CN111304297A - Method for analyzing circulating tumor DNA on single molecule level - Google Patents
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Abstract
The invention discloses an analysis method of circulating tumor DNA (ctDNA) on a single-molecule level, which takes the dimer proportion of quantum dots as a quantitative basis, avoids quantitative analysis by using the intensity change of signals such as light, electricity, magnetism, force and the like, and reduces the error caused by signal fluctuation, thereby quickly, sensitively and accurately detecting the ctDNA concentration in blood.
Description
Technical Field
The invention relates to an analysis method of circulating tumor DNA (ctDNA) on a single-molecule level, belonging to the technical field of biochemistry and medical detection.
Technical Field
Circulating tumor DNA (ctDNA) is a DNA fragment from tumor cell genome in human blood, tumor cells in the focus of a tumor patient continuously transfer, die and necrose in the human circulatory system, the circulating tumor DNA is released into the blood, and the circulating tumor DNA has the characteristics of tumor genome mutation, insertion, deletion, rearrangement, methylation and the like and carries biological information of tumor generation, development, transfer, relapse and the like, so the circulating tumor DNA has important values for diagnosis, treatment, prognosis evaluation and the like of tumors.
Tissue biopsy is the most common tumor detection means in clinical diagnosis at present, but tissue biopsy has the limitations of invasive intervention, radiation exposure, and the capability of providing information only at a certain moment of tumor. Liquid biopsy is used as a convenient and noninvasive method capable of dynamically reflecting tumor development and change, and plays an increasingly important role in accurate tumor treatment.
As a research hotspot in the field of current liquid biopsy, ctDNA detection can not only explain comprehensive genetic information of tumors, but also accurately reflect heterogeneity of tumor tissues and dynamically reflect important information of tumor development and change, and has extremely high scientific research value and clinical application prospect. However, ctDNA has extremely low content in peripheral blood, high background free DNA content and large individual difference, detection sites need to be pre-judged in advance, the analysis and amplification of amplification classes have the problem of false positive, and non-amplification classes are time-consuming and labor-consuming. Therefore, there is a great technical challenge to the analytical detection of ctDNA, and it is essential to develop a new technology to perform the detection analysis thereof.
At present, the great progress of scientific technology makes the ctDNA detection means greatly developed, and the typical result is as follows: real-time quantitative PCR, a targeted polygene next-generation sequencing technology, an electrochemical DNA clutch probe, an electrochemical luminescence method based on a DNA tetrahedron, a single-walled carbon nanotube based on a surface enhanced Raman scattering principle, a nano biosensor based on surface plasmon resonance and the like. These methods have problems in that the specificity is not high enough and the amount of sample used is large. Therefore, the development of a highly sensitive and highly specific assay in trace blood is of great importance for the detection of ctDNA.
Disclosure of Invention
The invention aims to provide an analysis method of circulating tumor DNA (ctDNA) on a single-molecule level, which is based on the quantitative proportion of quantum dot dimers, avoids the quantitative analysis by using the intensity change of signals such as light, electricity, magnetism, force and the like, reduces the error caused by signal fluctuation, and thus, quickly, sensitively and accurately detects the ctDNA concentration in blood.
In order to achieve the purpose, the invention provides the following technical scheme: a method for analyzing circulating tumor dna (ctdna) at a single molecule level, comprising the steps of:
1) modifying single-stranded DNA1 (ssDNA 1) on the surface of a quantum dot 1 (QD 1) according to a ctDNA sequence to be detected, adding single-stranded DNA2 (ssDNA 2) with a sequence at two ends complementary to the ssDNA1 to construct a DNA Triple Helix Molecular Switch (THMS), and modifying single-stranded DNA3(ssDNA3) on the surface of a quantum dot 2 (QD 2);
2) adding THMS-QD1 and ssDNA3-QD2 to a circulating tumor dna (ctDNA) sample to form a THMS-QD1@ ctDNA @ ssDNA3-QD2 dimer;
3) dripping the solution in the system on a glass slide, and imaging under a fluorescence spectrum microscope;
4) light emitted by the light source is irradiated on the glass slide at the excitation wavelength of the quantum dots, and the imaging condition of the quantum dots is recorded in the irradiation process;
5) in the process, two light spots corresponding to the first-level fringe spectrum are quantum dot dimers, and one light spot corresponding to the first-level fringe spectrum is a single quantum dot;
6) and recording the proportion of the quantum dot dimers in the total number of the light spots, and taking the proportion as a quantitative basis.
Carrying out ctDNA detection on the dimer structure in the step 2), wherein the detection limit molar concentration reaches picomolar level (pM).
