阅读说明：本技术 一种结核分枝杆菌cfp-10抗原免疫传感器及其制备方法和应用 (Mycobacterium tuberculosis CFP-10 antigen immunosensor and preparation method and application thereof ) 是由 李金龙 张永臣 张侠 许传军 胡凯 于 2019-08-27 设计创作，主要内容包括：本发明通过靶标循环策略和DNA模拟酶信号放大策略构建了一种可以检测结CFP10抗原的免疫传感器,使用该检测器的检测方法具有以下优势：由于适配体与CFP10较强的结合性能,因此本发明具有极强的特异性；由于靶蛋白的循环利用和DNA模拟酶的辣根过氧化物酶活性,实现了较低CFP-10抗原检测限(0.01ng.ml<Sup>-1</Sup>)；该方法没有使用到抗体及辣根过氧化物酶等,降低了检测成本。本发明免疫传感器利用该抗原的DNA适配体代替抗体,来实现该抗原的检测,该电化学免疫传感器利用DNA模拟酶作为信号放大元件,由于DNA空间结构较小,可以富集在金纳米颗粒表面,具有产生放大信号的作用,本发明所提出的CFP-10抗原检测方法在生物医学研究和临床诊断中有着广阔的应用前景。(The invention constructs an immunosensor capable of detecting the CFP10 antigen through a target circulation strategy and a DNA mimic enzyme signal amplification strategy, and the detection method using the immunosensor has the following advantages: the aptamer has strong binding performance with CFP10, so the invention has strong specificity; due to the recycling of target protein and the horseradish peroxidase activity of DNA mimic enzyme, the lower detection limit (0.01 ng.ml) of CFP-10 antigen is realized ‑1 ) (ii) a The method does not use antibody, horseradish peroxidase and the like, and reduces the detection cost. The immunosensor provided by the invention uses the DNA aptamer of the antigen to replace an antibody to realize the detection of the antigen, the electrochemical immunosensor uses DNA mimic enzyme as a signal amplification element, and the DNA space structure is smaller, so that the DNA mimic enzyme can be enriched on the surface of gold nanoparticles and has the effect of generating an amplification signal.)

1. The mycobacterium tuberculosis CFP-10 antigen immunosensor is characterized by being constructed by a target circulation strategy and a DNA mimic enzyme signal amplification strategy and being an immunosensor for detecting the CFP10 antigen.

2. The method for preparing the mycobacterium tuberculosis CFP-10 antigen immunosensor according to claim 1, comprising the following steps:

(1) preparation of AuNP_S-DNA complexes

1) Activating two different thiolated oligonucleotides by TCEP for 1-3h to obtain activated DNA solution;

2) adding 0.5-2mL AuNPs into the activated DNA solution obtained in the step 1), standing for 10-14h, adding NaCl, shaking at 37 ℃ to enable the concentration of sodium chloride to reach 0.3-0.8M, centrifuging for 10-30min, washing to remove unbound DNA, and thus obtaining the AuNP_S-storing the DNA complex at 4 ℃ until use;

(2) construction of an electrochemical immunosensor

1) Preparation of gold electrode

a) Polishing the gold electrode by using alumina powder to obtain a polished electrode;

b) soaking the electrode polished in the step 2-1) -a) in the goby solution for 2-20min to eliminate adsorbed organic matters, and thoroughly cleaning with deionized water;

c) soaking the electrode in 50% nitric acid for 10-30min, treating the electrode with ethanol and deionized water for 2-8min, blowing with nitrogen gas, soaking the electrode in 0.5M sulfuric acid, and scanning with Cyclic Voltammetry (CV) from 0 to 1.6V until stable signals are obtained;

2) immersing the gold electrode prepared in the step 1) into DBCO-DNA buffer solution with the thickness of 0.6-2 mu m for incubation for 8-16h, and then treating with aqueous solution containing 0.5-2mM MCH for 20 min;

3) further washing the electrode prepared in the step 2) for multiple times by deionized water, blowing and drying by nitrogen, gently immersing the electrode into a mixed solution containing 0.1-1 mu m of CFP-10 aptamer and capture-DNA for incubation for 0.5-2h, cleaning by deionized water, finally immersing the electrode into a solution containing different concentrations of Mycobacterium tuberculosis CFP-10 antigen and N3-DNA (0.25 mu m), and incubating for 40min at 37 ℃ to prepare the electrode for later use;

