99%), potassium dihydrogen phosphate (KH ₂ PO ₄ , 99.5%) and magnesium nitrate hexahydrate (Mg(NO ₃ ) ₂ ·6H ₂ O, 99%) (McLean Biochemical Co., Ltd.); HPLC-purified DNA chains and microRNAs (Table 1) were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; DNA powder was diluted with phosphate buffer (PBS, pH 7.4) containing 20 mM Na ₂ HPO ₄ , 20 mM KH ₂ PO ₄ and 5 mM Mg(NO ₃ ) ₂ ·6H ₂ O; DNA loading buffer (Beijing Dingguo Changsheng Biotechnology Co., Ltd.); polyacrylamide gel staining used GelRed dye (Shanghai Biyuntian Biotechnology Co., Ltd.). Ultrapure deionized water (18.2 MΩ) was obtained from an ATsrese ultrapure water machine.

In this embodiment, the fluorescence test was performed by setting the excitation slit width to 5 nm and the emission slit width to 10 nm, the voltage to 900 V, and collecting the two-dimensional emission spectrum at a wavelength of 580-700 nm by excitation at a wavelength of 550 nm. The real-time fluorescence was continuously monitored for 60 min, and the fluorescence intensity of S _5/4 -AgNC (synthesized by 5'-C ₅ ATATAC ₄ CATATCAAATTTATTATACTTCTT-3' as a template) at 600 nm was the maximum value, and the real-time fluorescence intensity of all samples was normalized.

Fluorescence test The fluorescence emission spectra were measured using a Hitachi F-7100 fluorescence spectrophotometer (Tokyo, Japan) and a BioTek Synergy H1 (California, USA), with the setting parameters of 900 V photomultiplier voltage, 5 nm excitation slit, and 10 nm emission slit.

In the examples, all logic circuits were built in PBS buffer using S _5/4 -AgNC as a fluorescent signal probe to achieve the establishment of a universal platform. Unless otherwise specified, the final concentration of input and S _5/4 -AgNC in all logic circuits was 1.0 μM. The basic elements of each basic logic gate in the examples were all based on existing technologies.

Example 1 Construction of logic circuit

1. Optimization of template sequence of S-AgNC

As shown in FIG1 , the template signal chain S used to generate silver nanoclusters is composed of a silver nanocluster template sequence at the 5' end and a hybridization sequence 1 at the 3' end. The template sequence contains a cytosine-rich sequence (C-rich sequence) and an AT linker (ATATA). The template signal chain S is incubated with AgNO ₃ and NaBH ₄ to form S-AgNC. Then, a guanine-rich signal enhancement chain GS chain is added. After the GS chain is hybridized with the S chain in the S-AgNC, an obvious fluorescence emission peak can be observed at 600nm through fluorescence excitation at 555nm. The GS used in this embodiment is composed of a hybridization sequence 2 at the 5' end and a guanine-rich sequence (G-rich sequence, GGGTGGGGTGGGGTGGGG) at the 3' end. The hybridization sequence 2 is complementary to the hybridization sequence 1 in the template signal chain S.

In order to obtain the best fluorescence performance, we optimized the template sequence in S-AgNC. In this example, the template sequence was designed to be Sn _{/m ( CnATATACm} ).

The Sn _/m and GS sequences in this example are shown in Table 1.

Table 1 Oligonucleotide base sequences used in the examples

As shown in FIG2 , among the tested Sn _/m -AgNCs (n/m=3/4, 4/4, 4/3, 3/3, 2/4, 4/2, 4/5, or 5/4), S5 _/4 -AgNCs exhibited superior fluorescence properties and had the potential to be used in logic circuit construction.

In this embodiment, the steps of hybridization of unlabeled S-AgNC silver nanoclusters with GS for fluorescence testing are as follows:

10 μL of AgNO ₃ (60 μM) prepared with ultrapure water and 10 μL of S chain (10 μM) were incubated in a dark environment at 4°C for 30 minutes. 10 μL of NaBH ₄ solution (60 μM) prepared with ultrapure water was quickly added to the incubated mixture and rapidly shaken for 60 seconds. After that, it was placed in a dark environment for 1.0 hour to generate S-AgNC. 10 μL of 10 μM GS was added to the previously synthesized S-AgNC solution and incubated at 37°C for 1.0 hour before fluorescence testing.

