A circulating tumor DNA biosensor and its construction method and application

Abstract

The present invention discloses a circulating tumor DNA biosensor and a construction method and application thereof, and belongs to the field of biosensor technology. The dual-mode ratio circulating tumor DNA biosensor of the present invention comprises a sensing substrate assembled with a triangular DNA probe, a probe solution and a nuclease exonuclease. The present invention uses a triangular DNA probe as an anchoring substrate to support target cyclic amplification, and utilizes the ECL-RET effect and enzyme-assisted target cyclic signal amplification strategy to effectively improve the sensitivity of the biosensor; the FL / ECL ratio is used as a dual-mode signal output strategy to improve the accuracy and feasibility of the detection technology. The biosensor of the present invention has high accuracy, high sensitivity and good selectivity, and can realize the quantitative detection of PIK3CA E545K ctDNA in the blood of breast cancer patients, and has important application value in the clinical diagnosis and drug research of breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a circulating tumor DNA biosensor and a construction method and application thereof. Background Art

[0002] Circulating tumor DNA (ctDNA) is extracellular DNA that is broken by tumor cells and enters body fluids such as blood, synovial fluid, and cerebrospinal fluid. Studies have shown that the concentration of ctDNA is related to tumor metastasis and malignant progression, and is used as an important liquid biopsy biomarker in clinical research and practice of tumors. At present, the high correlation between genetic (or epigenetic) abnormalities and cancer has been verified in ctDNA samples. For example, PIK3CA E545K ctDNA in the blood is an effective biomarker for the diagnosis of early breast cancer. However, the content of ctDNA in the blood is extremely low and the background interference is large, and the sensitive and accurate determination of ctDNA in the blood has encountered great challenges.

[0003] Researchers have designed a variety of signal amplification strategies based on nucleic acid chain reactions and conducted quantitative analysis of different ctDNAs. Among them, most nucleic acid chain reactions are carried out in homogeneous solutions, or the DNA chains are directly fixed to heterogeneous surfaces, which causes local crowding effects on linear DNA, thereby inhibiting the recognition and target binding capabilities of the DNA chains, and ultimately leading to reduced detection sensitivity. Therefore, it is very necessary to design nucleic acid chain reactions on the surface of triangular DNA by taking advantage of its mechanical rigidity, clear spacing, and specific orientation.

[0004] The dual-mode ratiometric sensor obtains two types of detection signals through different signal derivation technologies, and uses the ratio of the two signal intensities as the output mode to perform quantitative analysis of the target. This method not only effectively retains the unique performance of different detection methods and achieves complementary advantages, but also can be compared and verified through the detection results, effectively correcting minor changes in the detection environment and improving the accuracy of the detection results. Therefore, providing a dual-mode ratiometric biosensor for detecting circulating tumor DNA is of great significance for the diagnosis of early breast cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a circulating tumor DNA biosensor and its construction method and application to solve the problems existing in the above-mentioned prior art. The present invention uses a triangular DNA probe as an anchor substrate to support target cyclic amplification, and utilizes the ECL-RET effect and enzyme-assisted target cyclic signal amplification strategy to effectively improve the sensitivity of the biosensor; the FL / ECL ratio is used as a dual-mode signal output strategy to improve the accuracy and feasibility of the detection technology. The biosensor of the present invention has the advantages of high accuracy, high sensitivity, good selectivity, etc., and can realize the quantitative detection of PIK3CA E545K ctDNA in the blood of breast cancer patients, and has important application value in the clinical diagnosis and drug research of breast cancer.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a dual-mode ratiometric circulating tumor DNA biosensor, the biosensor comprising a sensing substrate assembled with triangular DNA probes, a probe solution and a nuclease exonuclease;

[0008] The triangular DNA probe is composed of L1 chain, L2 chain, L3 chain and L4 chain; the nucleotide sequence of the L1 chain is shown in SEQ ID NO.1; the nucleotide sequence of the L2 chain is shown in SEQ ID NO.2; the nucleotide sequence of the L3 chain is shown in SEQ ID NO.3; the nucleotide sequence of the L4 chain is shown in SEQ ID NO.4; the 5′ ends of the L1 chain, L2 chain and L3 chain are all modified with SH;

[0009] The probe solution is a mixed solution of probe 1-FAM and probe 2-Ag NPs; the nucleotide sequence of probe 1-FAM is shown in SEQ ID NO.5, and the 5' end is modified with FAM; the nucleotide sequence of probe 2-Ag NPs is shown in SEQ ID NO.6, and the 5' end is modified with SH.

