A biosensor for detecting telomerase activity, its preparation method and application

Abstract

The present invention discloses a biosensor for detecting telomerase activity, its preparation method and application, which relates to the technical field of biosensors. The preparation method includes the following steps: after the indium tin oxide electrode is cleaned, a Zn-MOFs@Au NPs composite is dropped onto its surface to obtain a Zn-MOFs@Au NPs / ITO electrode; the Zn-MOFs@Au NPs / ITO electrode is immersed in an LH:P double-stranded solution for an incubation reaction to obtain the biosensor; the Zn-MOFs@Au NPs composite is obtained by mixing and reacting gold nanoparticles and zinc-based metal-organic framework materials. The biosensor prepared by the present invention has advantages such as high accuracy, strong sensitivity and good selectivity, and can be applied to the accurate detection of telomerase in cervical cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a biosensor for detecting telomerase activity and a preparation method and application thereof. Background Art

[0002] Human telomerase, as a ribonucleoprotein reverse transcriptase, can maintain telomere length and protect genetic material by adding a repetitive short sequence (TTAGGG) to the end of chromosomes. In normal cells, as telomerase expression is inhibited or it is defective, telomeres gradually shorten during each round of cell division, leading to cell aging and death. In contrast, overexpressed telomerase in cancer cells effectively maintains the length of telomeres, leading to unlimited proliferation of cancer cells. Therefore, telomerase can be used as a biomarker for early cancer diagnosis. Given the association between telomerase activity and tumor progression, it is crucial to design an effective and sensitive method to assess telomerase activity.

[0003] Researchers have designed a variety of signal amplification strategies based on nucleic acid chain reactions and conducted quantitative analysis of telomerase activity. Among them, most nucleic acid chain reactions are carried out in homogeneous solutions, and the problem that the discarded DNA chain is difficult to separate cannot be ignored. Based on the enzyme-free and isothermal amplification characteristics of entropy-driven catalytic reactions and hybridization chain reactions, it is very necessary to design entropy-driven catalytic reactions at solid interfaces to stimulate hybridization chain reactions.

[0004] In recent years, metal organic frameworks (MOFs) formed by self-assembled site bonds between organic ligands and metal ions or metal clusters have attracted the attention of researchers. MOFs have porous crystal structures, special topological spaces, adjustable functionality, and moderate charge carrier recombination rates, which are beneficial to the construction of photoelectrochemical sensors. Nanomaterials have been used to improve conductivity, increase active sites, and catalyze the generation of signal enhancers or quenchers due to their efficient light absorption, conversion capabilities, and excellent catalytic properties. The quenchers generated by in situ catalysis can effectively aggregate and amplify local concentrations, thereby improving the sensitivity of the sensing system. Therefore, the present invention intends to construct a photoelectrochemical biosensor by combining MOFs with the in situ quenching strategy of nanomaterials, thereby realizing the detection of telomerase activity in cervical cancer cells. Summary of the invention

[0005] The purpose of the present invention is to provide a biosensor for detecting telomerase activity and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The biosensor prepared by the present invention has the advantages of high accuracy, strong sensitivity, good selectivity, etc., and can be applied to the accurate detection of telomerase in cervical cancer cells.

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

[0007] The present invention provides a method for preparing a biosensor for detecting telomerase activity in cervical cancer cells, comprising the following steps:

[0008] After the indium tin oxide electrode is cleaned, a Zn-MOFs@Au NPs composite is dropped onto its surface to obtain a Zn-MOFs@Au NPs / ITO electrode;

[0009] The Zn-MOFs@Au NPs / ITO electrode is immersed in an LH:P double-stranded solution for incubation reaction to obtain the biosensor;

[0010] The Zn-MOFs@Au NPs composite is obtained by mixing and reacting gold nanoparticles and zinc-based metal-organic framework materials;

[0011] The LH:P double-stranded solution contains an LH:P double strand obtained by incubating a thiol-activated LH strand with a P strand;

[0012] The nucleotide sequence of the LH strand is shown as SEQ ID NO.1; the nucleotide sequence of the P strand is shown as SEQ ID NO.2.