4) mu.l of AuNP prepared in step 1 to 8. mu.l_SDripping the DNA compound on the surface of the electrode prepared in the step 2-3), keeping the temperature at 37 ℃ for 0.5-2h, sequentially cleaning the electrode by PBS and deionized water, and drying the electrode by nitrogen;

5) dropwise adding the hemin solution on the surface of the electrode at 37 ℃ for 1-3h to form DNA mimic enzyme, and realizing the enrichment of the DNA mimic enzyme;

(3) detection of electrochemical immunosensors

1) The working electrode was gold, electrochemical measurements were performed on a 660E electrochemical analyzer, Differential Pulse Voltammetry (DPV) was performed in PBS, and Electrochemical Impedance Spectroscopy (EIS) experiments were performed in potassium ferricyanide complex solution and potassium nitrate, with the following experimental parameters: for the DPV experiments, the scan range was-0.1V to 0.2V.

3. The method for preparing a mycobacterium tuberculosis CFP-10 antigen immunosensor according to claim 2, wherein the sequences from 5 'to 3' ends are as follows:

SEQ ID NO.1：Dibenzocyclooctyne(DBCO)-DNA,

SH-CGTACAACCAAC-DBCO；

SEQ ID NO.2：CFP-10aptamer(CFP-10Apt)：TCCTGAAAGGGGCCTGCCCCACTATCTCACATGGGGTTCAGTTGGTTGTACG；

SEQ ID NO.3：Complementary probe(CP)：TGAACCCCATGTGAGATAGTGGGGCAGGCCCCTTTCAGGA；

SEQ ID NO.4：DNA 1,TGGGTAGGGCGGGTTGGGTTTTTT-SH；

SEQ ID NO.5：DNA 2,GGGGCAGGCCCCTTTCAGGATTTTTT-SH；

SEQ ID NO.6：azide(N₃)-DNA,N₃-TGAACCCCATGTGAGATAGT。

4. the method for preparing a mycobacterium tuberculosis CFP-10 antigen immunosensor of claim 2, wherein the immunosensor comprises:

the specific concentration, the dosage and the ratio of the two different thiolated oligonucleotides in the step 1-1) are as follows: 10 μm, 80-120 μ l, DNA 1: DNA2 molar ratio 1: 10;

the concentration of TCEP in the step 1-1) is 50 mm;

in the step 1-2), the centrifugal speed is 12000 rpm/min;

in step 1-2), washing with 10mM PBS, pH7.4;

the diameter of the gold electrode in the steps 2-1) -a) is 3 mm;

the solution of the tiger fish in the steps 2-1) -b) is [ V (H)₂SO₄)：(30％H₂O₂)＝3：1]；

The concentration of sulfuric acid in steps 2-1) -c) was 0.5M;

the concentration of the capture-DNA in the step 2-3) is 0.5 μm;

step 2-5) the hemin solution was [25mM HEPES,50mM KCl,200mM NaCl,12.5mM MgCl₂]The dosage is 8-15 mu L;

in the step 3-1), the PBS is 0.1m and contains 1.0mM of hydrogen peroxide and 0.2mM of p-phenylenediamine;

in the step 3-1), the level of the 5mM potassium ferricyanide composite solution is 5mM, and the potassium nitrate is 1M.

5. A method for detecting CFP-10 antigen using the Mycobacterium tuberculosis CFP-10 antigen immunosensor of claim 1.

6. Use of the mycobacterium tuberculosis CFP-10 antigen immunosensor of claim 1 and the detection method of claim 3 in biomedical research and clinical diagnostics.

