CN113341146B - Colorimetric sensor for detecting hepatitis B virus and preparation and application thereof - Google Patents
Colorimetric sensor for detecting hepatitis B virus and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a colorimetric sensor for detecting hepatitis B virus and preparation and application thereof. The sensor comprises a target recognition part and a signal output part, wherein the target recognition part consists of a hepatitis B surface antibody (Ab) and a biotin-modified hepatitis B surface antigen aptamer DNA (biotin-aptamer), when a target substance, namely hepatitis B surface antigen, is added, the hepatitis B surface antigen is captured by the Ab, the biotin-aptamer is combined with the antigen to form a sandwich compound, and the biotin can be combined with subsequently added streptavidin; the signal output part comprises a DM-Hemin/G4 compound formed by four modified DNA chains of S1, S2, S3 and S4, can catalyze TMB to develop color and emit blue light visible to naked eyes, biotin is modified on S4, and the biotin modified on S4 can be combined with streptavidin, so that the detection of hepatitis B surface antigen is realized. The scheme of the invention has the advantages of rapidness, simpleness, high sensitivity, universality and the like.
Description
Technical Field
The invention relates to the technical field of hepatitis B virus detection, in particular to a colorimetric sensor for detecting hepatitis B virus and preparation and application thereof.
Background
Hepatitis B Virus (HBV) infection is one of the major health problems worldwide, possibly leading to chronic hepatitis, cirrhosis and primary liver cancer, especially if the patient's diet and health are not well controlled. It is estimated that 20 hundred million people are serologically characterized by past or present HBV infection and 3.6 hundred million patients with chronic HBV-associated liver disease. According to the report of the REVEALHBV (increased viral load and associated risk assessment of liver disease/cancer-hepatitis B virus) panel, the risk of patients is particularly high when their serum viral load is > 105 copies/ml. Therefore, detection and monitoring at an early stage of HBV infection is very important. Hepatitis B is a common disease in China, and the determination of the virus concentration can provide important basis for diagnosis and treatment.
The most common HBV detection methods clinically used today are immunoassays and Polymerase Chain Reaction (PCR). The immunoassay is a serological method which takes virus antigen or antibody as a target spot, and has good selectivity and 100% accuracy. However, the immunoassay method is limited by serological reaction and cannot provide quantitative detection results. Hepatitis B Surface (HBs) antibodies are the most commonly used target for immunoassays, even several months after the acute phase of HBV infection. In clinical applications, PCR is a routine method of detection of HBV infection. Quantitative PCR has been used to estimate the initial amount of viral DNA by measuring the amount of amplified product. Real-time PCR is based on an assessment of the threshold period (CT) at which PCR product amplification is first detected. PCR consists of 20-40 cycles of repetition to achieve detectable DNA concentrations, thus requiring effective control of thermal cycling and therefore higher instrument costs. PCR also produces errors due to its hybridization mechanism. Therefore, in view of the above-mentioned drawbacks of the conventional clinical diagnosis methods, it is necessary to develop an HBV detection technology having the characteristics of high cost efficiency, fast response speed, portability, high sensitivity, and the like.
In addition to its biological function as a nucleic acid, ssDNA also has enzymatic activity similar to that of proteases under certain conditions. One of the dnases is called G-quadruplex dnase. In this DNase, guanine (G) -rich nucleic acid sequences are particularly prone to higher order structure formation and are found in K+, pb 2+ Or NH 4+ Folding into parallel or antiparallel G-quadruplexes in the presence of a ligand. Recent studies have shown that G-rich sequences can bind to Hemin to form a Hemin/G-quadruplex complex with horseradish peroxidase (HRP) -like activity, catalyzing H 2 O 2 Is reduced by (a). Therefore, the Hemin/G4 DNase has been widely used as a catalytic marker for replacing HRP, and provides a new way for amplifying detection and coupling of DNase and nano materials and design of biosensors or nano devices.
