阅读说明：本技术 一种纳米探针及其制备方法和应用 (Nano probe and preparation method and application thereof ) 是由 王卫 唐凯 宋志灵 罗细亮 于 2021-08-19 设计创作，主要内容包括：本发明涉及一种纳米探针及其制备方法和应用。端粒酶可以通过触发扩增反应保持端粒的长度,从而导致细胞永生和致癌作用。端粒酶在几乎所有癌细胞中均过表达。因此,开发灵敏检测端粒酶的技术对于肿瘤的早期诊断和治疗具有重要意义。本发明设计开发了一种基于阻断材料S1和S2的检测端粒酶活性的荧光探针。采用能够触发端粒酶重复扩增反应的端粒酶引物S1及能够与扩增产物发生多重杂交反应的S2作为多孔纳米载体的阻断材料模拟构建智能锁,实现了癌细胞中端粒酶活性的高特异、超灵敏检测及成像,克服了传统方法检测灵敏度低、样品需求量大、步骤繁琐、费用昂贵等缺点,为活体、细胞检测及原位成像提供了新的策略和方法。(The invention relates to a nano probe and a preparation method and application thereof. Telomerase can maintain telomere length by triggering an amplification reaction, leading to cell immortalization and carcinogenesis. Telomerase is overexpressed in almost all cancer cells. Therefore, the development of a technique for sensitively detecting telomerase is of great significance for the early diagnosis and treatment of tumors. The invention designs and develops a fluorescent probe for detecting telomerase activity based on blocking materials S1 and S2. A telomerase primer S1 capable of triggering repeated amplification reaction of telomerase and S2 capable of generating multiple hybridization reaction with an amplification product are used as blocking materials of the porous nano carrier to simulate and construct an intelligent lock, so that high-specificity and ultra-sensitive detection and imaging of the telomerase activity in cancer cells are realized, the defects of low detection sensitivity, large sample requirement, complex steps, high cost and the like of the traditional method are overcome, and a new strategy and method are provided for living body, cell detection and in-situ imaging.)

1. A nano fluorescent probe for detecting telomerase activity is characterized in that: the probe adopts S1 and S2 as blocking materials of the porous carrier to simulate and construct the intelligent lock, effectively inhibits the leakage of signal molecules in the carrier, and avoids the generation of false positive signals.

2. A nano fluorescent probe for detecting telomerase activity as claimed in claim 1, wherein: the S1 is a telomerase primer chain which can be triggered by telomerase to generate repeated amplification reactions, the S2 is a complementary long-chain molecule which can generate multiple hybridization reactions with amplification products, and the complementary long-chain molecule consists of 2 parts, wherein one part is the telomerase primer chain S1, and the other part is the complementary chain S3 of the amplification products of the telomerase primer chain.

3. A nano fluorescent probe for detecting telomerase activity as claimed in claim 1, wherein: the sequences of S1 and S2 are, from 5 'end to3' end: AATCCGTCGAGCAGAGTT, and AATCCGTCGAGCAGAGTTCCCTAACCCTAA.

4. A nano fluorescent probe for detecting telomerase activity as claimed in claim 1, wherein: the porous carrier is a hollow gold nano carrier with a porous surface.

5. A nano fluorescent probe for detecting telomerase activity as claimed in claim 1, wherein: the signal molecule is rhodamine B.

6. The method for preparing the nano fluorescent probe for detecting telomerase activity as claimed in claim 1, which is characterized by comprising the following steps:

(1) design and synthesize blocking materials S1 and S2, whose sequences from 5'to3' are: AATCCGTCGAGCAGAGTT and AATCCGTCGAGCAGAGTTCCCTAACCCTAA;

(2) respectively preparing S1 and S2 solutions with the concentration of 20 mu M;

(3) respectively transferring the 10 mu L S1 and S2 solutions prepared in the previous step, adding the solutions into 100 mu L of porous gold nano carrier solution loaded with rhodamine B signal molecules, and shaking overnight at 37 ℃;

(4) and (3) carrying out magnetic separation and cleaning to finally prepare the nano fluorescent probe for detecting the telomerase activity.

