Biosensor for telomerase activity detection as well as preparation method and application of biosensor

Abstract

The invention discloses a biosensor for telomerase activity detection and a preparation method and application thereof, and relates to the technical field of biosensors. The preparation method comprises the following steps that after an indium tin oxide electrode is cleaned, a Zn-MOFs (at) Au NPs compound is dropwise added to the surface of the indium tin oxide electrode, and a Zn-MOFs (at) Au NPs / ITO electrode is obtained; and immersing the Zn-MOFs (at) Au NPs / ITO electrode into an LH: P double-chain solution, and carrying out an incubation reaction to obtain the biosensor. The Zn-MOFs (at) Au NPs compound is obtained by carrying out mixed reaction on gold nanoparticles and a zinc-based metal organic framework material. The biosensor prepared by the invention has the advantages of high accuracy, strong sensitivity, good selectivity and the like, and can be applied to 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 complex is added dropwise to the surface of the indium tin oxide electrode to obtain a Zn-MOFs@Au NPs / ITO electrode;

[0009] Immersing the Zn-MOFs@Au NPs / ITO electrode in a LH:P double-chain solution for incubation reaction to obtain the biosensor;

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

[0011] 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;

[0012] 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.

[0013] Furthermore, the preparation method of the zinc-based metal organic framework material comprises the following steps:

[0014] 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.

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

[0016] Furthermore, the thiol-activated LH chain is obtained by mixing the LH chain 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 according to the above preparation method.

[0018] The present invention also provides the use of the above-mentioned biosensor in preparing 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-mentioned biosensor.

[0020] Furthermore, 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;

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

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

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

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

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

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

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

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

[0029] Furthermore, the kit also includes manganese dioxide nanoflowers;

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

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

[0032] The present invention constructs a photoelectrochemical biosensor to detect telomerase activity in cervical cancer cells. The biosensor uses magnetic bead separation technology to achieve signal transduction of telomerase activity in cervical cancer cells; uses Zn-MOFs as a photoelectrochemical active substrate and MnO2 NFs as an electron donor catalyst to perform in-situ quenching of photocurrent signals; and uses entropy-driven catalytic reactions to stimulate hybridization chain reactions to achieve cyclic amplification of target telomerase, thereby achieving high-sensitivity detection of telomerase activity in cervical cancer cells.

[0033] The present invention uses magnetic bead separation technology to perform signal transduction of telomerase activity in cervical cancer cells, thereby improving the selectivity of the sensing system; using Zn-MOFs as a photoelectrochemically active substrate, combined with in-situ quenching of MnO2 NFs, improves the output and response of the signal; based on a double DNA cycle signal amplification strategy, the accuracy and sensitivity of the biosensor are improved. 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 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.

[0035] Figure 1 Schematic diagram of the sensing mechanism of the photoelectrochemical biosensor for telomerase activity in cervical cancer cells of the present invention;

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

[0037] Figure 3 This is the X-ray photoelectron spectroscopy (XPS) diagram of Zn-MOFs;

[0038] Figure 4 The XPS graph of Zn 2p orbital electrons of Zn-MOFs;

[0039] Figure 5 SEM and element distribution diagram of Zn-MOFs;

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

[0041] Figure 7 is the particle size distribution diagram of MnO2 NFs;

[0042] Figure 8 The figure is a monitoring result of the double DNA cycle reaction process in Example 2; lanes 1 to 3 represent the S chain, LH chain and P chain transduced by telomerase, respectively, lane 4 represents a mixture of the LH chain and the P chain, lane 5 represents a mixture of the LH:P double chain and the released S chain, lane 6 represents the F chain, lane 7 represents a mixture of the LH:S double chain and F, lane 8 represents the H1 chain, lane 9 represents a mixture of the LH:F double chain and H1, and lane 10 represents a mixture of the LH:F double chain and the H1 chain, the H2 chain, the H3 chain and the H4 chain;

