A method and application of a double-stranded DNA colorimetric biosensor for detecting SARS-CoV-2 and its variants.
By combining a double-stranded DNA colorimetric biosensor with the Toehold strand displacement reaction, a portable colorimetric biosensor was constructed, which solved the problems of high cost and poor specificity in existing technologies, and realized low-cost, high-specificity detection of the novel coronavirus and its variants.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-31
- Publication Date
- 2026-04-03
AI Technical Summary
In existing technologies, protease-assisted identification of the novel coronavirus is costly and requires demanding experimental conditions, making it difficult to apply in remote areas or small clinics. Nucleic acid hybridization-based methods are unable to distinguish single-base mutations and cannot meet the demand for low-cost and high-specificity detection.
A double-stranded DNA colorimetric biosensor was designed, and a portable colorimetric biosensor was constructed using the Toehold strand displacement reaction (TSDR). The biosensor was used to detect the novel coronavirus and its variants by taking pictures with a smartphone and performing RGB analysis. The biosensor used reagents such as double-stranded DNA, heme chloride, and hydrogen peroxide to achieve enzyme-free specific recognition.
It enables highly specific detection of the novel coronavirus and its variants at low cost, and can quickly and accurately identify single base mismatches without the need for specific instruments. It is suitable for remote areas and small clinics, and is inexpensive and easy to operate.
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Figure CN116083424B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a method and application of a double-stranded DNA and colorimetric biosensor for detecting the novel coronavirus and its variants. Background Technology
[0002] The rapid mutation of SARS-CoV-2 poses significant challenges to the development of timely diagnosis, vaccines, and effective treatments for the virus. Therefore, there is an urgent need to develop a sensor platform with high specificity and low cost for detecting SARS-CoV-2.
[0003] Currently reported methods for specifically identifying SARS-CoV-2 mainly fall into two categories: protease-assisted recognition and methods based on specific hybridization. Protease-assisted recognition has been widely used, such as by endonucleases, polymerases, and ligases. However, inherent drawbacks such as unstable protease activity, high cost, and demanding experimental conditions limit its application in remote areas, small clinics, or community health facilities. Another strategy is based on nucleic acid hybridization, which can distinguish between perfect matches and single-base mutations without the need for additional protease assistance, meeting the specific needs of SARS-CoV-2 detection and aligning with the current demands of green chemistry. Furthermore, nucleic acid hybridization-based methods offer the advantages of simplicity and directness, further providing possibilities for detecting SARS-CoV-2 and its variants in real-world samples.
[0004] In recent years, the Toehold strand substitution reaction (TSDR) has been considered an effective strategy for controlling and constructing nucleic acid hybridizations. The principle behind this strategy is that, driven by thermodynamic entropy, the target sequence hybridizes with a region called the "Toehold" (composed of 5-8 bases) in the double-stranded DNA (dsDNA) sequence, resulting in the replacement of a shorter sequence from the dsDNA. Furthermore, the Toehold-mediated strand substitution reaction rate is 10 times that of the conventional strand substitution reaction. 6 This allows TSDRs to be applied to the construction of biosensors in fluorescence, colorimetry, and electrochemistry. Among these sensors, colorimetric biosensors have shone brightly in the field of precision nucleic acid diagnosis due to their advantages of simplicity, portability, and high specificity. Therefore, designing a portable colorimetric biosensor to detect variant strains of the SARS-CoV-2 virus is of great significance. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for detecting the novel coronavirus and its variants using a double-stranded DNA and colorimetric biosensor.
[0006] The objective of this invention is achieved through the following technical solution:
[0007] A double-stranded DNA, said double-stranded DNA being composed of DNA1 and DNA2, said DNA1 and DNA2 sequences being shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.
[0008] The present invention also provides a method for preparing the above-mentioned double-stranded DNA, comprising the following steps: adding the DNA1 and DNA2 to a buffer solution, reacting at 95°C for 4 minutes, and then annealing at a gradient to room temperature.
