CN114942223B - Ratio detection kit for circulating tumor cells and preparation method and application thereof - Google Patents
Ratio detection kit for circulating tumor cells and preparation method and application thereof Download PDFInfo
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Abstract
本发明公开了一种循环肿瘤细胞（CTCs）的比率式检测试剂盒及其制备方法和应用，该比率式检测试剂盒包括第一试剂Walker探针、第二试剂SERS探针、第三试剂硫酸锌溶液。所述试剂盒用于CTCs检测时，向含有待检测CTCs的样品中加入第一试剂、第二试剂和第三试剂，磁性分离后检测SERS光谱获取第二试剂SERS探针上拉曼分子DTNB和第一试剂中ROX分子的SERS特征峰强度的比值IR(IR=I1331/I1500)，实现对CTCs的比率式SERS定量检测。本发明公开的比率式检测试剂盒制备方法简单，检测灵敏度与特异性高，结果稳定性好，可满足血液中稀有CTCs可靠检测计数要求，具有突出的应用优势。
The present invention discloses a ratio detection kit for circulating tumor cells (CTCs), a preparation method and application thereof, wherein the ratio detection kit comprises a first reagent Walker probe, a second reagent SERS probe and a third reagent zinc sulfate solution. When the kit is used for CTCs detection, the first reagent, the second reagent and the third reagent are added to a sample containing CTCs to be detected, and after magnetic separation, the SERS spectrum is detected to obtain the ratio IR ( IR = I1331 / I1500 ) of the SERS characteristic peak intensity of the Raman molecule DTNB on the second reagent SERS probe and the ROX molecule in the first reagent, so as to realize the ratio SERS quantitative detection of CTCs. The ratio detection kit disclosed in the present invention has a simple preparation method, high detection sensitivity and specificity, good result stability, can meet the requirements for reliable detection and counting of rare CTCs in blood, and has outstanding application advantages.
Description
Technical Field
The invention belongs to the field of functional nano materials and biological detection, and particularly relates to a ratio type detection kit for circulating tumor cells, a preparation method and application thereof.
Background
The incidence, metastasis and mortality of cancer are still high, however, traditional tissue biopsy assessment, which is evaluated as a "gold standard" for tumor diagnosis, is usually based on techniques such as medical imaging to perform tumor detection, has great difficulty and risk in obtaining multiple and continuous tissues, and the tumors can evolve and be heterogeneous during treatment and development, which makes the assessment of primary tumor tissue by tissue biopsy not representative of the real-time status of molecular markers nor of the overall genomic status of the tumor. By performing minimally invasive liquid biopsies on biological liquids such as blood, saliva, urine and the like, a plurality of continuous samples are obtained, the overall or dynamic monitoring of cancer progression and treatment response is realized, and a good opportunity is provided for personalized treatment of cancer. Currently, markers such as Circulating Tumor Cells (CTCs), free circulating nucleic acids (cfDNAs), proteins and Exosomes (EVs) are widely used in liquid biopsies. The CTCs which shed from the primary tumor tissue retain the molecular characteristics of the primary tumor tissue, detect and analyze the CTCs which survive in peripheral blood, help to monitor the disease course of a patient in a real-time and dynamic manner, and have important significance in the aspects of diagnosis and treatment, drug evaluation, prognosis detection and the like of cancer patients. Whereas in a patient's blood, CTCs are very low in abundance, there are approximately 10 9-1010 erythrocytes and 10 6-107 leukocytes in a one milliliter whole blood sample, whereas CTCs are approximately only a few. The protein expression level will also vary from patient to patient on the surface of cancer cells in the same type of tumor. In addition, a portion of CTCs undergo epithelial-mesenchymal transition (EMT) during transfer, resulting in a decrease in cell surface epithelial characteristics and an increase in mesenchymal phenotypic characteristics. These characteristics make CTCs present in trace amounts in the blood highly dependent on the targeting molecule used in the detection method possible during the detection. Therefore, development of a detection technique having reliable recognition and ultrasensitive analysis is very necessary for improving sensitivity and specificity of CTCs assay.
In recent years, nucleic acid aptamer called as a chemical antibody has the advantages of effectively improving the efficiency and accuracy of targeted binding, having good programmability in molecular structure and designability of nucleic acid sequences, being convenient for binding to a nucleic acid-based signal amplification technology, and being widely applied to analysis of low-abundance substances to be detected. SERS, which is an ultrasensitive non-destructive analysis technique, has excellent resistance to photobleaching and photodegradation, and its detection sensitivity can even reach a single molecular level, and recent related studies have also shown that this technique has excellent sensitivity in CTCs detection. However, SERS techniques still face two problems in CTCs detection applications: on the one hand, the content of CTCs in the clinically collected blood sample is low, and the detection sensitivity cannot meet the detection requirement due to the fact that the concentration of the substance to be detected is low; on the other hand, due to the characteristic of exponential signal enhancement of the SERS technology, the anti-impurity interference performance of the SERS technology in low-purity complex sample detection is poor, and the error of the sensor and slight fluctuation of detection conditions can cause great deviation on detection results. The ratio-type SERS sensor can avoid influence of fluctuation of external conditions on SERS intensity to a certain extent, so that stability and reliability of SERS signal output are improved.
