CN109797200B - Ratio type telomerase activity quantitative detection method - Google Patents
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Abstract
The invention discloses a quantitative detection method of ratio type telomerase activity, which comprises the following steps: firstly, labeling methylene blue at the 5 'end of a hairpin DNA probe, labeling sulfydryl at the 3' end, and self-assembling on the surface of a gold electrode through a gold-sulfydryl bond; and hybridizing the TS primer of which the 5' end is marked with ferrocene with the hairpin DNA probe on the surface of the gold electrode, and using the obtained gold electrode modified with the hairpin DNA probe for detecting telomerase. The invention simultaneously utilizes two electrochemical probes to detect the telomerase activity, constructs a novel ratio type electrochemical biosensor, obviously improves the detection sensitivity, and overcomes the problems of poor repeatability, poor stability and the like in the prior art; according to the invention, the repetitive sequence generated by telomerase catalysis is complementary with the probe sequence, so that the detection specificity is enhanced; the method is simple and easy to implement, and has the characteristics of high sensitivity, rapidness, accuracy and low cost.
Description
Technical Field
The invention relates to the technical field of telomerase activity detection, and particularly relates to a ratio type telomerase activity quantitative detection method.
Background
Telomerase is a ribonucleoprotein complex with an endogenous RNA template that catalyzes the extension of DNA repeats. In most human cancers (e.g., lung, liver, colorectal and gastric cancers), telomerase expression is often upregulated, leading to unlimited proliferation of cancer cells. Telomerase can be used as a tumor marker for early screening of cancer, so that the development of a new method for detecting the activity of telomerase with high speed and high sensitivity has important value for early diagnosis and treatment of cancer. The traditional telomerase detection methods comprise a telomerase repetitive sequence amplification method, a telomerase repetitive sequence extension method, a fluorescein-labeled telomerase repetitive sequence amplification method and the like, and the methods have the defects of large sample requirement, long detection time, complex operation, false positive, high price and the like. With the development of the technology, the DNA electrochemical sensor is increasingly applied to the detection of tumor markers due to its advantages of simple operation, sensitive detection, low cost, low equipment requirement, etc. Therefore, there is a need for a method that can apply electrochemical methods to telomerase activity detection.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a quantitative detection method for ratio-type telomerase activity, aiming at the above-mentioned deficiencies in the prior art.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a quantitative detection method for ratio type telomerase activity comprises the following steps: firstly, labeling methylene blue at the 5 'end of a hairpin DNA probe, labeling sulfydryl at the 3' end, and self-assembling on the surface of a gold electrode through a gold-sulfydryl bond; then hybridizing the TS primer marked with ferrocene at the 5' end with a hairpin DNA probe on the surface of the gold electrode, and using the obtained gold electrode modified with the hairpin DNA probe for detecting telomerase; in the presence of telomerase, the telomerase catalyzes a TS primer extension reaction to generate a repeat sequence (TTAGGG) n, the TS primer extension reaction is hybridized with a hairpin DNA probe, a hairpin structure is opened, a methylene blue signal is far away from the surface of an electrode, an obtained electrochemical signal can be obviously changed, and a ferrocene signal on the TS primer is still kept on the surface of the electrode and is used as an internal reference; and (3) comparing the ratio of the methylene blue oxidation current peak and the ferrocene current peak to realize the detection of the telomerase activity.
Preferably, the method specifically comprises the following steps:
1) extracting telomerase;
2) preparing a hairpin DNA probe for detecting telomerase activity;
3) pretreating a working electrode, and modifying the hairpin DNA probe prepared in the step 2) on the working electrode;
4) performing activity detection on the extracted telomerase by using the working electrode prepared in the step 3);
5) and carrying out electrochemical analysis to obtain a telomerase activity detection result.
