阅读说明：本技术 一种比率型生物传感器及用于检测muc1的方法 (Ratio-type biosensor and method for detecting MUC1 ) 是由 胡蓉 谢发婷 杨通 杨云慧 于 2021-08-14 设计创作，主要内容包括：一种比率型生物传感器及用于检测MUC1的方法,属生物传感器检测技术领域。构建了一种基于DNA四面体的比率型电化学适体传感器并用于真实样品中MUC1的定量测定。以电解质溶液硫堇作为参比探针(I-(IR)),Co-MOFs标记的适配体作为信号探针(I-(SP)),构建比率型电化学传感器。传感器具有较强的抗干扰能力,仅需要与一种电活性物质结合。修改过程可以大大简化。具有很高的稳定性,即使在一个电极上进行十次重复扫描后,电流比(I-(SP)/I-(IR))仍保持不变,极大地减少了随机误差。此外,DNA NTH有效消除了非特异性吸附,提高了检测精度。具有较大比表面积和良好电导率的黑磷被用作传感界面,进一步提高检测灵敏度,检测极限为1.34fM。(A ratio type biosensor and a method for detecting MUC1 belong to the technical field of biosensor detection. A ratiometric electrochemical aptamer sensor based on DNA tetrahedra was constructed and used for quantitative determination of MUC1 in authentic samples. Using electrolyte solution thionine as reference probe (I) IR ) Co-MOFs labelled aptamers as signaling probes (I) SP ) And constructing a ratio type electrochemical sensor. The sensor has strong anti-interference capability and only needs to be combined with one electroactive substance. The modification process can be greatly simplified. Has high stability, even after ten times of repeated scanning on one electrode, the current ratio (I) SP /I IR ) Still remains unchanged, greatly reducing random errors. In addition, the DNA NTH effectively eliminates non-specific adsorption and improves the detection precision. Black phosphorus with large specific surface area and good conductivity is used as a sensing interface, so that the detection sensitivity is further improved, and the detection limit is 1.34 fM.)

1. A ratiometric biosensor, comprising the steps of:

(1) synthesizing DNA tetrahedron, and preparing Au NPs, Co-MOFs and BP absolute ethyl alcohol dispersion liquid;

(2) sequentially polishing the glassy carbon electrode GCE, cleaning the surface and drying;

(3) dripping 9-11 mu L of Au NPs @ BP dispersion liquid and chitosan solution which are mixed in the same volume to the surface of GCE, and airing at room temperature;

(4) dropping the synthesized tetrahedral DNA on the GCE electrode washed in the step (3), cultivating overnight at 25-38 ℃, and washing with PBS buffer solution;

(5) dripping MCH with the concentration of 0.9-1.1 mM and the volume of 9-11 mu L on the surface of the electrode prepared in the step (4), culturing at 36-38 ℃ for 8-12 min, and sealing the non-specific binding sites;

(6) dripping 9-11 mu L of MUC1 diluent with the concentration of 0.004 pM-400 pM on the surface of the electrode prepared in the step (5), and culturing for 0.8-1.2 h at 36-38 ℃;

(7) and (3) dripping 9-11 mu L of Au NPs @ Co-MOFs marked with signal probes on the surface of the electrode prepared in the step (6), and culturing at 36-38 ℃ for 0.8-1.2 h to finally obtain the DNA tetrahedron-based ratio type biosensor.

2. A ratiometric biosensor according to claim 1, prepared by the steps of:

(1) synthesizing DNA tetrahedron, and preparing Au NPs, Co-MOFs and BP absolute ethyl alcohol dispersion liquid;

(2) and the glassy carbon electrode GCE is coated on the chamois flannel with Al₂O₃Polishing the powder for at least 3 times, and sequentially adding V_{Water (W)}:V_{Nitric acid}1:1 HNO₃Washing the solution and absolute ethyl alcohol, carrying out ultra-pure water ultrasound for 2.5-3.5 min, and drying;

(3) dripping 9-11 mu L of Au NPs @ BP dispersion liquid and chitosan solution which are mixed in the same volume to the surface of GCE, and airing at room temperature;

(4) dropping the synthesized tetrahedral DNA on the GCE electrode washed in the step 3, incubating overnight at 37 ℃, and washing with PBS buffer solution;

(5) dripping 10 mu L MCH with the concentration of 1mM on the surface of the electrode prepared in the step (4), culturing for 10min at 37 ℃, and blocking the non-specific binding site;

(6) sequentially dripping 10 mu L of solution with the concentration of 0.004pM on the surface of the electrode prepared in the step (5); 0.02 pM; 0.04 pM; 0.4 pM; 2 pM; 4 pM; 20 pM; 40 pM; 200 pM; 400pM of MUC1 dilution, incubated at 37 ℃ for 1 h;

(7) and (3) dripping 10 mu L of Au NPs @ Co-MOFs marked with signal probes on the surface of the electrode prepared in the step (6), and culturing at 37 ℃ for 1h to obtain the DNA tetrahedron-based rate type biosensor.

3. The method of using a ratiometric biosensor of claim 1 to detect MUC1, wherein: the sensor was used to detect the Co-MOFs current in the label remaining on the electrode by differential pulse voltammetry DPV in thionine electrolyte.