Background

Mycobacterium Tuberculosis (MTB) infection is still serious in countries and regions with laggard economic development, has extremely high infection rate, and is easy to cause hidden infection, so the morbidity and the mortality are high. For the prevention and control of tuberculosis, besides the need of timely treatment measures, early diagnosis and early detection are very important. Therefore, establishing a method and a sensor which can sensitively and rapidly detect the mycobacterium tuberculosis is always a research hotspot and has important significance for human health. Researchers have developed a variety of diagnostic methods such as mycobacterium tuberculosis culture, tuberculin skin tests, and sputum smear microscopy, but all have a very clear determination of o. For example, the gold standard method for MTB diagnosis-Mycobacterium tuberculosis culture, which generally takes one or even several months, is extremely disadvantageous for the treatment of tuberculosis patients; tuberculin skin test, which has low specificity, is the most widely used method at present, but is not beneficial to the diagnosis of tuberculosis; the sputum smear microscopy method depends on the quality of the sputum specimen and the bacterial load of the sputum specimen, and has low sensitivity. Over the past few years, while efforts have been made to improve tuberculosis detection methods, sensitive, rapid tuberculosis detection methods have remained a necessity.

In order to overcome the disadvantages of the conventional methods, in recent years, a variety of new detection techniques for detecting mycobacterium tuberculosis antigens have been developed to diagnose infection with mycobacterium tuberculosis, such as Surface Plasmon Resonance (SPR) -based detection of mycobacterium tuberculosis antigens and immunosensor detection of mycobacterium tuberculosis antigens such as CFP 10. The electrochemical immunosensor generates electron transfer by utilizing the oxidation-reduction reaction of substances on the surface of an electrode, and quantifies the reactive substances on the surface of the electrode according to the quantity of the electron transfer.

Chinese patent CN201110056230.6 discloses a specific electrochemical immunosensor for serological diagnosis of tuberculosis, which is a novel biosensor constructed by combining immunoassay with highly sensitive sensing technology, applied to the analytical research of trace immunogenic substances, based on the previous antigen marker screening work, selecting mycobacterium tuberculosis Rv2175c gene encoding protein (hereinafter referred to as specific antigen) as specific antigen, constructing a high-sensitivity electrochemical immunosensor, and realizing the serological diagnosis of tuberculosis by detecting the antibody in human serum, which corresponds to the specific antigen. Through the current change difference of the immunosensor, the immunosensor can well distinguish healthy people from tuberculosis patients to achieve the purpose of serodiagnosis, but the detection application range of the invention is not wide enough, and the sensitivity is to be improved.

The immunological method is mainly used for detecting the mycobacterium tuberculosis through detecting antigens, the mycobacterium tuberculosis antigens are various, but the marked antigens mainly comprise: tubercle bacillus antigen 85A (Ag85A), tubercle bacillus antigen 85B (Ag85B), complex antigen composed of Ag85A, Ag85B and Ag85C, human culture filtrate protein 10(CFP10) and mycobacterium tuberculosis secretory protein (ESAT6), and the like, and the antigens have a common characteristic: the concentration is low and a method with high sensitivity is required for detection. Because the antigen level of the mycobacterium tuberculosis is very low, the existing traditional immune methods (such as ELISA methods) have the problem of detection limit, and the detection of the antigen with such low concentration is difficult, which is a great challenge for the early diagnosis of the tuberculosis, and in conclusion, a high-sensitivity method for detecting the antigen of the mycobacterium tuberculosis is urgently needed clinically to provide a basis for the early diagnosis of the tuberculosis.

Therefore, the invention constructs the electrochemical immunosensor for detecting the double-signal amplified mycobacterium tuberculosis antigen CFP10, and lays a foundation for early and rapid diagnosis of tuberculosis.

Disclosure of Invention

The principle of the invention is as follows: as shown in FIG. 1, CFP-10DNA aptamer was first modified on the surface of an electrode, the conformation of CFP-10APT was changed in the presence of CFP-10 antigen capable of binding to CFP-10APT, and complementary DNA hybridized with CFP1 was released, at which time DCBO group at the terminal of DCBO-DNA was exposed, and then click chemistry reaction was performed with azide group of N3-DNA, and subsequently, N3-DNA was subjected to N-click chemistry reaction₃DNA having the same base sequence as the released CP-DNA, capable of binding to CFP-10APT, releasing CFP-10 antigen in the next cycle, dipping into the next cycle of target protein, this cycle of target protein can be regarded as a first amplification step, with N₃After DNA pairing, the 5' overhang of CFP-10APT was exposed and hybridized to AuNPS-DNA complex, producing a significantly amplified electrochemical signal, so that a second amplification could be achieved by G-tetrad-heme-dnase.

