CN111534595A - Method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase - Google Patents
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Abstract
The invention belongs to the field of electrochemistry and analytical chemistry, and particularly relates to a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase. Under the condition that MB and p-NP exist, AuNPs @ ZnSeSnSs/GE generates strong photocurrent at 0V, has self-energizing property, and combines an in-situ recombinase polymerase amplification strategy to establish a new liquid biopsy method of prostate cancer circulating tumor DNA KLK 2. The method for determining KLK2 has high sensitivity, the detection limit is 30aM, and the method is simple.
Description
Technical Field
The invention belongs to the field of electrochemistry and analytical chemistry, and particularly relates to a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase.
Background
Circulating Tumor Cells (CTCs) and circulating tumor DNA (ctDNA) have been present in the peripheral blood of cancer patients before imaging shows clear evidence of cancer onset. ctDNA is free DNA (cell-free DNA, cfDNA) of tumor origin in blood of cancer patients. ctDNA is mainly derived from necrotic tumor cells, apoptotic tumor cells, circulating tumor cells and exosomes secreted by tumor cells, and has the highest content in plasma of tumor patients. The liquid biopsy is a non-invasive detection technology, and takes body fluid as a sample to realize an analysis method for measuring the content of a disease marker. The liquid biopsy technology is of great significance to the high-sensitivity determination of ctDNA in body fluid. However, ctDNA only accounts for 0.1% -5% of cfDNA, and ctDNA content in body fluids of cancer patients of different cancer species and different disease courses varies greatly. How to improve the sensitivity and selectivity of liquid biopsy for detecting markers such as trace tumor-derived nucleic acid remains a very challenging research topic. The establishment of the ctDNA liquid biopsy method with high sensitivity and strong specificity has important theoretical significance and practical value. Therefore, the invention provides a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase, which has the characteristics of simple method and high sensitivity.
Disclosure of Invention
The invention aims to invent a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase.
In view of the defects of the prior art, the invention aims to provide a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase.
The technical scheme for realizing the aim of the invention is as follows:
first, AuNPs @ ZnSeSs were modified to GE, and then FPs were fixed to AuNPs @ ZnSeSs/GE via Au-S bonds. After addition of the sample containing KLK2, the FP on the electrode surface and the RP in solution were subjected to RPA amplification in the presence of recombinase polymerase. The target is amplified on the electrode and the RP is captured at the electrode surface. After washing, St-ALP is bound to the electrode by the affinity interaction between biotin and streptavidin. ALP catalyzes the hydrolysis of p-NPP to p-NP. Due to the high catalytic activity of ALP, many p-NPs are produced. Strong photoelectric signals are generated under the action of MB and p-NP, and the content of KLK2 is determined according to the signal intensity of PEC. The high-sensitivity detection of the target is realized due to the co-regulation effect of MB and p-NP, the circulation of MB and the amplification of recombinase polymerase. Under the condition that MB and p-NP exist, AuNPs @ ZnSeSnSs/GE generates strong photocurrent at 0V, has self-energizing property, and combines an in-situ recombinase polymerase amplification strategy to establish a novel liquid biopsy method for prostate cancer circulating tumor DNAKLK 2. The method for determining KLK2 has high sensitivity and the detection limit is 30 aM. The self-powered photoelectrochemistry combined with the polymerase amplification sensing of the in situ recombinase opens up a new path for the development of the PEC analysis method. Provides a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase.
The invention is realized by the following measures: a method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase is characterized by comprising the following steps:
(1) preparing ZnSe nano-sheets;
(3) KLK2 detection;
(4) electrochemical and photoelectrochemical measurements.
Preferably, the preparation of the ZnSe nanosheet comprises the following steps:
and stripping by an ultrasonic stripping method to obtain ZnSe nanosheets (ZnSeSnSs). First, 0.1 to 450mg of ZnSe was added to 1 to 150mL of a DMF solution, and then the mixture was dispersed by sonication for 0.1 to 100 hours to obtain a yellow dispersion. Next, centrifugation was carried out at 14,000rpm for 20min, and exfoliated ZnSe was separated from the dispersion and washed 3 times with ethanol. Finally, the ZnSeNSs suspension was used for the next experiment.