The Chinese patent with the application number 201910807092.7 discloses a system and a method for detecting tumor cell markers miRNA-21 and tumor cells by combining 3D DNA Walker nanometer machine amplification and DNA tetrahedron nanometer probes. However, the method only carries out in-vitro quantitative detection on the tumor cell marker miRNA-21, and utilizes Raman and fluorescence imaging characterization to indicate that the tumor cell marker miRNA-21 exists in the cancer cell HeLa, so that the method has the capability of visually detecting the tumor cell, and the quantity of CTCs in a sample to be detected cannot be reflected through the content of miRNA-21. For circulating tumor cells which shed from solid tumor tissue and retain the integrity of the tumor cells, the cell-free DNA is more suitable for protein localization and cell morphology research of the tumor cells than cell-free DNA, and the number of the circulating tumor cells has important guiding significance for the detection of tumor metastasis, the prediction of prognosis of patients and the monitoring of cancer treatment results. In addition, the signal molecules adopted in the method are all marked at the tail end of the DNA, and the marking of the signal molecules at the tail end of the DNA is reduced, so that the cost of the detection kit can be effectively reduced. In order to solve the technical problems, the invention constructs an ultrasensitive lossless ratio type SERS sensing technology, realizes ratio type SERS quantitative detection of CTCs, and can meet the requirement of reliable detection and counting of rare CTCs in blood.
Disclosure of Invention
Aiming at the great demands of the current tumor molecular detection on the rapid and sensitive measurement of Circulating Tumor Cells (CTCs), in order to overcome the defects of low detection sensitivity of the low-abundance CTCs, poor anti-impurity interference performance of SERS technology in a low-purity detection system and the like, the invention researches and designs a strategy based on targeting CTCs by an aptamer and triggering DNA WALKER to assist in forming a Walker probe-SERS probe networking nano structure, provides a ratio type Surface Enhanced Raman Scattering (SERS) detection kit for detecting the circulating tumor cells, and a preparation method and application thereof, wherein the ratio type SERS detection kit is simple to prepare, does not need amplification and professional technician operation, has high sensitivity (the detection limit reaches the order of 1 cell/mL), has good specificity (can obviously distinguish nonspecific cells and has reliable recovery rate in a complex blood background), and has outstanding application advantages in the CTCs detection field.
The invention aims at realizing the following technical scheme:
a ratio detection kit for circulating tumor cells, comprising a first reagent, a second reagent and a third reagent;
The first reagent is a Walker probe, and the Walker probe is prepared by modifying a walking nucleic acid chain, a hairpin nucleic acid chain H1 containing an enzyme digestion response substrate and sulfhydryl polyethylene glycol methoxy SH-PEG-OMe on the surface of a magnetic core gold shell nanoparticle; the walking nucleic acid chain is formed by hybridizing a nucleic acid aptamer chain SYL3C-ROX with a Zn 2+ specific DNAzyme chain;
the second reagent is a SERS probe, and the SERS probe is prepared by modifying hairpin-type nucleic acid chain H2 and Raman signal molecules 5,5' -dithiobis (2-nitrobenzoic acid) on the surface of gold nanoparticles;
the third reagent is a zinc sulfate solution for assisting DNAzyme in specifically cutting a substrate nucleic acid chain.
Furthermore, the base sequences of the nucleic acid aptamer chain SYL3C-ROX, the Zn 2+ specific DNAzyme chain and the hairpin nucleic acid chain H1 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
Further, the base sequence of the hairpin nucleic acid chain H2 is shown as SEQ ID NO. 4.
Further, the particle size of the magnetic core gold shell nano-particles is 100-300 nm; the particle size of the gold nanoparticles is 15-100 nm.
The preparation method of the ratio detection kit for the circulating tumor cells comprises the following steps:
Preparation of first reagent, walker probe:
1) Mixing a nucleic acid aptamer chain SYL3C-ROX with a Zn 2+ specific DNAzyme chain to prepare a walking nucleic acid chain with a double-chain structure and a part structure of the enzyme chain protected by the nucleic acid aptamer, and cooling the walking nucleic acid chain to room temperature, wherein the molar ratio of the walking nucleic acid chain to a tricarboxyethyl phosphine solution is 1: 100-1: 1000 mixing room temperature reaction 4 h;
2) After annealing the hairpin nucleic acid strand H1, it was reacted with a tricarboxyethyl phosphine solution in a molar ratio of 1:100 to 1:1000, mixing and placing at room temperature for reaction 4 h;
3) The molar ratio was set to 1: 5-1: 10. Mu.L of the walking nucleic acid strand obtained by the reaction in the step 1) and 600. Mu.L of the hairpin nucleic acid strand H1 obtained by the reaction in the step 2) are added into 18 mL magnetic core-metal shell nano-particles to form a mixture, and the mixture is reacted overnight at room temperature;
4) 28 mM mercapto polyethylene glycol methoxy SH-PEG-OMe and magnetic core gold shell nano particles are mixed according to the volume ratio of 1: 100-1: 1000, adding the mixture into the mixture prepared in the step 3), reacting at room temperature for 10 min, centrifuging at 2000 rpm rotation speed for 10 min, cleaning, and finally dispersing the product into PBS buffer solution to prepare the Walker probe;
(II) preparation of the second reagent, i.e., SERS probe
1) After annealing the hairpin-shaped nucleic acid strand H2, it was reacted with a solution of tricarboxyethyl phosphine in a molar ratio of 1:100 to 1:1000, mixing and placing the mixture in room temperature for reaction 4 h;
2) 10 mu M hairpin nucleic acid chain H2 and gold nanoparticle are mixed according to the volume ratio of 1: 5-1: 10, mixing overnight at room temperature, and then adding 2M NaCl in small amounts for multiple times in 4 h to age the probe;
3) 100 mu M of Raman signal molecule 5,5' -dithiobis (2-nitrobenzoic acid) and gold nanoparticle are mixed according to the volume ratio of 1: 10-1: 20, adding the mixture obtained in the step 2) to react for 3 hours;
4) Centrifuging to remove supernatant, and dispersing centrifugal sediment by using PBS solution to obtain a SERS probe;
(III) preparation of a third reagent: zinc sulfate heptahydrate is dissolved in ultrapure water to prepare a zinc sulfate solution for assisting DNAzyme in specifically cutting a substrate nucleic acid chain.