[0043] Fig. 9 Electrochemical impedance spectroscopy (EIS) characterization diagram of 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 c reacts with the S chain transduced by telomerase, e represents the sensing substrate after d reacts with the fuel F chain, f represents the sensing substrate after e is incubated with a mixture containing H1 chain, H2 chain, H3 chain and H4 chain, g represents the sensing substrate after f further reacts with MnO2 NFs;

[0044] Fig.10 Photoelectrochemical response diagram of 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 b reacts with the S chain transduced by telomerase, d represents the sensing substrate after c reacts with the fuel F chain, e represents the sensing substrate after d is incubated with a mixture containing H1 chain, H2 chain, H3 chain and H4 chain, and f represents the sensing substrate after e further reacts with MnO2 NFs;

[0045] Fig.11 The photocurrent response curve of telomerase activity in cervical cancer cells with different concentrations; 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] Fig.12 It is a linear relationship diagram between the photocurrent change and the logarithmic value of the cervical cancer cell concentration;

[0047] Fig.13 is a bar graph of the photocurrent response of telomerase extracts in different cell lines;

[0048] Fig.14 This is a bar graph of the photocurrent response of telomerase activity after cervical cancer cells were treated with different concentrations of the drug catechin (EGCG);

[0049] Fig.15 This is a comparison of the results of detecting telomerase activity in blood samples of healthy volunteers (numbered 1 to 4) using biosensor and standard method ELISA. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0054] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0055] The present invention uses magnetic bead separation technology to convert the detection of telomerase activity in cervical cancer cells into nucleic acid analysis; Zn-MOFs are used as the photoelectrochemical sensing substrate to effectively improve the signal response and DNA assembly efficiency, and combined with the in-situ quenching of photocurrent by MnO2 NFs and the double DNA cycle signal amplification strategy, the accuracy and sensitivity of the biosensor are effectively improved (the sensing mechanism diagram of the biosensor is shown in Figure 1 ), which are 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, an indium tin oxide electrode (ITO) (2 cm long × 1 cm wide) was ultrasonically cleaned with acetone, ethanol, and deionized water for 5 minutes, and then dried with nitrogen. 30 μL of Zn-MOFs@Au NPs composite was added to its surface to obtain Zn-MOFs@Au NPs / ITO.

[0059] Next, 100 μL of LH chain (5.0 μM) was mixed with 2 μL of tris(2-carboxyethyl)phosphine hydrochloride solution (10 mM) to obtain thiol-activated LH chain, which was then incubated with an equal volume of (5.0 μM) P chain at 37 °C for 1 hour to form LH:P double chain. Zn-MOFs@Au NPs / ITO was immersed in the LH:P double chain 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 Zn-MOFs@Au NPs complex is as follows: 35 μL N,N-diethylethylenediamine, 14.3 mgZn(NO3)2⋅6H2O and 20 mg 9,10-di(4-carboxyphenyl)anthracene are dissolved in 3 mL N,N-dimethylformamide in sequence, and after 30 minutes of ultrasonic treatment, the above mixture is sealed in a high-pressure reactor and reacted at 120 °C for 12 hours to obtain zinc-based metal organic framework materials (Zn-MOFs). Then, 0.2 mg / mL gold nanoparticles (Au NPs) and 1 mg / mL Zn-MOFs are mixed in equal volumes and ultrasonicated for 5 minutes to obtain Zn-MOFs@Au NPs complex.