[0013] Further, the method for preparing the zinc-based metal-organic framework material comprises the following steps:

[0014] N,N-diethylethylenediamine, Zn(NO3)2·6H2O and 9,10-bis(4-carboxyphenyl)anthracene are sequentially dissolved in N,N-dimethylformamide, and after ultrasonic treatment, a mixed reaction is carried out at 120 °C to obtain the zinc-based metal-organic framework material.

[0015] Further, the cleaning treatment includes the steps of ultrasonic cleaning with acetone, ethanol and deionized water in sequence.

[0016] Further, the thiol-activated LH strand is obtained by mixing and reacting the LH strand with a tris(2-carboxyethyl)phosphine hydrochloride solution.

[0017] The present invention also provides a biosensor for detecting telomerase activity in cervical cancer cells prepared by the above preparation method.

[0018] The present invention also provides the application of the above biosensor in the preparation of a kit for detecting telomerase activity in cervical cancer cells.

[0019] The present invention also provides a kit for detecting telomerase activity in cervical cancer cells, comprising the above biosensor.

[0020] Further, the kit further includes an M chain with an amino group at the 3′ end, a telomerase primer, an S chain, an F chain, an H1 chain, an H2 chain, an H3 chain, and an H4 chain;

[0021] The nucleotide sequence of the M chain is as shown in SEQ ID NO.3;

[0022] The nucleotide sequence of the telomerase primer is as shown in SEQ ID NO.4;

[0023] The nucleotide sequence of the S chain is as shown in SEQ ID NO.5;

[0024] The nucleotide sequence of the F chain is as shown in SEQ ID NO.6;

[0025] The nucleotide sequence of the H1 chain is as shown in SEQ ID NO.7;

[0026] The nucleotide sequence of the H2 chain is as shown in SEQ ID NO.8;

[0027] The nucleotide sequence of the H3 chain is as shown in SEQ ID NO.9;

[0028] The nucleotide sequence of the H4 chain is as shown in SEQ ID NO.10.

[0029] Further, the kit further includes manganese dioxide nanoflowers;

[0030] The manganese dioxide nanoflowers are obtained by mixing oleic acid and KMnO4 for a reaction.

[0031] The present invention discloses the following technical effects:

[0032] The present invention constructs a photoelectrochemical biosensor and realizes the detection of telomerase activity in cervical cancer cells. This biosensor uses magnetic bead separation technology to achieve signal transduction of telomerase activity in cervical cancer cells; uses Zn-MOFs as the photoelectrochemical active substrate and MnO2 NFs as the electron-donating catalyst to quench the photocurrent signal in situ; and stimulates the hybridization chain reaction by means of entropy-driven catalytic reaction to achieve cyclic amplification of the target telomerase, thereby enabling highly sensitive detection of telomerase activity in cervical cancer cells.

[0033] The present invention conducts signal transduction of telomerase activity in cervical cancer cells by magnetic bead separation technology, improving the selectivity of the sensing system; uses Zn-MOFs as the optoelectrochemical activity substrate, combined with the in-situ quenching of MnO2 NFs, to improve signal output and response; and based on the dual DNA cyclic signal amplification strategy, improves the accuracy and sensitivity of the biosensor. Therefore, the biosensor prepared by the present invention has the advantages of high accuracy, strong sensitivity, good selectivity, etc., and can be applied to the accurate detection of telomerase in cervical cancer cells. Brief Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0035] Figure 1 It is a schematic diagram of the sensing mechanism of the optoelectrochemical biosensor for telomerase activity in cervical cancer cells of the present invention;

[0036] Figure 2 It is a scanning electron microscope (SEM) image of Zn-MOFs;

[0037] Figure 3 It is an X-ray photoelectron spectroscopy (XPS) image of Zn-MOFs;

[0038] Figure 4 It is an XPS image of the Zn 2p orbital electrons of Zn-MOFs;

[0039] Figure 5 It is an SEM and elemental distribution image of Zn-MOFs;

[0040] Figure 6 It is an SEM image of MnO2 NFs;

[0041] Figure 7 It is a particle size distribution image of MnO2 NFs;