The quantum dot dimers are judged by judging whether the first-order fringes corresponding to the zero-order light spots of the quantum dots are two, the light spots corresponding to the first-order fringes are dimers, and the light spots corresponding to the first-order fringes are single quantum dots.
The fluorescence microscope is equipped with a spectral imaging device.
Compared with the prior art: according to the method, THMS-QD and ssDNA-QD are added into a sample solution, a fluorescence microscope provided with a spectral imaging device is taken as a detection platform, the dimer proportion of quantum dots is sampled and observed, a standard curve is drawn according to the relation between the dimer proportion and the ctDNA concentration, and then the ctDNA in an unknown sample is quantified. The method takes the dimer proportion rather than the fluorescence intensity as the quantitative basis, avoids the error caused by the fluctuation of the fluorescence intensity, has the advantage of high detection sensitivity, and can reach the pM level.
The method does not need steps of fixing, separating, cleaning and the like; the amount of the used sample is small, and only a few microliters are needed; the intensity change of signals such as light, electricity, magnetism, force and the like is prevented from being used for quantitative analysis, and the error caused by signal fluctuation is reduced; imaging quantification is carried out on a single particle level, the detection limit is low, and the detection sensitivity is high.
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FIG. 1 is a schematic diagram of the analytical method of the present invention;
FIG. 2 is a comparison of the first order spectra of QD1, QD2, and quantum dot dimers of the present invention;
FIG. 3 correlation of different concentrations of ctDNA with quantum dot dimer ratios in aqueous solutions;
fig. 4 selectivity of the method for ctDNA and other common ions and molecules.
The specific implementation mode is as follows:
the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1, fig. 2, fig. 3 and fig. 4, the method for analyzing circulating tumor dna (ctdna) at a single-molecule level includes the following steps:
1) modifying single-stranded DNA1 (ssDNA 1) on the surface of a quantum dot 1 (QD 1) according to a ctDNA sequence to be detected, adding single-stranded DNA2 (ssDNA 2) with a sequence at two ends complementary to the ssDNA1 to construct a DNA Triple Helix Molecular Switch (THMS), and modifying single-stranded DNA3(ssDNA3) on the surface of a quantum dot 2 (QD 2);
2) adding THMS-QD1 and DNA3-QD2 to a sample of circulating tumor DNA (ctDNA) to form a dimer of THMS-QD1@ ctDNA @ ssDNA3-QD 2;
3) dripping the solution in the system on a glass slide, and imaging under a fluorescence spectrum microscope;
4) light emitted by the light source is irradiated on the glass slide at the excitation wavelength of the quantum dots, and the imaging condition of the quantum dots is recorded in the irradiation process. As shown in fig. 2, the left circular spot in fig. 2 is the zero-order spectrum of the quantum dot, the right stripe is the first-order spectrum of the quantum dot, the yellow is the QD1 spectrum, the red is the QD2 spectrum, the green is the quantum dot dimer spectrum, the distance between the QD1 zero-order spot and the first-order stripe is about 200 pixels, and the distance between the QD2 zero-order spot and the first-order stripe is about 223 pixels; it is obvious from the figure that under the effect of the grating, the first-order spectrum corresponding to the zero-order light spot of the dimer is split into two, and the first-order spectrum corresponding to the zero-order light spot of the single quantum dot is one;
5) judging whether the first-order stripes corresponding to the zero-order light spots of the quantum dots are dimers or not by observing whether the first-order stripes corresponding to the zero-order light spots of the quantum dots are two stripes, wherein the light spots corresponding to the first-order stripe spectrums are dimers of the quantum dots, and the light spots corresponding to the first-order stripe spectrums are single quantum dots;
6) and recording the proportion of the quantum dot dimers in the total number of the light spots, and taking the proportion as a quantitative basis. FIG. 3 is a quantitative standard curve of ctDNA, and it can be found by calculation that the detection limit of ctDNA reaches 5.36 pM at a signal-to-noise ratio of 3. FIG. 4 is the selectivity of the method for ctDNA and other common ions and molecules, wherein the concentrations of cations, anions, amino acids, glutathione, proteins, and total mismatch DNA are all 10 times the concentration of ctDNA, and the concentration of single mismatch DNA is equivalent to the concentration of ctDNA; as shown in the figure, the response values of the method to cations, anions, amino acids, glutathione, proteins and total mismatch DNA are all equivalent to blank values, and the response value to single mismatch DNA is 37.0 percent of ctDNA.
Carrying out ctDNA detection on the dimer structure in the step 2), wherein the detection limit molar concentration reaches picomolar level (pM).
