CN113186254B - Biosensor for ctDNA detection and preparation method thereof - Google Patents
Biosensor for ctDNA detection and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a biosensor for ctDNA detection, which comprises the following steps: preparing nano gold particles: mixing chloroauric acid and ultrapure water, heating, adding a sodium citrate solution, continuing heating and reacting, cooling to room temperature after the reaction is finished, filtering to obtain nano gold particles, and storing at low temperature for later use; modification of DNA: and annealing the DNA, uniformly mixing the annealed DNA with the prepared gold nanoparticles, standing, adding a sodium citrate solution, adding a phosphate buffer solution, standing, adding water, and centrifugally washing to obtain the biosensor for ctDNA detection.
Description
Technical Field
The invention relates to the technical field of DNA tumor analysis, in particular to a biosensor for ctDNA detection and a preparation method thereof.
Background
Tumors are a serious disease threatening the health of human beings. Currently, the subject of cancer diagnosis, staging and treatment decision is tissue biopsy, and although tissue biopsy is very useful, invasiveness and inherent selection preference of biopsy limit its usefulness as a real-time monitoring tool, are difficult to use for early diagnosis, and invasive tissue biopsy often causes great pain to patients. To overcome this drawback, various tumor biomarkers, such as protein markers, Circulating tumor cells, Circulating tumor DNA (ctDNA), and the like, have been sought. In recent years, research shows that tumor mutation information carried by ctDNA of tumor patients has good consistency with tumor tissues. Circulating tumor DNA has therefore attracted considerable attention.
Circulating tumor DNA (ctdna) refers to a DNA fragment that is secreted into the blood only by tumor cells, necrotic, apoptotic, or normal, and carries information on cancer-related genetic variations, which are detectable when variations occur at the tumor molecular level. ctDNA has a short half-life (about 2h), can dynamically assess cancer status, and can be clinically applied in multiple stages of tumor development: the detection accuracy of the ctDNA is high, the correlation degree between the ctDNA level and the size of the tumor is high, the tumor state can be well estimated by monitoring according to the mutation amount of the ctDNA, and early diagnosis and tumor staging are carried out; secondly, in the clinical treatment process, a tumor patient usually generates drug resistance, and whether the target site of the drug is changed or not can be dynamically monitored by detecting ctDNA (deoxyribonucleic acid) to carry out gene analysis, so that an optimal treatment scheme is screened out; three, several studies have shown that no ctDNA is detected after tumor resection, which correlates with no recurrence of the tumor, and therefore, detection of plasma ctDNA can be used as a prognostic biomarker for cancer patients, and to analyze the risk of recurrence in patients.
The research on ctDNA is still in the early stage, because the ctDNA concentration in peripheral blood is low, especially in early stage tumor, and because the sensitivity of the previous detection method is low, it hinders its rapid development and clinical application. Fortunately, advances in targeted selection and amplification methodology, as well as sequencing methods, have facilitated the detection of these traces of ctDNA. However, recent studies have shown that it is difficult to further improve the sensitivity of mutation detection by a simple biological method, and the improvement of detection sensitivity of biomolecules by the unique properties of nanomaterials in combination with molecular biological techniques has gradually become a hot spot in the field of biomedical research. Among them, gold nanoparticles (Au-NPs) have many advantages: in the development of a chemical biosensor, the introduction of gold nanoparticles can cause the amplification of current response signals, thereby greatly improving the sensitivity of the sensor; in addition, the nano flare utilizes the unique optical properties of gold nanoparticles (Au-NPs), and compared with a molecular quencher, the gold nanoparticles have higher quenching efficiency on fluorescence and longer distance, and can not effectively scatter visible light, which is important for designing an optical probe with minimum interference; and other advantages including ease of manufacture, greater oligonucleotide binding capacity, stability under a variety of conditions, and catalytic ability, while DNA-modified nanogold also provides several benefits including crossing cell membranes, protecting DNA probes from degradation, transporting additional cargo, encoding multiple functions using various functional cores.
On the other hand, studies have shown that there is some correlation between the ctDNA content in plasma and the type, size and stage of the tumor. One study of 640 tumor patients of different types and stages showed that the median ctDNA concentration at stage IV was 100-fold higher than that at stage I. Quantitative studies have shown ctDNA mutation copy numbers in late stage patients of less than 10 per 5mL of plasma, but around 100-1000 copy numbers in partially late stage patients. However, the current biosensor has a limitation: the physics of single-site binding produces a hyperbolic dose-response curve whose useful dynamic range spans a fixed change in target concentration. The dynamic range of the biosensor describes the range corresponding to the target concentration with a receptor occupancy between 10% and 90%. However, single-site binding is almost always characterized by a fixed hyperbolic relationship (Langmuir isotherm) between target concentration and receptor binding, with a dynamic range spanning an 81-fold range of target concentrations. This fixed dynamic range reduces the usefulness of ctDNA biosensors in clinical monitoring, as well as limiting their use when high sensitivity detection is required.