Technical Field

The invention belongs to the technical field of ratio type biosensor detection, and particularly relates to a ratio type sensor based on a DNA tetrahedron and a method for detecting MUC 1.

Background

In recent years, cancer has become one of the most serious diseases threatening the health of people around the world. Early diagnosis of cancer is critical for timely treatment and reduction of mortality. The presence of biomarkers can indicate the presence of certain diseases or infections, and has become increasingly important in clinical screening for cancer diseases. MUC1 is a transmembrane glycoprotein having a high molecular weight of more than 200KD [ Florea A, Taleat Z, Cristea C, et al. Label free MUC1 aptamers based on electrochemical displacement of gold nanoparticles on cultured electrons [ J ]. electrochemical Communications,2013,33:127 ions 130 ]. It has been used for the early diagnosis and biotherapy of Cancer because it is normally expressed abnormally on epithelial Cells of malignant tumor tissues, such as breast Cancer [ Jo H, Jin H, Ban C. Dual aptamer-functional silicon nanoparticles for the high sensitive detection of breast Cancer [ J ]. Biosensors & Bioelectronics,2015,71:129-, 1998,79(2):133-138.]. Because the content of MUC1 in the early stage of cell canceration is low and difficult to detect, the ultra-sensitive detection of MUC1 is very important for the early diagnosis of cancer, especially breast cancer.

Electrochemical biosensors have been widely used in various fields such as environmental analysis, food processing, and medical diagnosis. The instrument is widely concerned due to the characteristics of simple instrument, high sensitivity and the like. In recent years, rate-type sensors have become an intelligent choice for increasing the practical application of sensing systems, and can be used for specific assays between DNA and proteins. The ratiometric sensor provides a more accurate signal by eliminating the effects of variations in DNA loading concentration, temperature variations, and non-target induced DNA dissociation through an internal reference probe (IR), and the ratiometric response between Signaling Probes (SP) for built-in calibration. Currently, there are two types of operation of conventional ratiometric electrochemical DNA sensors. The first type immobilizes the signal molecule on the electrode. For example, Sheer et al designed a dual Signal Hairpin DNA-Based Ratiometric strategy for the detection of mucin 1 [ Deng C Y, Pi X M, Qian P, et al, high-Performance ratio metric electric Method Based on the Combination of Signal Probe and Inner Reference Probe in One Hairpin-Structured DNA [ J ]. Analytical chemistry,2017,89(1):966-973 ]. Ellington et al developed a ratiometric electrochemical DNA sensor for detecting Single Nucleotide Polymorphisms (SNPs) [ DuY, Lim B J, Li B L, et al. ratiometric electrochemical DNA sensors with improved robustness and reproducibility [ J ]. Analytical chemistry,2016,86(15): 8010-8016 ]. Two signal tags are generally required to label nucleic acids. One for IR and the other for SP. Typically, ferrocene and methylene blue are used as signal labels for ratiometric DNA electrochemical sensors. The second is the addition of IR molecules to the electrolyte solution. In contrast to the first type, only one electroactive species is required to be coupled to a detection probe (e.g., a nucleic acid). Therefore, the second type is more suitable for constructing a ratiometric electrochemical biosensor.

DNA is present in the nucleus in large amounts as a carrier of genetic information. According to the special space structure of DNA, self-assembly can be carried out according to the base complementary pairing principle, and meanwhile, the space structure has higher controllability and precision, so that the DNA nano material with various forms can be easily assembled. Compared with the traditional material, the DNA nano material has the advantages of easy penetration of negatively charged cell membranes, low toxicity, ribozyme resistance, high stability, abundant functional site modification according to needs and the like. The DNA tetrahedron is a DNA nanostructure composed of 4 paired single-stranded DNAs. It usually needs to design 4 single-stranded DNA base sequences, according to the base complementary pairing principle, 4 synthetic single-stranded DNA is added into buffer solution in equal amount, through one-step annealing operation, 4 single strands can be self-complementary hybridized to form a three-dimensional DNA structure with tetrahedral shape [ Chen X Q, Zhou G B, Song P, et al. ultrasensitive electronic detection of pro state-specific fluorescent by using anti-DNA and a DNA nanostructured scan [ J ]. Analytical chemistry,2014,86(15):7337 7342 ]. The DNA tetrahedron can effectively eliminate nonspecific adsorption of DNA and protein, and has strong anti-interference capability. The DNA tetrahedron with the three-dimensional (3D) scaffold can also adjust the space requirements of the probe, thereby facilitating the accessibility of the recognition probe while increasing the load capacity of the signal tag. And specific functional molecules can be modified at the 5 'or 3' end of the single strand of the DNA to realize the DNA tetrahedron functionalization.

Aptamers are single stranded RNA or DNA oligonucleotides that specifically and efficiently bind a range of proteins and cells [ Tuerk C, Gold L. systematic Evolution of Ligands by expression entity: RNA Ligands to Bacteriophage T4 DNA Polymerase [ J ] Science,1990,249(4968): 505) 510 ]. Compared with an antibody, the aptamer is more stable, stronger in adaptability and more flexible in modification. Therefore, it is desirable to develop a novel aptamer-based high sensitivity and selectivity quantitative MUC 1.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects of the prior art, a ratio type biosensor and a method for detecting MUC1 are provided, so that the sensitivity, reproducibility, accuracy and stability of MUC1 detection are improved.