[0010] Preferably, the method for constructing the triangular DNA probe comprises the following steps:

[0011] A1. The L1 chain, L2 chain, and L3 chain are mixed with a tris(2-carboxyethyl)phosphine hydrochloride solution and reacted for 50 to 80 minutes to obtain activated L1 chain, L2 chain, and L3 chain;

[0012] A2. The activated L1 chain, L2 chain, L3 chain and L4 chain are mixed in equal molar ratios, reacted at 90°C~100°C for 4 minutes~6 minutes, and then cooled to 20°C~25°C at a rate of 1°C / min to obtain the triangular DNA probe.

[0013] The present invention also provides a method for constructing the above-mentioned biosensor, comprising the following steps:

[0014] M1. Introducing gold nanoparticles onto graphite phase carbon nitride nanosheets to obtain Au-g-C3N4 NH;

[0015] M2. The glassy carbon electrode is polished, polished and cleaned, and the Au-g-C3N4 NH solution is added dropwise to the surface to obtain an Au-g-C3N4 NH / GCE electrode;

[0016] M3. Assembling the triangular DNA probe onto the surface of the Au-g-C3N4 NH / GCE electrode to obtain a sensing substrate assembled with the triangular DNA probe;

[0017] M4. Probe 2 is mixed with tri(2-carboxyethyl)phosphine hydrochloride solution to react, thereby obtaining activated probe 2; the activated probe 2 is mixed with Ag NPs and incubated in the dark, thereby obtaining probe 2-Ag NPs; probe 1-FAM and the probe 2-Ag NPs are mixed in an equal molar ratio, thereby obtaining a probe solution.

[0018] Preferably, in step M1, the preparation process of the Au-g-C3N4 NH is as follows: 0.1 mg / mL~0.2 mg / mL of graphite phase carbon nitride nanosheets, 8 mM~15 mM chloroauric acid solution, 8 mM~15 mM sodium borohydride solution and 8 mM~15 mM sodium citrate solution are mixed in a volume ratio of 100:6:15:6, and reacted for 40 minutes~60 minutes to obtain the Au-g-C3N4NH.

[0019] Preferably, in step M2, the concentration of the Au-g-C3N4NH solution is 0.5 mg / mL, and the amount of addition is 8 μL~15 μL.

[0020] Preferably, in step M3, the assembly method is: mixing the triangular DNA probe with tri(2-carboxyethyl)phosphine hydrochloride solution for 50 minutes to 80 minutes to obtain an activated triangular DNA probe; immersing the Au-g-C3N4 NH / GCE electrode in the activated triangular DNA probe solution and reacting at room temperature for 1.5 hours to 3 hours;

[0021] The concentration of the activated triangle DNA probe solution is 1 μM.

[0022] The present invention also provides an application of the above biosensor or the biosensor constructed by the above construction method in detecting circulating tumor DNA content. The method for detecting circulating tumor DNA content comprises the following steps:

[0023] S1. Mixing the pretreated sample to be tested with the nuclease exonuclease, and dropping it onto the sensor substrate assembled with the triangular DNA probe to perform a target cyclic amplification reaction;

[0024] S2. The sensing substrate after the reaction in step S1 is mixed with the probe solution and incubated. After the incubation is completed, the probe solution and the sensing substrate after the incubation are collected;

[0025] S3. Detect the fluorescence signal FL of the incubated probe solution under 490 nm excitation; use the incubated sensing substrate as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system to detect the electrochemiluminescence signal ECL in the K2S2O8 solution; calculate the FL / ECL ratio, and calculate the circulating tumor DNA content according to the standard curve.

[0026] Preferably, in step S1, the pretreatment method is to heat the sample to be tested at 90°C~100°C for 10 minutes~20 minutes; the mixing ratio of the nuclease and the pretreated sample to be tested is (0.3 U~0.5 U):1 μL; the amount of dripping is 80 μL~150 μL; the time of the target cycle amplification reaction is 40 minutes~60 minutes.

[0027] Preferably, in step S2, the concentration of the probe solution is 2 μM; and the incubation time is 30 minutes to 60 minutes.

[0028] Preferably, the circulating tumor DNA is PIK3CA E545K ctDNA.