Preferably, the step 1) specifically includes: hela cells were extracted and cultured in DMEM medium containing 10% fetal bovine serum in 5% CO2Culturing at 37 deg.C in the incubator of (1); counting the number of cells using a haemocytometer, collecting the cells during the exponential growth phase, 106Individual cells were collected in 1.5mL microcentrifuge tubes, washed twice with phosphate buffer, and resuspended in 200 μ L ice-cold CHAPS lysis buffer; the mixture was incubated on ice for 30 minutes and blown several times with a pipette to lyse the cells thoroughly; then, it was centrifuged at 12000rpm at 4 ℃ for 30 minutes to precipitate insoluble matters; collecting supernatant, subpackaging into new test tubes without RNA enzyme, and extracting to obtain telomerase.
Preferably, in step 1), the CHAPS lysis buffer has a pH of 7.5, and comprises: 10mM Tris-HCl, 1mM MgCl21mM EGTA, 0.1mM PMSF, 0.5% by mass CHAPS and 10% by volume glycerol.
Preferably, in the step 2), the hairpin DNA probe is labeled with methylene blue at the 5 'end and a sulfhydryl group at the 3' end, and the probe sequence is as follows: 5 ' - (MB) -CGCCCCTAACCCTAACCCTAACCCTAAAACTCTGCTGGGTTAGGGGCGTTT- (HS-SH) -3 ', then the disulfide bond at the 3 ' end of the hairpin DNA probe is opened, and the sulfhydryl group is obtained by reducing dithiothreitol.
Preferably, the step 2) specifically includes: dissolving the hairpin DNA probe labeled with methylene blue at the 5 'end and thiol at the 3' end in 100. mu.L of ultrapure water, and dissolving 3.5mg of dithiothreitol in 300. mu.L of 0.1M Tris-HCl buffer solution at pH 7.45; then fully mixing the two solutions at room temperature for 1 hour; mixing the mixed solution with 50 μ L of 3M sodium acetate and 1.5ml of 100% ethanol; then, centrifugation was carried out at 14000rpm for 30min, the supernatant was discarded, and the pellet was resuspended in 10mM Tris-HCl buffer solution to a final concentration of 2. mu.M to obtain a hairpin DNA probe for telomerase activity assay.
Preferably, in step 2), the pH of the Tris-HCl buffer solution during resuspension is 7.4, which comprises 1mM EDTA,10mM TCEP and 0.1M NaCl.
Preferably, the step 3) specifically includes:
3-1) pretreatment of a working electrode: firstly, mechanically polishing a working electrode by using P3000 silicon carbide paper, P5000 silicon carbide paper and alumina powder with various particle sizes; then ethanol and ultrapure water are used for ultrasonic treatment to remove residual alumina; then, completely immersing the electrode into the goby solution for 10 minutes, and then washing with pure water; finally, the fresh 0.5M H2SO4Performing electrochemical cleaning by cyclic potential in the solution until a stable cyclic voltammogram is obtained;
3-2) working electrode modification: the working electrode was washed with ultrapure water, and 30. mu.L of 2. mu.M hairpin DNA obtained in the above step 2) was added dropwise thereto and reacted for 16 hours to modify the DNA.
Preferably, the step 4) specifically includes: reacting the modified working electrode obtained in the step 3) with 200 mu L of 3 mu M TS primer solution at 37 ℃ for 2 hours, wherein the TS primer sequence is as follows: 5 '-Fc-TTTTTAATCCGTCGAGCAGAGTT-3', incubating the modified working electrode and the telomerase extracted in the step 1) at 37 ℃ for 1 hour, and analyzing the telomerase activity by using an electrochemical detection method.
