Fluorescence-colorimetric dual-mode biosensor and detection method and application of flap endonuclease 1

Abstract

The invention belongs to the technical field of biosensors, and particularly relates to a fluorescence-colorimetric dual-mode biosensor and a detection method and application of valvular endonuclease 1, in particular to a fluorescence-colorimetric dual-mode biosensor based on a cleavage-induced strand displacement reaction and an exponential rolling circle amplification reaction. Comprising a DNA branch structure of a 5'overhanging end, a hairpin substrate and a circular substrate, wherein the DNA branch structure of the 5'overhanging end is formed by hybridizing three DNA single chains D1, D2 and D3; the circular substrate is a circular substrate with three gap sites and a cytosine-rich sequence. The biosensor shows high sensitivity to FEN1, the fluorescence mode detection limit is 1.55 * 10 <-3 > UmL <-1 >, the colorimetric mode detection limit is 0.011 UmL <-1 >, the inhibition effect of an inhibitor can be effectively evaluated, the FEN1 activity in a cancer cell nuclear extract can be effectively detected, and the biosensor is a potential FEN1 activity analysis tool.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a fluorescence-colorimetry dual-mode biosensor and a detection method and application of flap-shaped nuclease 1. Background Art

[0002] DNA replication and repair processes are essential for maintaining genome integrity, and the DNA branch structures generated during these processes can damage genome integrity. Flap endonuclease 1 (FEN1) is a structure-specific nuclease that participates in the long-fragment DNA base excision repair pathway and DNA replication process by specifically recognizing and cleaving the 5' flap of the DNA branch structure, and plays an important role in maintaining telomerase stability and Okazaki fragment maturation. Studies have confirmed that FEN1 is overexpressed in breast cancer, lung cancer, liver cancer, prostate cancer, etc., and the expression level of FEN1 is related to the type and stage of cancer development, making it a promising biomarker for cancer diagnosis. Moreover, inhibiting the expression of FEN1 can inhibit cancer growth, making FEN1 an effective therapeutic target for cancer treatment. Therefore, the detection of FEN1 activity is crucial for studying the biological function of FEN1, early diagnosis, and treatment evaluation of cancer.

[0003] In the prior art, methods including Western blot, enzyme-linked immunosorbent assay and gel electrophoresis have been developed for quantitative and semi-quantitative detection of FEN1 activity. However, expensive antibodies and complex procedures limit their wide application. Recently, a variety of biosensors, including fluorescence, electrochemistry and SERS, have been developed for quantitative detection of FEN1 activity. Among them, fluorescent biosensors have the advantages of simple preparation, high sensitivity, rapid detection and good specificity, and have become a powerful tool in the field of sensing. In addition, colorimetric biosensors have the advantages of obvious color changes that can be distinguished by the naked eye and convenient operation, and have become another powerful tool in the field of sensing. Li et al. have developed a fluorescent biosensor based on gold nanostars for quantitative detection of FEN1 and imaging of FEN1 in living cells. Wang et al. have developed a fluorescent biosensor based on DNA nanofireworks for quantitative detection of FEN1 and imaging of FEN1 in living cells. Considering the low expression level of FEN1 in the early stage of cancer, the detection sensitivity of biosensors needs to be further improved at this stage. Signal amplification technologies such as rolling circle amplification reaction, CRISPR / Cas12a-assisted signal amplification reaction, hybridization chain reaction, and primer exchange reaction have signal amplification capabilities and have been applied to the development of fluorescent biosensors for FEN1 activity detection. Although these biosensors have improved the detection performance to a certain extent through the effective amplification of primary signal amplification technology and dual signal amplification technology, they still face new challenges. Most of the biosensors reported so far are based on single-mode detection, which is easily affected by fluctuations in different operators, instruments, and detection environments, reducing the reliability and accuracy of quantitative detection. Summary of the invention

[0004] The purpose of the present invention is to provide a fluorescence-colorimetric dual-mode biosensor and a detection method and application of flap-shaped nuclease 1, so as to overcome the shortcomings of the prior art. A fluorescence-colorimetric dual-mode biosensor is prepared based on cleavage-induced strand displacement reaction (CSDA) and exponential rolling circle amplification reaction (exRCA), which has high sensitivity and low detection limit for FEN1 activity detection.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a fluorescence-colorimetric dual-mode biosensor, comprising a DNA branch structure at a 5' overhang end, a hairpin substrate, and a circular substrate;

[0007] Wherein, the DNA branch structure at the 5' overhang end is formed by hybridization of three DNA single strands D1, D2 and D3;

[0008] The circular substrate is a circular substrate having three gap sites and a cytosine-rich sequence.

