CN109517880B - Method for detecting telomerase activity based on strand displacement reaction and DNA modified gold nanoparticles - Google Patents
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Abstract
The invention discloses a method for detecting telomerase activity based on strand displacement reaction (SDA) and DNA modified gold nanoparticles. In the method, when active telomerase exists, the extension product of the substrate can trigger the probe Hairpin and the Primer to carry out SDA reaction to form a long double-stranded DNA product with single strands at two ends. The SDA product can then hybridize to DNA-modified gold nanoparticle (DNA-AuNP) probes, causing the gold nanoparticles to aggregate. And (3) measuring the change of the dynamic light scattering signal (particle size) of the gold nanoparticles, and realizing high-sensitivity quantitative analysis on the activity of the end-particle enzyme. The method has the advantages of simple operation, low detection cost, less required samples, high sensitivity and good specificity.
Description
Technical Field
The invention relates to a telomerase activity detection method, belongs to the technical field of biosensing, and particularly relates to a method for detecting telomerase activity based on a strand displacement reaction and DNA modified gold nanoparticles.
Background
Telomeres are nucleic acids of contiguous repeats (TTAGGG) located at the ends of chromosomes. Telomerase is a ribonucleoprotein complex that maintains telomere length by adding TTAGGG repeats to the telomere end through its own RNA template and reverse transcriptase and related proteins. Telomerase activity is inhibited in most normal human tissues, and telomere length decreases with cell cycle division, leading to cell senescence and death. However, in more than 85% of malignant tumor cells, telomerase activity is activated and expressed, so that cancer cells can be continuously proliferated, and therefore, telomerase is considered as a biomarker and a therapeutic target for early cancer diagnosis.
In 1994, a Telomere Repeat Amplification Protocol (TRAP) was developed to detect telomerase activity and quantify telomerase in small tissue biopsies containing small numbers of cancer cells. PCR-based TRAP is the most widely used telomerase detection method at present, for example, TRAP quantified by real-time fluorescence signals is widely used in enzymes. While having a certain sensitivity, non-specific amplification with a strong background fluorescence signal is unavoidable.
Therefore, it is necessary to develop a simple and sensitive method for detecting telomerase activity, and the technical problem to be solved by those skilled in the art is urgent.
Disclosure of Invention
In view of the above problems and needs, an object of the present invention is to provide a method for detecting telomerase activity based on a Strand Displacement Assay (SDA) reaction and DNA-modified gold nanoparticles, which can achieve highly sensitive quantitative analysis of telomerase activity by measuring uv-visible absorbance and changes in gold nanoparticle dynamic light scattering signal (particle size).
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
A method for detecting telomerase activity based on strand displacement reaction (SDA) and DNA modified gold nanoparticles mainly comprises the following steps:
1) preparing a reagent: the kit comprises a DNA modified gold nanoparticle (DNA-AuNPs) probe, a telomerase Primer (TS Primer) to be detected, dNTPs, a Hairpin probe Hairpin, an SDA Primer (SDA Primer) and DNA extension enzyme;
2) and (3) extension reaction: adding a telomerase extracting solution to be detected into a primer (TS primer) containing the telomerase to be detected, dNTPs and an amplification buffer solution, and incubating at constant temperature to enable the TS primer to perform an extension reaction to obtain an extension reaction product solution;
3) SDA reaction: adding Hairpin probe Hairpin and Primer into the extension reaction product solution, mixing uniformly, and then carrying out SDA reaction to obtain a long double-stranded DNA product with single-stranded two ends;
4) adding the DNA-AuNP probe into the SDA product, aggregating the gold nanoparticles through DNA hybridization, determining the corresponding particle size change by colorimetry and dynamic light scattering, changing the solution color and increasing the average particle size.
In the method for detecting telomerase activity in urine, in step 1), the extension product of the TS primer can initiate an SDA reaction, and the TS primer has an amino acid sequence shown in SEQ ID NO: 1 and the sequence of the extension product is 5 '-AAT CCG TCG AGC AGA GTT AGGG (TTA GGG) n-3' (n is 1-10).
In the method for detecting the telomerase activity in the urine, in the step 3), the probe Hairpin comprises two parts, and one part participates in the SDA reaction; the other part can be hybridized with a DNA-AuNP probe, and the Hairpin probe Hairpin comprises an intermolecular arm (such as Spacer Cn (n is 1-10,000)) and is subjected to Spacer CnDividing the reaction solution into two parts, wherein one part initiates SDA reaction; the other part can be hybridized with the DNA-AuNP probe.