The materials used in the invention are as follows:

① CFP-10 antigen and ESAT-6 antigen were purchased from Cusabio (Houston, TX, USA);

② Bovine Serum Albumin (BSA), Hydroquinone (HQ), TCEP, EDTA, hemin, and 6-mercapto-1-hexanol (MCH) were purchased from Sigma-Aldrich Chemical Co.Ltd;

③ sputum specimen is obtained from the clinical laboratory of public health medical center of Nanjing, and the specimen is pretreated by taking 1ml of fresh sputum sample from a small measuring cup, adding the fresh sputum sample into a measuring cup containing 2ml of sputum sample treatment solution, shaking violently, and incubating for 10 minutes at room temperature;

④ the DNA oligonucleotides used in this experiment were synthesized by Shanghai Biotechnology Ltd;

the sequence is as follows (5 'to 3'):

SEQ ID NO.1：Dibenzocyclooctyne(DBCO)-DNA,

SH-CGTACAACCAAC-DBCO；

SEQ ID NO.2：CFP-10aptamer(CFP-10Apt)：

TCCTGAAAGGGGCCTGCCCCACTATCTCACATGGGGTTCAGTTGGTTGTACG；

SEQ ID NO.3：Complementary probe(CP)：

TGAACCCCATGTGAGATAGTGGGGCAGGCCCCTTTCAGGA；

SEQ ID NO.4：DNA 1,TGGGTAGGGCGGGTTGGGTTTTTT-SH；

SEQ ID NO.5：DNA 2,GGGGCAGGCCCCTTTCAGGATTTTTT-SH；

SEQ ID NO.6：azide(N₃)-DNA,N₃-TGAACCCCATGTGAGATAGT；

wherein: DNA1 is a ligation probe capable of binding to the sequence of the adapter end portion; DNA2 is a DNA rich in G base sequence; the sequence of italicized bases in CFP-10APT can bind to CFP-10 antigen.

In order to achieve the aim, the invention firstly provides the mycobacterium tuberculosis CFP-10 antigen immunosensor, and the immunosensor capable of detecting the mycobacterium tuberculosis CFP10 antigen is constructed through a target circulation strategy and a DNA mimic enzyme signal amplification strategy.

Then, the invention provides a preparation method of the mycobacterium tuberculosis CFP-10 antigen immunosensor, which comprises the following specific steps:

1. preparation of AuNP_S-DNA complexes

1) Activating two different thiolated oligonucleotides by TCEP for 1-3h to obtain activated DNA solution;

2) adding 0.5-2mL AuNPs into the activated DNA solution obtained in the step 1), standing for 10-14h, adding NaCl, slightly shaking at 37 ℃ to enable the concentration of sodium chloride to reach 0.3-0.8M, centrifuging for 10-30min, washing to remove unbound DNA, and thus obtaining the AuNP_S-storing the DNA complex at 4 ℃ until use;

2. construction of an electrochemical immunosensor

1) Preparation of gold electrode

a) Polishing the gold electrode by using alumina powder to obtain a polished electrode;

b) soaking the electrode polished in the step 2-1) -a) in the goby solution for 2-20min to eliminate adsorbed organic matters, and thoroughly cleaning with deionized water;

c) soaking the electrode in 50% nitric acid for 10-30min, treating the electrode with ethanol and deionized water for 2-8min, blowing with nitrogen gas, soaking the electrode in 0.5M sulfuric acid, and scanning with Cyclic Voltammetry (CV) from 0 to 1.6V until stable signals are obtained; 2) immersing the gold electrode prepared in the step 1) into DBCO-DNA buffer solution with the thickness of 0.6-2 mu m for incubation for 8-16h, and then treating with aqueous solution containing 0.5-2mM MCH for 20 min;

3) further washing the electrode prepared in the step 2) for multiple times by deionized water, blowing and drying by nitrogen, gently immersing the electrode into a mixed solution containing 0.1-1 mu m of CFP-10 aptamer and capture-DNA for incubation for 0.5-2h, cleaning by deionized water, finally immersing the electrode into a solution containing different concentrations of Mycobacterium tuberculosis CFP-10 antigen and N3-DNA (0.25 mu m), and incubating for 40min at 37 ℃ to prepare the electrode for later use;