[0009] Furthermore, the buffer solution is Tris-HCl, PBS, or HEPES buffer.
[0010] Furthermore, the molar ratio of DNA1 to DNA2 is in the range of 1:1:1.5:1, preferably 5.2:5.
[0011] The present invention also provides a colorimetric biosensor, comprising a molecular recognition reagent for SARS-CoV-2 strains and a signal conversion reagent, wherein the molecular recognition reagent for SARS-CoV-2 strains comprises the aforementioned double-stranded DNA, and the signal conversion reagent comprises heme chloride, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine.
[0012] Furthermore, the concentration range of the double-stranded DNA is 10 nM to 1 μM, with an optimal concentration of 50 nM; the concentration range of the heme chloride is 10 nM to 1 μM, with an optimal concentration of 50 nM; the concentration range of the hydrogen peroxide is 0.1 M to 100 M, with an optimal concentration of 1 M; and the concentration range of the 3,3',5,5'-tetramethylbenzidine is 0.1 mM to 10 mM, with an optimal concentration of 0.4 mM.
[0013] Furthermore, the heme chloride needs to be used in conjunction with a buffer solution, which includes a buffer solution formed by G-quadruplex;
[0014] The G-tetrachain forming buffer solution comprises 20 mM Tris-HCl, 200 nM hemin, 150 mM NaCl, 20 mM KCl, 0.01% (v / v) Triton X-100, and 1% (v / v) DMSO. The molar ratio of Tris-HCl, hemin, NaCl, and KCl is in the range of 100:1:750:100. The pH range of the G-tetrachain forming buffer solution is 6.0-8.0, preferably 7.
[0015] The present invention also provides a method for detecting the novel coronavirus, wherein the test object is mixed with the above-mentioned colorimetric biosensor, and when the color of the colorimetric biosensor turns blue, it is determined that the test object contains the novel coronavirus; when the color of the colorimetric biosensor remains unchanged, it is determined that the test object does not contain the novel coronavirus.
[0016] Furthermore, the method for determining the color change of the colorimetric biosensor is as follows: the RGB values of the solution are captured using a smartphone camera for determination.
[0017] The present invention also provides an application of the above-described double-stranded DNA, the above-described colorimetric biosensor, and the above-described detection method for detecting SARS-CoV-2 and SARS-CoV-2 variants.
[0018] Furthermore, the minimum detection limit is 1 nM.
[0019] The beneficial effects of this invention are:
[0020] Based on toehold-mediated DNA strand displacement reactions, this invention constructs a highly specific and cost-effective colorimetric sensor for detecting SARS-CoV-2. Without specific materials or scientific instruments, the color values of the solution can be easily and quickly read using smartphone photography and RGB analysis for qualitative analysis of SARS-CoV-2 target nucleic acids. This sensing system exhibits a highly specific and significant ability to recognize single-base mismatched nucleic acids. Furthermore, the colorimetric sensor for detecting SARS-CoV-2 target nucleic acids can be performed without any enzyme assistance, resulting in low detection costs. Attached Figure Description
[0021] Figure 1 A schematic diagram illustrating the principle of visually detecting the novel coronavirus with the naked eye;
[0022] Figure 2 This is a comparison chart of the target and single-base mutation samples;
[0023] Figure 3 Colorimetric plot (A) and RGB increment plot (B) of different concentrations of the RdRp gene at serial dilutions.
[0024] Figure 4 Colorimetric map (A) and RGB increment map (B) comparing the target in wastewater with a single-base mutation sample. Detailed Implementation
[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.