Compared with the traditional detection technology, the sensing technology developed based on the Hemin/G-quadruplex horseradish peroxidase (Hemin/G4-HRP enzyme) has the following advantages: (1) The catalyst has wide adaptability to the reaction temperature and has catalytic activity under the high-temperature condition; (2) The synthesis is simple and quick, and complicated purification steps of the traditional protease are not needed; (3) It has high specificity to nucleic acid sequence, and single base difference can produce obvious change in enzyme activity. However, the Hemin/G4-HRP enzyme has some limitations. Firstly, the catalytic efficiency of the Hemir/G4 enzyme in the current research generally cannot reach the efficiency of protease; meanwhile, most DNase detection methods can only be verified in a simple buffer system under laboratory conditions, and detection in complex biological samples is yet to be developed; furthermore, DNAzymes also function in the presence of only a few substrates.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a colorimetric sensor for detecting hepatitis b virus, and preparation and application thereof, wherein the colorimetric sensor of the present invention purposefully compiles and polymerizes Hemin/G4 into DNA micelles to form a catalytic unit, thereby realizing high-sensitivity detection of hepatitis b antigen.
To achieve the above and other objects, and in accordance with a first aspect of the present invention, there is provided a colorimetric sensor for detecting hepatitis b virus antigen, comprising a target recognition portion consisting of a hepatitis b surface antibody (Ab) and biotin-modified hepatitis b surface antigen aptamer DNA (biotin-aptamer) which is captured by the Ab when a target substance, hepatitis b surface antigen, is added, and the biotin-aptamer is combined with the antigen to form a sandwich complex, and the biotin is combined with streptavidin to be added later; the signal output part comprises a DNA micelle-Hemin/G4 (DM-Hemin/G4) compound formed by four modified DNA chains of S1, S2, S3 and S4, wherein the DM-Hemin/G4 compound can catalyze TMB to develop color and emit blue light visible to naked eyes, biotin (biotin) is modified on the S4, and the biotin modified on the S4 can be combined with streptavidin, so that the detection of hepatitis B surface antigen is realized.
Further, the nucleotide sequence of the single-stranded S1 is:
5’-CGTGATGAACGTATGACGTAT-chol-3’(SEQ ID NO.1)。
further, the nucleotide sequence of the single-stranded S2 is:
5′-chol-ATACGTCATACGTTCATCACGCCGTAAGTTAGTTGGAGACGTAGGAGGGTAGGGCGGGTTGGG-3′(SEQ ID NO.2)。
further, the nucleotide sequence of the single-stranded S3 is:
5′-hemin-CCTACGTCTCCAACTAACTTACGG-3′(SEQ ID NO.3)。
further, the nucleotide sequence of the single-stranded S4 is:
5′-biotin-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCTACGTCTCCAACTAACTTACGG-3′(SEQ ID NO.4)。
further, the nucleotide sequence of the target substance, i.e., hepatitis B surface antigen aptamer DNA, is:
5′-GGGAATTCGAGCTCGGTACCCACAGCGAACAGCGGCGGACATAATAGTGCTTACTACGACCTGCAGGCATGCAAGCTTGG-3′(SEQ ID NO.5)。
a second aspect of the present invention provides a method for preparing a colorimetric sensor according to the first aspect, comprising the steps of:
(a) Adding a reaction solution containing hepatitis B surface antigen into a cell culture plate for incubation, capturing target substances, washing the plate, adding streptavidin, and performing incubation;
(b) Mixing the four modified DNA chains S1, S2, S3 and S4 in proportion, and carrying out denaturation annealing to form a DNA micelle-Hemin/G4 (DM-Hemin/G4) compound;
(c) Adding DNA micelle-Hemin/G4 (DM-Hemin/G4) into a 96-well plate for light-proof incubation, washing the plate, adding TMB color development liquid, and performing light-proof incubation;
(d) And adding TMB stopping solution, and detecting the hepatitis B virus by measuring ultraviolet absorbance.
Further, in the step (a), the molar ratio of the hepatitis B surface antigen to the streptavidin is 1:1.
Further, in the step (a), the reaction solution containing hepatitis B surface antigen comprises: tris-HCL buffer and hepatitis B surface antigen.
Optionally, in the step (a), the reaction solution containing hepatitis b surface antigen comprises: 45mM Tris-HCl buffer, 1. Mu.M hepatitis B surface antigen.
Further, in the step (a), the incubation temperature is 37 ℃, and the incubation time is 1-3 hours, preferably 2 hours.
Further, in the step (a), after the addition of streptavidin, the incubation is performed at room temperature for 1 to 3 hours, preferably 2 hours.
Further, in the step (b), the molar ratio of S1, S2, S3 and S4 is (4-6): (3-5): 1, preferably 5:5:4:1.