[0061] The nucleotide sequence of the LH chain (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 method of using the biosensor is as follows: telomerase extract is separated from HeLa cells and transferred to a primer: M: S / magnetic bead complex for signal transduction, and the released S chain is collected after magnetic separation; then, LH: F / Zn-MOFs@AuNPs / ITO is reacted with the released S chain, F chain and a mixed solution containing H1 chain, H2 chain, H3 chain and H4 chain in turn; after that, the sensing substrate is further placed in a MnO2 NFs solution, and after the incubation, the sensing substrate is collected; finally, the incubated sensing substrate is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire is used as the auxiliary electrode to form a three-electrode system to 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 0.1V, and the interval between the xenon lamp switch 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: the carboxylated magnetic beads (1 mg / mL) were treated 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 surface carboxyl-activated magnetic beads; the activated magnetic beads were added to 500 μL of 3′-terminally amino-labeled M chains (5 μM) and reacted at 25°C for 2 hours. The M chains were assembled onto the surface of the magnetic beads through amide bonds to form M / magnetic beads, and 2% mass fraction of bovine serum albumin blocked the nonspecific sites on the surface of the magnetic beads. After being separated by a magnet and washed with a buffer solution, the M / magnetic beads were further placed in a mixed solution containing telomerase primers (5 μM) and S chains (5 μM) and reacted at 25°C for 1 hour to form a primer:M:S / magnetic bead complex. After the primer:M:S / magnetic bead complex was separated by a magnet and washed with a buffer solution, it was added to 500 μL of telomerase reaction solution containing different amounts of telomerase extracts extracted from cervical cancer cells, deoxynucleoside triphosphates, 0.005% Tween and 1 mM 3,6-dioxa-1,8-octanediaminetetraacetic acid, reacted at 37°C for 2 hours, and separated by a magnet to obtain the signal transduction S chain of the target telomerase activity.

[0066] Among them, the nucleotide sequence of the M chain (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 chain (SEQ ID NO. 5) is: 5′-CGTTGTGATAGAGTATTGAGGATTGGGATT-3′.

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

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

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

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

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

[0074] The nucleotide sequence of the H3 chain (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) The electrode after the hybridization chain reaction obtained in step (2) was immersed in a 0.2 mg / mL amino-modified manganese dioxide nanoflower (MnO2 NFs) solution, and the reaction was incubated at 25°C for 2 hours to assemble the MnO2 NFs onto the electrode surface through amide bonds. After the incubation, the sensing substrate was collected; finally, the incubated sensing substrate was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire was used as the auxiliary electrode to form a three-electrode system, and the photocurrent signal was detected in the ascorbic acid solution. According to the standard curve, the telomerase activity level in different numbers of cervical cancer cells was calculated. The test instrument was a CHI760e electrochemical workstation equipped with a 500 W xenon lamp, the applied potential was 0.1 V, and the interval between the xenon lamp on and off was 10 seconds.

[0077] Among them, MnO2 NFs were prepared by reducing KMnO4 with oleic acid: 0.5 g KMnO4 was added to 250 mL ultrapure water and stirred at 25°C for 30 minutes. Subsequently, 5 mL oleic acid was slowly injected to reduce KMnO4 and form a stable emulsion. The reaction was continued for 4 hours, and the brown-black MnO2 NFs product was collected after centrifugation at 8000 rpm for 10 minutes. Then, 0.25 g MnO2 NFs was added to 1 mL 3-aminopropyltriethoxysilane (10% v / v) and stirred at 120°C for 6 hours in a nitrogen atmosphere to assemble amino groups on the surface of MnO2 NFs.

[0078] Example 2

[0079] This example tests the performance of the biosensor prepared in Example 1, 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 catalyzes the electron donor ascorbic acid to achieve in-situ signal quenching. In order to verify the feasibility of the photoelectrochemical in-situ quenching, the Zn-MOFs and MnO2 NFs prepared in Example 1 were characterized. Figure 2 It can be seen that Zn-MOFs have an ultra-thin layered nanoflower-like structure. Figure 3-Figure 5 It shows that the material contains C, N, O and Zn elements. In addition, Figure 6-Figure 7 It was shown that MnO2 NFs had 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 the double DNA cycle signal amplification reaction. Therefore, the construction process of the sensing interface and the feasibility of the double DNA cycle signal amplification are monitored and verified. Figure 8 As shown, lanes 1 to 3 represent the S chain, LH chain and P chain transduced by telomerase, respectively. Lane 4 represents a mixture of LH chain and P chain, and the delayed new band represents the formation of LH:P double chain. Lane 5 represents a mixture of LH:P double chain and released S chain. It can be seen that the P chain can be replaced by the S chain. Lane 6 represents the F chain, and lane 7 represents a mixture of LH:S double chain and F. Two new bands can be observed, belonging to the S chain and LH:F double chain, indicating that the S chain can be replaced by the F chain, and the released S chain participates in the reaction again, activating the entropy-driven catalytic reaction. Lane 8 represents the H1 chain, lane 9 represents a mixture of LH:F double chain and H1, and lane 10 represents a mixture of LH:F double chain and H1 chain, H2 chain, H3 chain and H4 chain. It can be found that the significantly delayed band proves the formation of long-chain DNA polymers.