[0042] Figure 8 It is a monitoring result image of the dual DNA cyclic reaction process in Example 2; Lanes 1-3 represent the S strand, LH strand, and P strand transduced by telomerase respectively, lane 4 represents the mixture of the LH strand and the P strand, lane 5 represents the mixture of the LH:P double strand and the released S strand, lane 6 represents the F strand, lane 7 represents the mixture of the LH:S double strand and F, lane 8 represents the H1 strand, lane 9 represents the mixture of the LH:F double strand and H1, lane 10 represents the mixture of the LH:F double strand and the H1 strand, H2 strand, H3 strand, and H4 strand;

[0043] Figure 9 Electrochemical impedance (EIS) characterization diagrams for the construction process of the sensing substrate interface. a represents bare ITO, b represents Zn-MOFs@Au NPs / ITO, c represents LH:P / Zn-MOFs@Au NPs / ITO, d represents the sensing substrate after the reaction of c with the S strand transduced by telomerase, e represents the sensing substrate after the reaction of d with the fuel F strand, f represents the sensing substrate after incubation of e with a mixture containing H1 strand, H2 strand, H3 strand and H4 strand, and g represents the sensing substrate after further reaction of f with MnO2 NFs;

[0044] Figure 10 Photocurrent response diagrams for the construction process of the sensing substrate interface. a represents Zn-MOFs@Au NPs / ITO, b represents LH:P / Zn-MOFs@Au NPs / ITO, c represents the sensing substrate after the reaction of b with the S strand transduced by telomerase, d represents the sensing substrate after the reaction of c with the fuel F strand, e represents the sensing substrate after incubation of d with a mixture containing H1 strand, H2 strand, H3 strand and H4 strand, and f represents the sensing substrate after further reaction of e with MnO2 NFs;

[0045] Figure 11 Photocurrent response curves of telomerase activity in cervical cancer cells at different concentrations; among them, the concentrations corresponding to a~i are 0, 50, 1×10 2 、5×10 2 、1×10 3 、5×10 3 、1×10 4 、1×10 5 and 1×10 6 cells / mL;

[0046] Figure 12 Linear relationship diagram between the change in photocurrent and the logarithm of the cervical cancer cell concentration;

[0047] Figure 13 Bar chart of photocurrent response of telomerase extracts in different cell lines;

[0048] Figure 14 Bar chart of photocurrent response of telomerase activity after the action of different concentrations of the drug epigallocatechin gallate (EGCG) on cervical cancer cells;

[0049] Figure 15 Comparison diagram of the results of detecting telomerase activity in blood samples (numbered 1~4) of healthy volunteers using a biosensor and the standard method ELISA. Detailed implementation manners

[0050] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0051] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0052] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0053] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0054] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0055] With the help of magnetic bead separation technology, the detection of telomerase activity in cervical cancer cells is converted into nucleic acid analysis in the present invention; using Zn-MOFs as the optoelectrochemical sensing substrate effectively improves the signal response and DNA assembly efficiency. Combining with the in-situ quenching of photocurrent by MnO2 NFs and the double DNA cycling signal amplification strategy, the accuracy and sensitivity of the biosensor are effectively improved (for the schematic diagram of the sensing mechanism of this biosensor, see Figure 1 ) and are specifically described in detail as follows:

[0056] Example 1

[0057] The construction process of a biosensor for detecting telomerase activity in cervical cancer cells is as follows:

[0058] First, the indium tin oxide electrode (ITO) (2 cm long × 1 cm wide) was ultrasonically cleaned with acetone, ethanol, and deionized water for 5 minutes in sequence and then dried with nitrogen. 30 μL of the Zn-MOFs@Au NPs complex was dropped onto its surface to obtain Zn-MOFs@Au NPs / ITO.

[0059] Next, 100 μL of the LH strand (5.0 μM) was mixed and reacted with 2 μL of tris(2-carboxyethyl)phosphine hydrochloride solution (10 mM) to obtain the thiol-activated LH strand. Then, it was incubated with the P strand (5.0 μM) in an equal volume at 37 °C for 1 hour to form the LH:P double strand. The Zn-MOFs@Au NPs / ITO was immersed in the LH:P double strand solution and incubated at 37 °C for 2 hours to obtain LH:P / Zn-MOFs@Au NPs / ITO.