Example 2 Construction of logic gate based on S _5/4 -AgNC

Thanks to the excellent performance of S _5/4 -AgNC, we use it to construct various basic logic gates. This embodiment takes OR logic gate and AND and INH logic circuits as examples.

1. OR logic gate

The OR logic gate construction process is as follows:

In PBS buffer, S _5/4 -AgNC (final concentration 1.0 μM) was used as a fluorescent signal probe to establish an OR logic gate. Specifically, 10 μL of AgNO ₃ (60 μM) prepared with ultrapure water was incubated with S _5/4 chain (10 μM, 10 μL) in a dark environment at 4°C for 30 minutes. 10 μL of NaBH ₄ solution (60 μM) prepared with ultrapure water was quickly added to the incubated mixture and rapidly shaken for 60 seconds. After that, it was placed in a dark environment for 1.0 h to generate S _5/4 -AgNC. The input containing the G-rich fragment was added to the previously synthesized S-AgNC solution and incubated at 37°C for 1.0 h. The final concentration of the input was 1.0 μM.

The inputs of the OR logic gate of this embodiment are: IN2 and IN3. IN2 consists of a hybridization sequence three at the 5' end and a G-rich sequence (GGGTGGGGTGGGGTGGGG) at the 3' end, and IN3 consists of a hybridization sequence four at the 5' end and a G-rich sequence at the 3' end. Both the hybridization sequence three and the hybridization sequence four can hybridize with the hybridization sequence one in S _5/4 . The sequences of IN2 and IN3 are shown in Table 1.

The OR logic gate outputs high signal when any input exists. The working principle, truth table and logic circuit symbol of the OR logic gate of the present embodiment are shown in Fig. 3 A and Fig. 3 B. As can be seen from the PAGE image of the different DNAs of Fig. 3 C, IN2, IN3 and S _5/4 swimming lanes demonstrate single DNA bands, and IN2, S _5/4 mixed swimming lanes and IN3, S _5/4 mixed swimming lanes demonstrate single bands higher than IN2, IN3 and S _5/4 single-stranded positions, indicating the formation of IN2/S _5/4 and IN3/S _5/4 double-strands. Bands similar to IN2, S _5/4 mixed swimming lanes and IN3, S _5/4 mixed swimming lanes have appeared in S _5/4 , IN2 and IN3 mixed swimming lanes, indicating that IN2/S _5/4 and IN3/S _5/4 exist simultaneously.

The fluorescence detection results showed that the input IN2 and/or IN3 containing G-rich fragments can hybridize with S _5/4 -AgNC to form S _5/4 /IN2 double strands and/or S _5/4 /IN3 double strands, bringing the G-rich fragments close to S _5/4 -AgNC, thereby enhancing the fluorescence (Figure 3D and E). This characterization result is consistent with the PAGE results. In addition, real-time fluorescence showed fast reaction kinetics (Figure 3F). Corresponding to different input conditions, the corresponding results can be observed in the TIRFM image of S _5/4 -AgNC (Figure 3G).

The above results demonstrate the successful construction of the OR logic gate.

2. AND and INH logic circuits with three inputs

The construction process of AND and INH logic circuits is as follows:

In PBS buffer, S _5/4 -AgNC (final concentration 1.0 μM) was used as a fluorescent signal probe to construct an AND and INH logic circuit with three inputs. The circuit consists of two basic logic gates, AND and INH, connected in series. Specifically, 10 μL of AgNO ₃ (60 μM) prepared with ultrapure water was incubated with S _5/4 chain (10 μM, 10 μL) in a dark environment at 4°C for 30 minutes. 10 μL of NaBH ₄ solution (60 μM) prepared with ultrapure water was quickly added to the incubated mixture, and rapidly shaken for 60 seconds, and placed in a dark environment for 1.0 hour to generate S _5/4 -AgNC. The three inputs were added to the previously synthesized S-AgNC solution and incubated at 37°C for 1.0 hour. The final concentration of the inputs was 1.0 μM.