In the method for detecting the telomerase activity in the urine, in the step 3), the Primer contains two parts, and one part participates in the SDA reaction; the other part can be hybridized with a DNA-AuNP probe, and is characterized in that: the Primer contains an intermolecular arm (e.g., Spacer Cn (n: 1 to 10,000)).
The method for detecting telomerase activity comprises the steps of preparing a DNA-AuNP probe (having a nucleotide sequence 5 '-SH-TTTTTTATCACATCA-3' shown in SEQ ID NO: 4 and a nucleotide sequence 5 '-SH-TTTTTTAAGGAGTGT-3' shown in SEQ ID NO: 5), adding the DNA-AuNP probe into an SDA product, and aggregating gold nanoparticles through DNA hybridization.
In the method for detecting the telomerase activity, in the step 4), the DNA sequence modified by the gold nanoparticles is designed according to complementary segments of the probe Hairpin and the Primer; according to the ultraviolet visible absorbance and the dynamic light scattering signal (particle size) of the gold nanoparticles, the telomerase activity in the sample is determined.
In the method for detecting the activity of telomerase in urine, in the step 4), the ultraviolet visible absorbance and the change of a gold nanoparticle dynamic light scattering signal (particle size) are measured, so that the activity of the telomerase is analyzed.
The method for detecting the telomerase activity in the urine has the lower detection limit of 3 cells.
According to the method for detecting the activity of the telomerase in the urine, the extraction process of the telomerase in the urine is as follows:
fresh urine samples were collected and centrifuged at 850rpm for 10 minutes at 4 ℃, once washed with PBS and then centrifuged at 2300rpm for 5 minutes at 4 ℃, after which the pellet was dissolved in 200 μ l lysis buffer and incubated on ice for 30 minutes and finally centrifuged at 12000rpm for 20 minutes at 4 ℃, and the supernatant was transferred and stored at-80 ℃ until use.
By means of the technical scheme, the invention has the following advantages and beneficial technical effects:
1) when active telomerase exists, the method of the invention leads the extension product of the Primer (TS Primer) to initiate SDA reaction, the SDA product can be hybridized with the DNA-AuNP probe to cause the gold nanoparticles to gather, and high-sensitivity quantitative analysis on the telomerase activity is realized by measuring the ultraviolet visible absorbance and the change of the dynamic light scattering signal (particle size) of the gold nanoparticles.
2) The method has the advantages of simple operation, high sensitivity, good specificity, low cost and the like.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention.
FIG. 1 is a schematic diagram of a method for detecting telomerase activity in urine based on strand displacement reaction (SDA) and DNA modified gold nanoparticles according to the present invention.
FIG. 2 is a graph of absorbance of different samples (uninitiated SDA and initiated SDA) analyzed by UV-Vis spectrophotometry according to the present invention.
FIGS. 3a and 3b show particle size distribution plots for different samples (un-SDA-initiated and SDA-initiated) analyzed by dynamic light scattering according to the present invention.
FIG. 4 is a graph of the size of the telomerase activity of different numbers of MCF-7 cells detected by the present invention, with the inset being a standard curve for 5 to 1000 MCF-7 cells.
FIG. 5 is a graph showing the particle size of the urine telomerase activity of different cancer patients tested by the present invention.
Detailed Description
Strand displacement reaction (SDA) has attracted attention as an isothermal nucleic acid amplification technique, in which a hairpin probe and a primer undergo a cascade hybridization reaction under the induction of a priming Strand to form a long double-helix DNA with single strands at both ends. SDA can be combined with a plurality of probes, gold nanoparticles (AuNPs) are the most commonly used nano materials, and are widely used for constructing a sensing platform due to large specific surface area, unique optical properties and good biocompatibility. The AuNPs change the particle size with the dispersion/aggregation state.
Thus, AuNPs can be bound to SDA, and the dispersed/aggregated state of gold nanoparticles and gold nanoparticle size can be converted to the amount of SDA product.