Preferably, the synthesis of AuNPs @ ZnSeSnSs comprises the following steps:
AuNPs @ ZnSeSnSs are prepared by an in-situ growth method. 0.1-30 mL of ZnSeSnSs and 0.1-10 mL of LHAuCl4·4H2O was added to the flask and heated to 115 ℃ by an oil bath with stirring. The solution was continued to reflux and stirred for 3 minutes. Then, 0.1mL to 10mL of 1.66mg/mL sodium citrate solution was dropped into the flask. The mixture was refluxed at 115 ℃ for 3 minutes to give black AuNPs @ ZnSeSnSs. The final black precipitate was collected by centrifugation at 14,000rpm for 10 min. In the synthesis of AuNPs @ ZnSeSnss, different masses of chloroauric acid influence PECA signal. The mass ratio of ZnSeSnSs to chloroauric acid is 1:0.2, 1:0.33, 1:0.66, 1:1 and 1:1.5 respectively.
Preferably, the KLK2 detection comprises the following steps:
first, 1. mu.L to 60. mu.L of AuNPs @ ZnSeSnSs were dropped onto the surface of the gold electrode, and naturally dried in the air. Then, 1. mu.L to 60. mu.L of 10mM TCEP was added to 1. mu.L to 60. mu.L of 10. mu.M FP solution, and the mixture solution was incubated at 37 ℃. After 60 minutes, 1 to 60 μ L of the mixture was added to the surface of AuNPs @ ZnSeNs/GE and incubated at 37 ℃ for 2 hours to give FP/AuNPs @ ZnSeNs/GE. Then, FP/AuNPs @ ZnSeSnSs/GE was immersed in a 1mM MCH solution for 1h to eliminate non-specific adsorption. Next, RPA solution was prepared for surface amplification of KLK2 gene. The RPA reaction solution was prepared from 2.4. mu.L of 10. mu.M RP, 13.2. mu.L of DNase-free water, 31.9. mu.L of recombinase polymerase buffer solution and 2.5. mu.L of 280mM magnesium acetate solution. Then 1. mu.L to 50. mu.L of this RPA solution and 1. mu.L to 50. mu.L of a sample containing KLK2 were added to the surface of FP/AuNPs @ ZnSeNSs/GE. After 30 min of amplification at 37 ℃, the electrodes were washed with 0.1M PBS. Finally, the electrodes were immersed in 2. mu.L-60. mu.L of 1. mu.M St-ALP solution, incubated at 37 ℃ for 30 minutes, washed with 0.1M PBS, and inserted into a solution of p-NPP and MB, which catalyzes the hydrolysis of p-NPP to p-NP. Strong photoelectric signals are generated under the action of MB and p-NP, and the content of KLK2 is determined according to the signal intensity of PEC.
Preferably, the electrochemical and photoelectrochemical measurements comprise the steps of:
in the presence of 0.1M KCl at 5.0mM [ Fe (CN)6]3 -4Measuring Electrochemical Impedance Spectroscopy (EIS) response. The PEC signal was measured in 0.1M phosphoric acid buffer solution at pH 7.4 containing p-NPP and MB, and the light source was a 10W LED lamp. Voltage 0V and on-off lamp time interval 10 s.
Advantages and effects of the invention
When the concentration of KLK2 ranged from 5.0 × 10-17M to 2.0 × 10-15When M changes, the PEC signal increases significantly (fig. 2 (B)). A linear regression curve was plotted showing that the PEC signal is proportional to the logarithm of the KLK2 concentration. The regression equation is expressed as I ═ 2027.logc +2185 (R)20.9992), c is KLK2 concentration (aM). The limit of detection (LOD) of the method was 30 aM.
To evaluate the specificity of the method, the interference of KLK2 with the mixture containing PCA3, SChLAP1, PSA gene, TMPRSS2-ERG (500 aM concentration, respectively) was examined, as shown in FIG. 3(A), and only KLK2 and KLK2 had a higher PEC response. All other substances did not significantly interfere with the determination of KLK2, demonstrating the high selectivity of the PEC assay for KLK2 detection.