[0060] Among them, the preparation method of the Zn-MOFs@Au NPs complex: 35 μL of N,N-diethylethylenediamine, 14.3 mg of Zn(NO3)2·6H2O, and 20 mg of 9,10-bis(4-carboxyphenyl)anthracene were dissolved in 3 mL of N,N-dimethylformamide in sequence. After ultrasonic treatment for 30 minutes, the above mixture was sealed in a high-pressure reaction kettle and reacted at 120 °C for 12 hours to obtain the zinc-based metal-organic framework material (Zn-MOFs). Then, 0.2 mg / mL of gold nanoparticles (Au NPs) and 1 mg / mL of Zn-MOFs were mixed in an equal volume and ultrasonicated for 5 minutes to obtain the Zn-MOFs@Au NPs complex.

[0061] The nucleotide sequence of the LH strand (SEQ ID NO.1) is: 5′-SH-GCTTCATCTTCATCTCCGACACTCGAGATGAAGATGAAGCGGGTAATACTCAATACTCTATCACAACG-3′;

[0062] The nucleotide sequence of the P strand (SEQ ID NO.2) is: 5′-TAGACTATTGAGTATTACCC-3′.

[0063] The usage method of this biosensor is as follows: Isolate the telomerase extract from HeLa cells and transfer it to the primer:M:S / magnetic bead complex for signal transduction. After magnetic separation, collect the released S strand; Then, react LH:P / Zn-MOFs@AuNPs / ITO with the released S strand, F strand, and a mixed solution containing H1 strand, H2 strand, H3 strand, and H4 strand in sequence; After that, further immerse the sensing substrate in the MnO2 NFs solution. After the incubation ends, collect the sensing substrate; Finally, use the incubated sensing substrate as the working electrode, Ag / AgCl electrode as the reference electrode, and platinum wire as the auxiliary electrode to form a three-electrode system, and detect the photocurrent signal in the ascorbic acid solution. The test instrument is a CHI760e electrochemical workstation equipped with a 500 W xenon lamp. The applied potential is 0.1 V, and the interval time for turning the xenon lamp on and off is 10 seconds. The specific details are as follows:

[0064] (1)Signal transduction of telomerase activity in cervical cancer cells.

[0065] The specific operation is as follows: Treat carboxylated magnetic beads (1 mg / mL) with a mixture of N-hydroxysuccinimide (10 mg / mL) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL) at 25 °C for 1 hour to obtain magnetic beads with activated surface carboxyl groups; Add the activated magnetic beads to 500 μL of 3'-amino-terminated M strand (5 μM), and react at 25 °C for 2 hours. Through the amide bond, the M strand is assembled onto the surface of the magnetic beads to form M / magnetic beads, and the non-specific sites on the surface of the magnetic beads are blocked with 2% (mass fraction) bovine serum albumin. After magnetic separation and washing with buffer solution, the M / magnetic beads are further immersed in a mixed solution containing telomerase primer (5 μM) and S strand (5 μM), and react at 25 °C for 1 hour to form the primer:M:S / magnetic bead complex; After magnetic separation and washing with buffer solution, the primer:M:S / magnetic bead complex is added to 500 μL of telomerase reaction solution, which contains telomerase extracts from different numbers of cervical cancer cells, deoxynucleoside triphosphates, 0.005% Tween, and 1 mM 3,6-dioxaoctane-1,8-diaminetetraacetic acid, and react at 37 °C for 2 hours. After magnetic separation, the signal transduction S strand of the target telomerase activity is obtained.

[0066] Among them, the nucleotide sequence of the M strand (SEQ ID NO.3) is: 5'-AACCCTAACCCTAACCCTAACCCTAACTCTGCTCGACGGATT-NH2-3';

[0067] The nucleotide sequence of the telomerase primer strand (SEQ ID NO.4) is: 5'-AATCCGTCGAGCAGAGTT-3';

[0068] The nucleotide sequence of the S strand (SEQ ID NO.5) is: 5′-CGTTGTGATAGAGTATTGAGGATTGGGATT-3′.