Disclosure of Invention
In view of the above, the present invention provides a biosensor to meet the detection requirements of ctDNA in clinical samples with different concentrations.
The invention provides a preparation method of a biosensor for ctDNA detection, which comprises the following steps:
s1, preparing nano gold particles: mixing chloroauric acid and ultrapure water, heating, adding a sodium citrate solution, continuing heating and reacting, cooling to room temperature after the reaction is finished, filtering to obtain nano gold particles, and storing at low temperature for later use;
s2, modified DNA: and annealing the DNA, uniformly mixing the annealed DNA with the gold nanoparticles prepared in the step S1, standing, adding a sodium citrate solution, adding a phosphate buffer solution, standing, adding water, and centrifuging and washing to obtain the biosensor for ctDNA detection.
Further, in step S1, the preparation process of the gold nanoparticles specifically includes: heating chloroauric acid and ultrapure water at an initial temperature of 120 ℃, adding a sodium citrate solution after the solution begins to boil, heating and reacting at a temperature of 130 ℃, cooling to room temperature after the reaction is finished, filtering by using a filter screen to obtain nano gold particles, and storing at a temperature of 4 ℃ for later use; the preparation of the nano gold particles is a crystallization process, and any impurities can cause the nonuniformity of the sizes of the gold particles, so the introduction of the impurities needs to be avoided in the preparation process, and the particles are uniform and have better monodispersity only by fully stirring in the preparation process. The gold nanoparticles (Au-NPs) have the advantages of easy manufacture, strong oligonucleotide binding capacity, high stability under various conditions, excellent catalytic capacity and optical properties and the like.
Further, in step S2, the nucleotide sequence of the DNA used is shown in SEQ ID No.1, specifically: ACTATTGAGA TGCGGTGGTA ATAGGAAAAA AAAAAAAAAA are provided.
Further, in step S2, the nucleotide sequence of the DNA used is shown in SEQ ID No.2, specifically: ACTCTCGAGA TGCGGTGGTG AGAGGAAAAA AAAAAAAAAA is added.
Further, in step S2, the nucleotide sequence of the DNA used is shown in SEQ ID No.3, specifically: ACTATTGAGA TGCGGTGGTA ATAGGAAAAA AAAAAAAAAA is added.
Further, in step S2, the concentration of the sodium citrate solution was 0.5mol/L and the concentration of the phosphate buffer was 0.2 mol/L.
The invention also provides a biosensor for ctDNA detection, which is prepared by the method.
The technical scheme provided by the invention has the beneficial effects that: according to the invention, the DNA is modified on the nanogold to prepare the biosensor, and the biosensor is specifically combined with ctDNA and is suitable for ctDNA detection; the dynamic range of the sensor can be adjusted and controlled by modifying the DNA of different stem-loop structures.
Drawings
FIG. 1 is a transmission electron microscope image of the gold nanoparticles prepared in example 1 of the present invention.
FIG. 2 is a comparative synthesis diagram of the biosensor prepared in example 1.
FIG. 3 is a graph showing the results of the signal-to-noise ratio of biosensors obtained in examples 1 to 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings and examples.
Example 1:
embodiment 1 of the present invention provides a method for preparing a biosensor for ctDNA detection, including the steps of:
step S1, preparing gold nanoparticles: adding chloroauric acid and ultrapure water into a double-mouth flask, setting the heating temperature of an oil bath to be 300 ℃, setting a sensor to be 130 ℃, heating the oil bath kettle to 120 ℃ at the rotating speed of 600rpm, adjusting the rotating speed to be 1100rpm, then placing the double-mouth flask filled with chloroauric acid and ultrapure water into the oil bath kettle, starting oil bath heating, starting boiling the solution in the double-mouth flask after 10min, adding a sodium citrate solution, boiling for 3min, and indicating that crystal nuclei begin to form; 5min later, the whole solution is obviously purple; 8min, the solution is purple red, the crystal nucleus grows up, and the solution turns red; 9min, wine red, with more particles, the red deepens; the red color deepens after 12 min; the solution has no obvious change after 20 min; at this time, the heating was turned off, the rotation speed was maintained at 1100rpm, and the mixture was cooled at room temperature for 30 min. After that, the stirring was stopped, the mixture was cooled to room temperature, filtered through a 0.22 μm filter, and stored at 4 ℃. The prepared nano gold particles are characterized, and the TEM result of a transmission electron microscope is shown in figure 1, and as can be seen from figure 1, the prepared nano gold particles are uniform, good in dispersity and free of obvious agglomeration phenomenon;
step S2, modifying DNA: rapidly heating 5 mu L of DNA with a nucleotide sequence shown as SEQ ID No.1 to 95 ℃ and maintaining for 10 minutes, then rapidly cooling to 4 ℃ and maintaining for more than 7 minutes to finish annealing treatment, mixing and shaking the annealed DNA with 1mL of gold nanoparticle AuNPs prepared in the step S1 uniformly, and standing for 15 minutes; adding 20 μ L of 0.5mol/L sodium citrate, and standing for 15 min; then adding 50 mu L of 0.2mol/L phosphate buffer solution, and standing for 15 min; finally, 200. mu.L of water was added, and after washing away unbound DNA by three centrifugation, a biosensor for ctDNA detection was obtained, which was suspended in 0.01mol/L phosphate buffer.