The inputs of the AND logic gate are IN4 and IN10; the inputs of the INH logic gate are IN9 and the output of the AND logic gate. IN4 consists of a hybridization sequence V at the 5' end and a G-rich sequence at the 3'end; the hybridization sequence V of IN4 can complementarily hybridize with a partial sequence of IN10, IN9 can complementarily hybridize with a partial sequence of IN10, and the hybridization sequence I of S _5/4 can complementarily hybridize with a partial sequence of IN10. The specific sequences of IN4, IN9, and IN10 are shown in Table 1.

As shown in Figure 4A, in the absence or presence of only one input, the hybridization complex of IN9/IN10 and S _5/4 will not be formed, making it impossible for S _5/4 -AgNC to approach the G-rich sequence. Therefore, no obvious signal change is observed. When there are two inputs (IN4/IN9, IN4/IN10 or IN9/IN10), only the IN4/IN10 group can observe fluorescence. However, when three inputs (IN4/IN9/IN10) are input together, the fluorescence is inhibited due to the chain migration between IN9 and S _5/4 -AgNC. The corresponding logic circuit symbol and truth table are shown in Figure 4B. In addition, the mutual hybridization of the logic circuit DNA chains was verified by PAGE (Figure 4C). IN4, S _5/4 , IN9 and IN10 all have their own unique bands. A higher band appeared in the mixture of IN4/IN10 and S _5/4 /IN10, respectively, and the position of the band after the mixture of IN4, IN10 and S _5/4 was higher than that after the mixture of any two of them, indicating that the assembly of the AND logic gate was successful. Then, after the introduction of IN9 into IN4, IN10 and S _5/4 , brighter and higher bands appeared, as well as a band similar to that in the single lane of S _5/4 , indicating that IN9 can replace S _5/4 .

The fluorescence detection results showed that the fluorescence emission spectrum and the corresponding emission intensity were consistent with the expected results of the designed AND-INH (Figure 4D, E, and F), and its real-time fluorescence spectrum indicated a rapid response process and the corresponding results were similar to the emission spectrum.

The above results demonstrate the successful construction of AND and INH logic circuits with three inputs.

Example 3 Construction of miRNA Biosensor

The biosensor of this embodiment contains a series/parallel circuit (AND-INH/AND) combining three logic circuit elements, which is composed of two basic logic gates AND and INH connected in series and connected in parallel with another AND logic gate. It can realize the analysis of dual targets (miR-21 and miR-141, which are overexpressed in malignant tumors) with only one fluorescence wavelength excitation (Figure 5).

The logic circuit involved in the biosensor of this embodiment is constructed in a PBS buffer using S _5/4 -AgNC (final concentration 1.0 μM) as a fluorescent signal probe. miR-21 and GS1, miR-141 and GS2 are the inputs of two AND logic gates, respectively. Specifically, the silver nanocluster solution (30 μL, 3.33 μM) synthesized with the S1 chain as the template signal chain is mixed with 10 μL of the guanine-rich signal enhancement chain GS1 (10 μM). After that, 10 μL of the miR-21 (10 μM) and miR-141 (10 μM) mixture is introduced to react for 1.0 h, and then the fluorescence is measured. Next, the silver nanocluster solution (30 μL, 3.33 μM) synthesized with the S2 chain as the template signal chain and 10 μL of the guanine-rich signal enhancement chain GS2 (10 μM) are added to the above solution, and the fluorescence is measured after incubation for 1.0 h. The synthesis method of the silver nanocluster solution is the same as that in Example 1.

In the above logic circuit, the template signal chain S1 is composed of a C-rich sequence (CCCCCATATACCCC) and a hybridization sequence A (CTGATAAGCTA) (5'-3'), and its hybridization sequence A is complementary to the 5' end of miR-21. The signal enhancement chain GS1 is composed of a hybridization sequence B (CAAGAGTC), a hybridization sequence C (TCAACATCAGT), and a G-rich sequence (GGGTGGGGTGGGGTGGGG) (5'-3'), and its hybridization sequence C is complementary to the 3' end of miR-21.