[0009] In some other embodiments, the nucleotide sequence of D1 is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of D2 is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of D3 is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the hairpin substrate is shown in SEQ ID NO.4;

[0013] The nucleotide sequence of the circular substrate is shown in SEQ ID NO.5.

[0014] In some other embodiments, flap endonuclease 1, T4 DNA ligase, Phi29 DNA polymerase and Nt.BbvCI are also included.

[0015] In a second aspect, the present invention provides the use of the fluorescence-colorimetry dual-mode biosensor described in the first aspect in detecting flap-shaped nuclease 1.

[0016] In a third aspect, the present invention provides a method for detecting flap-shaped nuclease 1, using the fluorescence-colorimetric dual-mode biosensor described in the first aspect, comprising the following steps:

[0017] (1) Mixing the sample to be tested with the DNA branch structure at the 5' overhang end, adding dNTPs, KF polymerase, Nt.BbvCI and hairpin substrate to induce strand displacement reaction, then adding T4 ligase and circular substrate to carry out ligation reaction to obtain a circular probe;

[0018] (2) reacting the circular probe, dNTPs, NMM, Phi29 polymerase and Nt.BbvCI to generate a fluorescent amplification signal;

[0019] (3) After the circular probe, dNTPs, heme, Phi29 polymerase and Nt.BbvCI were reacted, H 2 O 2 and TMB for oxidation reaction, adding H 2 SO 4 The reaction is terminated and a UV-visible absorption amplification signal is generated.

[0020] In some other embodiments, in step (1), the mixing time is 0.2-2 hours; the induced strand displacement reaction time is 0.3-1.5 hours; the exponential rolling circle amplification reaction time is 1-5 hours;

[0021] Preferably, the mixing time is 1.5 h; the inducing strand displacement reaction time is 1.0 h;

[0022] The time of exponential rolling circle amplification reaction was 3.5 h.

[0023] In some other embodiments, in step (2), the concentration of the Phi29 polymerase is 0.5-2.0U; the concentration of the Nt.BbvCI is 1-5U;

[0024] The concentration of the NMM is 1-7 μM;

[0025] Preferably, the concentration of the Phi29 polymerase is 1.5U; the concentration of the Nt.BbvCI is 4U;

[0026] The concentration of NMM was 3 μM.

[0027] In some other embodiments, in step (3), the H 2 O 2 The concentration of is 2-18 mM; the concentration of TMB is 0.2-1.6 mM; the pH of the oxidation reaction is 3-5;

[0028] Preferably, the H 2 O 2 The concentration of is 12 mM; the concentration of TMB is 1 mM; and the pH of the oxidation reaction is 4.

[0029] In a fourth aspect, the present invention provides the use of the fluorescence-colorimetry dual-mode biosensor described in the first aspect and / or the detection method of flap-shaped endonuclease 1 described in the third aspect in flap-shaped endonuclease 1 related drug screening and / or flap-shaped endonuclease 1 analysis of biological samples;

[0030] The flap endonuclease 1 related drugs include flap endonuclease 1 inhibitors and flap endonuclease 1 activators;

[0031] The biological samples include ex vivo blood, body fluids, tissues and cells.

[0032] In a fifth aspect, the present invention provides a flap endonuclease 1 kit comprising the fluorescence-colorimetric dual-mode biosensor described in the first aspect.

[0033] Beneficial effects of the present invention:

[0034] (1) The fluorescence and colorimetric dual-mode biosensor prepared by the present invention based on cleavage-induced strand displacement reaction (CSDA) and exponential rolling circle amplification reaction (exRCA) is a dual-mode biosensor that can not only give full play to the advantages of each biosensor, but also mutually verify and calibrate the measurement results from different modes, thereby improving the accuracy and reliability of the detection results. In addition, the dual-mode biosensor has two optional signal modes, which can flexibly adapt to more detection conditions and expand the detection range, so it has a wide range of applicability.

[0035] (2) The fluorescence and colorimetric dual-mode biosensor prepared by the present invention exhibits high sensitivity to FEN1, and the detection limit of the fluorescence mode is 1.55×10 -3 U m -1 The detection limit of the colorimetric mode is 0.011U mL -1 , the results are better than those reported previously.

[0036] (3) The fluorescence and colorimetric dual-mode biosensor prepared by the present invention can effectively evaluate the inhibitory effect of inhibitors. The results confirmed that the dual-mode biosensor is a potential tool for FEN1 activity analysis.