[0026] I. Experimental Conditions
[0027] Materials and reagents: Dimethyl sulfoxide (DMSO) (Shanghai Sinopharm Chemical Reagent Co., Ltd.); sodium chloride, potassium chloride, magnesium chloride hexahydrate, Triton X-100, tris(hydroxymethyl)aminomethane, hemin chloride, and 3,3',5,5'-tetramethylbenzidine (TMB) (Shanghai Aladdin Biochemical Technology Co., Ltd.); 30% H2O2 (Chengdu Kelon Chemical Co., Ltd.); all reagents were of analytical grade. The nucleic acid sequences required for this invention were designed using NUPACK software. All nucleic acid sequences were purchased from Shanghai Sangon Biotech Co., Ltd., and the specific nucleic acid sequences are shown in Table 1.
[0028] Table 1. Nucleic acid names and specific sequences used in this invention.
[0029]
[0030] Experimental instruments: pipette, electronic analytical balance, pH meter, PCR instrument, centrifuge, vortex mixer, smartphone (iPhone 13 Pro).
[0031] Characterization Method: Data acquisition in this invention takes place in a sealed environment with an LED surface light source. The sample is placed below the light source, and a smartphone is used to take pictures directly in front of the sample to obtain colorimetric experimental results. Simultaneously, the data images taken by the smartphone are processed using the software Corlor Picker to extract the R, G, and B values of the blue areas. The RGB increment (ΔRGB) value of the blank control sample with added target nucleic acid compared to the blank control sample without added target nucleic acid is calculated using the following formula: (Where R, G, and B represent values with added target nucleic acid; R', G', and B' represent values without added target nucleic acid).
[0032] II. Experimental Procedures and Principles
[0033] (1) Preparation of double-stranded DNA probes:
[0034] DNA1 (5.2 μM) and DNA2 (5.0 μM) were added to a buffer (20 mM Tris-HCl, 100 mM NaCl) and the final volume was adjusted to 50 μL. The mixture was then reacted at 95 °C for 4 minutes and then annealed to room temperature in a gradient manner to obtain a double-stranded DNA probe (i.e., dsDNA) composed of DNA1 and DNA2.
[0035] (2) Colorimetric detection of SARS-CoV-2 target nucleic acid:
[0036] Different concentrations of SARS-CoV-2 target nucleic acid were added to dsDNA for TSDR reaction. The TSDR product was then added to a G-quadruplex-hemin DNase buffer (20 mM Tris-HCl, 200 nM hemin, pH 7.0, 150 mM NaCl, 20 mM KCl, 0.01% (v / v) Triton X-100, 1% (v / v) DMSO) and reacted at room temperature for 30 minutes to form G-quadruplex-hemin DNase. Afterwards, TMB and H2O2 were added, and the solution immediately turned blue.
[0037] (3) Experimental principle:
[0038] Based on the Toehold chain substitution reaction (TSDR), this invention constructs a simple and portable colorimetric biosensor for detecting SARS-CoV-2 (e.g., Figure 1 The target nucleic acid binds to the Toehold region on the dsDNA probe, composed of DNA1 and DNA2, triggering a strand displacement reaction. This displaces DNA1, releasing the intact G-rich sequence from DNA2, which then binds to hemin chloride to form a G-quadruplex / hemin complex. This complex catalyzes the oxidation of TMB by hydrogen peroxide, resulting in a blue solution. However, in the absence of the target nucleic acid, the dsDNA probe remains stable, and the G-rich sequence in DNA2 is blocked by DNA1, making the solution colorless. As the concentration of SARS-CoV-2 target nucleic acid increases, the color depth of the solution gradually increases. Finally, the solution color value is read using smartphone photography and image analysis for analysis of SARS-CoV-2 target nucleic acid. This method offers advantages such as low cost, high accuracy, and portability.