7. Use of a nanofluorescent probe according to claim 1, characterized by the in situ imaging of telomerase in cancer cells by the following method:

(1) respectively transferring 10 mu L of 20 mu M S1 and S2 solutions, adding the solutions into 100 mu L of porous gold nano carrier solution loaded with rhodamine B signal molecules, shaking overnight at 37 ℃, carrying out magnetic separation and cleaning;

(2) dispersing the product in 200. mu.L PBS buffer solution;

(3) mixing 150 μ L of LHeLa cells with 10 μ L of dNTPs and 20 μ L of the fluorescent probe solution obtained in the previous step at 37 deg.C and 5% CO₂Incubating for 2 h;

(4) placing the cells on a laser confocal microscope for fluorescence imaging, selecting an excitation wavelength: 488 nm.

Technical Field

The invention relates to detection of biomarkers, in particular to a nano fluorescent probe for detecting telomerase activity in cancer cells, a preparation method and application thereof, and belongs to the fields of photoelectrochemistry biosensing analysis and biomedical clinical diagnosis.

Background

Research shows that telomeres and telomerase are closely related to cell senescence, cell division, cell immortalization, cell degeneration and the like and are important factors of senescence and tumors. Telomeres are a stretch of nucleotide repeats present at the ends of chromosomes in most eukaryotic cells, and in humans, the telomere sequence (TTAGGG) n repeats about 2500 times. The telomeres themselves become shorter in length as the number of cell divisions increases, and the extremely short length causes the cells to stop dividing and to enter the apoptotic and senescent stages. Therefore, telomeres act as caps on the ends of chromosomes, and the length of the telomeres is reduced continuously to protect important genetic information in the chromosomes from being lost during replication at the sacrifice. Telomerase is a special ribonucleoprotein polymerase with reverse transcription activity in a human body, and can catalyze and synthesize telomere repetitive sequences by using an RNA component of the telomerase as a template, maintain the length of the telomere, increase the division times of cells and enable the cells to have the capability of unlimited proliferation. Cancer cells are able to divide indefinitely and survive because telomeres of their chromosomes are protected by telomerase, and their length is continued by telomerase-triggered amplification reactions.

Except for telomerase being positive in a small number of cells such as germ cells testis, ovary, placenta and fetal cells, expression of telomerase activity is undetectable in normal human cells, as its activity is inhibited in normal cells. However, in tumor cells and most tumor tissues, telomerase activity is abnormally and highly expressed, so that cancer cells have the capability of unlimited proliferation. Telomerase positive tumors include ovarian cancer, lymphoma, acute leukemia, breast cancer, colon cancer, lung cancer, and the like. Researches suggest that the aim of selectively killing cancer cells can be achieved by inhibiting the activity of telomerase, and the anticancer therapy taking the telomerase as a target has potential application value. Meanwhile, the sensitive detection of telomerase activity also provides a basis for early diagnosis of cancer. Therefore, telomerase has been widely studied and studied as a specific tumor biomarker for early cancer diagnosis and treatment.

Therefore, the development of a technology for sensitively detecting telomerase activity is of great significance to the early diagnosis and treatment of tumors. The traditional detection methods such as a radioactive labeling method, a telomere repeat amplification scheme (TRAP) and other technologies have the defects of large sample demand, low detection sensitivity, tedious processing, time consumption, high cost, high residual pollution risk, generation of false positive/negative signals and the like, and simultaneously, the biggest problem is that the technologies cannot completely quantitatively detect the activity of telomerase in cells, and bring certain difficulty to practical clinical application. In order to overcome the defects and shortcomings of the traditional detection technology of telomerase, the development of a simple, sensitive, specific and high-accuracy fluorescence detection technology is urgently needed to meet the actual requirements of early diagnosis and treatment of cancers in the field of biological medicine. Compared with other methods, the detection technology based on the fluorescent probe is widely researched and applied due to the advantages of simple and convenient operation, high sensitivity, low sample demand, suitability for high-throughput detection and the like.