The 12 cycle switching lamp test investigated the stability of the stability method, as shown in fig. 3 (B). The PEC response remained nearly constant under continuous on/off cycling illumination, indicating that the response of the self-powered PEC analysis with co-regulation of MB and p-NP was sufficiently stable. The constructed electrodes were stored at 4 degrees celsius and measured every 5 days. The PEC reaction in fig. 3(C) was not significantly changed. The results show that the MB and p-NP co-regulated self-powered PEC assay has good stability and repeatability.
Drawings
FIG. 1(A) is a PAGE analysis. +, positive sample; no T, No target. (B) At 0.1M KCl, 5.0mM [ Fe (CN)6]3-/4-EIS response in (1): naked GCE (a), AuNPs @ ZnSeNs/GE (b), FP/AuNPs @ ZnSeNs/GE (c), MCH/FP/AuNPs @ ZnSeNs/GE (d), and after recombinase polymerase amplification (e, f).
FIG. 2 shows the magnitude of PEC signal (A) for different concentrations of KLK2, and KLK2 concentrations from a to j were 0aM, 30aM, 50aM, 70aM, 100aM, 200aM, 500aM, 700aM, 1000aM, and 2000 aM. Linear regression (B) of KLK2 was examined, inset is a linear regression curve of the logarithm of the concentration versus the PEC signal at KLK2 concentrations between 50aM and 2000 aM.
FIG. 3 specificity, stability and reproducibility of the assay. (A) Alternatively, the concentration of the interferent was 5.0fM and the concentration of KLK2 was 500aM, respectively. (B) Stability studies were performed under continuous on/off. Reproducibility of the constructed electrode after 20 days of storage. The concentration of KLK2 was 1.0fM for stability and reproducibility measurements.
Detailed Description
The following examples further illustrate the method of operation of the present invention, but are not to be construed as further limiting the invention.
Example 1: characterization of the MB and p-NP co-regulated self-powered PEC analytical detection method.
In the PEC KLK2 assay, RPA was used to amplify the target. The feasibility of target amplification was verified by polyacrylamide gel electrophoresis (PAGE) (fig. 1 (a)). The gel electrophoresis results show that the KLK2 amplification process was successful. It is well known that Electrochemical Impedance Spectroscopy (EIS) can also be used to monitor the manufacture of modified electrodes. To further characterize the PEC biosensor construction procedure, in 5.0mM [ Fe (CN) ] containing 0.1M KCl6]3-/4-EIS measurements were performed in solution. As shown in fig. 1(B), GE shows a smaller Ret value (curve a). When AuNPs @ ZnSeNs are modified onto the GE (curve b), the Ret values are improved compared to the naked GE. After the FP was modified onto AuNPs @ ZnSeSnSs/GE (curve c), the Ret value increased. After the electrode had reacted with MCH, the Ret value increased further (curve d). Finally, Ret increased significantly after recombinase polymerase amplification on AuNPs @ ZnSeSnSs/GE (curve e). The EIS results from curves a to e show that the construction of the analytical method was successful.
Example 2: sample detection
Tris (hydroxymethyl) aminomethane (Tris), Mercaptoethylamine (MCH), Tris (2-carboxyethyl) phosphine (TCEP), streptavidin-labeled alkaline phosphatase (St-ALP), N-Dimethylformamide (DMF) were provided by Sangon Biotech co. Twist Amp Basic RT-RPA kit (Twist-DX), recombinase polymerase buffer solution purchased from Babraham (UK). Biotin-labeled Reverse Primer (RP), thiolated Forward Primer (FP) were synthesized by Sangon Biotech Co., Ltd. (China, Shanghai),
the sequences of the forward and reverse primers are as follows:
KLK2 Forward Primer (FP): 5'-SH-GGGGGTCCACTTGTCTGTAA-3'
KLK2 Reverse Primer (RP)5'-GGTGAGTTCCAAGCTTCAGG-biotin-3'
KLK2 gene: 5'-GGGGGTCCA CTTGTCTGTA ATGGTGTGCT TCAAGGTATC ACATCATGGGGCCCTGAGCC ATGTGCCCTG CCTGAAAAGC CTGCTGTGTA CACCAAGGTG GTGCATTACC GGAAGTGGATCAAGGACACC ATCGCAGCCA ACCCCTGAGT GCCCCTGTCC CACCCCTACC TCTAGTAAAT TTAAGTCCACCTCACGTTCT GGCATCACTT GGCCTTTCTG GATGCTGGAC ACCTGAAGCT TGGAACTCAC C-3'
Polyacrylamide gel electrophoresis (PAGE) analysis was performed using an ADYCP-31E electrophoresis apparatus (WoDeLife sciences Instrument Co., Ltd., China). All photo-electrochemical (PEC) measurements were performed on a CHI660E electrochemical workstation (china, shanghai chenhua instruments ltd).