[0037] The overall inventive concept adopted by the present invention is: a fluorescent and colorimetric dual-mode biosensor for highly sensitive detection of FEN1 activity based on cleavage-induced strand displacement reaction (CSDA) and exponential rolling circle amplification reaction (exRCA), a DNA branch structure with a 5′flap is designed, which is then recognized and cleaved by FEN1 to release the 5′flap. The released 5′flap triggers CSDA to generate oligonucleotide fragments. With the assistance of T4 ligase, Phi29 polymerase and Nt.BbvCI, the generated oligonucleotide fragments hybridize with circular substrates as primers, and trigger ligation reactions and exRCA in turn to produce abundant guanine (G)-rich sequences. The G-rich sequences are assembled into G-quadruplexes, which bind to heme to form DNAzymes with horseradish peroxidase mimicking activity, and in H 2 O 2 In the presence of β-catenin, it catalyzes the oxidation of TMB to obtain an exponential UV-visible absorption signal output, and combines with NMM to obtain an exponential fluorescence signal output. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0039] Figure 1 The principle of the fluorescence and colorimetric dual-mode biosensor for highly sensitive detection of FEN1 activity based on CSDA and exRCA in the embodiment of the present invention;

[0040] Figure 2 The feasibility of FEN1 determination in the examples of the present invention was investigated by fluorescence emission spectroscopy, polyacrylamide gel electrophoresis and UV-visible absorption spectroscopy, wherein (A) FEN1 determination fluorescence spectroscopy, (B) polyacrylamide gel electrophoresis of FEN1 cleavage process, (C) polyacrylamide gel electrophoresis of FEN1 determination, (D) UV-visible absorption spectroscopy of FEN1 determination;

[0041] Figure 3 Optimized FEN1 assay experimental conditions in the present invention, including (A) FEN1 cleavage time, (B) CSDA reaction time, (C) exRCA reaction time, (D) Phi29 polymerase concentration, (E) Nt.BbvCI concentration, (F) NMM concentration, (G) H 2 O 2 concentration, (H) TMB concentration, (I) pH value; Figure 4 The fluorescence signal and UV-visible absorption signal of different concentrations of FEN1 in the embodiments of the present invention are used to study the detection sensitivity of FEN1 determination, wherein (A) fluorescence spectra of different FEN1 concentrations, (B) curves of different FEN1 concentrations and fluorescence signals, and linear curves of lgC and ΔF, (C) UV-visible absorption spectra of different FEN1 concentrations, (D) curves of different FEN1 concentrations and UV-visible absorption signals, and linear curves of lgC and ΔA;

[0042] Figure 5 The selectivity of FEN1 determined by different enzymes in the examples of the present invention, wherein (A) is the fluorescence signal and (B) is the UV-visible absorption signal;

[0043] Figure 6 In the examples of the present invention, the results of the inhibitory effects of inhibitors on FEN1 activity were studied, including (A) the inhibitory effects of FEN1-IN-1 and ATA on FEN1 activity, and (B) the inhibitory effects of different concentrations of ATA on FEN1 activity. DETAILED DESCRIPTION

[0044] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Specific conditions are not specified in the examples, and the conventional conditions or conditions recommended by the manufacturer are used. The components used without indicating the manufacturer are all conventional products available on the market.

[0045] Example 1

[0046] 1. Preparation of Fluorescence and Colorimetric Dual-mode Biosensors

[0047] (1) Reagents

[0048] Dam methyltransferase, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, Klenow fragment (3′-5′exo-) polymerase (KF polymerase), Nt.BbvCI, and exonuclease I (Exo I) were purchased from New England Biolabs (USA). EcoRI, deoxynucleotide triphosphates (dNTPs), and Phi29 DNA polymerase were purchased from Sangon Biotech (Shanghai) Co., Ltd. (China). FEN1-IN-1 was purchased from MedChemExpress (USA). 3,3′,5,5′-Tetracarboxyl-2,2′-dihydroxybenzidine (ATA) was purchased from Sigma-Aldrich (USA). N-Methylporphyrin dipropionic acid IX (NMM) was purchased from Beijing J&K Technology Co., Ltd. (Beijing, China). The nucleotide sequences (Table 1) were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd. (China). All other reagents were of analytical grade and no additional pretreatment was required. Ultrapure water (>18.25 MΩ) was used to prepare buffer solutions in the experiments.

[0049] Buffers include ThermoPol buffer, CutSmart buffer, T4 DNA ligase buffer and HEPES buffer. The components of the buffer are as follows:

[0050] ThermoPol buffer: 20 mM Tris-Ac, 10 mM (NH 4 ) 2 SO 4 ,10 mM KCl, 10 mM MgSO 4 , 0.1% Triton X-100, pH 8.8.

[0051] CutSmart Buffer: 100 μg mL -1 BSA, 10mM Mg(Ac)2, 50mM KAc, 20mM Tris-Ac, pH7.9.

[0052] T4 Ligase Buffer: 10 mM MgCl 2 , 50mM Tris-HCl, 1mM ATP, 10mM DTT, pH 7.5.