Further, in the step (b), S1, S2, S3, S4 are added to NEBuffer3.0 buffer to carry out denaturation annealing.
Optionally, in the step (b), the reaction solution includes: 10. Mu.L of 10 XNEBuffer 3.0 buffer, 1. Mu.L of 100. Mu. M S1 strand, 1. Mu.L of 100. Mu. M S2 strand, 8. Mu.L of 10. Mu. M S3 strand, 2. Mu.L of 10. Mu. M S4 strand.
Further, in said step (b), the denaturation temperature is 90-100 ℃, preferably 95 ℃; the denaturation time is 5-10min, preferably 5min.
Further, in the step (b), the slow annealing is performed under the ice water bath condition.
Further, in the step (b), the reaction solution obtained after annealing is ultrafiltered and centrifuged to obtain a purified DNA micelle-Hemin/G4 (DM-Hemin/G4) complex.
Further, in the step (b), the ultrafiltration centrifugation conditions are 300 to 500rpm, 5 to 10min, preferably 300rpm, 5min.
Further, in the step (c), the TMB color developing solution is added in an amount of 40 to 100. Mu.L, preferably 50. Mu.L.
Further, in the step (c) (d), the addition amounts of the TMB color developing solution and the TMB terminating solution are equal.
Further, in the step (c), the DNA micelle-Hemin/G4 is added to a 96-well plate and incubated at room temperature for 0.5 to 1.5 hours, preferably 1 hour, in a dark place.
Further, in the step (c), after the TMB color developing solution is added, the incubation temperature is 37 ℃, and the incubation time is 10-20min, preferably 15min.
Further, in the step (d), the TMB terminator is added in an amount of 40 to 100. Mu.L, preferably 50. Mu.L.
Further, in the step (d), ultraviolet absorbance is measured at 450 nm.
In a third aspect, the present invention provides a method for detecting hepatitis b virus antigen, the colorimetric sensor according to the first aspect being further used for the colorimetric sensor according to the second aspect.
Further, the colorimetric sensor detects as follows:
(1) Setting parameters: setting a detection channel com3, wherein the detection wavelength is 450nm;
(2) Adding a sample, and placing a 96-well plate with a reaction lath into an enzyme labeling instrument;
(4) And (3) detecting: click detection to obtain a signal.
As described above, the colorimetric sensor for detecting hepatitis B virus, and the preparation and application thereof have the following beneficial effects:
the invention innovatively provides a colorimetric sensor and a colorimetric method for detecting hepatitis B surface antigen by using a Hemin/G4 efficient catalytic signal probe based on DNA micelle growth. The technical scheme of the invention has the following advantages:
(1) The method of the invention is rapid and simple, and can detect the target substance under the naked eye condition by outputting signals through colorimetric reaction of oxidation of TMB by hydrogen peroxide.
(2) Compared with the traditional DNase, the high sensitivity of the Hemin/G4-DNA micelle integrates free Hemin/G4, thereby greatly enhancing the catalytic efficiency.
(3) The multi-channel can coat antibodies corresponding to hepatitis B surface antigen (HBsAg), core antigen (HBcAg) and e antigen (HBEAg) in different micropores, so that accurate detection of hepatitis B is realized.
(4) The detection method has universality, and can realize detection of various target substances such as nucleic acid, protein, vesicle and the like by replacing antibodies coated on the 96-micro-pore plate.
Drawings
Fig. 1 shows a schematic diagram of the detection of the present invention.
FIG. 2 is a graph showing fluorescence signal spectra obtained by verifying the feasibility of the colorimetric sensor in example 2.
FIG. 3 shows a bar graph of fluorescence signals obtained by verifying the feasibility of colorimetric sensors in example 2.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The invention provides a colorimetric sensor for detecting hepatitis B virus, which is used for realizing high-sensitivity detection of hepatitis B antigen by purposefully editing and polymerizing Hemin/G4 into DNA micelle to form a catalytic unit. The colorimetric sensor constructed by the invention has the advantages of simple preparation and detection method, low cost, effective prevention of non-specific reaction, good stability and reproducibility, and is expected to be popularized and used in the detection analysis and application research of hepatitis B virus.