A non-diagnostic use of a ratiometric SERS detection kit for detection of circulating tumor cells as described above in the detection of breast cancer circulating tumor cells MCF-7, the steps of applying comprising:
1) Adding MCF-7 cells into PBS buffer solution to obtain a sample solution for later use; wherein the concentration range of MCF-7 cells in the obtained sample solution is 5-1000 cells/mL;
2) Mixing the first reagent, the second reagent and the third reagent with sample solutions containing different numbers of cells to be detected MCF-7;
3) Performing SERS test on the mixed sample obtained in the step 2) after being magnetically separated and washed for a plurality of times by using a PBS buffer solution, detecting to obtain SERS spectrums of MCF-7 of different numbers of cells to be detected, obtaining 5,5' -dithiobis (2-nitrobenzoic acid), namely, the ratio I R(IR =I1331 /I1500 of the characteristic peak signal intensity I 1331 of the DTNB at 1331 cm -1 and the characteristic peak signal intensity I 1500 of the ROX at 1500 cm -1, and calculating to obtain the detection limit (S/N=3) of the SERS detection kit for detecting the MCF-7 cells according to the working curve of the SERS detection kit.
Further, the co-cultivation condition in the step 3) is that the culture is carried out for 60 minutes in a constant temperature mixer at 37 ℃ and 300 rpm.
Further, the applying step further includes:
Adding MCF-7 cells into a peripheral blood extraction sample to obtain a sample solution for later use, wherein the concentration range of the MCF-7 cells in the obtained sample solution is 75-600 cells/mL; mixing the first reagent, the second reagent and the third reagent with sample solutions containing different numbers of cells to be detected MCF-7; performing SERS test after the mixed sample obtained by the treatment is magnetically separated and washed for a plurality of times by using PBS buffer solution, detecting to obtain SERS spectrum, obtaining the ratio I R(IR =I1331 /I1500 of the signal intensity I 1331 of the characteristic peak of the DTNB at 1331 cm -1 and the signal intensity I 1500 of the characteristic peak of the ROX at 1500 cm -1), and calculating to obtain the concentration of the circulating tumor cells in the sample according to the working curve of the SERS detection kit.
Further, the nucleotide sequence of the nucleic acid aptamer SYL3C-ROX chain is shown as SEQ ID NO.1, the nucleotide sequence of the Zn 2+ -specific DNAzyme chain is shown as SEQ ID NO.2, the nucleotide sequence of the hairpin nucleic acid chain H1 is shown as SEQ ID NO.3, and the nucleotide sequence of the hairpin nucleic acid chain H2 is shown as SEQ ID NO. 4.
Further, in the step 2), the volume ratio of the first reagent, the second reagent and the third reagent is 1:0.2: 1-1: 2:1 are mixed with sample solutions containing different numbers of cells to be examined MCF-7.
Principle of reaction (for example, detection of breast cancer cell MCF-7): the walking chain formed by annealing the aptamer chain SYL3C-ROX and DNAzyme chain and the hairpin-shaped nucleic acid chain H1 are modified on the magnetic core-gold shell nano-particle GMNPs through an Au-S covalent bond, and a first reagent (Walker probe) is prepared by modifying sulfhydryl polyethylene glycol methoxy SH-PEG-OMe on the surface of the magnetic core-gold shell nano-particle; sequentially fixing hairpin-type nucleic acid chains H2 and DTNB on the surface of Au NPs to prepare a second reagent (SERS probe); the third reagent (ZnSO 4) was prepared by dissolving zinc sulfate heptahydrate in ultrapure water.