[0029] The present invention discloses the following technical effects:

[0030] The present invention constructs a dual-mode ratiometric circulating tumor DNA biosensor, which realizes the clinical detection of PIK3CA E545K ctDNA in the blood samples of breast cancer patients. The construction process and signal output principle of the biosensor of the present invention are shown in the figure. Figure 1As shown, the present invention hybridizes four single-stranded DNAs (L1, L2, L3 and L4) to form a triangular DNA probe, and the vertex of each triangle contains SH-(CH2)6- and a hanging DNA fragment (i.e., capture DNA). Then, the triangular DNA probe is orderly assembled on the Au-g-C3N4-NH modified sensing substrate through Au-S bonds. When the target ctDNA is present, the ctDNA is partially complementary to the capture DNA, and the formed double-stranded DNA is cleaved by the Exo III enzyme. After cleavage by the Exo III enzyme, the target ctDNA is released and hybridizes with the end portion of another capture DNA to form a new cleavage site, thereby triggering target cyclic signal amplification and retaining a large number of short fragments of capture DNA on the surface of the sensing substrate. Based on the competitiveness of DNA hybridization, the carboxyfluorescein-labeled probe 1-FAM and the silver nanosphere-modified probe 2-Ag-NPs selectively compete with the end portion and short fragment of the capture DNA, respectively. Based on the ECL-RET effect, the ECL emission of Au-g-C3N4-NH was absorbed by the Ag NPs plasma, resulting in the quenching of the ECL signal of Au-g-C3N4 NH (ECL off); at the same time, the FL signal of the remaining single-stranded probe 1-FAM in the solution was restored (FL on). By measuring the signal ratio of FL / ECL, a dual-mode ratiometric biosensor that can accurately measure ctDNA was obtained.

[0031] The present invention uses a triangular DNA probe as a supporting substrate to improve the orderly arrangement and distribution of DNA chains; utilizes the ECL-RET effect and Exo III enzyme-assisted target cycle signal amplification capability to significantly improve the sensitivity of the biosensor; based on the self-correction capability of the FL / ECL dual-mode detection technology for complex environments, the accuracy and feasibility of the ratio biosensor are improved. Therefore, the dual-mode ratio biosensor prepared by the present invention has the advantages of high accuracy, high sensitivity, good selectivity, etc., and can be applied to the accurate determination of PIK3CA E545K ctDNA in the blood of breast cancer patients, and has important application value in the clinical diagnosis and drug research of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 Schematic diagram of the construction process and signal output principle of the dual-mode ratiometric circulating tumor DNA biosensor of the present invention;

[0034] Figure 2 Characterization diagrams of Au-g-C3N4 NH and Ag NPs prepared in Example 1; wherein A is the TEM image of g-C3N4 NS; B is the TEM image of Au-g-C3N4 NH; C is the TEM image of Ag NPs; D is the UV-vis absorption spectrum of Ag NPs and the ECL spectrum of Au-g-C3N4 NH;

[0035] Figure 3 Graphs showing the monitoring results of the triangular DNA probe construction process, the target cyclic amplification reaction process, and the sensing substrate interface construction process in Example 2; wherein, A is a graph showing the monitoring results of the triangular DNA probe construction process, lanes 1 to 4 represent L1 chain, L2 chain, L3 chain, and L4 chain, respectively, lane 5 represents a mixture of L1 chain, L2 chain, and L3 chain, and lane 6 represents a triangular DNA probe; B is a graph showing the monitoring results of the target cyclic amplification reaction process, lane 1 represents a triangular DNA probe, lane 2 represents ctDNA, lane 3 represents a mixture of a triangular DNA probe and ctDNA, lane 4 represents a supernatant after the triangular DNA probe, ctDNA, and ExoIII enzyme are mixed and centrifuged, lane 5 represents probe 1-FAM, lane 6 represents probe 2-Ag NPs, and lane 7 represents a sample after the supernatant of lane 4, probe 1-FAM, and probe 2-Ag NPs are incubated; C is an electrochemical impedance spectroscopy (EIS) characterization graph of the sensing substrate interface construction process, a represents GCE, and b represents Au-g-C3N4 NH / GCE, c represents triangle DNA / Au-g-C3N4 NH / GCE, d represents the sensing substrate after reaction with PIK3CA E545K ctDNA and Exo III, and e represents the sensing substrate after incubation with the probe solution; D is the ECL response diagram of the sensing substrate interface construction process, a represents Au-g-C3N4 NH / GCE, b represents triangle DNA / Au-g-C3N4 NH / GCE, c represents the sensing substrate after reaction with PIK3CA E545K ctDNA and Exo III, and d represents the sensing substrate after incubation with the probe solution;