The quantum dot dimers are judged by judging whether the first-order fringes corresponding to the zero-order light spots of the quantum dots are two, the light spots corresponding to the first-order fringes are dimers, and the light spots corresponding to the first-order fringes are single quantum dots. The invention carries out the analysis of the circulating tumor DNA by counting the proportion of the quantum dot dimers, can be used for the quantitative detection of the circulating tumor DNA in the blood sample, and has the following specific detection data:
experimental example:
a. mu.L of 8. mu.M stock solutions of carboxylated QD585 and QD655, respectively, were added to 14. mu.L of NaHCO3(50 mM, pH9.0) buffer, and then adding cold NaHCO3(50 mM, pH9.0) 4. mu.L of freshly prepared EDC (1 mg/mL) was incubated at room temperature in the dark for 25 min.
ssDNA1 (primary amino group modified at 5' -end) is immobilized on the surface of QD585 by reacting its primary amino group with the carboxyl group of QD 585. mu.L of ssDNA1 (12.2. mu.M) and 20. mu.L of the above activated QD585 solution were mixed well and incubated at room temperature for 4 h in the absence of light. ssDNA3 (modified primary amino group at 5' end) is immobilized on the surface of QD655 by reaction of its primary amino group with the carboxyl group of QD 655. mu.L of ssDNA3 (16.9. mu.M) and 20. mu.L of the above activated QD655 solution were mixed well and incubated at room temperature for 4 h in the absence of light.
c. ssDNA1-QD585 and ssDNA2 were added to PBS (10 mM, pH6.8, containing 300 mM NaCl) buffer at a concentration ratio of 1:6, heated in a water bath at 50 ℃ for 5 minutes, and slowly cooled to 18 ℃.
d. THMS-QD585 and ssDNA3-QD655 are mixed in a ratio of 2:3 and then added into ctDNA solutions with different concentrations, the solution is heated in a water bath at 50 ℃ for 5 minutes, slowly cooled to 18 ℃, taken out and placed in a refrigerator at 4 ℃ for overnight, and a THMS-QD585@ ctDNA @ ssDNA3-QD655 complex is formed.
e. And (3) dripping 1.8 mu L of the solution on a glass slide, and placing the glass slide on a fluorescence spectrum microscope for imaging, observation and shooting.
f. Quantitative experiments: and (3) carrying out fluorescence imaging on the compound of the ctDNA with different concentrations obtained by the step (e), processing the shot data by using imageJ, and calculating the polymerization ratio of THMS-QD585@ ctDNA @ ssDNA3-QD655 under different concentrations. The concentration of ctDNA was plotted on the abscissa and the ratio of dimers was plotted on the ordinate (FIG. 3), which was linear in the range of 10 to 100 pM and had a linear correlation coefficient of 0.9834.
g. Selective experiments: the implementation process is the same as the steps a-c of the experimental steps, and the solution to be tested is changed from ctDNA into single mismatching DNA with equal concentration and cation, anion, amino acid, glutathione, protein and total mismatching DNA with 10 times concentration respectively. The invention has high selectivity for ctDNA detection (see fig. 4).
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any minor modifications, equivalent replacements and improvements made to the above embodiment according to the technical spirit of the present invention should be included in the protection scope of the technical solution of the present invention.
Claims (4)
1. A method for analyzing circulating tumor DNA at the single molecule level, comprising: the method comprises the following steps:
1) modifying single-stranded DNA1 (ssDNA 1) on the surface of a quantum dot 1 (QD 1) according to a ctDNA sequence to be detected, adding single-stranded DNA2 (ssDNA 2) with a sequence at two ends complementary to the ssDNA1 to construct a DNA Triple Helix Molecular Switch (THMS), and modifying single-stranded DNA3(ssDNA3) on the surface of a quantum dot 2 (QD 2);
2) adding THMS-QD1 and ssDNA3-QD2 to the ctDNA sample to form a dimer of THMS-QD1@ ctDNA @ ssDNA3-QD 2;
3) dripping the solution in the system on a glass slide, and imaging under a fluorescence spectrum microscope;
4) irradiating the glass slide with the excitation wavelength of the quantum dots, and recording the imaging condition of the quantum dots in the irradiation process;
5) the light spots split into two by the first-order fringe spectrum are quantum dot dimers;
the first-level stripe spectrum is not split into single quantum dots;
and recording the proportion of the quantum dot dimers in the total number of the light spots, and taking the proportion as a quantitative basis.
2. The method for analyzing circulating tumor DNA at a single molecular level as claimed in claim 1, wherein the dimer structure in step 2) is subjected to circulating tumor DNA detection, and the detection limit molar concentration reaches picomolar level.
3. The method of claim 1, wherein the quantum dots are capable of spectral blue-shifting, and include but are not limited to semiconductor core-shell quantum dots.
4. The method according to claim 1, wherein the quantum dot dimer is determined by whether the first order fringe corresponding to the quantum dot zero order spot is split, and the spectrally split spot is a dimer.
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