The aptamer chain SYL3C-ROX on the walking chain in the first reagent binds to the cell surface protein in the presence of the MCF-7 cell of interest, resulting in release of the DNAzyme chain. The hairpin nucleic acid chain H1 is sheared by the Zn 2+ in the ZnSO 4 solution, and the H1 fragment remained on the GMNPs surface after the cutting can be hybridized with H2 on the second reagent SERS probe, so that a network nano structure of the Walker probe-SERS probe containing rich SERS hot spots is formed. At the same time, the completed sheared DNAzyme is released for recycling. Finally, by testing the Raman signal of the networked nanostructure of the Walker probe-SERS probe, the rapid, specific and high-sensitivity detection of the circulating tumor cell MCF-7 can be realized. In contrast, without MCF-7, the mixture of the first and second reagents failed to form nanoparticle network aggregates. Only a single first reagent Walker probe exists in the product obtained through magnetic separation, only a more obvious ROX molecular signal exists in a Raman detection result, and no obvious signal is output from DTNB molecules in the second reagent. The ratio SERS signal I R(IR =I1331 /I1500 can be obtained by analyzing the ratio of DTNB and ROX signals in the networked nanostructure of the Walker probe-SERS probe to the cell number, and a linear working curve of I R to cell number (S/n=3) is constructed.
The beneficial effects of the invention are as follows:
The invention provides a ratio type detection kit for circulating tumor cells, a preparation method and application thereof, wherein the SERS kit comprises a first reagent, a second reagent and a third reagent. The first reagent is a Walker probe, namely a walking nucleic acid chain formed by hybridizing a nucleic acid aptamer SYL3C-ROX and a Zn 2+ specific DNAzyme on the surface, a hairpin nucleic acid chain H1 containing an enzyme digestion response substrate, and a magnetic core gold shell nanoparticle (GMNPs) of sulfhydryl polyethylene glycol methoxy SH-PEG-OMe for improving stability, the second reagent is a SERS probe, namely a gold nanoparticle modified with the hairpin nucleic acid chain H2 and a Raman signal molecule DTNB, and the third reagent is a zinc sulfate solution for assisting DNAzyme in cutting the substrate. The ratio type Surface Enhanced Raman Scattering (SERS) detection kit for detecting the circulating tumor cells is simple to prepare, does not need amplification and operation of professional technicians in detection, has high sensitivity (the detection limit reaches the order of 1 cell/mL), has good specificity (can obviously distinguish nonspecific cells and has reliable recovery rate in complex blood background), can realize ratio type SERS quantitative detection on CTCs, meets the requirement of reliable detection and counting of rare CTCs in blood, and has outstanding application advantages in the fields of CTCs detection, early diagnosis and the like.
Drawings
FIG. 1 is a schematic diagram of a preparation method of a ratio SERS detection kit for detecting circulating tumor cells according to the invention;
FIG. 2a is a gel electrophoresis characterization of the DNA hybridization process involved in the ratiometric SERS detection kit described in example 2 of the present invention;
FIG. 2b is an absorption spectrum characterization of the feasibility of the target MCF-7 trigger DNA WALKER to assist in forming a Walker probe-SERS probe network-like plasmon nanostructure, as referred to in example 2 of the present invention;
FIG. 2c is a dynamic light scattering characterization of the feasibility of the target MCF-7 trigger DNA WALKER to assist in forming a Walker probe-SERS probe network-like plasmon nanostructure, as referred to in example 2 of the present invention;
FIG. 2d is a Raman characterization of the feasibility of the target MCF-7 trigger DNA WALKER to assist in forming a Walker probe-SERS probe network-like plasmon nanostructure, as referred to in example 2 of the present invention;
FIG. 2e is a TEM characterization of the MCF-7 induced formation of Walker probe-SERS probe network structure involved in example 2 of the present invention
FIG. 3a is a chart showing SERS spectra corresponding to different concentrations of MCF-7 detected by the ratio SERS detection kit according to example 3 of the present invention;
FIG. 3b is a linear working curve of the ratio SERS detection kit according to the embodiment 3 of the invention, wherein the ratio I R(IR =I1331 /I1500 of the signal intensity of the characteristic peak of the DTNB at 1331 cm -1 and the signal intensity of the characteristic peak of the ROX at 1500 cm -1) to the cell number in each spectral line of the SERS spectrogram corresponding to different concentrations of MCF-7;
FIG. 4a is a chart showing SERS spectra corresponding to different types of cells detected by the ratio SERS detection kit according to example 4 of the present invention;
FIG. 4b is a graph showing the ratio of the signal intensity of the characteristic peak of the DTNB at 1331 cm -1 to the signal intensity of the characteristic peak of the ROX at 1500 cm -1 I R(IR =I1331 /I1500) in each spectral line of the SERS spectrogram corresponding to different types of cells detected by the ratio SERS detection kit according to the embodiment 4 of the invention;
FIG. 5 is a chart showing SERS spectra corresponding to different concentrations of MCF-7 cells in a blood sample detected by the ratio-type SERS detection kit according to example 5 of the present invention.
Description of sequence Listing
SEQ ID NO.1: the base sequence of the aptamer strand SYL 3C-ROX;
SEQ ID NO.2: base sequence of Zn 2+ -specific DNAzyme strand;
SEQ ID NO.3: base sequence of hairpin nucleic acid strand H1;
SEQ ID NO.4: base sequence of hairpin nucleic acid strand H2.
Detailed Description
For better understanding of the content of the patent of the present invention, the following detailed description of the embodiments of the present invention is provided for the purpose of providing detailed embodiments and specific operation procedures on the premise of the technical solution of the present invention, but the content of the present invention is not limited to the following examples.