The product ratio type biosensor is a sensor based on a DNA tetrahedron, and is characterized by being prepared by the following steps:

1. synthesizing DNA tetrahedron, and preparing Au NPs, Co-MOFs and BP absolute ethyl alcohol dispersion liquid.

2. Polishing the glassy carbon electrode GCE in sequence, cleaning the surface and drying; preferably, Al is used on chamois flannel₂O₃Polishing the powder for at least 3 times, and sequentially adding V_{Water (W)}:V_{Nitric acid}1:1 HNO₃Washing with a solution, washing with absolute ethyl alcohol, performing ultrasonic treatment with ultrapure water for 2.5-3.5 min, and drying.

3. And dropwise adding 9-11 mu L of Au NPs @ BP dispersion liquid and chitosan solution which are mixed in an equal volume to the surface of the GCE, and airing at room temperature.

4. Dropping the synthesized tetrahedral DNA on the GCE electrode washed in the step 3, cultivating overnight at 25-38 ℃, and washing with PBS buffer solution; preferably, the incubation is carried out at 37 ℃ overnight.

5. Dripping MCH with the concentration of 0.9-1.1 mM and the volume of 9-11 mu L on the surface of the electrode prepared in the step 4, culturing at 36-38 ℃ for 8-12 min, and sealing the non-specific binding sites; preferably, 10. mu.L MCH at a concentration of 1mM is added dropwise to the electrode surface prepared in step 4, incubated at 37 ℃ for 10min, and non-specific binding sites are blocked.

6. Dripping 9-11 mu L of MUC1 diluent with the concentration of 0.004 pM-400 pM on the surface of the electrode prepared in the step 5, and culturing for 0.8-1.2 h at the temperature of 36-38 ℃; preferably, 10 mu L of the electrode with the concentration of 0.004pM is sequentially dripped on the surface of the electrode prepared in the step 5; 0.02 pM; 0.04 pM; 0.4 pM; 2 pM; 4 pM; 20 pM; 40 pM; 200 pM; 400pM of MUC1 dilution was incubated at 37 ℃ for 1 h.

7. And (3) dripping 9-11 mu L of Au NPs @ Co-MOFs marked with signal probes on the surface of the electrode prepared in the step (6), and culturing at 36-38 ℃ for 0.8-1.2 h to finally obtain the DNA tetrahedron-based ratio type biosensor. Preferably, 10. mu.L of Au NPs @ Co-MOFs labeled with signal probes is dropped on the surface of the electrode prepared in step 6 and incubated at 37 ℃ for 1 h.

The methods for synthesizing DNA tetrahedrons and preparing Au NPs, Co-MOFs and BP absolute ethanol dispersions are the prior art reported in the prior publications.

The method for detecting MUC1 by the ratio type biosensor of the invention comprises the following steps: the prepared sensor was used to detect the Co-MOFs current in the label remaining on the electrode by differential pulse voltammetry DPV in thionine electrolyte.

The invention constructs a ratio-type electrochemical aptamer sensor based on DNA tetrahedron and is used for quantitative determination of MUC1 in real samples. The invention takes electrolyte solution thionine as a reference probe (I)_IR) Co-MOFs labelled aptamers as signaling probes (I)_SP) And constructing a ratio type electrochemical sensor. The ratio type electrochemical aptamer sensor has strong anti-interference capability, and can effectively eliminate interference caused by factors such as different DNA load densities, environmental influences, instrument efficiency and the like. The DNA aptamer is compared with a conventional ratio-type aptamer sensorThe sensor need only be bound to one electroactive species. The modification process can be greatly simplified. The constructed proportional aptamer sensor has high stability, even after ten repeated scans on one electrode, the current ratio (I)_SP/I_IR) Still remain unchanged. Thus, random errors are greatly reduced. In addition, DNANTH effectively eliminates nonspecific adsorption and improves detection precision. Black phosphorus having a large specific surface area and good conductivity is used as a sensing interface to further improve detection sensitivity. The biosensor has high sensitivity and a detection limit of 1.34 fM. Due to these superior properties, the proportional aptamer sensor achieves high reproducibility, accuracy, stability and sensitivity. Such a universal ratiometric sensor may be used to detect other analytes of interest based on the induced change in the aptamer.

The invention has the advantages that: compared with the traditional stem-loop or linear DNA probe, the invention utilizes the DNA tetrahedron, can increase the loading capacity of signal substances and effectively eliminate the nonspecific adsorption of DNA and protein. When three primer amplification strands H1/H2/DNA2 are introduced, the sensitivity of the biosensor is remarkably improved, and the characteristic can be used for constructing a novel double-signal amplification type biosensor. The experimental result shows that compared with the traditional method, the biosensing method has the advantages of improving the sensitivity, the reproducibility, the accuracy and the stability and being simple to operate.