The invention provides an ultrasensitive fluorescent probe detection system based on a multiple signal amplification strategy, aiming at overcoming the defects of low detection sensitivity, large sample demand, complex steps, high cost and the like of the traditional technology. The invention adopts a hollow porous gold nano-carrier to construct a telomerase activity detection system. Due to the structural characteristics of the gold nano-carrier material, signal molecules can be easily loaded in the gold nano-carrier material, so that the gold nano-carrier material is an ideal choice for realizing a controllable release strategy. Nevertheless, how to optimize the blocking strategy on the surface of the gold nanoporous carrier material, preventing false positive/negative results caused by signal molecule leakage remains a challenge. The invention synthesizes a biomolecule S1 based on a telomerase primer chain, and the biomolecule S1 is used as a blocking material of a nano controllable release carrier. Only when telomerase exists, the repeated amplification reaction of the primer chain of the telomerase can be triggered to separate the primer chain from the surface of the carrier, which destroys the blocking effect of the primer chain on signal molecules, generates a signal enhancement result and accordingly realizes the detection of the activity of the telomerase. Due to the fact that the design of the blocking material simulates the effect of the intelligent lock, only the target molecules can be identified by the blocking material, controllable release of signal molecules is achieved, and the detection signals which are amplified remarkably are obtained. The signal amplification effect is no longer 1 to 1 of the traditional technology, but more signal molecules can be obtained by 1 target molecule, and the sensitivity is obviously improved. In order to further improve the sensitivity, the invention designs a multiple hybridization amplification strategy on the basis of amplification of an intelligent lock-controllable release signal, namely, complementary long-chain molecules S2 with more excellent blocking performance are designed, synthesized and introduced into a blocking material, the synthesized molecules not only have more excellent blocking performance, but also can generate hybridization reaction with an amplification product of a telomerase primer chain to form a double-chain compound, and finally the double-chain compound is separated from the surface of a carrier, the blocking effect is destroyed, so that more intelligent locks are opened, the released signal molecules are exponentially increased, and the sensitivity is further improved. The great advantage of this strategy is that the designed and synthesized complementary long-chain molecule S2 is composed of 2 parts, one part is telomerase primer chain S1, and the other part is the complementary chain S3 of the amplification product of the telomerase primer chain, and this part can be hybridized with the amplification product of the telomerase primer chain to form a double-stranded complex. Due to the repeated amplification of the primer strands, the amplification product of each primer strand can bind to multiple complementary long-chain molecules S2, again achieving a 1-to-many strategy. Thus, the fluorescence signal is significantly enhanced. Finally, in order to realize the ultra-sensitive detection of telomerase activity and reduce the sample demand to the maximum extent, the invention applies an exonuclease III (EXO III) mediated enzyme assisted circulation amplification strategy to the system on the basis of amplification-multiple hybridization signal amplification. Because the EXO III can crack the formed double-stranded complex, the amplified primer chain can be released from the double-stranded complex and continuously circulates, so that more signal molecules are released; meanwhile, the cleavage reaction also generates more telomerase primer chains S1, which provides continuous raw material molecules for the cyclic amplification reaction. Therefore, the combination of the amplification-multiplex hybridization strategy and the cycling enzyme amplification strategy can realize the ultra-sensitive detection of the telomerase activity.

Based on the detection mechanism, the specific recognition and response of the blocking material to the target molecule are realized, a large amount of signal molecules are released, so that a remarkably enhanced fluorescent signal is obtained, and due to the amplification reaction characteristics of the primer chain and telomerase, the primer chain is circularly amplified, so that more blocking materials are hybridized with the blocking material, and meanwhile, the effect of the target molecule is circularly amplified by combining the enzyme-assisted circular amplification effect mediated by exonuclease III, so that even a trace amount of telomerase can open more intelligent locks through a continuous amplification-multiple hybridization-shearing circulation mode, so that more signal molecules are continuously released, the fluorescent signal is exponentially enhanced, and the ultra-sensitive detection of the target molecule is finally realized.

The invention designs and develops a controllable release fluorescent probe for detecting telomerase activity based on blocking materials S1 and S2 based on the strategy. The telomerase primer S1 capable of triggering repeated amplification reaction of telomerase and the S2 capable of generating multiple hybridization reaction with the amplification product are used as blocking materials of the porous nano carrier to simulate and construct an intelligent lock, so that leakage of signal molecules is effectively inhibited, generation of false positive signals is avoided, and controllable release of the signal molecules is realized. Compared with other traditional fluorescence detection strategies, the fluorescent probe based on the amplification-multiple hybridization-cycling enzyme amplification strategy has the advantages that the selective response to telomerase can be realized through simple modification, and excellent specificity is shown. More importantly, the system does not need any external stimulation (such as radiation or pH value), and can release a large number of signal molecules only by triggering of the target, so that the sensitivity is greatly improved. Therefore, the fluorescent probe provides a new strategy for detecting telomerase activity and imaging in situ. So far, no literature report is available for constructing a fluorescence probe for detecting telomerase activity based on an amplification-multiple hybridization-cycle enzyme amplification strategy by using S1 and S2 as blocking materials of porous nano carriers.