The invention discloses a method for detecting telomerase activity based on strand displacement reaction (SDA) and DNA modified gold nanoparticles. In the method, when active telomerase exists, the extension product of the substrate can trigger the probe Hairpin and the Primer to carry out SDA reaction to form a long double-stranded DNA product with single strands at two ends. The SDA product can then hybridize to DNA-modified gold nanoparticle (DNA-AuNP) probes, causing the gold nanoparticles to aggregate. The change of the dynamic light scattering signal (particle size) of the gold nanoparticle is measured, and the high-sensitivity quantitative analysis of the activity of the nanoparticle enzyme is realized.
FIG. 1 shows a schematic diagram of the method for detecting telomerase activity based on DNA-modified gold nanoparticles and SDA according to the present invention. When no telomerase exists or the telomerase is inactivated, the gold nanoparticles keep a dispersed state; and when active telomerase exists, the gold nanoparticles are aggregated, and the particle size of the gold nanoparticles is obviously increased.
In the method, when active telomerase exists, the extension product of the substrate can trigger a probe Hairpin and a Primer to perform SDA reaction to form a long double-stranded DNA product with single strands at two ends. The SDA product can then hybridize to DNA-modified gold nanoparticle (DNA-AuNP) probes, causing the gold nanoparticles to aggregate. And (3) measuring the ultraviolet visible absorbance and the change of the gold nanoparticle dynamic light scattering signal (particle size), and realizing high-sensitivity quantitative analysis on the activity of the end-particle enzyme.
The method has the advantages of simple operation, low detection cost, less required samples, high sensitivity and good specificity. Meanwhile, the detection limit of the method is 3 cells.
The present invention will be described in more detail below with reference to specific preferred embodiments and drawings, but the present invention is not limited to the following embodiments.
As shown in FIG. 1, the present invention relates to a method for detecting telomerase activity in urine based on DNA modified gold nanoparticles and strand displacement reaction (SDA). The method comprises the following specific steps:
1. based on the property that telomerase can add TTAGGG repeated sequences at the end of telomerase primer (TS primer):
the SDA reaction is initiated by the extension product of TS primer, and the TS primer has the sequence shown in SEQ ID NO: 1 and the sequence of the extension product is 5 '-AAT CCG TCG AGC AGA GTT AGGG TTA GGG-3';
2. SDA reaction:
the extension product was reacted with Hairpin probe Hairpin (having the nucleotide sequence 5' -ACACTCCTT spacer18 TCTTGGAC shown in SEQ ID NO: 2)TAACCCTAACCCTAAAACTTAGTCCAAGA-3') and Primer (having the sequence of SEQ ID NO: 3, 5 '-TGA TGT GAT spacer18 TCT TGG AC-3') to carry out SDA reaction (the underlined part is the part participating in the SDA reaction), and a long SDA product with single strands at both ends is obtained. Wherein, spacer18 is formed by connecting DNA with C18 polyethylene glycol, namely C18 polyethylene glycol 1 phosphoric acid, and the specific structural formula is as follows:
3. and (3) hybridization:
preparing a DNA-AuNP probe (having a nucleotide sequence 5 '-SH-TTTTTTATCACATCA-3' shown in SEQ ID NO: 4; having a nucleotide sequence 5 '-SH-TTTTTTAAGGAGTGT-3' shown in SEQ ID NO: 5), adding the DNA-AuNP probe into the SDA product, and aggregating the gold nanoparticles through DNA hybridization;
4. colorimetric and dynamic light scattering analysis: and (3) measuring the ultraviolet visible absorbance and the change of the dynamic light scattering signal (particle size) of the gold nanoparticles, and realizing the analysis of the activity of the end-particle enzyme.
As shown in fig. 2, fig. 2 is a graph showing an absorbance distribution of different samples analyzed by ultraviolet-visible spectrophotometry according to the present invention, when no telomerase or telomerase is inactivated, SDA reaction cannot be initiated, and the absorbance is about 0.7; when active telomerase exists, the extension product of the primer can initiate SDA reaction, and the absorbance is about 0.5.
As shown in fig. 3, fig. 3 is a distribution diagram of particle sizes of different samples analyzed by dynamic light scattering according to the present invention, when no telomerase or telomerase is inactivated (3a), SDA reaction cannot be initiated, and the particle size of gold nanoparticles is 41.3 nm; when active telomerase exists (3b), the extension product of the primer can initiate SDA reaction, and the particle size of the gold nanoparticle is increased to 435.5 nm.