Template signal chain S2 consists of a C-rich sequence, a hybridization sequence D (AGACAGTGTTA), a hybridization sequence E (ACTGATGTTGA) and a hybridization sequence F (GACTCTTG) (5'-3'), wherein the hybridization sequence D is complementary to the 5' end of miR-141, and signal enhancement chain GS2 consists of a hybridization sequence G (CCATCTTTACC) and a G-rich sequence (5'-3'), wherein the hybridization sequence G is complementary to the 3' end of miR-141, the hybridization sequence B of GS1 is complementary to the hybridization sequence F of S2, and the hybridization sequence C of GS1 is complementary to the hybridization sequence E of S2. The sequences of miR-21, miR-141, S1, GS1, S2 and GS2 are shown in Table 1.

In the above biosensor, when miR-21 is present, miR-21 can complement the template signal chain S1 (S1) and signal enhancement chain 1 (GS1) that generate S _5/4 -AgNC, respectively, to generate miR-21/S1/GS1, and obtain enhanced fluorescence signal A. When miR-141 is present, miR-141, template signal chain 2 (S2) and signal enhancement chain 2 (GS2) are hybridized to obtain similar enhanced fluorescence (signal B). At the same time, since the introduction of S2 can displace GS1 from the hybridization product of miR-21/S1/GS1, S _5/4 -AgNC is separated from the G-rich fragment (G-rich fragment), and the A signal disappears. From the corresponding logic circuit symbol, the AND logic gate (miR-21 and GS1) is connected in series to the INH logic gate, and the INH logic gate is connected in parallel to the AND logic gate (miR-141 and S2).

In addition, we simplified the reaction route through two modular schematic diagrams, including four reaction results and circuit diagrams (Figure 5) to better illustrate the applicability of the dual-response AND-INH/AND logic gate to miRNAs analysis.

As shown in Figure 6A, GS1 and S1, which can output signal A, are first added to the initial system (miR-21 or miR-141), and then S2 and GS2 are introduced to monitor the corresponding output signal B by enhancing fluorescence. Therefore, there are four possible signal output situations (Figure 6B). Finally, the signal of the corresponding S _5/4 -AgNC signal probe is read out, and the logic "0" or "1" indicates that the fluorescence intensity is lower or higher than the threshold. When the input miR-21 is "1", A is true; if the input miR-141 is "1", B is true, otherwise it is false. The obtained truth table is shown in Figure 6C. The fluorescence kinetics show that the signal readout time is very short (t < 30min) (Figures 6D and 6E), indicating its rapid response ability. From the fluorescence spectra (Figures 6F and 6G) and the corresponding intensity histogram (Figure 6H), it can be found that the double input (1,1) can have obvious double response fluorescence signals, the single input ((0,1) or (1,0)) can produce independent strong fluorescence signals, and there is no obvious fluorescence signal when there is no input (0,0), indicating that the series/parallel circuit based on S _5/4 -AgNC can handle different input conditions well and output the expected results. Therefore, the series/parallel circuit based on S _5/4 -AgNC can be used to prepare biosensors for detecting miRNA.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims (10)