Disclosure of Invention

In order to overcome the defects of the prior art, no report is found in the literature on constructing a fluorescence probe for detecting telomerase activity based on an amplification-multiplex hybridization-cycle enzyme amplification strategy by using S1 and S2 as blocking materials of a porous carrier, and therefore, the first object of the invention is that: providing a novel nano fluorescent probe for detecting telomerase activity, which is constructed by using blocking materials of S1 and S2 as porous carriers based on an amplification-multiple hybridization-cyclic enzyme amplification strategy; second object of the invention: providing a preparation method of the nano fluorescent probe provided by the invention; the third object of the present invention: provides a method for detecting telomerase activity by using the nano fluorescent probe provided by the invention. The invention designs and synthesizes S1 and S2, the intelligent lock is constructed by taking the S1 and S2 as blocking materials of the porous carrier through simple modification simulation, the controllable release of signal molecules in the porous carrier is realized, the leakage of the signal molecules is effectively inhibited, and the generation of false positive signals is avoided. The detection principle is shown in figure 1. Wherein, S1 is a telomerase primer chain which can be triggered by telomerase to generate repeated amplification reaction, S2 is a complementary long-chain molecule which can generate multiple hybridization reaction with an amplification product, and the complementary long-chain molecule consists of 2 parts, one part is the telomerase primer chain S1, the other part is the complementary chain S3 of the amplification product of the telomerase primer chain, and the part can generate hybridization reaction with the amplification product of the telomerase primer chain to form a double-chain compound. Due to the existence of telomerase, primer chains are repeatedly amplified, and an amplification product of each primer chain can be combined with a plurality of complementary long-chain molecules S2 to form a double-chain compound, so that more intelligent locks are opened, released signal molecules are exponentially increased, the sensitivity is greatly improved, and the detection of the telomerase activity is realized by detecting the enhanced fluorescent signals. The invention has the following advantages: the selective response to telomerase is realized by simple modification by using designed and synthesized S1 and S2, and excellent specificity is shown; more importantly, the release of a large number of signal molecules triggered by the target can be achieved without any external condition stimuli such as radiation or pH. Due to the amplification reaction characteristics of the primer chain and the telomerase, the primer chain is circularly amplified, so that more blocking materials S2 are hybridized with the primer chain, and simultaneously, the amplification products of the primer chain and the primer chain are circularly utilized through the enzyme-assisted circular amplification effect mediated by exonuclease III, and finally the effect of a target molecule is circularly amplified, so that even a trace amount of telomerase can open more intelligent locks through a continuous amplification-multiple hybridization-shearing circular mode, more signal molecules are released, a fluorescent signal is exponentially enhanced, and the ultra-sensitive detection of the target molecule is finally realized. Because the intelligent lock is constructed by adopting the simulation of S1 and S2, the effect of the target is circularly amplified by the cyclic mode of amplification, multiple hybridization and shearing, so that a detection system is optimized and improved, the defects of low detection sensitivity, large sample demand, complex steps, high cost and the like of the traditional method are overcome, and the intelligent lock has important significance on living body and cell detection and practical application thereof.

The invention has the advantages of improving the detection sensitivity to the maximum extent, simplifying the detection system, reducing the detection cost and the sample usage amount, widening the application field, solving the practical difficulties of complex in vivo and cell imaging technology, high price, insufficient specificity and sensitivity and the like, and having potential application value in the fields of in vitro and in vivo detection, in vivo and cell imaging and the like.