As shown in FIG. 4, a graph of the size of the granules for detecting telomerase activity in different numbers of MCF-7 cells according to the present invention is shown in FIG. 4, with the standard curve for 5 to 1000 MCF-7 cells being inserted.
The cell number of MCF-7 and the grain diameter of the gold nanoparticles are in good linear relation in the range of 10 to 1000, and the detection limit is 3 cells. As shown in FIG. 5, a particle size graph for detecting the urine telomerase activity of different cancer patients according to the present invention is shown in FIG. 5.
Only the urine samples of bladder cancer patients have obviously increased grain size, and the grain size of the urine samples of normal persons and other cancer patients has no obvious change compared with the grain size of blank samples (without telomerase), which indicates that the invention has specific response to bladder cancer.
Example 1
A method for detecting telomerase activity in urine based on strand displacement reaction (SDA) and DNA modified gold nanoparticles comprises the following steps:
1) extraction of telomerase
Extraction of telomerase from cells:
MCF-7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100. mu.g/ml penicillin and 100. mu.g/ml streptomycin. After extraction of the cells with trypsin, 7X 10 cells were collected6And (4) cells. Cells were dispersed in 1.5 ml EP tubes, centrifuged at 1000rpm for 5min, and washed twice with ice-cold PBS. The extract was then suspended in 200. mu.L of lysis buffer and frozen on iceAfter incubation for 30 minutes, the cells were centrifuged at 12000rpm at 4 ℃ for 20 minutes. Transferring the supernatant, diluting, and storing at-80 deg.C.
Extracting telomerase in urine:
fresh urine samples were collected and centrifuged at 850rpm for 10 minutes at 4 ℃, once washed with PBS and then centrifuged at 2300rpm for 5 minutes at 4 ℃, after which the pellet was dissolved in 200 μ l lysis buffer and incubated on ice for 30 minutes and finally centrifuged at 12000rpm for 20 minutes at 4 ℃, and the supernatant was transferred and stored at-80 ℃ until use.
2) DNA-modified gold nanoparticle preparation
AuNPs were prepared according to the classical sodium citrate reduction method with appropriate adjustments. 2mL of freshly prepared 38.8mM sodium citrate solution was added rapidly to 20mL of boiled 1mM HAuCl chloroaurate4In the solution, the reaction solution changed from light yellow to black, then changed to purple, and finally changed to wine red, and the solution was continuously heated under reflux and stirred for 10 minutes. Finally, the reaction was allowed to cool to room temperature and kept stirring, then filtered through a 0.45 micron nylon filter and stored in a refrigerator at 4 ℃ until use.
AuNPs surface-modified DNA:
thiol-modified DNA was first treated with tris (2-chloroethyl) phosphate TCEP, followed by treatment with 200: 1 was added to the previously prepared AuNPs solution and incubated at room temperature for 16 hours. Then, NaCl was added in several portions over the next 44 hours to carry out salt aging to a final concentration of 0.1M. Finally, the reaction solution was centrifuged at 13800rpm for 30 minutes to remove free DNA (repeated 3 times), and the resulting oily precipitate was dissolved in 10mM PBS buffer (pH 7.4, 0.1M NaCl) and stored in a refrigerator at 4 ℃ until use.
3) Implementation of the SDA reaction
A solution containing telomerase extract, 1.5. mu.M TS primer, 1mM dNTPs, 0.2mg/mL BSA, 0.4U/mL RNase inhibitor was incubated at 37 ℃ for 1.0h, and Hairpin was heated at 95 ℃ for 5min and cooled to room temperature. The solution containing the Hairpin (1. mu.M), Primer (1. mu.M) and the telomerase extension product was then reacted for 1.5h at 37 ℃.
The TS primer has the sequence shown in SEQ ID NO: 1 and the sequence of the extension product is 5 '-AAT CCG TCG AGC AGA GTT AGGG (TTA GGG) n-3' (n is 1-10).
The probe Hairpin contains an intermolecular arm (such as Spacer C)n(n is 3 to 10,000)) and is supported by intermolecular arms (e.g., Spacer C)n(n is 3-10,000)) into two parts, and one part participates in SDA reaction; the other part can be hybridized with the DNA-AuNP probe.