[0069] (2) A double DNA cyclic signal amplification was assembled at the LH:P / Zn-MOFs@Au NPs / ITO electrode interface to obtain a sensing substrate assembled with a DNA long-chain polymer.

[0070] The LH:P / Zn-MOFs@Au NPs / ITO electrode was incubated with the released S strand and 100 μL of the F strand (2.5 μM) at 37 °C for 50 minutes to activate the entropy-driven catalytic reaction, obtaining LH:F / Zn-MOFs@Au NPs / ITO. Then, after the carboxyl groups modified at the ends of the H1 strand, H2 strand, H3 strand, and H4 strand were activated with a mixture of N-hydroxysuccinimide (10 mg / mL) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL), the LH:F / Zn-MOFs@Au NPs / ITO was placed into 100 μL of a mixed solution containing 6 µM of the H1 strand, 6 µM of the H2 strand, 6 µM of the H3 strand, and 6 µM of the H4 strand, and reacted at 37 °C for 1 h to initiate a hybridization chain reaction, forming a DNA long-chain polymer at the electrode interface.

[0071] The nucleotide sequence of the F strand (SEQ ID NO.6) is: 5′-ATGACTATTGAGTATTACCCGCTTCATCTTCATCTC-3′;

[0072] The nucleotide sequence of the H1 strand (SEQ ID NO.7) is: 5′-COOH-GAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCATCTTCATCTCCG-3′;

[0073] The nucleotide sequence of the H2 strand (SEQ ID NO.8) is: 5′-COOH-GCTTCATCTTCATCTCCGGTTTTGCGGAGATGAAGATGAAGCACGATG-3′;

[0074] The nucleotide sequence of the H3 strand (SEQ ID NO.9) is: 5′-COOH-CAAAACCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCG-3′;

[0075] The nucleotide sequence of the H4 chain (SEQ ID NO.10) is: 5′-COOH-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA-3′.

[0076] (3) Immerse the electrode after the hybridization chain reaction obtained in step (2) into a 0.2 mg / mL solution of amino-functionalized manganese dioxide nanoflowers (MnO2 NFs), incubate at 25 °C for 2 hours, and assemble MnO2 NFs onto the electrode surface through amide bonds. After incubation, collect the sensing substrate; finally, use the incubated sensing substrate as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode to form a three-electrode system, and detect the photocurrent signal in an ascorbic acid solution. According to the standard curve, calculate the telomerase activity level in different numbers of cervical cancer cells. The test instrument is a CHI760e electrochemical workstation equipped with a 500 W xenon lamp, the applied potential is 0.1 V, and the interval time for turning the xenon lamp on and off is 10 seconds.

[0077] Among them, MnO2 NFs are prepared by reducing KMnO4 with oleic acid: Add 0.5 g of KMnO4 to 250 mL of ultrapure water and stir at 25 °C for 30 minutes. Subsequently, slowly inject 5 mL of oleic acid to reduce KMnO4 and form a stable emulsion. Continuously react for 4 hours, and collect the brownish-black MnO2 NFs product after centrifuging at 8000 rpm for 10 minutes. Then, add 0.25 g of MnO2 NFs to 1 mL of 3-aminopropyltriethoxysilane (10% v / v), and stir at 120 °C for 6 hours in a nitrogen atmosphere to assemble amino groups on the surface of MnO2 NFs.

[0078] Example 2

[0079] In this example, the performance of the biosensor prepared in Example 1 was detected, and the specific process is as follows:

[0080] (1) The biosensor of the present invention uses Zn-MOFs to improve the photoelectrochemical signal response, and MnO2 NFs catalyze the electron-donating body ascorbic acid to achieve in-situ signal quenching. To verify the feasibility of this in-situ photoelectrochemical quenching, the Zn-MOFs and MnO2 NFs prepared in Example 1 were characterized. From Figure 2 It can be seen that Zn-MOFs have an ultrathin layer-like nanoflower structure, Figures 3 - 5 indicating that the material contains C, N, O, and Zn elements. In addition, Figures 6 - 7 it is shown that MnO2 NFs have a spherical nanoflower-like structure with a particle size of approximately 150 nm.