The invention achieves the purpose through the following technical scheme. Specifically, molecules S1 and S2 with excellent blocking performance are designed and synthesized, and an intelligent lock is constructed on the surface of a porous carrier loaded with a large number of signal molecules through simple modification by utilizing S1 and S2. On one hand, the leakage of signal molecules can be blocked, the blank is reduced, false positive signals are avoided, and on the other hand, when target molecules exist, the intelligent lock can be opened, the signal molecules can be released, and controllable release can be realized. The S1 and S2 are used as blocking materials, so that the preparation process of the probe is simplified, the optimal blocking effect is obtained, and controllable release and cyclic amplification of signals are realized. The intelligent lock constructed in a simulation way only generates specific response to the target molecule telomerase, and in addition, the intelligent lock does not respond to other substances such as NaCl, KCl, L-cysteine, glutathione, glucose or bovine serum albumin, and shows excellent selectivity and specificity. When telomerase of different activities is added, there is a strong correlation between the increase in fluorescence signal and telomerase activity. Thus, detection of telomerase activity is achieved based on the enhanced fluorescence signal. The result shows that the detection system can obtain sensitivity far higher than that of the traditional technology, and most importantly, the detection system provided by the invention can realize specific controllable release, and can generate cyclic amplification of signals without any external condition stimulation due to the synthesized special blocking material, thereby providing a new method and technology for applications such as living body and cell imaging.

The preparation method for preparing the novel nano fluorescent probe for detecting the telomerase activity, which is provided by the invention, by using the blocking materials S1 and S2 as porous carriers to construct a strategy based on amplification-multiple hybridization-cyclic enzyme amplification, comprises the following steps:

(1) s1 and S2 were designed and synthesized, the sequences of which from 5'to3' are: AATCCGTCGAGCAGAGTT and AATCCGTCGAGCAGAGTTCCCTAACCCTAA;

(2) respectively preparing S1 and S2 solutions with the concentration of 20 mu M;

(3) respectively adding the prepared 10 mu LS1 and S2 solutions into a porous gold nano carrier solution loaded with rhodamine B signal molecules, and shaking overnight at 37 ℃;

(4) magnetic separation and cleaning are carried out, and finally the nano fluorescent probe for detecting the telomerase activity is prepared;

wherein, the porous gold nano-carrier solution carrying the rhodamine B signal molecule is prepared by adding PBS solution with 20 mu M RhB final concentration into the porous gold nano-carrier and shaking overnight at 37 ℃.

The invention has the beneficial effects that: the nano fluorescent probe provided by the invention adopts designed and synthesized S1 and S2 to simulate and construct an intelligent lock, circularly amplifies the action of a target in a cyclic mode of amplification-multiple hybridization-shearing, provides a specific and efficient multiple cyclic amplification detection technology for detecting the activity of telomerase, overcomes the defects of low detection sensitivity, large sample requirement, complicated steps, high cost and the like of the traditional method, provides a new technology and method for detecting and imaging living bodies and cells, and has potential application value in the fields of biomedicine and clinical diagnosis. Compared with the prior art, the telomerase-triggered multiple signal cyclic amplification is realized without any external conditions such as stimulation of temperature, laser irradiation, pH and the like, and a simple and efficient amplification strategy is provided for detection and imaging of living bodies and cells. The invention has the advantages that: the method not only optimizes and improves the detection method, but also improves the detection sensitivity to the utmost extent, simplifies the detection process, reduces the detection cost, widens the application field, solves the problems of complex living body and cell imaging technology, high price, insufficient sensitivity and the like, and provides a new method and technology for the application of living body and cell imaging and the like.

The nano fluorescent probe for detecting telomerase activity provided by the invention has the advantages of high efficiency, sensitivity, stable performance, simple structure, easy preparation, good biocompatibility, wide application range and the like, shows high specificity and selectivity, and cannot be interfered by other substances such as NaCl, KCl, L-cysteine, glutathione, glucose or bovine serum albumin. The experimental result shows that the telomerase activity in the cervical cancer cells is detected by adopting the nano fluorescent probe provided by the invention, the sensitivity and the selectivity which are far higher than those of the prior art are shown, in addition, a wider cell detection range is obtained, the detection limit is as low as 46 HeLa cells, as shown in figure 2, the size of the telomerase activity in a HeLa cell lysate is detected through experiments, and the relation between the telomerase activity of a target object and the fluorescence signal enhancement is determined by observing the relation between lysates with different cell numbers and fluorescence signals. In the range of 0 to 10000 HeLa cells, the fluorescence signal is enhanced along with the increase of the cell number, and in the range of 70 to 1100 cells, the fluorescence signal and the cell number show a good linear relation, and the linear equation is as follows: f ═ 0.38n +194.18, R²0.997. F is the fluorescence intensity and n is the cell number.

