阅读说明：本技术 一种铜纳米簇作为电化学信号探针的β-淀粉样蛋白寡聚体传感器 (Beta-amyloid oligomer sensor with copper nanocluster as electrochemical signal probe ) 是由 周艳丽 吕玉冰 董辉 刘澜涛 徐茂田 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种铜纳米簇作为电化学信号探针的β-淀粉样蛋白(Aβ)寡聚体传感器,属于新型功能纳米复合材料及生物传感器检测技术领域。本发明以金纳米粒子修饰的垂直石墨烯/碳布膜作为基底电极,以聚胸腺嘧啶为模板原位合成铜纳米簇作为电化学信号探针。通过Au-S键将朊蛋白固定在基底电极表面,作为Aβ寡聚体的受体；核酸适配体-T30-铜纳米簇偶联物通过核酸适配体识别Aβ寡聚体后引入了铜纳米簇作为电化学信号探针。实验表明：该方法构建的电化学生物传感器灵敏度高、检测限低、特异性高且稳定性好,在人血清样本中的检测中表现出可行性,对阿尔茨海默病的早期诊断和治疗具有重要意义。(The invention relates to a beta-amyloid (Abeta) oligomer sensor with a copper nanocluster as an electrochemical signal probe, belonging to the technical field of novel functional nano composite materials and biosensor detection. According to the invention, a gold nanoparticle modified vertical graphene/carbon cloth membrane is used as a substrate electrode, and poly-thymine is used as a template to synthesize a copper nano-cluster in situ as an electrochemical signal probe. The prion protein is fixed on the surface of a substrate electrode through an Au-S bond and is used as a receptor of the Abeta oligomer; the aptamer-T30-copper nanocluster conjugate introduces the copper nanoclusters as electrochemical signaling probes after the aptamer recognizes A beta oligomers. Experiments show that: the electrochemical biosensor constructed by the method has high sensitivity, low detection limit, high specificity and good stability, shows feasibility in detection in human serum samples, and has important significance for early diagnosis and treatment of Alzheimer's disease.)

1. An electrochemical sensor for detecting beta-amyloid oligomers, which is prepared by the following method:

(1) depositing gold nanoparticles on a vertical graphene-carbon cloth electrode: immersing a clean carbon cloth electrode which is vertical to the growth of the graphene into a chloroauric acid solution for constant potential electrodeposition;

(2) immobilization of aptamers and recognition of a β oligomers: immersing the gold nanoparticle modified vertical graphene/carbon cloth electrode prepared in the step (1) into a sulfhydrylation prion solution for incubation at room temperature, and sealing the surface of the electrode by using a 1-sulfhydrylation hexanol solution to eliminate non-specific binding sites to prepare a prion modified electrode; at room temperature, respectively incubating the prepared prion protein modified electrode with different concentrations of A beta oligomer solutions, and identifying the A beta oligomers;

(3) introducing a probe molecule: mixing aptamer-T30 with L-ascorbic acid at room temperature, and adding CuSO₄·5H₂Stirring the solution O at room temperature in a dark place to obtain an aptamer-poly T-copper nanocluster conjugate solution, and incubating the solution and the electrode after the A beta oligomer is identified in the step (2) together to obtain the A beta oligomer electrochemical sensor;

the amino acid sequence of the thiolated prion protein in the step (2) is as follows: HS-ThrHisSerGlnTrpAsnLysProSerLysProLysThrAsnMetLys;

the DNA sequence of the aptamer-T30 in the step (3) is as follows: GCCTGTGGTGTTGGGGCGGGTGCG-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT.

2. The electrochemical sensor for detecting oligomers of β -amyloid protein according to claim 1, wherein: the time and the potential for the gold nano-particle electrochemical deposition in the step (1) are respectively 100s and-0.1V, and the concentration of the chloroauric acid solution is 20 mmol.L^-1。

3. The electrochemical sensor for detecting oligomers of β -amyloid protein according to claim 1, wherein: the concentration of prion protein in the step (2) is selected to be 4 mu mol. L^-1。

Technical Field

The invention relates to an electrochemical biosensor for ultrasensitive detection of beta-amyloid oligomer and a preparation method thereof, in particular to an electrochemical sensor prepared by taking gold nanoparticle modified vertical graphene/carbon cloth as a substrate electrode and taking polythymine as a copper nanocluster of a synthetic template as a signal probe, belonging to the technical field of biosensing and electroanalytical chemical detection.

Background

Alzheimer's Disease (AD), one of the most common progressive neurodegenerative diseases, has become a very serious social health problem. The pathological features of AD are caused by the deposition of extracellular plaques consisting of beta-amyloid (a β). Small molecule soluble oligomers are the major neurotoxic species in different aggregation states of a β. Therefore, Α β oligomers are considered as reliable molecular biomarkers for early diagnosis of AD.

Accurate detection of a β oligomers is a great challenge due to the extremely low concentration of a β oligomers in human body fluids and the tendency to form heterogeneous mixtures. In order to solve these problems and to improve analytical performance, many novel functional nanomaterials having excellent properties, such as metal oxides, carbon nanoparticles, quantum dots, and magnetic nanomaterials, are introduced into electrochemical, fluorescent, colorimetric, and other analytical methods. Particularly, copper-based materials are used as electrode modifications of electrochemical sensors because of their reasonable price, abundant reserves, and unique electrochemical properties. Despite the unique electrochemical properties of copper-based metal organic frameworks or copper oxides, signal transduction remains a significant problem for copper-based materials in electrochemical sensor construction applications. Therefore, starting from the design and controllable preparation of the copper-based material, the electrochemical sensor for detecting the A beta oligomer with high sensitivity is constructed, and the realization of the detection of the A beta oligomer in body fluid has important significance for early diagnosis and disease control of AD.