[0081] (2)The biosensor of the present invention uses Zn-MOFs as the sensing substrate, and further improves the detection sensitivity through a double DNA cyclic signal amplification reaction. Therefore, the construction process of the sensing interface and the feasibility of the double DNA cyclic signal amplification are monitored and verified. As Figure 8 shown, lanes 1-3 represent the S strand, LH strand, and P strand transduced by telomerase, respectively. Lane 4 represents the mixture of the LH strand and the P strand, and the lagging new band represents the formation of the LH:P double strand. Lane 5 represents the mixture of the LH:P double strand and the released S strand. It can be seen that the P strand can be replaced by the S strand. Lane 6 represents the F strand, and lane 7 represents the mixture of the LH:S double strand and the F strand. Two new bands can be observed, attributed to the S strand and the LH:F double strand, indicating that the S strand can be replaced by the F strand, and the released S strand participates in the reaction again, activating the entropy-driven catalytic reaction. Lane 8 represents the H1 strand, lane 9 represents the mixture of the LH:F double strand and the H1 strand, and lane 10 represents the mixture of the LH:F double strand and the H1 strand, H2 strand, H3 strand, and H4 strand. It can be found that the significantly lagging band proves the formation of the DNA long-chain polymer.

[0082] Next, EIS and photocurrent were used to characterize the construction of the sensing interface, and the results are as Figures 9 - 10 shown. As Figure 9 shown by curve a in et , on the bare ITO surface, the electron transfer impedance (R et ) is about 232.9 Ω. As Figure 9 shown by curve b in et , when Zn-MOFs@Au NPs are assembled onto the ITO surface, the R et of the obtained Zn-MOFs@Au NPs / ITO electrode increases to 862.7 Ω. At the same time, an obvious photocurrent intensity of 858 nA can be observed, as shown by curve a in Figure 10 . When the modified electrode is incubated with the LH:P double strand (1450.2 Ω, Figure 9 shown by curve c in Figure 9 ), the released S strand (1952.8 Ω, Figure 9 shown by curve d in Figure 9 ), the F strand (2406.7 Ω, Figure 9 shown by curve e in Figure 9 ), and the mixture containing the H1 strand, H2 strand, H3 strand, and H4 strand (5342.0 Ω, Figure 9 shown by curve f in et ), the R et gradually increases, attributed to the negatively charged phosphate backbone in the DNA molecular structure hindering the interfacial charge transfer. The corresponding photocurrent intensity gradually decreases to 798 nA ( Figure 10 shown by curve b in Figure 10 ), 762 nA ( Figure 10 shown by curve c in Figure 10 ), 735 nA ( Figure 10 shown by curve d in Figure 10The curve e) in. Subsequently, after the assembled electrode was further placed in MnO2 NFs for reaction, R et increased to 7070.2 Ω ( Figure 9 The curve g) in, and the photocurrent intensity decreased to 256 nA ( Figure 10 The curve f) in, which proved the successful assembly of MnO2 NFs and the quenching of the photocurrent signal. The above EIS and photocurrent results effectively proved the construction process of the electrode sensing interface.

[0083] (3) Telomerase extracts in cervical cancer cells at different concentrations (0, 50, 1×10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 1×10 5 , 1×10 6 cells / mL) were used to verify the detection performance of the biosensor according to the usage method described in Example 1.

[0084] The photocurrent response curves of telomerase extracts in cervical cancer cells at different concentrations are as Figure 11 shown. It can be seen that as the concentration of cervical cancer cells increases, the photocurrent intensity gradually decreases. The linear relationship between the logarithm of the concentration of cervical cancer cells and the change in photocurrent intensity (the difference in photocurrent values before and after target incubation ΔI) is as Figure 12 shown, and the linear regression equation is . According to the 3-fold relative standard deviation rule, the detection limit was calculated to be 18 cells / mL. From Figure 13 it can be found that compared with breast cancer and lung cancer cells, cervical cancer cells have higher telomerase activity. When cervical cancer cells were heat-treated, the telomerase activity was inactivated, indicating that the sensor has good selectivity. Then, after treating cervical cancer cells with the catechin (EGCG) drug, the inhibitory effect of the constructed biosensor on intracellular telomerase activity was detected, and the results are as Figure 14 shown. As the concentration of EGCG increases, the photocurrent intensity gradually increases, indicating that EGCG has a good inhibitory effect on intracellular telomerase activity, and it also proves the significance of this biosensor for clinical diagnosis and treatment.