The aptamer strands SYL3C-ROX, zn 2+ -specific DNAzyme strand, hairpin nucleic acid strand H1 and hairpin nucleic acid strand H2 used in the examples were as follows:
SYL3C-ROX：5’-ROX-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3’
DNAzyme：5'-SH-(CH2)6-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTG ACG ATC TAG TTG AGC TGT CAG ACG CAA CCT CTG TAG TG-3'
H1：5'-SH-(CH2)6-TTT TTT ATA ACG ACG ATT GAA ATA GTC TGA CGT TGA AGG ATC GTC TTA GCA GGC GCG TCG TTA T-3'
H2：5'-SH-(CH2)6-TTT TTT TCA ACG TCA GAC TAT TTC AAT CGT CGT TAT TTT TTT TTA CGA TTG AAA TAG TCT GAC GTT GA-3'.
Taking breast cancer cell MCF-7 as an example, the DNA base sequence fragments used in the invention are all synthesized by human beings and are synthesized by the division of biological engineering (Shanghai).
Example 1: preparation of ratio type detection kit for circulating tumor cells
1. The first reagent, walker probe preparation:
(1) Mixing a nucleic acid aptamer chain SYL3C-ROX with a Zn 2+ specific DNAzyme chain, reacting for 5 minutes at 95 ℃ to prepare a walking chain with a double-chain structure, wherein the part structure of the enzyme chain is protected by the nucleic acid aptamer, cooling the walking chain to room temperature, and mixing the walking chain with a TCEP solution (i.e. a tricarboxyethyl phosphine solution) in a molar ratio of 1:100 to 1:1000 mixture at room temperature 4 h.
(2) After annealing the hairpin nucleic acid strand H1 (heating to 95℃for 5 minutes and cooling to room temperature), it was reacted with a TCEP solution in a molar ratio of 1:100 to 1:1000 were mixed and placed in room temperature for reaction 4 h.
(3) The molar ratio was set to 1: 5-1: 10. Mu.L of walking strand obtained by the reaction in step (1) and 600. Mu.L of hairpin nucleic acid strand H1 of step (2) were added to 18 mL GMNPs to form a mixture, and reacted overnight at room temperature.
(4) And (3) adding 28 mM mercapto polyethylene glycol methoxy SH-PEG-OMe and magnetic core-gold shell nano particles into the mixture prepared in the step (3) according to the volume ratio of 1: 100-1: 1000, after reacting at room temperature for 10min, centrifuging at 2000: 2000 rpm, washing by 10: 10min, and finally dispersing the product in 1.8: 1.8 mL PBS to obtain the Walker probe.
2. Preparation of the second reagent, the SERS probe:
(1) After annealing the hairpin nucleic acid strand H2, the hairpin nucleic acid strand H2 is reacted with a TCEP solution in a molar ratio of 1:100 to 1:1000, mixing and placing the mixture in room temperature for reaction 4 h;
(2) 10 mu M hairpin nucleic acid chain H2 and gold nanoparticle are mixed according to the volume ratio of 1: 5-1: 10, mixing overnight at room temperature, and then adding 2M NaCl in small amounts for multiple times in4 h to age the probe;
(3) 100 mu M of Raman signal molecule DTNB and gold nanoparticles are mixed according to the volume ratio of 1: 10-1: 20, adding the mixture obtained in the step (2) to react for 3 hours;
(4) Finally, removing the supernatant by centrifugation, and dispersing the centrifugal sediment by using PBS solution to obtain the SERS probe.
3. Preparation of the third reagent: zinc sulfate heptahydrate is dissolved in ultrapure water to prepare a zinc sulfate solution for assisting DNAzyme in specifically cutting a substrate nucleic acid chain.
Example 2: ratio SERS detection kit feasibility characterization
The non-diagnostic application of the above ratio detection kit for circulating tumor cells is as follows (taking detection of breast cancer cells MCF-7 as an example):
Adding a certain concentration of circulating tumor cells MCF-7 into 900 mu L of PBS buffer solution to obtain a sample solution for later use; mixing 50 mu L of the first reagent, 50 mu L of the second reagent and 5 mu L of the third reagent with a sample solution of the cell MCF-7 to be detected with a certain concentration; a blank was prepared without adding MCF-7 cells, without adding a third reagent, and without adding a mixture of MCF-7 cells and a third reagent. The incubation time was 60 minutes in a constant temperature mixer at 37℃and 300 rpm. The processed mixed sample is magnetically separated and washed for a plurality of times by using PBS buffer solution, then dispersed in 50 mu LPBS, enriched by using an external magnetic field and subjected to SERS test after air drying.