Preferably, a conventional three-electrode system comprising a gold electrode as a working electrode, a platinum wire counter electrode and a saturated calomel reference electrode is used in the step 4); wherein, in 5mM [ Fe (CN) ] with 1M KCl6]3-/4-Performing electrochemical impedance spectroscopy and cyclic voltammetry analysis in buffer solution, and performing square wave voltammetry experiment in 20mM Tris-HCl (Tris-HCl)pH 7.4, which comprises 140mM NaCl, 5mM MgCl2。
The beneficial effects of the invention are: the invention simultaneously utilizes two electrochemical probes to detect the activity of telomerase, constructs a novel ratio type electrochemical biosensor, obviously improves the detection sensitivity, and overcomes the problems of poor repeatability, poor stability and the like in the prior art; according to the invention, the repetitive sequence generated by telomerase catalysis is complementary with the probe sequence, so that the detection specificity is enhanced; meanwhile, the method adopts the double-signal ratio as the detection basis, the background interference is basically eliminated, and the method is simple, convenient and feasible, has the characteristics of high sensitivity, rapidness, accuracy and low cost, and has good popularization and application values.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the quantitative detection method of ratio-type telomerase activity of the present invention;
FIG. 2 is an impedance profile of various modified electrodes in an example of the invention;
FIG. 3 is a cyclic voltammogram of different modified electrodes in an example of the invention;
FIG. 4 is a square wave voltammogram obtained before and after telomerase reaction in an example of the present invention;
FIG. 5 is a square wave voltammogram for the detection of telomerase in an example of the invention;
FIG. 6 shows a diagram IMB/IFcA linear plot of telomerase concentration.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention designs a ratio type electrochemical sensor based on a hairpin DNA probe and a Telomerase Substrate (TS) primer.
A ratio type of the present embodimentA method for quantitatively detecting telomerase activity comprises the following steps: firstly, labeling Methylene Blue (MB) at the 5 'end of a hairpin DNA probe, labeling sulfydryl at the 3' end, and self-assembling on the surface of a gold electrode through a gold-sulfydryl bond; then hybridizing a Telomerase Substrate (TS) primer with ferrocene (Fc) labeled at the 5' end with a hairpin DNA probe on the surface of the gold electrode, and using the obtained gold electrode modified with the hairpin DNA probe for telomerase detection; extracting telomerase from HeLa cells, which catalyzes TS primer extension reaction and generates repetitive sequences (TTAGGG) in the presence of telomerasenWhen the probe is hybridized with a hairpin DNA probe, the hairpin structure is opened, the methylene blue signal is far away from the surface of the electrode, the obtained electrochemical signal can be obviously changed, and the ferrocene signal on the TS primer is still kept on the surface of the electrode to be used as an internal reference, so that the limitation of a single signal output strategy is avoided. By comparing the oxidation current peaks (I) of MBMB) And Fc Current Peak (I)Fc) The ratio of (a) to (b), the activity of telomerase can be sensitively detected. Through data analysis, the ratio and the telomerase activity have a linear relation in the range of 0.2 to 200 cells/mu L, and the detection limit is 0.02 cell/mu L. According to the invention, the repetitive sequence generated by telomerase catalysis is complementary with the probe sequence, so that the detection specificity is enhanced; meanwhile, the method adopts the ratio of double signals as the detection basis, and basically eliminates background interference.
Referring to fig. 1, a schematic diagram of the detection principle of the method for quantitative detection of ratio-type telomerase activity of the present invention is shown, wherein the labeled values, Hairpin DNA: hairpin DNA, TS primer: TS primer, Telomerase: telomerase, Fc: ferrocene, MB: methylene blue, MCH mercaptoethanol, HeLa cell: heila cells.
FIG. 2 is an impedance profile of different modified electrodes; FIG. 3 is a cyclic voltammogram of different modified electrodes. In fig. 2 and 3 (a): bare gold electrode, (b): hairpin DNA-modified electrode, (c): electrode after TS primer hybridization, (d): and (3) carrying out telomerase extension reaction.
The electrode profiles in fig. 2 and 3 were used to verify the feasibility of the telomerase detection system. FIG. 2 shows the results of electrochemical impedance spectroscopy of various modified gold electrodes. The bare gold electrode exhibits a very small semicircular area (curve a), indicating a fast charge transfer process. While the gold electrode surface modified with hairpin DNA will repel [ Fe (CN)6]3-/4-Anions, resulting in an increase of the surface impedance of the electrode (curve b). The electrodes after incubation of the TS primer further produced a significantly enhanced impedance value (curve c). When telomerase exists, the electrode modified with the probe and the primer react to generate a large number of repeated sequences, and the corresponding charge transfer resistance value is obviously increased (curve d). To further verify the electrode step-wise modification process, Cyclic Voltammetry (CV) was used for further electrochemical characterization. As shown in fig. 3, a pair of distinct peaks (curve a) can be observed in the CV plot of the bare gold electrode. After hairpin DNA modification of the electrode, the peak current decreased (curve b). After modification with the TS primer, the peak current decreased accordingly (curve c). Further processing by telomerase results in more negatively charged sequences being produced, and thus, the peak current continues to decrease (curve d). These results are in full agreement with the impedance results.