The specific implementation process of the invention is as follows:
example 1
Construction of colorimetric sensor and detection of hepatitis B Virus
1. Material
Cholesterol-modified DNA sequences S1, S2, S3 were purchased from Takara corporation. HPLC purification of the biotin-modified DNA sequence S4 was synthesized by Shanghai. The specific sequences are shown in Table 1 below:
TABLE 1
2. Detection instrument
An enzyme-linked immunosorbent assay instrument.
3. Principle of detection
As shown in FIG. 1, under the condition of denaturation annealing, two sequences S1 and S2 can be complemented into double chains, and simultaneously, as the same ends of the complemented sequences are both modified with hydrophobic substance cholesterol, the sequences can spontaneously aggregate in a homogeneous hydrophilic environment, so that spherical nucleic acid formed by DNA sequences, also called DNA micelle, is formed. And S3 modified with Hemin can be complementary with the other end of S2, and Hemin/G4 is formed through the ortho effect, so that the efficient peroxidase mimic complex is constructed.
4. Preparation and detection process
(a) 100. Mu.L of a reaction solution containing 1. Mu.M hepatitis B surface antigen (prepared from 90. Mu.L of 50mM Tris-HCl buffer, 10. Mu.L of 10. Mu.M hepatitis B surface antigen) was added to a 96-well plate, incubated at 37℃for 2 hours, the target substance was captured, then the plate was washed, and 10. Mu.L of streptavidin at a concentration of 1. Mu.M prepared from 0.05mol/L sodium bicarbonate buffer (pH 9.6) was added, and incubated at room temperature for 2 hours.
(b) Mixing four modified DNA chains of S1, S2, S3 and S4 according to the molar ratio of 5:5:4:1, wherein the reaction solution comprises the following components: 10. Mu.L of 10 XNEBuffer 3.0 buffer, 1. Mu.L of 100. Mu. M S1 strand, 1. Mu.L of 100. Mu. M S2 strand, 8. Mu.L of 10. Mu. M S3 strand, 2. Mu.L of 10. Mu. M S4 strand, denaturation at 95℃for 5min, and slow annealing under ice-water bath conditions to form a DNA micelle-Hemin/G4 (DM-Hemin/G4) complex; the obtained reaction liquid is ultrafiltered and centrifuged (300 rpm, 5 min) to obtain purified DM-Hemin/G4 for later use.
(c) Purified DM-Hemin/G4 was added to a 96-well plate and incubated at room temperature in the dark for 1h, after washing the plate, TMB color development solution was added in 50. Mu.L, and incubated at 37℃in the dark for 15min.
(d) 50. Mu.L of TMB stop solution was added thereto, and the ultraviolet absorbance was measured at 450 nm. The method specifically comprises the following steps:
(1) Setting parameters: setting a detection channel com3, wherein the detection wavelength is 450nm;
(2) Adding a sample, and placing a 96-well plate with a reaction lath into an enzyme labeling instrument;
(4) And (3) detecting: click detection to obtain a signal.
Example 2
Verifying feasibility of colorimetric sensor for detecting hepatitis B virus
Mixing S1, S2, S3 and S4 to form a DNA micelle-Hemin/G4 compound (DM-Hemin/G4), purifying and separating a macromolecular product DM-Hemin/G4 and an unreacted free nucleic acid chain, respectively adding TMB chromogenic solution and TMB stop solution, and measuring ultraviolet absorbance at 450 nm. FIGS. 2 and 3 show fluorescence signal spectra and fluorescence signal histograms of micelle groups, free nucleic acid groups without synthesized micelles, and blank control groups, respectively.