[0082] Next, EIS and photocurrent were used to characterize the construction of the sensing interface. The results are as follows Figure 9-10 As shown. Fig. 9 As shown in curve a in Figure 2, on the bare ITO surface, the electron transfer impedance (R et ) is about 232.9 Ω. Fig. 9 As shown in curve b in Figure 2, when Zn-MOFs@Au NPs are assembled onto the ITO surface, the R et It increases to 862.7 Ω. At the same time, a significant photocurrent intensity of 858 nA can be observed. Fig.10 Curve a in Figure 1. When the modified electrode is sequentially connected with LH:P double chain (1450.2 Ω, Fig. 9 Curve c in the figure), the released S chain (1952.8 Ω, Fig. 9 Curve d in the figure), F chain (2406.7Ω, Fig. 9 Curve e in Figure 2) and a mixture containing H1, H2, H3, and H4 chains (5342.0 Ω, Fig. 9 Curve f) After incubation, R et The photocurrent intensity gradually increases, which is 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 ( Fig.10 curve b), 762 nA ( Fig.10 Curve c in Figure ), 735 nA ( Fig.10 Curve d) and 603 nA ( Fig.10Then, the assembled electrode was further placed in MnO2 NFs for reaction, and R et Increased to 7070.2 Ω ( Fig. 9 The photocurrent intensity decreases to 256 nA ( Fig.10 The curve f in Figure 1 proves the successful assembly of MnO2NFs and the quenching of the photocurrent signal. The above EIS and photocurrent results effectively prove the construction process of the electrode sensing interface.

[0083] (3) Using different concentrations of cervical cancer cells (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) was used to verify the detection performance of the biosensor according to the method described in Example 1.

[0084] The photocurrent response curves of telomerase extracts from cervical cancer cells at different concentrations are shown in Figure 2. Fig.11 As shown, it can be seen that as the concentration of cervical cancer cells increases, the photocurrent intensity gradually decreases. The linear relationship between the logarithmic value of the concentration of cervical cancer cells and the change in photocurrent intensity (the difference in photocurrent value before and after target incubation ΔI) is as follows: Fig.12 As shown, the linear regression equation is According to the 3-fold relative standard deviation rule, the detection limit was 18 cells / mL. Fig.13 It can be found that cervical cancer cells contain higher telomerase activity than breast cancer and lung cancer cells. When cervical cancer cells are heat treated, telomerase activity is inactivated, indicating that the sensor has good selectivity. Next, after EGCG drug treatment of cervical cancer cells, the constructed biosensor was used to detect the inhibitory effect of intracellular telomerase activity. The results are as follows Fig.14 As shown in the figure, with the increase of EGCG concentration, the photocurrent intensity gradually increased, indicating that EGCG has a good inhibitory effect on intracellular telomerase activity. It also proves that the biosensor is of great significance for clinical diagnosis and treatment.

[0085] Example 3

[0086] This example uses healthy volunteer blood samples (numbered 1 to 4) to verify the detection performance of the biosensor in Example 1. The specific process is as follows:

[0087] The blood samples were diluted 10-fold with phosphate buffered saline and then 10 4 , 5×104 , 10 5 and 5×10 5 Telomerase extracted from cervical cancer cells, primer: M: S / magnetic bead complex and deoxynucleoside triphosphate were used to detect the released S chain. The linear regression equation of Example 2 was used to calculate the concentration of telomerase. The result is as follows: Fig.15 Compared with the results of ELISA test using a standard commercial kit, the concentration error is less than 5%, indicating that the biosensor designed by the present invention has good detection capability and application even in complex blood samples.

[0088] 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 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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