Primer contains intermolecular arms (e.g., Spacer C)n(n is 3 to 10,000)), and is coated with spacer CnDividing the reaction solution into two parts, wherein one part initiates SDA reaction; the other part can be hybridized with the DNA-AuNP probe.
4) The SDA product was mixed with a DNA-AuNP probe and the particle size change was detected.
The DNA sequence modified on the gold nanoparticles is designed according to complementary segments of the probes Hairpin and Primer, so that the DNA-AuNPs probe is hybridized with the SDA product to cause the change of color and dynamic light scattering signals (particle size), and the telomerase activity in the sample is measured.
Adding a proper amount of DNA-AuNP probe into the SDA product, aggregating the gold nanoparticles through DNA hybridization reaction, and determining the corresponding particle size change (the solution color is changed from red to blue, and the average particle size is changed from small to large) by colorimetric and dynamic light scattering.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, so that any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Sequence listing
<110> Zhongshan university
<120> method for detecting telomerase activity in urine based on strand displacement reaction and DNA modified gold nanoparticles
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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Claims (1)
1. A method for detecting telomerase activity based on strand displacement reaction and DNA modified gold nanoparticles, which is a non-disease diagnostic method, comprising the steps of:
1) preparation of reagents: the kit comprises a DNA modified gold nanoparticle DNA-AuNPs probe, a telomerase primer TS primer to be detected, dNTPs, a Hairpin probe Hairpin, an SDA primer and DNA extension enzyme;
the SDA reaction is triggered by the extension product of the TS primer, and the TS primer is shown as SEQ ID NO: 1, and the sequence of the extension product is 5 '-AAT CCG TCG AGC AGA GTT AGGG (TTA GGG) n-3', wherein n is 1-10;
the DNA-AuNP probe has a nucleotide sequence of 5 '-SH-TTTTTTATCACATCA-3'; and has the nucleotide sequence 5 '-SH-TTTTTTAAGGAGTGT-3';
2) and (3) extension reaction: adding a telomerase extracting solution to be detected into a buffer solution containing a telomerase primer TS primer to be detected, dNTPs and the amplification, and incubating at constant temperature to enable the TS primer to generate an extension reaction to obtain an extension reaction product solution;
3) SDA reaction: adding Hairpin probe Hairpin and SDA primer into the extension reaction product solution, mixing uniformly and then carrying out SDA reaction to obtain a long double-stranded DNA product with single strands at two ends;
the nucleotide sequence of the SDA primer is 5' -ACACTCCTT spacer18 TCTTGGACTAACCCTAACCCTAAAACTTAGTCCAAGA-3'; the sequence of the probe Hairpin is 5 '-TGA TGT GAT spacer18 TCT TGG AC-3';
the concrete structural formula of the spacer18 is as follows:
4) adding the DNA-AuNP probe into the SDA product, aggregating the gold nanoparticles through DNA hybridization, and determining corresponding particle size change by adopting colorimetry and dynamic light scattering; telomerase activity in the sample is measured.
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| CN111235228B (en) * | 2020-01-22 | 2023-04-14 | 中山大学 | Method for detecting CA125 based on polymerase chain reaction and dynamic light scattering |
| CN112698020B (en) * | 2020-11-12 | 2022-08-26 | 中山大学 | Multimodal coupling analysis method of cross response system based on DNA-AuNP coding |
| CN112553304A (en) * | 2020-12-25 | 2021-03-26 | 山东大学 | Detection test paper, preparation method and application thereof in detecting telomerase activity |
| CN112795565B (en) * | 2021-02-04 | 2024-01-23 | 南京邮电大学 | Detection probe, kit and method for directly detecting telomerase activity |
| CN113234800B (en) * | 2021-05-13 | 2023-01-10 | 中山大学 | Detection method of aflatoxin M1 and application thereof |
| CN113481279A (en) * | 2021-07-01 | 2021-10-08 | 广州博徕斯生物科技股份有限公司 | Method for detecting telomerase activity |
| CN113667723B (en) * | 2021-08-17 | 2023-08-25 | 西北工业大学 | PSA detection method based on CRISPR/Cas12a and strand displacement amplification reaction |
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| Fluorescence detection of telomerase activity in high concentration of cell lysates based on strand-displacement mediated recycling;Xiaoqian Li;《Analyst》;20160324;第141卷(第8期);2388-2391 * |
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