[0036] Figure 4 : The detection result diagram of the dual-mode ratiometric circulating tumor DNA biosensor for PIK3CAE545K ctDNA samples with different concentrations in Example 2; wherein A is the ECL response curve diagram of PIK3CA E545K ctDNA samples with different concentrations, B is the FL response curve diagram of PIK3CA E545K ctDNA samples with different concentrations, and the concentrations corresponding to 1 to 9 are 0, 5×10 -17 , 10 -16 , 5×10 -16 , 5×10-15 , 10 -14 , 10 -13 , 10 -12 and 10 -11 M; C is a curve diagram showing the relationship between the FL / ECL ratio and the logarithm of the PIK3CAE545K ctDNA concentration, and the inset is a linear relationship diagram between the logarithm of the FL / ECL ratio and the logarithm of the PIK3CA E545K ctDNA concentration; D is a bar graph showing the FL and ECL response values ​​of different DNA samples, where a represents a blank solution, b represents a DNA sample with a three-base mismatch, c represents a DNA sample with a single-base mismatch, and d represents a PIK3CA E545K ctDNA sample;

[0037] Figure 5 This is a graph showing the test results of blood samples from healthy volunteers and blood samples from breast cancer patients in Example 3; wherein the left column is the ECL value, and the right column is the FL value. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] This example prepares a dual-mode ratiometric circulating tumor DNA biosensor, which includes a sensing substrate assembled with triangular DNA probes, a probe solution, and a nuclease. The construction process is as follows:

[0045] N1. Design and synthesize triangular DNA probes. The specific operation is as follows: To open the disulfide bond, the 5′-end SH-modified L1 chain, L2 chain, and L3 chain were mixed with tri(2-carboxyethyl)phosphine hydrochloride solution (2 μM) for 60 minutes, and then filtered and purified using a DNA centrifugal ultrafiltration tube (3 KD) to remove tri(2-carboxyethyl)phosphine hydrochloride to obtain activated L1 chain, L2 chain, and L3 chain. Then, the activated L1 chain, L2 chain, L3 chain, and L4 chain were mixed in an equimolar ratio, reacted at 95°C for 5 minutes, and then cooled to 25°C at a rate of 1°C / min to obtain a triangular DNA probe. The three vertices of the triangular DNA probe are modified with capture DNA and SH-(CH2)6-.

[0046] Among them, the sequence of the L1 chain is: 5′-SH-TTT TCA CCA GGC AGT AAC CTG CAC TAG CAT ACTCTC TCT AAA ATC ACT GAG-3′ (SEQ ID NO. 1);

[0047] The sequence of the L2 chain is: 5′-SH-TTT GTG TAG CAA GCT AAC CTG CAC TAG CAT ACT CTCTCT AAA ATC ACT GAG-3′ (SEQ ID NO. 2);

[0048] The sequence of the L3 chain is: 5′-SH-TTT ATG CGA GGT CAA AAC CTG CAC TAG CAT ACT CTCTCT AAA ATC ACT GAG-3′ (SEQ ID NO. 3);

[0049] The sequence of the L4 chain is: 5′-TTG ACC TCG CAT AGC TTG CTA CAC ACT GCC TGG TGA-3′ (SEQ ID NO. 4).

[0050] N2. Construct Au-g-C3N4 NH / GCE electrode, assemble triangular DNA probes in order on the surface of Au-g-C3N4 NH / GCE electrode, and obtain a sensing substrate assembled with triangular DNA probes. The specific operation is as follows: First, the glassy carbon electrode (GCE) is polished and polished with Al2O3, and after ultrasonic cleaning, 10 μL of Au-g-C3N4 NH solution (concentration of 0.5 mg / mL) is drop-coated on the surface of GCE. After natural drying, the Au-g-C3N4 NH functionalized GCE electrode (Au-g-C3N4 NH / GCE) is obtained. Then, the triangular DNA probe was mixed with tri(2-carboxyethyl)phosphine hydrochloride solution for 60 minutes and filtered to remove tri(2-carboxyethyl)phosphine hydrochloride to obtain the thiol-activated triangular DNA probe. The Au-g-C3N4 NH / GCE was immersed in 100 μL of thiol-activated triangular DNA probe solution (concentration was 1 μM), and the triangular DNA probe was orderly assembled onto the surface of Au-g-C3N4 NH / GCE using Au−S bonds to obtain a sensing substrate assembled with triangular DNA probes (referred to as triangular DNA / Au-g-C3N4 NH / GCE).