[0053] HEPES buffer: 25 mM HEPES, 200 mM NaCl, 10 mM KCl, 0.05% Triton X-100, pH 4.0.

[0054] Table 1 Nucleotide sequences

[0055]

[0056] (2) FEN1 activity assay

[0057] D1, D2, and D3 were annealed at 95°C for 5 min, gradually cooled to 25°C and maintained for 2 h to form a stable DNA branch structure with a 5′ flap. Similarly, the hairpin substrate formed a stable hairpin structure under the same conditions.

[0058] First, 20 nM DNA branch structure and different concentrations of FEN1 were reacted in ThermoPol buffer at 37°C for 1.5 h to cleave the 5′ flap. Subsequently, 0.6 mM dNTPs, 1 U KF polymerase, 2 U Nt.BbvCI and 40 nM hairpin substrate were added and reacted in CutSmart at 37°C for 1 h to amplify the oligonucleotide fragment. Then, 80 U T4 ligase and 8 μM circular substrate were added and reacted in T4 ligase buffer at 37°C for 1 h to form a circular probe.

[0059] For the fluorescent biosensor, 1 mM dNTPs, 3 μM NMM, 1.5 U Phi29 polymerase, and 4 UNt.BbvCI were added and reacted in CutSmart buffer at 37 °C for 3.5 h to generate a fluorescent amplification signal.

[0060] For the colorimetric biosensor, 1 mM dNTPs, 0.2 μM heme, 1.5 U Phi29 polymerase, and 4 U t.BbvCI were added and reacted in CutSmart buffer at 37 °C for 3.5 h. Then, 12 mM H 2 O 2 and 1 mM TMB, reacted in HEPES buffer at 37°C for 5 min, and 1 M H 2 SO 4 The oxidation reaction is terminated and a UV-visible absorption amplification signal is generated.

[0061] (3) Selective determination

[0062] 20nM 5′flap DNA branching structure and different enzymes (2U mL -1 FEN1, 20U mL -1 T4 PNK, 20U mL -1 T4 DNA ligase, 20 U mL -1 EcoRI, 20U mL -1 Exo I or 20U mL -1 Dam MTase) was reacted in ThermoPol buffer at 37°C for 1.5 hours. The subsequent experimental steps were consistent with the FEN1 activity assay in step (2). The selectivity was studied by recording the fluorescence and UV-visible absorption signals of different samples.

[0063] (4) Inhibition assay

[0064] Different concentrations of ATA (0-5 μM) or 60 μM FEN1-IN-1 and 2 U mL -1 FEN1 was reacted in ThermoPol buffer at 37°C for 30 min, and then 20 nM 5′flap DNA branch structure was added and reacted at 37°C for 1.5 h. The subsequent experimental steps were consistent with the FEN1 activity determination in step (2). The inhibitory effect was studied by recording the fluorescence and UV-visible absorption signals of different samples.

[0065] (5) FEN1 assay principle

[0066] The principle of the fluorescence and colorimetric dual-mode biosensor for highly sensitive detection of FEN1 activity based on CSDA and exRCA is as follows Figure 1 As shown. In this biosensor, a DNA branch structure with a 5′flap, a hairpin substrate, and a circular substrate were designed. Three DNA chains D1, D2, and D3 were designed, and a DNA branch structure with a 5′flap for FEN1 recognition was obtained by hybridizing D1 and D2 with D3 respectively. The hairpin substrate used for the CSDA reaction consists of a protruding region complementary to the 5′flap, a primer complementary sequence that triggers exRCA, and a cleavage site of Nt.BbvCI. The circular substrate used for the exRCA reaction consists of three cleavage sites of Nt.BbvCI, two cytosine-rich sequences, and a complementary primer sequence. In the presence of FEN1, the 5′flap of the DNA branch structure is specifically recognized and cut. Then, the 5′flap with a 3′OH end hybridizes with the protruding end of the hairpin substrate and initiates a polymerization extension reaction along the hairpin substrate with the assistance of KF polymerase. After the polymerization extension reaction, a double-stranded DNA product with a complete cleavage site is generated for the cleavage reaction. After cutting by Nt.BbvCI, new polymerization extension and cutting reaction incisions are generated, and oligonucleotide fragments are generated. After multiple cycles, a large number of oligonucleotide fragments are generated, hybridized with the circular substrate, and the ligation reaction of the circular substrate is triggered with the assistance of T4 ligase to generate a circular probe. Then, the generated oligonucleotide fragments act as primers along the circular probe to trigger RCA with the assistance of Phi29 polymerase, generating a large molecular weight DNA chain with many cutting sites. The DNA chain hybridizes with the circular substrate to generate a double-stranded DNA with many complete cutting sites. After cutting by Nt.BbvCI, new primers are provided for the next round of RCA and cutting reactions to generate G-rich sequences. After exponential cycles, abundant G-rich sequences are generated exponentially. The G-rich sequences assemble into G-quadruplexes, bind to NMM to produce exponential fluorescence signal output, and bind to heme to form a DNAzyme with horseradish peroxidase mimetic activity, which is activated in H 2 O2 In the presence of FEN1, it catalyzes the oxidation of TMB and produces an exponential UV-visible absorption signal output. In the absence of FEN1, the DNA branched structure with 5′ flap did not trigger a subsequent exponential reaction, and the fluorescence and UV-visible absorption signals were extremely weak. By integrating CSDA and exRCA, the dual-mode biosensor showed high sensitivity to FEN1.