Due to excellent biocompatibility, the nano fluorescent probe provided by the invention can realize in-situ imaging of telomerase in cells, and well solves the problem that the traditional technology is easily interfered by other enzymes and biological matrixes. Therefore, the method also provides a new approach and strategy for developing a cell in situ imaging probe of a high-sensitivity tumor biomarker.

Drawings

FIG. 1 is a detection schematic;

FIG. 2 (A) is a fluorescence spectrum of the probe of the present invention after reacting with different amounts of HeLa cell lysates, the cell amounts are: 0. 70, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10000; (B) linear plot of fluorescence signal versus number of HeLa cells.

Detailed Description

The following are specific examples related to the present invention, and further description is made on the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

An experimental instrument: THZ-82A gas bath constant temperature oscillator (gold jar medical instrument factory); f-4600 Fluorospectrophotometer (Hitachi, Japan); magnetic separation rack (Tianjin double Si le chromatographic development center); TDL-40B 4000 rpm centrifuge (Shanghai' an Tint scientific Instrument factory); TGL-16B 10000 rpm centrifuge (Shanghai' an pavilion scientific instruments factory); SCIEWTZ-10N lyophilizer (Ningbo Xinzhi Biotech Co., Ltd.); leica TCS SP5II confocal laser scanner (lycra, germany).

Experimental reagent: polyvinylpyrrolidone K-30(PVP), acetone (C)₃H₆O), sodium chloride (NaCl), Ethylene Glycol (EG) purchased from chinese medicinal chemical company; silver nitrate (AgNO)₃) Sodium sulfide (Na) nonahydrate₂S·9H₂O), 3 '-azido-3' -deoxythymidine (AZT), dimethyl sulfoxide (DMSO), Phosphate Buffered Saline (PBS) from Admas-beta; chloroauric acid tetrahydrate (HAuCl)₄·4H₂O) purchased from Sigma-Aldrich; cervical cancer Hela cells (university of Qingdao); secondary water, etc. and all the reagents are analytically pure. Rhodamine B (RhB), diethylene glycol diacrylate phthalate (Pdda) obtained from Shanghai Aladdin Biotech GmbH; exonuclease III (EXO III), nucleotides (dNTPs) from Thermo Scientific; the 3-4 mu m sulfydryl modified magnetic beads are obtained from a BeisLe chromatographic technology development center in Tianjin; the S1 and S2 sequence designs used in this experiment are shown in Table 1: both are entrusted with Shanghai Biotechnology, Inc.

Table 1 detailed deoxyribonucleotide sequence (5'to3')

Example 1:

the preparation method for preparing the novel nano fluorescent probe for detecting the telomerase activity of the cancer cells, which is provided by the invention and uses the blocking materials S1 and S2 as porous carriers to construct a strategy based on amplification-multiple hybridization-cyclic enzyme amplification, comprises the following steps:

(1) s1 and S2 were designed and synthesized, the sequences of which from 5'to3', respectively: AATCCGTCGAGCAGAGTT and AATCCGTCGAGCAGAGTTCCCTAACCCTAA;

(2) respectively preparing S1 and S2 solutions with the concentration of 20 mu M;

(3) respectively transferring the 10-mu LS1 and S2 solutions prepared in the previous step, adding the solutions into 100-mu L of porous gold nano carrier solution loaded with rhodamine B signal molecules, and shaking overnight at 37 ℃;

(4) magnetic separation and cleaning are carried out, and finally the nano fluorescent probe for detecting the activity of cancer cell telomerase is prepared;

(5) dispersing the product in 200 mu L PBS buffer solution to obtain fluorescent probe solution;

wherein, the porous gold nano-carrier solution carrying the rhodamine B signal molecule is prepared by adding PBS solution with 20 mu MRhB final concentration into the porous gold nano-carrier and shaking overnight at 37 ℃; the porous gold nano-carrier is prepared according to a literature method (Sun, Y.; Xia, Y. Science2002,298,2176.), and then the prepared porous gold nano-carrier is reacted with thiol magnetic beads according to the literature method and then surface electropositive modification is carried out to obtain the porous gold nano-carrier (Analytica Chimica Acta,2018,998, 45-51.).