[0085] Example 3

[0086] In this example, blood samples from healthy volunteers (numbered 1 to 4) were used to verify the detection performance of the biosensor in Example 1. The specific process is as follows:

[0087] The blood samples were diluted 10 times with phosphate buffer, and then 10 4 , 5×104 , 10 5 and 5×10 5 Telomerase, primer: M:S / magnetic bead complex, and deoxynucleoside triphosphate extracted from cervical cancer cells were used to detect the released S strand. The concentration of telomerase was calculated using the linear regression equation of Example 2, and the results are as follows Figure 15 shown. Compared with the results detected by the standard method commercial kit ELISA, the concentration error was less than 5%. It shows that the biosensor designed by the present invention has good detection ability and application even in complex blood samples.

[0088] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a biosensor for detecting telomerase activity in cervical cancer cells, characterized in that: The following steps are involved: After the indium tin oxide electrode was cleaned, a Zn-MOFs@Au NPs complex was added dropwise to its surface to obtain a Zn-MOFs@AuNPs / ITO electrode; Immersing the Zn-MOFs@Au NPs / ITO electrode in a LH:P double-chain solution for incubation reaction to obtain the biosensor; The Zn-MOFs@Au NPs composite is obtained by mixing gold nanoparticles and zinc-based metal organic framework materials; The LH:P double chain contained in the LH:P double chain solution is obtained by incubating a thiol-activated LH chain with a P chain; The nucleotide sequence of the LH chain is shown in SEQ ID NO.1; the nucleotide sequence of the P chain is shown in SEQ ID NO.

2.

2. The preparation method according to claim 1, characterized in that: The preparation method of the zinc-based metal organic framework material comprises the following steps: N,N-diethylethylenediamine, Zn(NO3)2⋅6H2O and 9,10-di(4-carboxyphenyl)anthracene are dissolved in N,N-dimethylformamide in sequence, and after ultrasonic treatment, mixed reaction is carried out at 120° C. to obtain the zinc-based metal organic framework material.

3. The preparation method according to claim 1, characterized in that: The cleaning process comprises the steps of ultrasonic cleaning with acetone, ethanol and deionized water in sequence.

4. The preparation method according to claim 1, characterized in that: The thiol-activated LH chain is obtained by mixing the LH chain with a tris(2-carboxyethyl)phosphine hydrochloride solution.

5. A biosensor for detecting telomerase activity in cervical cancer cells prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the biosensor according to claim 5 in preparing a kit for detecting telomerase activity in cervical cancer cells.

7. A kit for detecting telomerase activity in cervical cancer cells, characterized in that: Comprising the biosensor according to claim 5.

8. The kit according to claim 7, characterized in that The kit also includes a 3′-terminally amino-terminated M chain, a telomerase primer, an S chain, an F chain, an H1 chain, an H2 chain, an H3 chain, and an H4 chain; The nucleotide sequence of the M chain is shown in SEQ ID NO.3; The nucleotide sequence of the telomerase primer is shown in SEQ ID NO.4; The nucleotide sequence of the S chain is shown in SEQ ID NO.5; The nucleotide sequence of the F chain is shown in SEQ ID NO.6; The nucleotide sequence of the H1 chain is shown in SEQ ID NO.7; The nucleotide sequence of the H2 chain is shown in SEQ ID NO.8; The nucleotide sequence of the H3 chain is shown in SEQ ID NO.9; The nucleotide sequence of the H4 chain is shown in SEQ ID NO.

10.

9. The kit according to claim 8, characterized in that The kit also includes manganese dioxide nanoflowers; The manganese dioxide nanoflowers are obtained by mixing oleic acid and KMnO4.

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