[0067] 2. Performance Verification

[0068] (1) Feasibility study

[0069] The feasibility of FEN1 determination was investigated by fluorescence emission spectroscopy, polyacrylamide gel electrophoresis, and UV-visible absorption spectroscopy. Figure 2 As shown, (A) FEN1 fluorescence spectrum. (a) 3μM NMM, (b) 20nM DNA branch structure + 2U Nt.BbvCI + 3μM NMM, (c) 20nM DNA branch structure + 6U Nt.BbvCI + 3μM NMM, (d) 20nM DNA branch structure + 2U mL -1 FEN1+2U Nt.BbvCI+3μM NMM, (e) 20nM DNA branch structure+2U mL -1 FEN1+6U Nt.BbvCI+3μM NMM. (B) Polyacrylamide gel electrophoresis of FEN1 cleavage process. Band 1, 1U mL -1 FEN1 + 40nM DNA branch structure; Band 2, 40nM DNA branch structure; Band 3, 200nM D1; Band 4, 200nM D2; Band 5, 200nM D3. (C) Polyacrylamide gel electrophoresis of FEN1 assay. Band 1, 200nM DNA branch structure; Band 2, 200nM hairpin substrate; Band 3, 200nM circular substrate; Band 4, 200nM DNA branch structure + 200nM hairpin substrate + 200nM circular substrate; Band 5, 2UmL -1 FEN1+200nM DNA branch structure+200nM hairpin substrate+200nM circular substrate. (D)(a) 12mM H 2 O 2 +1mM TMB, (b) 20nM DNA branch structure + 2U Nt.BbvCI + 12mM H 2 O 2 +1mM TMB, (c) 20nM DNA branch structure + 6UNt.BbvCI + 12mM H 2 O 2 +1mM TMB, (d) 20nM DNA branching structure + 2U mL -1FEN1+2U Nt.BbvCI+12mMH 2 O 2 +1mM TMB, (e)20nM DNA branching structure +2U mL -1 FEN1+6U Nt.BbvCI+12mM H 2 O 2 +1mM TMB.

[0070] Figure 2 A in the figure shows an extremely low fluorescence signal in the presence of NMM (curve a). The fluorescence signal slightly increased in the absence of FEN1 (curve c), which is the background signal, indicating that the subsequent CSDA and exRCA reactions cannot be triggered. However, the fluorescence signal increased significantly in the presence of FEN1 (curve e), indicating that FEN1 catalyzes and cuts the 5′flap to trigger the subsequent CSDA and exRCA reactions to produce a strong fluorescence signal. To confirm the exponential amplification effect, the fluorescence signal of the CSDA and RCA cascade reaction (curve d) was significantly enhanced compared with the corresponding background signal (curve b), indicating that FEN1 catalyzes the cutting of the 5′flap to trigger the subsequent CSDA and RCA reactions to produce fluorescence signals. In addition, the fluorescence signal of the cascade amplification reaction (curve d) is significantly lower than that of the exponential amplification reaction (curve e), confirming that the exponential amplification reaction has a better amplification effect than the linear amplification reaction.

[0071] Figure 2 The polyacrylamide gel electrophoresis results of B in the figure show that the three low molecular weight DNA single strands D1 (band 3), D2 (band 4) and D3 (band 5) hybridize to form a stable DNA branch structure with a 5′ flap and a large molecular weight (band 1). After FEN1 cleavage, a new bright band with a high migration speed (band 2) appears, indicating that the 5′ flap of the DNA branch structure is cleaved by FEN1 to produce low molecular weight double-stranded DNA, confirming the feasibility of the FEN1 cleavage process.

[0072] Figure 2 C in Figure 1 shows three obvious bright bands (bands 1-3), indicating the formation of a DNA branch structure with a 5′flap (band 1), a hairpin substrate (band 2), and a circular substrate (band 3). In the absence of FEN1, a new band of high molecular weight appeared, and the band of the template substrate became darker (band 4), which may be due to the hybridization of the 5′flap with the hairpin substrate to form a high molecular weight double-stranded DNA. In the presence of FEN1, a large number of high molecular weight bright bands (band 5) appeared, indicating that FEN1 triggered the CSDA and exRCA reactions to generate DNA fragments of different molecular weights, confirming the feasibility of the amplification process.