1. A silver nanocluster fluorescent signal probe, characterized in that the silver nanocluster fluorescent signal probe is formed by incubating a template signal chain of a silver nanocluster with AgNO ₃ and NaBH ₄ , wherein the silver nanocluster template sequence in the template signal chain is C _n ATATAC _m , n/m=3/4, 4/4, 4/3, 3/3, 2/4, 4/2, 4/5 or 5/4. 2. A silver nanocluster fluorescent signal probe according to claim 1, characterized in that the template signal chain contains a silver nanocluster template sequence and a hybridization sequence. 3 . The silver nanocluster fluorescent signal probe according to claim 1 , wherein n/m=5/4. 4. Use of the silver nanocluster fluorescent signal probe according to any one of claims 1 to 3 in constructing a programmable DNA logic circuit. 5. A programmable DNA logic circuit constructed using the silver nanocluster fluorescent signal probe according to any one of claims 1 to 3, characterized in that the logic circuit comprises a silver nanocluster fluorescent signal probe and a fluorescent signal enhancement chain; the logic circuit uses whether the fluorescent signal enhancement chain can be complementary paired with the template signal chain of the silver nanocluster to perform logical operations and signal output; If the fluorescence signal enhancement chain in the logic circuit can complementarily pair with the template signal chain of the silver nanoclusters and the fluorescence enhancement is higher than the threshold, the output signal is true; otherwise, it is false. 6. The programmable DNA logic circuit according to claim 5 is characterized in that the template signal chain of the silver nanocluster contains a silver nanocluster template sequence and a hybridization sequence 1 connected in sequence by 5'-3', and the fluorescent signal enhancement chain contains a hybridization sequence 2 and a guanine-rich sequence connected in sequence by 5'-3', and the hybridization sequence 2 is complementary to the hybridization sequence 1. 7. Use of the silver nanocluster fluorescent signal probe according to any one of claims 1 to 3 in constructing a biosensor for detecting miRNA. 8. A biosensor based on silver nanocluster fluorescent signal probe, characterized in that it is constructed using the logic circuit described in claim 5 or 6, and the biosensor is used to detect dual-target miRNA, and the logic circuit is an AND-INH/AND logic circuit. 9. The biosensor based on silver nanocluster fluorescent signal probe according to claim 8, characterized in that the biosensor contains silver nanocluster fluorescent signal probe 1, fluorescent signal enhancement chain 2, silver nanocluster fluorescent signal probe 2, fluorescent signal enhancement chain 2, target miRNA 1 and target miRNA 2; The silver nanocluster fluorescent signal probe 1 includes a silver nanocluster template sequence and a hybridization sequence A connected in sequence from 5' to 3', and the fluorescent signal enhancement chain 1 includes a hybridization sequence B, a hybridization sequence C and a guanine-rich sequence connected in sequence from 5' to 3', the hybridization sequence A is complementary to the 5' end of miRNA1, and the hybridization sequence C is complementary to the 3' end of miRNA1; The silver nanocluster fluorescent signal probe 2 includes a silver nanocluster template sequence, a hybridization sequence D, a hybridization sequence E and a hybridization sequence F connected in sequence from 5' to 3', and the fluorescent signal enhancement chain 2 includes a hybridization sequence G and a G-rich sequence connected in sequence from 5' to 3'. The hybridization sequence D is complementary to the 5' end portion of miRNA2, the hybridization sequence G is complementary to the 3' end portion of miRNA2, the hybridization sequence B is complementary to the hybridization sequence F, and the hybridization sequence C is complementary to the hybridization sequence E. 10. The biosensor constructed based on silver nanocluster fluorescent signal probe according to claim 9, characterized in that: The silver nanocluster fluorescent signal probe 1 is: CCCCCATATACCCCCTGATAAGCTA; hybridization sequence A: CTGATAAGCTA; Fluorescence signal enhancement chain 1: CAAGAGTCTCAACATCAGTGGGTGGGGTGGGGTGGGG; hybridization sequence B: CAAGAGTC, hybridization sequence C: TCAACATCAGT. Silver nanocluster fluorescent signal probe 2: CCCCCATATACCCCAGACAGTGTTAACTGATGTTGAGACTCTTG; hybridization sequence D: AGACAGTGTTA, hybridization sequence E: ACTGATGTTGA, hybridization sequence F: GACTCTTG; Fluorescence signal enhancement chain 2: CCATCTTTACCGGGTGGGGTGGGGTGGGG, hybridization sequence G: CCATCTTTACC. C-rich sequence: CCCCCATATACCCC, G-rich sequence: GGGTGGGGTGGGGTGGGG; miRNA1: UAGCUUAUCAGACUGAUGUUGA; miRNA2:UAACACUGUCUGGUAAAGAUGG.