[0051] The preparation method of Au-g-C3N4 NH solution is as follows: 0.5 mL of graphite phase carbon nitride nanosheets (0.15 mg / mL) is mixed with 30 μL of chloroauric acid solution (10 mM), 75 μL of sodium borohydride solution (10 mM) and 30 μL of sodium citrate solution (10 mM), and reacted for 50 minutes to obtain the Au-g-C3N4 NH. Graphite phase carbon nitride nanosheets (abbreviated as g-C3N4 NS) have a lamellar structure, such as Figure 2 The structure of Au-g-C3N4 NH is shown in Figure 2 As shown in B, the −NH2 and −NH groups carried by g-C3N4 NS and NaBH4 are both beneficial to AuCl4 − reduction, while the addition of sodium citrate effectively improved the stability of Au-g-C3N4 NH.

[0052] N3. Mix probe 1-FAM and probe 2-Ag NPs to obtain a probe solution. The specific operation is as follows: Mix probe 2 labeled with SH at the 5′ end with tri(2-carboxyethyl)phosphine hydrochloride solution for 60 minutes to open the disulfide bond, filter and purify, remove tri(2-carboxyethyl)phosphine hydrochloride, and then add it dropwise to Ag NPs, incubate for 2 hours in the dark, and further centrifuge (10000 rpm, 8 minutes) to purify to obtain probe 2-Ag NPs. Then, mix probe 1-FAM and probe 2-Ag NPs in equal molar ratios to obtain a probe solution.

[0053] Among them, Ag NPs were prepared by reducing AgNO3 with NaBH4, and their morphology was characterized by transmission electron microscopy. Figure 2 As shown in Figure 3C, the spherical Ag NPs are well dispersed with an average size of 10 nm.

[0054] Among them, the sequence of probe 1-FAM is 5′-FAM-CTC AGT GAT TTT AGA GAG AGT ATG CTA G-3′ (SEQ ID NO.5); the sequence of probe 2-Ag NPs is 5′-SH-TTT TTT TAT GCT AGT GCA GGT T-3′ (SEQID NO.6).

[0055] N4. The method of using the biosensor is as follows: the sample to be tested is heated at 95°C for 15 minutes, followed by the addition of 0.4 U / μL Exo III enzyme, and 100 μL is added to the surface of the triangular DNA / Au-g-C3N4 NH / GCE sensing substrate for 50 minutes to perform a target cyclic amplification reaction. The sensing substrate after the reaction is mixed with the probe solution and incubated for 40 minutes. After the incubation is completed, the remaining probe solution and the sensing substrate after incubation are collected.

[0056] The remaining probe solution was detected by Hitachi F-7000 fluorescence spectrophotometer with an excitation wavelength of 490 nm, and its fluorescence (FL) signal was recorded. The three-electrode system was composed of Ag / AgCl electrode as reference electrode, platinum wire as auxiliary electrode, and the incubated sensing substrate as working electrode. The electrochemiluminescence (ECL) signal of the three-electrode system in K2S2O8 solution was detected by Chenhua CHI 660E electrochemical workstation. The FL / ECL ratio was calculated.

[0057] Example 2

[0058] This example tests the performance of the biosensor prepared in Example 1, and the specific process is as follows:

[0059] N1. The biosensor of the present invention utilizes the ECL-RET effect to improve the detection sensitivity. Au-g-C3N4 NH is the ECL donor and Ag NPs is the energy acceptor, forming an ECL-RET sensing system. In order to verify the efficiency of the ECL-RET sensing system, the Ag NPs and Au-g-C3N4 NH prepared in Example 1 were subjected to spectral detection. The results are shown in FIG. Figure 2 As shown in Figure d, Ag NPs show a characteristic absorption peak at 425 nm, which shows a good overlap with the ECL emission spectrum of Au-g-C3N4 NH at 440 nm.

[0060] N2. The biosensor of the present invention uses a triangular DNA probe as a supporting substrate, and further improves the detection sensitivity through a target cyclic amplification reaction. Therefore, the triangular DNA probe construction process, the target cyclic amplification reaction process and the sensing substrate interface construction process are monitored and verified.