[0073] Figure 2 D shows TMB and H2 O 2 In the presence of FEN1, there is a very low UV-visible absorption signal (curve a). The UV-visible absorption signal in the absence of FEN1 increases slightly (curve c), which is the background signal. However, the UV-visible absorption signal in the presence of FEN1 increases significantly (curve e), indicating that FEN1 cuts the 5′flap, triggering the subsequent CSDA and exRCA reactions, generating strong absorption signals. In addition, the UV-visible absorption signal of the CSDA and RCA cascade reaction (curve d) is significantly enhanced compared with the corresponding background signal (curve b), indicating that FEN1 cuts the 5′flap to trigger the subsequent CSDA and RCA reactions to produce absorption signals, and the UV-visible absorption signal of the cascade amplification reaction (curve d) is significantly lower than the absorption signal of the exponential amplification reaction (curve e), confirming that the exponential amplification reaction has a better amplification effect than the linear amplification reaction. All results confirm the feasibility of FEN1 determination.

[0074] (2) Optimize experimental conditions

[0075] Experimental conditions directly affect FEN1 determination and need to be optimized to obtain the best analytical results. In this biosensor, the conditions mainly include FEN1 cleavage time, SDA reaction time, exRCA reaction time, Phi29 polymerase concentration, Nt.BbvCI concentration, NMM concentration, heme concentration, H 2 O 2 The results of the optimization experiments of concentration, TMB concentration and pH value are shown in Figure 3 As shown, (A) FEN1 cutting time, (B) CSDA reaction time, (C) exRCA reaction time, (D) Phi29 polymerase concentration, (E) Nt.BbvCI concentration, (F) NMM concentration, (G) H 2 O 2 concentration, (H) TMB concentration, (I) pH value; error bars are from three parallel experiments.

[0076] The FEN1 cleavage time affects the cleavage efficiency, and we first optimized it. The fluorescence increment (ΔF = F FEN1 -F 不含FEN1 ) increases effectively with the increase of cutting time and reaches the maximum platform at 1.5h ( Figure 3 A) is the optimal cutting time. The amplification reaction time of CSDA and exRCA directly affects the amplification efficiency, and they were optimized in turn. ΔF increased effectively with the increase of CSDA and exRCA reaction time, and at 1h CSDA ( Figure 3 B) and 3.5h exRCA ( Figure 3C) reached the maximum platform, which was the optimal amplification reaction time. In addition, the concentrations of Phi29 polymerase, Nt.BbvCI and NMM were also optimized. ΔF increased effectively with the increase of enzyme concentration. Figure 3 D) and 4U Nt.BbvCI( Figure 3 E) reaches the maximum platform, which is the optimal enzyme concentration, and ΔF increases first and then decreases with the increase of NMM concentration ( Figure 3 F), reaching a maximum at 3 μM NMM, which is the optimal dye concentration. The result is that a small amount of NMM is not enough to interact with the generated oligonucleotide to produce signal output, and excess NMM self-quenches to reduce the fluorescence signal. Then optimize H 2 O 2 and TMB concentration. ΔA(ΔA=A FEN1 -A 不含FEN1 ) With the hemoglobin, H 2 O 2 and TMB concentrations, and at 12 mM H 2 O 2 ( Figure 3 G) and 1 mM TMB ( Figure 3 H) reaches the maximum platform, which is the optimal enzyme concentration. The pH value of the oxidation reaction is optimized. ΔA increases first and then decreases with the increase of pH value ( Figure 3 I). It reaches its maximum value at 4.0, which is the optimal pH value.

[0077] (3) Sensitivity, selectivity, precision and repeatability of FEN1 detection

[0078] The detection sensitivity of FEN1 assay was studied by detecting the fluorescence signal and UV-visible absorption signal of FEN1 at different concentrations. Figure 4 As shown, (A) fluorescence spectra of different FEN1 concentrations. (B) curves of different FEN1 concentrations and fluorescence signals, as well as linear curves of lgC and ΔF. (C) UV-visible absorption spectra of different FEN1 concentrations. (D) curves of different FEN1 concentrations and UV-visible absorption signals, as well as linear curves of lgC and ΔA; error bars are from three parallel experiments.