[0039] III. Experimental Results
[0040] To verify the feasibility of the construction method of this invention, we explored the use of the DNA fragment corresponding to the RdRp gene (Target sequence) of the novel coronavirus. For example... Figure 2 As illustrated in the illustration, in the absence of target nucleic acid (Blank sample), the DNA1 / DNA2 probe remains stable. Because DNA1 blocks the portion of DNA2 that can form G-quadruplexes, G-quadruplex-hemin DNase cannot form, and TMB cannot be catalyzed; the solution is colorless in this case. However, in the presence of target nucleic acid (Target sample), driven by thermodynamic entropy, the target nucleic acid binds to the toehold and initiates the TSDR reaction, subsequently displacing DNA1 to form the target / DNA2 complex, fully exposing the complete G-rich sequence. In K... +With the help of hemin, the target / DNA2 complex can form a G-quadruplex / heme DNase, which catalyzes the oxidation of TMB by H2O2, turning the solution from colorless to blue. Furthermore, as... Figure 2 As shown in the blue bar chart, the blue solution with added target nucleic acid produces a significant ΔRGB value compared to the solution without added target nucleic acid, proving that this method can be used for the detection of target nucleic acid.
[0041] First, a series of different concentrations of the RdRp gene (1-20 nM) were prepared using a serial dilution method. The experimental results are shown in Figure 3A. The color of the solution deepened with the increase of the target nucleic acid concentration, and the lowest level of target nucleic acid that could be detected by the naked eye was 1 nM. Figure 3 B shows that the ΔRGB value increases with increasing target concentration, which is consistent with the color change of the reaction solution.
[0042] Researchers have reported the presence of SARS-CoV-2 nucleic acid in wastewater, food packaging, and on the surface of fruit. We added the sequences of the target nucleic acid (Target sample) and the single-base mutation (SM sample) to wastewater to further validate the applicability of this method for detecting SARS-CoV-2 nucleic acid in the environment. Figure 4 As shown in Figure A, visual observation reveals that the color depth of the target nucleic acid sample solution is significantly stronger than that of the sample with a single base mismatch. Furthermore, as... Figure 4 As shown in Figure B, the ΔRGB value generated by the target nucleic acid sample is 3.0 times that of the single-base mismatch sample, further demonstrating the strong specificity of our constructed colorimetric sensing system in actual sample detection. Therefore, this method has clinical application value in detecting SARS-CoV-2 variants in complex biological samples.
[0043] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described in the present invention through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.
Claims
1. A double-stranded DNA, characterized in that, The double-stranded DNA consists of DNA1 and DNA2, and the sequences of DNA1 and DNA2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
2. The method for preparing double-stranded DNA according to claim 1, characterized in that, The process includes the following steps: adding DNA1 and DNA2 to a buffer solution, reacting at 95°C for 4 minutes, and then annealing at a gradient to room temperature.
3. The preparation method according to claim 2, characterized in that, The buffer solution is Tris-HCl, PBS, or HEPES buffer.
4. The preparation method according to claim 2, characterized in that, The molar ratio of DNA1 to DNA2 ranges from 1:1 to 1.5:
1.
5. A colorimetric biosensor, characterized in that, The reagent includes a molecular recognition reagent for SARS-CoV-2 strains and a signal conversion reagent. The molecular recognition reagent for SARS-CoV-2 strains includes the double-stranded DNA described in claim 1, and the signal conversion reagent includes heme chloride, potassium ions, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine.
6. A colorimetric biosensor according to claim 5, characterized in that, The concentration range of the double-stranded DNA is 10 nM-1 μM, the concentration range of the heme chloride is 10 nM-1 μM, and the concentration range of the hydrogen peroxide is: The concentration range of 3,3',5,5'-tetramethylbenzidine is 0.1 mM-10 mM.
7. A colorimetric biosensor according to claim 5, characterized in that, The heme chloride and potassium ions need to be used in conjunction with a buffer solution, which includes a buffer solution formed by G-quadruplex. The G-tetrachain-forming buffer solution comprises 20 mM Tris-HCl, 200 nM hemin, 150 mM NaCl, 20 mM KCl, 0.01% v / v Triton X-100, and 1% v / v DMSO. The molar ratio of Tris-HCl, hemin, NaCl, and KCl is in the range of 100:1:750:
100. The pH range of the G-tetrachain-forming buffer solution is 6-8.