[0061] First, the construction process of the triangular DNA probe was detected by polyacrylamide gel electrophoresis (PAGE). Figure 3 As shown in A, lanes 1 to 4 represent L1, L2, L3 and L4 chains, respectively, lane 5 represents a mixture of L1, L2 and L3 chains, and lane 6 represents a triangular DNA probe. It can be seen that only the band corresponding to the position of L1 chain (or L2 chain, L3 chain) is observed in lane 5, indicating that no hybridization reaction occurs between L1, L2 and L3 when L4 is missing. When the mixture of L1 chain, L2 chain and L3 chain is heated and annealed, a new lagging band appears in lane 6, indicating that a triangular DNA probe has been formed, which has a larger molecular weight and exhibits a slower electrophoretic mobility.

[0062] Next, the enzyme-assisted target cyclic amplification reaction process was monitored by PAGE. Figure 3 As shown in Figure B, when the triangular DNA probe and ctDNA were mixed (lane 3), a slower migrating band was observed compared with the triangular DNA probe (lane 1) and ctDNA (lane 2), proving the successful hybridization of ctDNA with the captured DNA and assembly onto the triangular DNA probe. After the addition of Exo III enzyme and centrifugation, a new band appeared in the supernatant (lane 4), indicating that the formed duplex was cleaved under the shearing of ExoIII enzyme, and the faster migrating band corresponded to the released ctDNA. Lanes 5 and 6 represent 1-FAM and probe 2-Ag NPs, respectively. When 1-FAM and probe 2-Ag NPs were incubated with the supernatant of lane 4, their bands disappeared, and new bands were observed in lane 7. Therefore, these results verified the feasibility of target circular amplification on triangular DNA.

[0063] Finally, in order to verify the construction process of the sensing substrate interface, EIS and ECL were used to characterize the signal response of different sensing substrate interfaces. Figure 3 As shown in C and D, on the bare GCE surface, the electron transfer impedance (Ret) is about 476 Ω ( Figure 3 C, curve a). When Au-g-C3N4 NH was assembled onto the GCE surface, the Ret of the resulting Au-g-C3N4 NH / GCE electrode was significantly reduced to 78 Ω ( Figure 3 C, curve b), and at the same time, an obvious ECL intensity of 14650 au ( Figure 3D, curve a). When the triangular DNA probe was assembled onto the surface of Au-g-C3N4 NH / GCE electrode, Ret increased to 2112 Ω ( Figure 3 C, curve c), the ECL intensity decreases to 13570 au ( Figure 3 D, curve b). This is attributed to the negatively charged phosphate backbone in the DNA molecular structure hindering the interfacial charge transfer. Subsequently, after the triangle DNA / Au-g-C3N4 NH / GCE was reacted in a mixture of PIK3CA E545K ctDNA and Exo III, Ret decreased to 1741 Ω ( Figure 3 C, curve d), the ECL intensity increases to 13910 a.u. ( Figure 3 D, curve c), which proves that the presence of target ctDNA drives the continuous cleavage reaction of Exo III, and a large number of DNA fragments are dissociated from the surface of the sensing substrate. Finally, the sensing substrate after the reaction is incubated in the probe solution. Ag NPs have good conductivity and can effectively accelerate electron transfer, so the Ret value is further reduced to 870 Ω ( Figure 3 At the same time, based on the ECL-RET effect between Au-g-C3N4 NH and Ag NPs, the ECL intensity dropped to 6240 au ( Figure 3 The above EIS and ECL results effectively demonstrate the construction process of the electrode sensing interface.

[0064] N3. Using different concentrations of PIK3CA E545K ctDNA samples (0, 5×10 -17 , 10 -16 , 5×10 -16 , 5×10 -15 , 10 -14 , 10 -13 , 10 -12 and 10 -11 M), the detection performance of the biosensor was verified according to the method described in step (4) of Example 1.

[0065] ECL signals of PIK3CA E545K ctDNA samples with different concentrations are shown in Figure 2. Figure 4 As shown in A, the FL signal is Figure 4 As shown in B, the concentrations corresponding to 1 to 9 are 0, 5×10 -17 , 10 -16 , 5×10 -16 , 5×10 -15 , 10 -14 , 10 -13 , 10 -12 and 10 -11M. It can be seen that with the increase in the concentration of PIK3CA E545K ctDNA samples, the ECL intensity gradually decreased and the FL signal significantly increased.