[0079] As the concentration of FEN1 increased from 0.002 U mL -1 Increase to 2U mL -1 , the fluorescence signal increased significantly, but as the FEN1 concentration increased from 5 U mL -1 Further increase to 10 U mL -1 , the fluorescence signal did not increase significantly or even change ( Figure 4A and B). The results show that low concentrations of FEN1 are insufficient to completely cleave the 5′flap of the DNA branch structure, and the cleaved 5′flap increases with the increase in FEN1 concentration, resulting in a gradual increase in the signal. However, high concentrations of FEN1 completely cleave the 5′flap of the DNA branch structure, and the signal is only slightly affected or does not change with further increases in FEN1 concentration. The regression equation of lgC (logarithm of FEN1 concentration) and ΔF is fitted as: ΔF = 2968.03 + 1051.88lgC, R 2 =0.9968. The fluorescence biosensor has a high sensitivity to FEN1, with a detection limit of 1.55×10 -3 U m -1 As the concentration of FEN1 increased from 0.02 U mL -1 Increase to 2U mL -1 , the UV-visible absorption signal increased significantly, but as the FEN1 concentration increased from 5 U mL -1 Further increase to 10 U mL -1 , the UV-visible absorption signal did not significantly increase or even change ( Figure 4 C and D). The regression equation of lgC (logarithm of FEN1 concentration) and ΔA is fitted as: ΔA = 0.65 + 0.33lgC, R 2 =0.9976. The colorimetric biosensor has a high sensitivity to FEN1, with a detection limit of 0.011 UmL -1 The results were better than those reported previously (Table 2).

[0080] Table 2 Comparison of different FEN1 detection methods

[0081]

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[0087] [5]XHLi,

[0088] [6]YHTang,DDZhang,Y.Lu,SQLiu,J.Zhang,YPPu,W.Wei,Fluorescenceimaging of FEN1 activity in living cells based on controlled-release offluorescence probe from mesoporous silica nanoparticles,Biosens.Bioelectron.214(2022)114529.https: / / doi.org / 10.1016 / j.bios.2022.114529.

[0089] The selectivity of the FEN1 assay was investigated by using ten-fold different enzyme concentrations. Figure 5 As shown, (A) is the fluorescence signal, (B) is the UV-visible absorption signal, and the error bars are from three parallel experiments. In contrast, although the concentrations of other enzymes were 10 times higher than that of FEN1, they (T4 PNK, T4 DNA ligase, EcoRI, Exo I, or Dam MTase) caused the same low fluorescence signal and UV-visible absorption signal as the blank group without FEN1, confirming the high selectivity of the FEN1 assay. In addition, by detecting the low (0.02 U mL -1 ), medium (0.2U mL -1 ) and high (2U mL -1 ) concentration, and its precision and repeatability were studied. The intra-day and inter-day relative standard deviations were less than 3.09% and 5.00%, respectively, confirming that the FEN1 determination had reasonable precision and repeatability.

[0090] (4) Inhibition assay

[0091] The inhibitory effect of the inhibitor on FEN1 activity was studied. Figure 6 As shown, in order to facilitate inhibitor screening. Here, ATA and FEN1-IN-1 were selected as models for study. (A) The inhibitory effect of FEN1-IN-1 and ATA on FEN1 activity. (B) The inhibitory effect of different concentrations of ATA on FEN1 activity.

[0092] The study found that the inhibitor significantly reduced FEN1 activity ( Figure 6 A). The inhibitory effect was further studied by detecting FEN1 activity under different concentrations of ATA. The study found that the inhibitory effect of ATA on FEN1 activity was dose-dependent, and FEN1 activity decreased significantly with the increase of ATA concentration ( Figure 6 B), the calculated maximum half inhibitory concentration (IC50) of ATA was 0.54 μM, which is competitive with the reported results ([7] HTYang, CCWang, ESXu, W.Wei, Y.Liu, SQLiu, Dual-modeFEN1 activity detection based on Nt.BstNBI-induced tandem signal amplification, Anal.Chem. 93 (2021) 6567–6572. https: / / doi.org / 10.1021 / acs.analchem.1c00829. and [8] B.Zhou, L.Lin, BZLi, Exponential amplificationreaction-based fluorescent sensor for the sensitive detection of tumorbiomarker flap endonuclease 1, Sensor.Actuat.B-Chem.346(2021)130457.https: / / doi.org / 10.1016 / j.snb.2021.130457.), confirming the potential of FEN1 assay for screening inhibitory drugs and evaluating their efficacy.