The feasibility of DNA hybridization to form Walker probe-SERS probe network-like plasmon nanostructure was aided by characterizing the target MCF-7 trigger DNA WALKER with 10% polyacrylamide gel electrophoresis. As shown in FIG. 2a, lanes 1 and 2 are SYL3C and DNAzyme, respectively, and the migration rate of the newly occurring band in lane 3 is significantly lower than that of the two single-stranded DNAs, which are assigned to the hybridization products (i.e., walking chains) of SYL3C and DNAzyme. Lane 4 is a mixture of SYL3C, DNAzyme and H1 with Zn 2+ added, the band intensity of H1 in lane is substantially identical to H1 alone in lane 6, indicating that DNAzyme is blocked by aptamer in the absence of target cells and substrate cleavage is not possible even with Zn 2+ added. Lane 5 is the addition of Zn 2+ to the hybridization product of DNAzyme and H1, where the aptamer chain was captured in the presence of mock target cells, and the naked DNAzyme cleaved H1 under the action of Zn 2+. The newly appearing band below in the lane demonstrates that DNAzyme can catalyze cleavage of substrate H1 with the aid of Zn 2+. Lane 8 is a mixed solution of H1 and H2, from which overlap of bands is clearly observed compared to the H1 band alone (lane 6) and the H2 band (lane 7), because the number of bases of H1 and H2 is relatively close, and the appearance of a lighter band above the lane indicates that only a small amount of H1 and H2 hybridize, indicating that intact H1 and H2 exhibit a more stable hairpin structure and do not readily hybridize. Lane 9 shows a distinct slower migration band compared to lane 5 in the presence of Zn 2+ added to the mixture of DNAzyme, H1 and H2 to mimic the target cells, indicating that the remaining fragment after H1 cleavage can successfully hybridize to H2. Lane 10 shows that when Zn 2+ is added to the mixture of SYL3C, DNAzyme, H1 and H2, i.e. the mock target cells are not present, DNAzyme is blocked by the aptamer and cannot start working even if Zn 2+ is added, no new band appears in lane 10, which is consistent with expectations, indicating that the designed working mechanism has better specificity.
The feasibility of the target MCF-7 trigger DNA WALKER to assist in forming the Walker probe-SERS probe network-like plasmon nanostructure was further characterized by absorption spectroscopy (fig. 2 b), dynamic light scattering (fig. 2 c), raman (fig. 2 d), TEM (fig. 2 e), etc. EpCAM positive MCF-7 cells were selected as CTCs model to be tested. Here, three blank controls containing only Walker probe and SERS probe (1 #), walker probe, SERS probe and Zn 2+ (2 #), walker probe, SERS probe and MCF-7 cells (3 #) in the reaction system, and an experimental group containing Walker probe, SERS probe, zn 2+ and MCF-7 cells (4 #) in the reaction system were set. Comparing the absorption peak value, the hydration particle size and the Raman spectrum result of the experiment of 4 groups, the absorption peak value of the system is obviously shifted to 728 nm when the Walker probe, the SERS probe, zn 2+ and MCF-7 cells are simultaneously added into the reaction system, the hydration particle size value is increased to 880.2 nm, the characteristic peaks of DTNB at 1331 cm -1 and ROX at 1500 cm -1 and 1646 cm -1 appear in the SERS spectrum, and the more obvious nanoparticle assembly phenomenon can be observed after TEM characterization of the product. The results show that the designed network-like plasma nanostructure based on CTCs targeting specificity can be used for detecting target CTCs.
Example 3: working curve and detection limit for detecting MCF-7 by ratio SERS detection kit
Mixing 50 mu L of the first reagent, 50 mu L of the second reagent, 5 mu L of the third reagent and 900 mu L of PBS containing MCF-7 (5-1000 cells/mL) of cells to be detected, culturing in a 300 rpm constant-temperature mixer at 37 ℃ for 60 minutes, and magnetically separating and cleaning for multiple times by using PBS buffer solution. After the product is enriched and air-dried by using an external magnetic field, SESR test (Raman test conditions: scanning time 1s, laser power 10%, objective lens magnification 20x, accumulated times 10 times, excitation light wavelength 633: 633 nm) is carried out, SERS spectrum and characteristic signal intensity values thereof are obtained, a working curve is made by taking the concentration of the cell MCF-7 to be detected as an abscissa and the characteristic peak intensity value of the SERS probe as an ordinate, and the detection limit of the SERS detection kit for detecting the MCF-7 is calculated according to the working curve. FIG. 3a shows the SERS spectra obtained from the detection of MCF-7 cells with different concentrations, and FIG. 3b shows the ratio SERS signal I R(IR =I1331 /I1500 obtained by analyzing the characteristic peak signal intensity of each line at 1331 cm -1 at DTNB and the characteristic peak signal intensity of ROX at 1500 cm -1. For detection of MCF-7, a working curve of I R=6.97×10-3CMCF-7+0.486（R2 = 0.945 was obtained, with a calculated limit of detection of 1 cell/mL.
Example 4: specific characterization of ratio SERS detection kit for detecting MCF-7
MCF-7 was selected as positive control, heLa, LO2, HUVEC, hs578Bst, etc. cells were selected as negative control, and a reaction buffer without any additional biomolecules was used as a blank control. 50 mu L of the first reagent, 50 mu L of the second reagent and 5 mu L of the third reagent are respectively mixed with 900 mu L of PBS containing different types of cell sample solutions to be detected. After the above mixed solution was incubated in a 300 rpm constant temperature mixer at 37℃for 60 minutes, it was magnetically separated and washed with PBS buffer several times. And (3) enriching and air-drying the product by using an external magnetic field, and then performing SESR test to obtain an SERS spectrum and a characteristic peak intensity value thereof. FIG. 4a shows SERS spectra of samples of different biomolecules, and FIG. 4b shows the analysis of the characteristic peak signal intensity of each line at 1331 cm -1 at DTNB and the characteristic peak signal intensity of ROX at 1500 cm -1 to yield a ratio SERS signal I R(IR =I1331 /I1500). As is evident from the statistical results in FIG. 4b, the ratio SERS signal I R obtained by the non-specific cell experimental group is similar to that of the blank sample and is far lower than that of the target cell MCF-7 to be detected, which indicates that the ratio SERS detection kit has good specificity and reliability.