[0066] The relationship curve between the FL / ECL ratio of PIK3CA E545K ctDNA samples with different concentrations and the logarithmic value of sample concentration is shown in the figure. Figure 4 As shown in C, it can be seen that at 5×10 -17 M to 10 -11 Within the concentration range of M, there was a good linear relationship between the logarithm of the FL / ECL ratio and the logarithm of the PIK3CAE545K ctDNA concentration (R 2 = 0.995), the linear regression equation is lgFL / ECL = 0.257 lg c + 3.443. According to the calculation formula LOD = 3S b / m, the detection limit (LOD) of this method was 20 aM, where S b is the standard deviation of the blank response, and m is the slope of the calibration curve.

[0067] N4. Use different DNA samples, including blank solution, PIK3CA E545K ctDNA sample (10 -13 M), DNA samples with three base mismatches and DNA samples with one base mismatch, and the selectivity and specificity of the biosensor were verified according to the method described in step (4) of Example 1.

[0068] Test results such as Figure 4 As shown in D. It can be seen that compared with the blank solution (column a), the target ctDNA can cause a significant decrease in the ECL signal and a significant increase in the FL signal (column d), while the ECL and FL responses of the three-base mismatched DNA (column b) and the single-base mismatched DNA (column c) are similar to those of the blank solution (column a). Therefore, the biosensor of the present invention has good selectivity and specificity for ctDNA detection.

[0069] Example 3

[0070] This example uses blood samples from healthy volunteers (numbered H1 to H6) and blood samples from breast cancer patients (numbered C1 to C6) to verify the detection performance of the biosensor in Example 1. The specific process is as follows:

[0071] Blood samples from healthy subjects (numbered H1~H6) and blood samples from breast cancer patients (numbered C1~C6) were collected from Xuzhou Central Hospital on June 1, 2023.

[0072] N1. Dilute the sample to be tested 10 times with phosphate buffer, heat at 95°C for 15 minutes, then add 0.4U / μL Exo III enzyme, take 100 μL and drop it on the surface of the triangular DNA / Au-g-C3N4 NH / GCE sensor substrate for 50 minutes to perform target cyclic amplification reaction.

[0073] N2. Mix the reacted sensing substrate with 100 μL of 2 μM probe solution and incubate for 40 minutes. After the incubation, collect the remaining probe solution and the incubated sensing substrate. Place the remaining probe solution under 490 nm excitation to detect the fluorescence (FL) signal; use the incubated sensing substrate as the working electrode, and form a three-electrode system with the Ag / AgCl reference electrode and the platinum wire auxiliary electrode, and place it in a phosphate buffer solution containing K2S2O8. Use Chenhua CHI 660E electrochemical workstation to detect the electrochemiluminescence (ECL) signal of the three-electrode system.

[0074] Test results such as Figure 5 As shown, the ECL signal intensity of blood samples from breast cancer patients (C1, C2, C3, C4, C5 and C6) was significantly lower than that of blood samples from healthy volunteers (H1, H2, H3, H4, H5 and H6), while the FL response showed a completely opposite trend.

[0075] The average concentration of PIK3CA E545K ctDNA in the blood samples of healthy volunteers was 0.85 fM, while the average concentration of PIK3CA E545K ctDNA in the blood samples of breast cancer patients was 39.83 fM, calculated using the linear regression equation of Example 2. The high expression level of PIK3CA E545K ctDNA in the blood of breast cancer patients is consistent with current literature reports, indicating that the dual-mode ratiometric biosensor of the present invention has good detection capabilities even in complex blood samples.

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A dual-mode ratiometric circulating tumor DNA biosensor, characterized in that: The biosensor comprises a sensing substrate assembled with a triangular DNA probe, a probe solution and a nuclease; The triangular DNA probe is composed of L1 chain, L2 chain, L3 chain and L4 chain; the nucleotide sequence of the L1 chain is shown in SEQ ID NO.1; the nucleotide sequence of the L2 chain is shown in SEQ ID NO.2; the nucleotide sequence of the L3 chain is shown in SEQ ID NO.3; the nucleotide sequence of the L4 chain is shown in SEQ ID NO.4; the 5′ ends of the L1 chain, L2 chain and L3 chain are all modified with SH; The probe solution is a mixed solution of probe 1-FAM and probe 2-Ag NPs; the nucleotide sequence of probe 1-FAM is shown in SEQ ID NO.5, and the 5' end is modified with FAM; the nucleotide sequence of probe 2-Ag NPs is shown in SEQ ID NO.6, and the 5' end is modified with SH.