4) mu.l of AuNP prepared in step 1 to 8. mu.l_SDripping the DNA compound on the surface of the electrode prepared in the step 2-3), keeping the temperature at 37 ℃ for 0.5-2h, sequentially cleaning the electrode by PBS and deionized water, and drying the electrode by nitrogen;

5) dropwise adding the hemin solution on the surface of the electrode at 37 ℃ for 1-3h to form DNA mimic enzyme, and realizing the enrichment of the DNA mimic enzyme;

3. detection of electrochemical immunosensors

1) The working electrode was gold, electrochemical measurements were performed on a 660E electrochemical analyzer, Differential Pulse Voltammetry (DPV) was performed in PBS, and Electrochemical Impedance Spectroscopy (EIS) experiments were performed in potassium ferricyanide complex solution and potassium nitrate, with the following experimental parameters: for the DPV experiment, the scan range was-0.1V to 0.2V;

preferably, the specific concentrations, amounts and ratios of the two different thiolated oligonucleotides used in step 1-1) are: 10 μm, 80-120 μ l, DNA 1: DNA2 molar ratio 1: 10;

preferably, the concentration of TCEP in step 1-1) is 50 mm;

preferably, in the step 1-2), the centrifugal speed is 12000 rpm/min;

preferably, in step 1-2), washing with 10mM PBS, pH 7.4;

preferably, the gold electrode in step 2-1) -a) has a diameter of 3 mm;

the solution of the tiger fish in the steps 2-1) -b) is [ V (H)₂SO₄)：(30％H₂O₂)＝3：1]；

Preferably, the concentration of sulfuric acid in steps 2-1) -c) is 0.5M;

preferably, the capture-DNA concentration in step 2-3) is 0.5 μm;

step 2-5) the hemin solution was [25mM HEPES,50mM KCl,200mM NaCl,12.5mM MgCl₂]The dosage is 8-15 mu L;

preferably, the PBS in step 3-1) is 0.1m, and contains 1.0mM hydrogen peroxide and 0.2mM p-phenylenediamine;

preferably, the 5mM potassium ferricyanide complex solution in the step 3-1) has the level of 5mM and the potassium nitrate is 1M;

secondly, the invention provides a detection method for detecting CFP-10 antigen by using the mycobacterium tuberculosis CFP-10 antigen immunosensor.

Finally, the invention provides the application of the mycobacterium tuberculosis CFP-10 antigen immunosensor and the detection method thereof in biomedical research and clinical diagnosis.

The invention has the advantages of

(1) Because the concentration of the CFP-10 antigen is very low, the invention can realize extremely low detection limit by applying a dual signal amplification strategy;

(2) the invention does not need CFP10 antibody, thus avoiding the purchase of expensive antibody and reducing the detection cost;

(3) the analytical process of the present invention is relatively simple because the detection method can be operated under a constant temperature condition without a protease system.

Drawings

FIG. 1 is a schematic diagram of the design of an immunosensor.

FIG. 2 is a graph showing the results of characterization of AuNPs-DNA in Experimental example 1.

FIG. 3 is a graph showing the results of characterization of the electrode modification process in Experimental example 2.

FIG. 4 is a graph showing the effect of DBCO-DNA concentration in Experimental example 3.

FIG. 5 is a graph showing the effect of CFP-10Apt concentration in Experimental example 3.

FIG. 6 shows the effect of the ratio of DNA1/DNA 2 in Experimental example 3.

FIG. 7 shows the effect of the pH of TE buffer in Experimental example 3.

FIG. 8 is a graph showing the effect of incubation time for CFP-10 in Experimental example 3.

FIG. 9 is a graph showing the effect of the incubation time of Hemin in Experimental example 3.

FIG. 10 is a DPV graph of CFP-10(ng.ml-1) at various concentrations in Experimental example 4.

FIG. 11 is a graph comparing the peak DPV current in the presence of BSA, ESAT-6 and CFP-10 in Experimental example 5.

FIG. 12 is a graph showing the results of DPV obtained on the modified electrode cultured with a biological sample in Experimental example 6.

FIG. 13 is a comparison of the method of Experimental example 6 with an enzyme-linked immunosorbent assay

Detailed Description