Example 2:

the method for detecting the telomerase activity in the cancer cells by using the nano fluorescent probe provided by the invention comprises the following steps:

(1)2.0mL of 1.0X 10 cells containing HeLa cells⁷cell/mL, washing with PBS buffer (pH 7.4) for three times, centrifuging at 2000rpm for 5min, adding 200. mu.L cell lysis buffer, performing ice bath for 30min, and performing cell lysis with ice bathCentrifuging at 4 deg.C and 12000rpm for 20min to obtain cell lysate;

(2) in order to detect the activity of telomerase in a HeLa cell lysate, the obtained cell lysate is diluted step by step to respectively obtain solutions containing different cell numbers, wherein the specific cell numbers comprise: 70. 100, 200, 400, 600, 800, 1000, 1100, 2000, 4000, 6000, 8000, 10000 cells;

(3) the diluted cell lysate was mixed with 20. mu.L of a probe solution (10nM), 1.0. mu.L of EXO III cleaving enzyme (20U/. mu.L) and 10. mu.L of dNTPs (10mM), respectively, and diluted to 100. mu.L with PBS buffer;

(4) after incubation for 80min at 37 ℃, detection is carried out by adopting an F-4600 type fluorescence spectrometer, and the excitation wavelength is as follows: 488nm, slit width 5.0 nm.

The experimental result shows that the fluorescence signal is enhanced along with the increase of the cell number in the range of 0 to 10000 HeLa cells, and the fluorescence signal and the cell number present a good linear relation in the range of 70 to 1100 cells, and the linear equation is as follows: f ═ 0.38n +194.18, R²0.997, detection limit was as low as 46 HeLa cells. The probe of the invention can be used for detecting the telomerase activity in cancer cells, and the method is proved to have the advantages of high sensitivity and high specificity.

Example 3:

(1) respectively transferring 10 mu L of 20 mu M S1 and S2 solutions, adding the solutions into 100 mu L of porous gold nano carrier solution loaded with rhodamine B signal molecules, shaking overnight at 37 ℃, carrying out magnetic separation and cleaning;

(2) dispersing the product in 200 mu L PBS buffer solution to obtain fluorescent probe solution;

(3) mu.L of HeLa cells were mixed with 10. mu.L of dNTPs (10mM) and 20. mu.L of a fluorescent probe solution at 37 ℃ and 5% CO₂Incubating for 2 h;

(4) placing the cells on a laser confocal microscope for fluorescence imaging, selecting an excitation wavelength: 488 nm;

wherein, the porous gold nano-carrier solution carrying the rhodamine B signal molecule is prepared by adding a PBS solution with the final concentration of 20 MuM RhB into the porous gold nano-carrier and shaking overnight at 37 ℃; the porous gold nano-carrier is prepared according to a literature method (Sun, Y.; Xia, Y. Science2002,298,2176.), and then is directly subjected to surface electropositive modification to obtain the porous gold nano-carrier (Analytica Chimica Acta,2018,998, 45-51.).

Experimental results show that after cells are incubated together by the probes and the base dNTPs, the cells enter the cells, because telomerase exists in the cells, a telomerase primer chain on the probes is triggered to generate a circular amplification reaction, and along with the amplification of the primer chain, S2 on the probes and an amplification product generate multiple hybridization reactions, so that all the reactions can cause the intelligent lock to be opened, and a large number of fluorescent molecules are released. Therefore, the fluorescence signal in the cell is significantly enhanced. The fluorescent probe adopted by the invention is proved to be capable of specifically and sensitively realizing the in-situ fluorescence imaging of telomerase in cells.

The nano fluorescent probe provided by the invention provides a high-efficiency, simple and economic multiple-cycle amplification technology for detecting the telomerase activity in cancer cells and in-situ imaging. The intelligent lock is constructed by adopting designed and synthesized S1 and S2 simulation, an amplification-multiple hybridization-shearing circulation mode is formed, the effect of the target is circularly amplified, the defects of low detection sensitivity, large sample requirement, complex steps, high cost and the like of the traditional method are overcome, compared with the traditional technology, the multiple circulation signal amplification triggered by the target molecule is realized without any external conditions such as temperature, laser irradiation, pH and the like, and a simple and efficient amplification strategy is provided for living body, cell detection and imaging. The invention has the advantages that: the method not only optimizes and improves the detection method, but also improves the detection sensitivity to the maximum extent, simplifies the detection process, reduces the detection cost, widens the application field, solves the problems of complex living body and cell imaging technology, high price, insufficient sensitivity and the like, provides a new method and technology for the application of living body and cell imaging and the like, and has potential application value in the fields of biomedicine and clinical diagnosis.