2. The biosensor according to claim 1, characterized in that The method for constructing the triangular DNA probe comprises the following steps: A1. The L1 chain, L2 chain, and L3 chain are mixed with a tris(2-carboxyethyl)phosphine hydrochloride solution and reacted for 50 to 80 minutes to obtain activated L1 chain, L2 chain, and L3 chain; A2. The activated L1 chain, L2 chain, L3 chain and L4 chain are mixed in equal molar ratios, reacted at 90°C~100°C for 4 minutes~6 minutes, and then cooled to 20°C~25°C at a rate of 1°C / min to obtain the triangular DNA probe.

3. A method for constructing a biosensor according to claim 1 or 2, characterized in that: The steps include: M1. Introducing gold nanoparticles onto graphite phase carbon nitride nanosheets to obtain Au-g-C3N4 NH; M2. The glassy carbon electrode is polished, polished and cleaned, and the Au-g-C3N4 NH solution is added dropwise to the surface to obtain an Au-g-C3N4 NH / GCE electrode; M3. Assembling the triangular DNA probe onto the surface of the Au-g-C3N4 NH / GCE electrode to obtain a sensing substrate assembled with the triangular DNA probe; M4. Probe 2 is mixed with tri(2-carboxyethyl)phosphine hydrochloride solution to react, thereby obtaining activated probe 2; the activated probe 2 is mixed with Ag NPs and incubated in the dark, thereby obtaining probe 2-Ag NPs; probe 1-FAM and the probe 2-Ag NPs are mixed in an equal molar ratio, thereby obtaining a probe solution.

4. The construction method according to claim 3, characterized in that: In step M1, the preparation process of the Au-g-C3N4 NH is as follows: 0.1 mg / mL~0.2 mg / mL of graphite phase carbon nitride nanosheets, 8 mM~15 mM chloroauric acid solution, 8 mM~15 mM sodium borohydride solution and 8 mM~15 mM sodium citrate solution are mixed in a volume ratio of 100:6:15:6, and reacted for 40 minutes~60 minutes to obtain the Au-g-C3N4 NH.

5. The construction method according to claim 3, characterized in that: In step M2, the concentration of the Au-g-C3N4 NH solution is 0.5 mg / mL, and the amount of addition is 8 μL~15 μL.

6. The construction method according to claim 3, characterized in that: In step M3, the assembly method is: mixing the triangular DNA probe with tri(2-carboxyethyl)phosphine hydrochloride solution for 50 minutes to 80 minutes to obtain an activated triangular DNA probe; immersing the Au-g-C3N4 NH / GCE electrode in the activated triangular DNA probe solution and reacting at room temperature for 1.5 hours to 3 hours; The concentration of the activated triangle DNA probe solution is 1 μM.

7. Use of the biosensor according to claim 1 or 2 or the biosensor constructed by the construction method according to any one of claims 3 to 6 in detecting circulating tumor DNA content, characterized in that: The method for detecting circulating tumor DNA content comprises the following steps: S1. Mixing the pretreated sample to be tested with the nuclease exonuclease, and dropping it onto the sensor substrate assembled with the triangular DNA probe to perform a target cyclic amplification reaction; S2. The sensing substrate after the reaction in step S1 is mixed with the probe solution and incubated. After the incubation is completed, the probe solution and the sensing substrate after the incubation are collected; S3. Detect the fluorescence signal FL of the incubated probe solution under 490 nm excitation; use the incubated sensing substrate as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system to detect the electrochemiluminescence signal ECL in the K2S2O8 solution; calculate the FL / ECL ratio, and calculate the circulating tumor DNA content according to the standard curve.

8. The use according to claim 7, characterized in that: In step S1, the pretreatment method is to heat the sample to be tested at 90°C~100°C for 10 minutes~20 minutes; the mixing ratio of the nuclease and the pretreated sample to be tested is (0.3 U~0.5 U):1 μL; the amount of dropwise addition is 80 μL~150 μL; and the time of the target cyclic amplification reaction is 40 minutes~60 minutes.

9. The use according to claim 7, characterized in that: In step S2, the concentration of the probe solution is 2 μM; and the incubation time is 30 minutes to 60 minutes.

10. The use according to any one of claims 7 to 9, characterized in that: The circulating tumor DNA is PIK3CAE545K ctDNA.

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