FIG. 4 is a square wave voltammogram obtained before and after telomerase reaction, wherein 1 is a hairpin DNA modified electrode; 2 is a curve after hybridization with the TS primer; curve 3 is after further incubation with telomerase.
To further verify the feasibility of the proposed ratiometric detection of telomerase, the present invention performed Square Wave Voltammetry (SWV) measurements. As shown in fig. 4, SWV of the hairpin DNA-modified electrode showed MB signal without Fc signal in the absence of TS primer and telomerase. In the presence of the Fc-labeled TS primer, two peaks for MB and Fc can be observed at 0.256 and-0.267V, respectively, indicating successful hybridization of the hairpin DNA and TS primer, demonstrating that MB and Fc signals remain near the electrode surface. However, after introduction of the telomerase, the hairpin structure is opened by the extension reaction to generate the repeat sequence. In the experiment, the peak current of Fc was almost unchanged, while the peak current of MB was sharply decreased, confirming the telomerase-induced conformational change. By comparing electrochemical signals, sensitive measurement of telomerase activity can be achieved.
FIGS. 5 and 6 are graphs showing electrochemical detection results of telomerase: FIG. 5 is a square wave voltammogram used to detect telomerase. The concentration is 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 pieces of fine powderCell/. mu.L (from top to bottom, 1-10). FIG. 6 is IMB/IFcLinear relationship to telomerase concentration. The inset shows the linear range.
The invention monitors MB and Fc (I)MBAnd IFc) To study the analytical performance of the designed sensing system. As shown in FIG. 5, I can be clearly observedMBThe concentration of the telomerase is increased and is reduced within the range of 0.2-200 cells/mu L. FIG. 6 shows the use of IMBAnd IFcCalibration curve for telomerase assay of ratio values. The results showed that the signal ratio values were linear with the cell log and the regression equation was 0.8079-0.1329x (R)20.992). The detection limit was calculated as 0.02 cells/. mu.l (S/N-3).
The following provides a specific embodiment of the method for quantitative determination of ratio-type telomerase activity, which specifically comprises the following steps:
1) and (3) extracting telomerase:
hela cells were extracted and cultured in DMEM medium containing 10% fetal bovine serum in 5% CO2Culturing at 37 deg.C in the incubator of (1); counting the number of cells using a hemocytometer, collecting the cells during the exponential growth phase, and counting 106The cells were collected in 1.5mL microcentrifuge tubes, washed twice with phosphate buffer (PBS, pH 7.4), and resuspended in 200. mu.L of ice-cold CHAPS lysis buffer (10mM Tris-HCl, pH 7.5, 1mM MgCl)21mM EGTA, 0.1mM PMSF, 0.5% (w/v) CHAPS and 10% (v/v) glycerol); the mixture was incubated on ice for 30 minutes and blown several times with a pipette to lyse the cells thoroughly; then, it was centrifuged at 12000rpm at 4 ℃ for 30 minutes to precipitate insoluble matters; collecting supernatant, subpackaging into new test tubes without RNA enzyme, and extracting to obtain telomerase.
2) Preparation of hairpin DNA probes for telomerase activity detection:
the disulfide bond of the DNA probe is reduced by labeling methylene blue at the 5 ' end of the hairpin DNA probe, labeling a sulfhydryl group at the 3 ' end, then opening the disulfide bond at the 3 ' end of the hairpin DNA probe, and reducing dithiothreitol to obtain the sulfhydryl group.