Human serum is taken, KLK2 in a healthy human serum sample is detected by adopting a standard addition method, and the concentration of KLK2 determined by the method is compared with the concentration determined by qRT-PCR. As can be seen from Table 1, when KLK2 was added to serum samples at concentrations of 0.1fM, 1.0fM, 5.0fM, and 10.0fM, the recovery rate was 96.7% to 103.8% and the Relative Standard Deviation (RSD) was between 1.7% and 3.8%. Compared with the standard method qRT-PCR, the PEC determination result is better matched with the qRT-PCR result. The results demonstrate the feasibility and accuracy of the method in the analysis of actual samples.
Table 1 determination of KLK2 in serum of healthy persons (n ═ 9)
aSerum samples from healthy humans were diluted 10-fold with 10mM PBS
bfM。
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<213>Artificial
<400>3
gggggtccac ttgtctgtaa tggtgtgctt caaggtatca catcatgggg ccctgagcca 60
tgtgccctgc ctgaaaagcc tgctgtgtac accaaggtgg tgcattaccg gaagtggatc 120
aaggacacca tcgcagccaa cccctgagtg cccctgtccc acccctacct ctagtaaatt 180
taagtccacc tcacgttctg gcatcacttg gcctttctgg atgctggaca cctgaagctt 240
Claims (2)
1. A method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase is characterized in that AuNPs @ ZnSeNs are modified on GE, and then FP is fixed on the AuNPs @ ZnSeNs/GE through Au-S bonds; after adding a sample containing KLK2, performing RPA amplification on FP on the surface of the electrode and RP in the solution in the presence of recombinase polymerase; the target is amplified on the electrode, and RP is captured on the surface of the electrode; after washing, the St-ALP is linked to the electrode by the affinity interaction between biotin and streptavidin; ALP catalyzes the hydrolysis of p-NPP to p-NP; due to the high catalytic activity of ALP, many p-NPs are produced; strong photoelectric signals are generated under the action of MB and p-NP, and the content of KLK2 is determined according to the signal intensity of PEC; the high-sensitivity detection of a target object is realized due to the co-regulation and control effect of MB and p-NP, the circulation of MB and the amplification of recombinase polymerase; under the condition that MB and p-NP exist, AuNPs @ ZnSeSnSs/GE generates strong photocurrent at 0V, has self-energizing characteristic, and combines an in-situ recombinase polymerase amplification strategy to establish a new prostate cancer circulating tumor DNA KLK2 liquid biopsy method; the method comprises the following specific steps:
the preparation of the ZnSe nano-sheet comprises the following steps:
stripping by an ultrasonic stripping method to obtain ZnSe nanosheets; firstly, adding 0.1-450 mg of ZnSe into 1-150 mL of DMF solution, and then ultrasonically dispersing the mixture for 0.1-100 h to obtain yellow dispersion liquid; next, centrifugation was performed at 14,000rpm for 20min, and exfoliated ZnSe was separated from the dispersion and washed 3 times with ethanol; finally, the ZnSeNSs suspension was used for the next experiment;
the synthesis of AuNPs @ ZnSeSnSs comprises the following steps:
preparing AuNPs @ ZnSeSnSs by an in-situ growth method; 0.1-30 mL of ZnSeSnSs and 0.1-10 mL of LHAuCl4·4H2O was added to the flask and heated to 115 ℃ by an oil bath with stirring; the solution was continuously refluxed and stirred for 3 minutes; then, 0.1-10 mL of 1.66mg/mL sodium citrate solution is dripped into the flask; the mixture was refluxed at 115 ℃ for 3 minutes to give black AuNPs @ ZnSeNSs; centrifuging at 14,000rpm for 10min, and collecting the final black precipitate; in the synthesis method of AuNPs @ ZnSeSnSs, chloroauric acids of different masses affect PEC signals; the mass ratio of ZnSeSnSs to chloroauric acid is 1:0.2, 1:0.33, 1:0.66, 1:1 and 1:1.5 respectively;
the KLK2 detection comprises the following steps:
dripping 1-60 muL of AuNPs @ ZnSeSnSs onto the surface of the gold electrode, and naturally drying in the air; then 1-60 μ L of 10mM TCEP was added to 1-60 μ L of 10 μ M FP solution, and the mixture solution was incubated at 37 ℃; after 60 minutes, adding 1-60 microliter of the mixture to the surface of AuNPs @ ZnSeNs/GE, and incubating for 2 hours at 37 ℃ to obtain FP/AuNPs @ ZnSeNs/GE; then, the FP/AuNPs @ ZnSeSnSs/GE was immersed in 1mM MCH solution for 1h to eliminate non-specific adsorption; next, RPA solution was prepared for surface amplification of KLK2 gene; the RPA reaction solution was prepared from 2.4. mu.L of 10. mu.M RP, 13.2. mu.L of DNase-free water, 31.9. mu.L of recombinase polymerase buffer solution and 2.5. mu.L of 280mM magnesium acetate solution; then adding 1-50 mul of the RPA solution and 1-50 mul of a sample containing KLK2 to the surface of the FP/AuNPs @ ZnSeSnSs/GE; after 30 min of amplification at 37 ℃, the electrodes were washed with 0.1M PBS; finally, the electrode was immersed in 2. mu.L-60. mu.L of 1. mu.M St-ALP solution, incubated at 37 ℃ for 30 minutes, washed with 0.1M PBS, and inserted into a solution of p-NPP and MB, which catalyzes the hydrolysis of p-NPP to p-NP; strong photoelectric signals are generated under the action of MB and p-NP, and the content of KLK2 is determined according to the signal intensity of PEC.
The sequences of the forward primer, the reverse primer and the KLK2 gene are as follows:
KLK2 Forward Primer (FP): 5'-SH-GGGGGTCCACTTGTCTGTAA-3'
KLK2 Reverse Primer (RP)5'-GGTGAGTTCCAAGCTTCAGG-biotin-3'
KLK2 gene: 5'-GGGGGTCCA CTTGTCTGTA ATGGTGTGCT TCAAGGTATC ACATCATGGGGCCCTGAGCC ATGTGCCCTG CCTGAAAAGC CTGCTGTGTA CACCAAGGTG GTGCATTACC GGAAGTGGATCAAGGACACC ATCGCAGCCA ACCCCTGAGT GCCCCTGTCC CACCCCTACC TCTAGTAAAT TTAAGTCCACCTCACGTTCT GGCATCACTT GGCCTTTCTG GATGCTGGAC ACCTGAAGCT TGGAACTCAC C-3' are provided.
2. The method of claim 1, wherein the reagents and apparatus used in the method of amplifying photoelectrochemical circulating tumor DNA biopsy are as follows:
tris (hydroxymethyl) aminomethane (Tris), Mercaptoethylamine (MCH), Tris (2-carboxyethyl) phosphine (TCEP), streptavidin-labeled alkaline phosphatase (St-ALP), N-Dimethylformamide (DMF) were provided by Sangon Biotech co., Ltd. (china, shanghai); twist Amp Basic RT-RPA kit (Twist-DX), recombinase polymerase buffer solution purchased from Babraham (UK); biotin-labeled Reverse Primer (RP), thiolated Forward Primer (FP) were synthesized by Sangon Biotech Co., Ltd. (China, Shanghai),
polyacrylamide gel electrophoresis analysis was performed using an ADYCP-31E electrophoresis apparatus, available from WoDeLife sciences Instrument Co., Ltd.; all photo-electrochemical measurements were performed on the CHI660E electrochemical workstation, purchased from chenhua instruments ltd, shanghai.
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