Example 5: characterization of recovery rate of detection MCF-7 by SERS detection kit
A peripheral blood sample of 4mL healthy individuals was taken, placed in a centrifuge tube containing 2mL Histopaque-1119 and 2mL Histopaque-1077 lymphocyte isolates, centrifuged at 700 g at room temperature for 30 min, the isolated monocytes (white blood cells and) were removed and dispersed in PBS, and centrifuged at 200 g for 10 min and the cells were resuspended at 4mL PBS. Different numbers of MCF-7 cells are dispersed in 1.16X10 6 white blood cell backgrounds, 50 mu L of first reagent, 50 mu L of second reagent, 5 mu L of third reagent and 900 mu L of PBS are mixed with sample solutions containing different concentrations of MCF-7 cells to be detected (75-600 cells/mL), and after the mixture is cultured in a 300: 300 rpm constant temperature mixer at 37 ℃ for 60 minutes, the mixture is subjected to magnetic separation and washing for multiple times by using PBS buffer solution. And enriching and air-drying the product by using an external magnetic field, and then performing SESR test. The recovery rate of MCF-7 is detected by calculating the concentration of CTCs in the peripheral blood sample through a standard curve and calculating a ratio SERS detection kit, and the experimental result is shown in figure 5, so that the ratio SERS detection kit designed in the invention has good accuracy; table 1 shows the recovery rate results obtained by calculation of the ratio I R(IR =I1331 /I1500) of the signal intensity of the characteristic peak of the DTNB at 1331 cm -1 and the signal intensity of the characteristic peak of the ROX at 1500 cm -1 in each spectral line of the SERS spectrogram corresponding to the MCF-7 cells with different concentrations detected by the SERS detection kit.
TABLE 1
| Sample number | Cell number/number of addition | Cell number/number of detected cells | Recovery/% | Relative standard deviation/% |
| 1 | 75 | 78.4 | 104.5 | 6.8 |
| 2 | 150 | 148.3 | 98.9 | 4.6 |
| 3 | 300 | 308.4 | 102.8 | 7.0 |
| 4 | 600 | 564.1 | 94.0 | 4.7 |
The above description is only a preferred embodiment of the present invention and the above embodiments are not intended to limit the scope of the present invention, but the present invention is not limited to the above embodiments, and all equivalent modifications, equivalent substitutions and improvements made by those skilled in the art based on the present disclosure should be included in the scope of the present invention.
Sequence listing
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Claims (8)
1. A ratio detection kit for circulating tumor cells, characterized in that the detection kit comprises a first reagent, a second reagent and a third reagent;
The first reagent is a Walker probe, and the Walker probe is prepared by modifying a walking nucleic acid chain, a hairpin nucleic acid chain H1 containing an enzyme digestion response substrate and sulfhydryl polyethylene glycol methoxy SH-PEG-OMe on the surface of a magnetic core gold shell nanoparticle; the walking nucleic acid chain is formed by hybridizing a nucleic acid aptamer chain SYL3C-ROX with a Zn 2+ specific DNAzyme chain; the base sequences of the nucleic acid aptamer chain SYL3C-ROX, the Zn 2+ specific DNAzyme chain and the hairpin nucleic acid chain H1 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
The second reagent is a SERS probe, and the SERS probe is prepared by modifying hairpin-type nucleic acid chain H2 and Raman signal molecules 5,5' -dithiobis (2-nitrobenzoic acid) on the surface of gold nanoparticles; the base sequence of the hairpin-type nucleic acid chain H2 is shown as SEQ ID NO. 4;
the third reagent is a zinc sulfate solution for assisting DNAzyme in specifically cutting a substrate nucleic acid chain.
2. The kit for ratiometric detection of circulating tumor cells according to claim 1, wherein the magnetic core gold shell nanoparticle has a particle size of 100-300 nm; the particle size of the gold nanoparticles is 15-100 nm.