The method specifically comprises the following steps: dissolving hairpin DNA probe labeled with methylene blue at 5 'end and sulfhydryl at 3' end in 100. mu.L of ultrapure water, dissolving 3.5mg of dithiothreitol in 300. mu.L of 0.1M Tris-HCl buffer solution with pH 7.45; then fully mixing the two solutions at room temperature for 1 hour; mixing the mixed solution with 50 μ L of 3M sodium acetate and 1.5ml of 100% ethanol; then, it was centrifuged at 14000rpm for 30min, the supernatant was discarded, and the pellet was resuspended in 10mM Tris-HCl buffer solution (1mM EDTA,10mM TCEP and 0.1M sodium chloride, pH 7.4) to a final concentration of 2. mu.M to obtain a hairpin DNA probe for telomerase activity assay. The hairpin DNA probe sequence is as follows:
5’-(MB)-CGCCCCTAACCCTAACCCTAACCCTAAAACTCTGCTGGGTTAGGGGCGTTT-(HS-SH)-3’。
3) pretreatment and modification of a working electrode:
pretreatment of a working electrode: firstly, mechanically polishing a working electrode by using P3000 silicon carbide paper, P5000 silicon carbide paper and alumina powder with various particle sizes (1, 0.3 and 0.05 mu m); then carrying out ultrasonic treatment by using ethanol and ultrapure water to remove residual alumina; then the electrode was completely immersed in the goby solution (H)2SO4：H2O23: 1, note that: highly corrosive) for 10 minutes, followed by rinsing with pure water; finally passing through the fresh 0.5M H2SO4Performing electrochemical cleaning by cyclic potential in the solution until a stable cyclic voltammogram is obtained;
modification of a working electrode: the working electrode was washed with ultrapure water, and 30. mu.L of 2. mu.M hairpin DNA obtained in the above step 2) was added dropwise thereto and reacted for 16 hours to modify the DNA.
4) Detecting telomerase activity:
reacting the modified working electrode obtained in the step 3) with 200 mu L of 3 mu M TS primer solution at 37 ℃ for 2 hours, wherein the TS primer sequence is as follows: 5 '-Fc-TTTTTAATCCGTCGAGCAGAGTT-3', incubating the modified working electrode and the telomerase extracted in the step 1) at 37 ℃ for 1 hour, and analyzing the telomerase activity by using an electrochemical detection method.
5) Carrying out electrochemical analysis to obtain a telomerase activity detection result:
all electrochemical experiments were performed by a computer controlled electrochemical workstation (CHI 660D, CH Instruments). Wherein a conventional three-electrode system was used, comprising a gold electrode (2mm diameter) as working electrode, an auxiliary platinum electrode and a saturated calomel reference electrode; wherein, in 5mM [ Fe (CN) with 1M KCl6]3-/4-In buffer, Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) analyses were performed in 20mM Tris-HCl (140mM NaCl, 5mM MgCl)2pH 7.4) was performed.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
The invention name is as follows: ratio type telomerase activity quantitative detection method
Hairpin DNA probes: 5 '- (MB) -CGCCCCTAACCCTAACCCTAACCCTAAAACTCTGCTGGGTTAGGGGCGTTT- (HS-SH) -3'
TS primer: 5 '-Fc-TTTTTAATCCGTCGAGCAGAGTT-3'
Claims (4)
1. A quantitative detection method for ratio-type telomerase activity, which is characterized in that the detection method is not used for diagnosing diseases, and the method comprises the following steps: firstly, labeling methylene blue at the 5 'end of a hairpin DNA probe, labeling sulfydryl at the 3' end, and self-assembling on the surface of a gold electrode through a gold-sulfydryl bond; then hybridizing the TS primer marked with ferrocene at the 5' end with a hairpin DNA probe on the surface of the gold electrode, and using the obtained gold electrode modified with the hairpin DNA probe for detecting telomerase; in the presence of telomerase, it catalyzes the TS primer extension reaction and produces a repeat sequence (TTAGGG)nHybridizing with a hairpin DNA probe, opening a hairpin structure, keeping a methylene blue signal away from the surface of an electrode, obviously changing an obtained electrochemical signal, and keeping a ferrocene signal on a TS primer on the surface of the electrode as an internal reference; by comparisonDetecting the activity of telomerase by the ratio of the methylene blue oxidation current peak to the ferrocene current peak;