In order to prove that the DNA is successfully modified on the gold nanoparticles, 20 μ L of phosphate buffer solution with the concentration of 0.1mol/L is respectively added into the gold nanoparticles and the prepared biosensor, and after the phosphate buffer solution is added, the gold nanoparticles (AuNP) turn black, while the color of the biosensor (DNA-AuNP) of successfully modified DNA is not obviously changed (see figure 2), which is enough to prove that the DNA is successfully modified on the gold nanoparticles in example 1.
Example 2:
example 2 the procedure for preparing the biosensor differs from example 1 only in that: the nucleotide sequence of the DNA used in example 2 is shown in SEQ ID No.2, and the rest is substantially the same as in example 1.
Example 3:
example 3 the procedure for preparing the biosensor differs from example 1 only in that: the nucleotide sequence of the DNA used in example 3 is shown in SEQ ID No.3, and the rest is substantially the same as in example 1.
Using the biosensors prepared in examples 1 to 3, 100nM ctDNA was detected and the SNR F/F was recorded0As shown in FIG. 3, it can be seen from FIG. 3 that the signal-to-noise ratios of the biosensors with different stem-loop structures to 100nM ctDNA are different, and the dynamic range of the sensor can be controlled based on the difference.
The DNAs used in examples 1 to 3 were all artificially synthesized.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> China geological university (Wuhan)
<120> biosensor for ctDNA detection and preparation method thereof
<141> 2021-06-30
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213> Artificial Synthesis (Artificial)
<400> 1
actattgaga tgcggtggta ataggaaaaa aaaaaaaaaa 40
<210> 2
<211> 40
<212> DNA
<213> Artificial Synthesis (Artificial)
<400> 2
actctcgaga tgcggtggtg agaggaaaaa aaaaaaaaaa 40
<210> 3
<211> 40
<212> DNA
<213> Artificial Synthesis (Artificial)
<400> 3
actattgaga tgcggtggta ataggaaaaa aaaaaaaaaa 40
Claims (7)
1. A preparation method of a biosensor for ctDNA detection is characterized by comprising the following steps:
s1, preparing nano gold particles: mixing chloroauric acid and ultrapure water, heating, adding a sodium citrate solution, continuing heating and reacting, cooling to room temperature after the reaction is finished, filtering to obtain nano gold particles, and storing at low temperature for later use;
s2, modified DNA: and annealing the DNA, uniformly mixing the annealed DNA with the gold nanoparticles prepared in the step S1, standing, adding a sodium citrate solution, adding a phosphate buffer solution, standing, adding water, and centrifuging and washing to obtain the biosensor for ctDNA detection.
2. The method of claim 1, wherein in step S1, the gold nanoparticles are prepared by the following steps: heating chloroauric acid and ultrapure water at an initial temperature of 120 ℃, adding a sodium citrate solution after the solution begins to boil, heating and reacting at a temperature of 130 ℃, cooling to room temperature after the reaction is finished, filtering by using a filter screen to obtain the gold nanoparticles, and storing at a temperature of 4 ℃ for later use.
3. The method of manufacturing a biosensor for ctDNA detection as claimed in claim 1, wherein in step S2, the nucleotide sequence of the DNA used is shown in SEQ ID No. 1.
4. The method for manufacturing a biosensor in use for detecting ctDNA as claimed in claim 1, wherein in step S2, the nucleotide sequence of the DNA used is shown in SEQ ID No. 2.
5. The method of manufacturing a biosensor for ctDNA detection as claimed in claim 1, wherein in step S2, the nucleotide sequence of the DNA used is shown in SEQ ID No. 3.
6. The method of manufacturing a biosensor for ctDNA detection as claimed in claim 1, wherein in step S2, the concentration of sodium citrate solution is 0.5mol/L and the concentration of phosphate buffer is 0.2 mol/L.
7. A biosensor for ctDNA detection prepared by the method according to any one of claims 1 to 6.
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| CN110029150A (en) * | 2019-03-28 | 2019-07-19 | 天津大学 | For detecting the preparation method of the small molecule metal-chelator labeled oligonucleotide probe of Microrna |
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| CN101245387A (en) * | 2008-01-22 | 2008-08-20 | 中国科学院上海应用物理研究所 | Nano-gold signal probe for DNA detection, production method and method for detecting DNA |
| CN102080131A (en) * | 2010-12-10 | 2011-06-01 | 郑州大学 | Hairpin type DNA modified gold colloid nano particles and synthesizing method thereof |
| CN110029150A (en) * | 2019-03-28 | 2019-07-19 | 天津大学 | For detecting the preparation method of the small molecule metal-chelator labeled oligonucleotide probe of Microrna |
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