It can be seen from FIGS. 2 and 3 that only the micelle group had a distinct signal, whereas none of the free nucleic acid groups that did not synthesize micelles had a signal with the blank group. The result shows that the colorimetric sensor constructed by the invention can detect hepatitis B virus.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
SEQUENCE LISTING
<110> university of Chongqing medical science
<120> colorimetric sensor for detecting hepatitis B virus, preparation and use thereof
<130> PCQYK2110479-HZ
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Claims (20)
1. A colorimetric sensor for detecting hepatitis B virus is characterized by comprising a target recognition part and a signal output part, wherein the target recognition part is formed by a hepatitis B surface antibody Ab and a biotin-modified hepatitis B surface antigen aptamer
DNA biotin-aptamer, which is captured by Ab when the target substance hepatitis B surface antigen is added, and combined with antigen to form sandwich complex, biotin can be combined with subsequently added streptavidin; the signal output part comprises a DNA micelle-Hemin/G4 compound formed by four modified DNA chains of S1, S2, S3 and S4, wherein the DNA micelle-Hemin/G4 compound can catalyze TMB to develop color and emit blue light visible to naked eyes, biotin is modified on the S4, and biotin modified on the S4 can be combined with streptavidin, so that the detection of hepatitis B surface antigen is realized;
the nucleotide sequence of the S1 is as follows:
the nucleotide sequence of the S2 is as follows:
the nucleotide sequence of the S3 is as follows:
the nucleotide sequence of the S4 is as follows:
the nucleotide sequence of the target substance, namely hepatitis B surface antigen aptamer DNA is as follows:
2. the method of preparing a colorimetric sensor according to claim 1, comprising the steps of:
(a) Adding a reaction solution containing hepatitis B surface antigen into a cell culture plate for incubation, capturing target substances, washing the plate, adding streptavidin, and performing incubation;
(b) Mixing the four modified DNA chains S1, S2, S3 and S4 in proportion, and carrying out denaturation annealing to form a DNA micelle-Hemin/G4 complex;
(c) Adding the DNA micelle-Hemin/G4 complex into a 96-well plate for light-proof incubation, washing the plate, adding TMB color development liquid, and performing light-proof incubation;
(d) And adding TMB stopping solution, and detecting the hepatitis B virus by measuring ultraviolet absorbance.
3. The preparation method according to claim 2, characterized in that: in the step (a), the molar ratio of the hepatitis B surface antigen to the streptavidin is 1:1.
4. The preparation method according to claim 2, characterized in that: in the step (a), the reaction solution containing the hepatitis B surface antigen comprises Tris-HCL buffer solution and the hepatitis B surface antigen.
5. The preparation method according to claim 2, characterized in that: in step (a), the incubation temperature is 37 ℃ and the incubation time is 1-3h.
6. The preparation method according to claim 2, characterized in that: in step (a), after the addition of streptavidin, incubation is carried out for 1-3h at room temperature.
7. The preparation method according to claim 2, characterized in that: in step (b), the molar ratio of S1, S2, S3 and S4 is (4-6): (4-6): (3-5): 1.
8. the preparation method according to claim 2, characterized in that: in step (b), S1, S2, S3 and S4 are added to NEBuffer3.0 buffer for denaturation annealing.
9. The preparation method according to claim 2, characterized in that: in the step (b), the denaturation temperature is 90-100 ℃ and the denaturation time is 5min.
10. The preparation method according to claim 2, characterized in that: in step (b), the slow annealing is performed under ice-water bath conditions.
11. The preparation method according to claim 2, characterized in that: in the step (b), the reaction liquid obtained after annealing is ultrafiltered and centrifuged to obtain the purified DNA micelle-Hemin/G4 complex.
12. The method of manufacturing according to claim 11, wherein: the ultrafiltration and centrifugation conditions are 300-500rpm, 5-10min.
13. The preparation method according to claim 2, characterized in that: in the step (c), TMB color developing solution is added in an amount of 40-100. Mu.L.
14. The preparation method according to claim 2, characterized in that: in the step (c), the DNA micelle-Hemin/G4 is added into a 96-well plate, and incubated for 0.5-1.5h at room temperature in a dark place.
15. The preparation method according to claim 2, characterized in that: in the step (c), after TMB color development liquid is added, the incubation temperature is 37 ℃ and the incubation time is 10-20min.
16. The preparation method according to claim 2, characterized in that: in the step (d), the TMB stop solution is added in an amount of 40 to 100. Mu.L.
17. The preparation method according to claim 2, characterized in that: in the step (c) and (d), the TMB color developing solution and the TMB stopping solution are added in the same amount.
18. The preparation method according to claim 2, characterized in that: in step (d), the ultraviolet absorbance is measured at 450 nm.
19. A method for detecting hepatitis b virus employing the colorimetric sensor of claim 1.
20. The method of claim 19, wherein the colorimetric sensor detects as follows:
(1) Setting parameters: setting a detection channel com3, wherein the detection wavelength is 450nm;
(2) Adding a sample, and placing a 96-well plate with a reaction lath into an enzyme labeling instrument;
(4) And (3) detecting: click detection to obtain a signal.
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