3. The method for preparing the ratio detection kit for circulating tumor cells according to claim 2, which is characterized by comprising the following steps:
Preparation of first reagent, walker probe:
1) Mixing a nucleic acid aptamer chain SYL3C-ROX with a Zn 2+ specific DNAzyme chain to prepare a walking nucleic acid chain with a double-chain structure and a part structure of the enzyme chain protected by the nucleic acid aptamer, and cooling the walking nucleic acid chain to room temperature, wherein the molar ratio of the walking nucleic acid chain to a tricarboxyethyl phosphine solution is 1: 100-1: 1000 mixing room temperature reaction 4 h;
2) After annealing the hairpin nucleic acid strand H1, it was reacted with a tricarboxyethyl phosphine solution in a molar ratio of 1:100 to 1:1000, mixing and placing at room temperature for reaction 4 h;
3) The molar ratio was set to 1: 5-1: 10. Mu.L of the walking nucleic acid strand obtained by the reaction in the step 1) and 600. Mu.L of the hairpin nucleic acid strand H1 obtained by the reaction in the step 2) are added into 18 mL magnetic core-metal shell nano-particles to form a mixture, and the mixture is reacted overnight at room temperature;
4) 28 mM mercapto polyethylene glycol methoxy SH-PEG-OMe and magnetic core gold shell nano particles are mixed according to the volume ratio of 1: 100-1: 1000, adding the mixture into the mixture prepared in the step 3), reacting at room temperature for 10 min, centrifuging at 2000 rpm rotation speed for 10 min, cleaning, and finally dispersing the product into PBS buffer solution to prepare the Walker probe;
(II) preparation of the second reagent, i.e., SERS probe
1) After annealing the hairpin-shaped nucleic acid strand H2, it was reacted with a solution of tricarboxyethyl phosphine in a molar ratio of 1:100 to 1:1000, mixing and placing the mixture in room temperature for reaction 4 h;
2) 10 mu M hairpin nucleic acid chain H2 and gold nanoparticle are mixed according to the volume ratio of 1: 5-1: 10, mixing overnight at room temperature, and then adding 2M NaCl in small amounts for multiple times in 4 h to age the probe;
3) 100 mu M of Raman signal molecule 5,5' -dithiobis (2-nitrobenzoic acid) and gold nanoparticle are mixed according to the volume ratio of 1: 10-1: 20, adding the mixture obtained in the step 2) into the mixture, and reacting for 3 hours;
4) Centrifuging to remove supernatant, and dispersing centrifugal sediment by using PBS solution to obtain a SERS probe;
(III) preparation of a third reagent: zinc sulfate heptahydrate is dissolved in ultrapure water to prepare a zinc sulfate solution for assisting DNAzyme in specifically cutting a substrate nucleic acid chain.
4. The non-diagnostic use of the ratio assay kit for circulating tumor cells of claim 2 in the detection of breast cancer circulating tumor cells MCF-7, wherein the applying step comprises:
1) Adding MCF-7 cells into PBS buffer solution to obtain a sample solution for later use; wherein the concentration range of MCF-7 cells in the obtained sample solution is 5-1000 cells/mL;
2) Mixing the first reagent, the second reagent and the third reagent with sample solutions containing different numbers of cells to be detected MCF-7;
3) And 2) performing SERS test on the mixed sample obtained in the step 2) after magnetic separation and washing for a plurality of times by using PBS buffer solution, detecting to obtain SERS spectrums of different numbers of cells to be detected MCF-7, and obtaining 5,5' -dithiobis (2-nitrobenzoic acid), namely, the ratio I R =I1331 /I1500 of the DTNB characteristic peak signal intensity I 1331 at 1331 cm -1 and the ROX characteristic peak signal intensity I 1500 at 1500 cm -1, and calculating to obtain the detection limit of the SERS detection kit for detecting the MCF-7 cells according to the working curve of the SERS detection kit.
5. The non-diagnostic use of the kit for the ratiometric detection of circulating tumor cells according to claim 4, wherein the co-culture conditions in step 3) are culturing for 60 minutes in a thermostatic mixer at 37 ℃ and 300 rpm.
6. The non-diagnostic use of a kit for the ratiometric detection of circulating tumor cells according to claim 5, wherein the step of applying further comprises,
Adding MCF-7 cells into a peripheral blood extraction sample to obtain a sample solution for standby, wherein the concentration range of the MCF-7 cells in the obtained sample solution is 75-600 cells/mL; mixing the first reagent, the second reagent and the third reagent with sample solutions containing different numbers of cells to be detected MCF-7; and carrying out SERS test on the mixed sample obtained by the treatment after being magnetically separated and washed for a plurality of times by using a PBS buffer solution, detecting to obtain a SERS spectrum, obtaining the ratio I R =I1331 /I1500 of the signal intensity I 1331 of the characteristic peak of the DTNB at 1331 cm -1 and the signal intensity I 1500 of the characteristic peak of the ROX at 1500 cm -1, calculating to obtain the concentration of circulating tumor cells in a simulated clinical sample according to a working curve obtained by the SERS detection kit in a PBS buffer solution sample, and characterizing to obtain the practical performance of the detection kit.
7. The non-diagnostic use of the kit for the ratiometric detection of circulating tumor cells according to any one of claims 4 to 6 in the detection of breast cancer circulating tumor cells MCF-7, wherein the first reagent, the second reagent and the third reagent in step 2) are in a volume ratio of 1:0.2: 1-1: 2:1 are mixed with sample solutions containing different numbers of cells to be examined MCF-7.
8. The non-diagnostic use of the ratio detection kit for circulating tumor cells according to claim 7 in the detection of breast cancer circulating tumor cells MCF-7, wherein the nucleotide sequence of the aptamer SYL3C-ROX strand is shown in SEQ ID No.1, the nucleotide sequence of the Zn 2+ -specific DNAzyme strand is shown in SEQ ID No.2, the nucleotide sequence of the hairpin nucleic acid strand H1 is shown in SEQ ID No.3, and the nucleotide sequence of the hairpin nucleic acid strand H2 is shown in SEQ ID No. 4.
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