the ratio type telomerase activity quantitative detection method specifically comprises the following steps:
1) extracting telomerase;
2) preparing a hairpin DNA probe for detecting telomerase activity;
3) pretreating a working electrode, and modifying the hairpin DNA probe prepared in the step 2) on the working electrode;
4) performing activity detection on the extracted telomerase by using the working electrode prepared in the step 3);
5) carrying out electrochemical analysis to obtain a telomerase activity detection result;
in the step 2), the 5 'end of the hairpin DNA probe is labeled with methylene blue, and the 3' end of the hairpin DNA probe is labeled with sulfydryl, wherein the probe sequence is as follows:
5 ' - (MB) -CGCCCCTAACCCTAACCCTAACCCTAAAACTCTGCTGGGTTAGGGGCGTTT- (HS-SH) -3 ', then opening the disulfide bond at the 3 ' end of the hairpin DNA probe, and reducing by dithiothreitol to obtain a sulfhydryl group;
the step 3) specifically comprises the following steps:
3-1) pretreatment of a working electrode: firstly, mechanically polishing a working electrode by using P3000 silicon carbide paper, P5000 silicon carbide paper and alumina powder with various particle sizes; then carrying out ultrasonic treatment by using ethanol and ultrapure water to remove residual alumina; then, completely immersing the electrode into the goby solution for 10 minutes, and then washing with pure water; finally, the fresh 0.5M H2SO4Performing electrochemical cleaning by cyclic potential in the solution until a stable cyclic voltammogram is obtained;
3-2) working electrode modification: washing the working electrode with ultrapure water, and dropwise adding 30 mu L of 2 mu M hairpin DNA obtained in the step 2) for reacting for 16 hours for modification;
the step 4) specifically comprises the following steps: reacting the modified working electrode obtained in the step 3) with 200 mu L of 3 mu M TS primer solution at 37 ℃ for 2 hours, wherein the TS primer sequence is as follows: 5 '-Fc-TTTTTAATCCGTCGAGCAGAGTT-3', incubating the modified working electrode and the telomerase extracted in the step 1) at 37 ℃ for 1 hour, and analyzing the telomerase activity by using an electrochemical detection method;
the traditional three-electrode system is used in the step 4), and comprises a gold electrode serving as a working electrode, a platinum wire counter electrode and a saturated calomel reference electrode; wherein, in 5mM [ Fe (CN) ] with 1M KCl6]3-/4-In buffer, electrochemical impedance spectroscopy and cyclic voltammetry analysis were performed, square wave voltammetry experiments were performed in 20mM Tris-HCl, pH 7.4, which included 140mM NaCl, 5mM MgCl2。
2. The method for quantitative detection of ratiometric telomerase activity of claim 1, wherein in step 1), the CHAPS lysis buffer has a pH of 7.5, which comprises: 10mM Tris-HCl, 1mM MgCl21mM EGTA, 0.1mM PMSF, 0.5% by mass CHAPS and 10% by volume glycerol.
3. The method for quantitative determination of ratiometric telomerase activity according to claim 2, wherein said step 2) specifically comprises: dissolving hairpin DNA probe labeled with methylene blue at 5 'end and sulfhydryl at 3' end in 100. mu.L of ultrapure water, dissolving 3.5mg of dithiothreitol in 300. mu.L of 0.1M Tris-HCl buffer solution with pH 7.45; then fully mixing the two solutions at room temperature for 1 hour; mixing the mixed solution with 50 μ L of 3M sodium acetate and 1.5ml of 100% ethanol; then, centrifugation was carried out at 14000rpm for 30min, the supernatant was discarded, and the pellet was resuspended in 10mM Tris-HCl buffer solution to a final concentration of 2. mu.M to obtain a hairpin DNA probe for telomerase activity assay.
4. The method for quantitative detection of ratio-type telomerase activity as claimed in claim 3, wherein in step 2), the pH value of Tris-HCl buffer solution in the process of resuspension is 7.4, which comprises 1mM EDTA,10mM TCEP and 0.1M NaCl.
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