[0093] In summary, a fluorescent and colorimetric dual-mode biosensor for highly sensitive detection of FEN1 activity was developed based on CSDA and exRCA reactions. In this biosensor, FEN1 cuts the 5′flap of the DNA branch structure to trigger CSDA and exRCA, generating abundant G-quadruplexes, which have NMM-specific interactions to achieve exponential fluorescence signal output, and have heme-assisted horseradish peroxidase-mimicking activity to catalyze TMB oxidation to achieve exponential UV-vis absorption signal output. This biosensor uses a hairpin substrate for CSDA reaction and a circular substrate for exRCA reaction, which effectively avoids nonspecific amplification caused by linear substrates and improves detection sensitivity. In addition, this biosensor uses the specific interaction of G-quadruplexes with NMM for signal output, effectively avoiding high background signals caused by nonspecific dyes, or the expensive and cumbersome synthesis of fluorescent group-labeled oligonucleotides. Taking advantage of these advantages, this dual-mode biosensor exhibits high sensitivity to FEN1, and the detection limit of the fluorescent biosensor is 1.55×10 -3 U m -1 , the detection limit of the colorimetric biosensor was 0.011 U mL -1 , and the inhibitory effects of inhibitors were effectively evaluated, confirming that the biosensor is a potential tool for FEN1 activity analysis.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescence-colorimetric dual-mode biosensor, characterized in that: Including DNA branching structures at 5' overhangs, hairpin substrates, and circular substrates; Wherein, the DNA branch structure at the 5' overhang end is formed by hybridization of three DNA single strands D1, D2 and D3; The circular substrate is a circular substrate having three gap sites and a cytosine-rich sequence.

2. The fluorescence-colorimetric dual-mode biosensor according to claim 1, characterized in that: The nucleotide sequence of D1 is shown in SEQ ID NO.1; The nucleotide sequence of D2 is shown in SEQ ID NO.2; The nucleotide sequence of D3 is shown in SEQ ID NO.3; The nucleotide sequence of the hairpin substrate is shown in SEQ ID NO.4; The nucleotide sequence of the circular substrate is shown in SEQ ID NO.

5.

3. The fluorescence-colorimetric dual-mode biosensor according to claim 1, characterized in that: Also included are flap endonuclease 1, T4 DNA ligase, Phi29 DNA polymerase, and Nt.BbvCI.

4. Use of the fluorescence-colorimetric dual-mode biosensor according to any one of claims 1 to 3 in detecting flap-shaped nuclease 1.

5. A method for detecting flap endonuclease 1, characterized in that: The fluorescence-colorimetric dual-mode biosensor according to any one of claims 1 to 3 comprises the following steps: (1) Mixing the sample to be tested with the DNA branch structure at the 5' overhang end, adding dNTPs, KF polymerase, Nt.BbvCI and hairpin substrate to induce strand displacement reaction, then adding T4 ligase and circular substrate to carry out ligation reaction to obtain a circular probe; (2) reacting the circular probe, dNTPs, NMM, Phi29 polymerase and Nt.BbvCI to generate a fluorescent amplification signal; (3) After the circular probe, dNTPs, heme, Phi29 polymerase and Nt.BbvCI are reacted, H2O2 and TMB are added for oxidation reaction, and H2SO4 is added to terminate the reaction to generate a UV-visible absorption amplification signal.

6. The method for detecting flap endonuclease 1 according to claim 5, characterized in that: In step (1), the mixing time is 0.2-2 hours; the induced strand displacement reaction time is 0.3-1.5 hours; and the exponential rolling circle amplification reaction time is 1-5 hours; Preferably, the mixing time is 1.5 h; the inducing strand displacement reaction time is 1.0 h; The time of exponential rolling circle amplification reaction was 3.5 h.

7. The method for detecting flap endonuclease 1 according to claim 5, characterized in that: In step (2), the concentration of the Phi29 polymerase is 0.5-2.0 U; the concentration of the Nt.BbvCI is 1-5 U; The concentration of the NMM is 1-7 μM; Preferably, the concentration of the Phi29 polymerase is 1.5U; the concentration of the Nt.BbvCI is 4U; The concentration of NMM was 3 μM.

8. The method for detecting flap endonuclease 1 according to claim 5, characterized in that: In step (3), the concentration of H2O2 is 2-18 mM; the concentration of TMB is 0.2-1.6 mM; the pH of the oxidation reaction is 3-5; Preferably, the concentration of H2O2 is 12 mM; the concentration of TMB is 1 mM; and the pH of the oxidation reaction is 4.

9. Application of the fluorescence-colorimetric dual-mode biosensor according to any one of claims 1 to 3 and / or the method for detecting flap-shaped endonuclease 1 according to any one of claims 5 to 8 in flap-shaped endonuclease 1 related drug screening and / or flap-shaped endonuclease 1 analysis of biological samples; The flap endonuclease 1 related drugs include flap endonuclease 1 inhibitors and flap endonuclease 1 activators; The biological samples include ex vivo blood, body fluids, tissues and cells.

10. A flap endonuclease 1 kit, characterized in that: A fluorescence-colorimetric dual-mode biosensor comprising any one of claims 1 to 3.

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