The process for synthesizing the copper nanocluster by using the poly-thymine (poly-T) as the template is simple, and the synthesized copper nanocluster has fluorescence and electrochemical activity. On the other hand, successful screening of aptamers to a β oligomers offers the possibility of specificity of detection in the construction of a β oligomer detection sensors. The aptamer is obtained by in vitro screening in an exponential enrichment mode and is a single-stranded oligonucleotide consisting of 20-60 bases. Therefore, through the design of the aptamer, a poly-T sequence can be modified, and the poly-T is further used as a template to synthesize the electrochemically active copper nanocluster, and the connected aptamer can realize the recognition of the A beta oligomer.

Therefore, the design of aptamer containing poly-T modification for copper nanocluster synthesis, the application of the aptamer in detecting signal amplification in A beta oligomer electrochemical biosensor, and the preparation of electrochemical biosensor with high sensitivity, high selectivity and high stability are important for understanding the role of A beta oligomer as AD marker.

Disclosure of Invention

Based on the prior art, the invention provides a sensor for detecting the Abeta oligomer with high sensitivity and high selectivity; another object is to provide a process for the preparation thereof.

In order to realize the purpose of the invention, the gold nanoparticle modified vertical graphene/carbon cloth film is used as a substrate electrode, and the in-situ synthesized copper nanocluster is used as a signal probe to construct the electrochemical sensor for double amplification signals for the ultrasensitive detection of the A beta oligomer.

The following technical scheme is adopted specifically:

(1) immersing a clean carbon cloth for vertical graphene growth into a chloroauric acid solution for constant potential electrodeposition to obtain a gold nanoparticle modified vertical graphene/carbon cloth film which is used as a substrate electrode;

(2) immersing the substrate electrode prepared in the step (1) into a sulfhydrylation prion solution for incubation at room temperature, and after the prion and the gold nanoparticles are combined through Au-S bonds, sealing the electrode by using a 1-sulfhydryl hexanol solution to eliminate non-specific binding sites; respectively incubating the prepared modified electrode with Abeta oligomers with different concentrations at room temperature, and realizing specific recognition of the Abeta oligomers by utilizing the specificity of the prion protein to a target substance;

(3) mixing aptamer-T30 with L-ascorbic acid at room temperature, and adding CuSO₄·5H₂O solution, chamberStirring in a warm and dark place to obtain an aptamer-poly T-copper nanocluster conjugate solution;

(4) and (3) incubating the electrode after the A beta oligomer is identified in the step (2) with the aptamer-poly T-copper nano cluster conjugate solution, and introducing the copper nano cluster to the surface of the electrode through the combination of the aptamer and the A beta oligomer on the surface of the electrode to prepare the A beta oligomer electrochemical sensor.

When the method is applied, differential pulse voltammetry measurement is carried out, and quantitative determination of the A beta oligomer is realized through the relation between the redox peak current change value of the copper nanocluster and the concentration of the A beta oligomer. Preferably 0.1 mol. L at pH 7.4^-1Phosphate buffered solution.

Further, the gold nanoparticles modified vertical graphene/carbon cloth in the step (1) has a constant potential of-0.1V and a time of 100s, and the concentration of the chloroauric acid solution is 20 mmol.L^-1。

Further, the sequence of the A beta oligomer aptamer-T30 is GCCTGTGGTGTTGGGGCGGGTGCG-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT, and the sequence of the prion protein is: HS-ThrHisSerGlnTrpAsnLysProSerLysProLysThrAsnMetLys utilizes the strong interaction and high specificity of the two and the A beta oligomer, and can improve the selectivity of detecting the A beta oligomer.

Furthermore, the experimental conditions are optimized, and the concentration of copper particles is 0.5 mmol.L when the copper nanocluster synthesis is carried out in the step (3)^-1(ii) a The concentration of prion protein in the step (2) is 4 mu mol. L when being fixed^-1。

Compared with the prior art, the invention has the following beneficial effects:

(1) the gold nanoparticle modified vertical graphene/carbon cloth substrate prepared by the method has a large surface area (3 times of that of a planar electrode), so that the electronic conduction is promoted, and an amplified electrochemical signal is provided.

(2) According to the invention, the copper nanocluster taking poly-T as a template is taken as a signal probe, so that secondary signal amplification can be realized, and the detection has good sensitivity.

(3) The invention utilizes the high affinity of the prion protein and the aptamer to the Abeta oligomer, so that the Abeta oligomer can be captured in a sandwich structure with high selectivity, and the high specificity of the sensing strategy is ensured by double recognition of the prion protein and the aptamer to a target molecule, and the use of expensive enzyme-labeled antibody reagents is avoided.

(4) The electrochemical sensor adopts a flexible carbon cloth electrode substrate, and is easy to miniaturize; and the electrochemical signals of the substrate electrode and the copper nanocluster have high stability, so that the substrate electrode and the copper nanocluster have high repeatability and stability.

(5) The electrochemical sensor realizes the high-sensitivity and high-selectivity determination of the A beta oligomer, and the detection limit is 3.5 pmol.L^-1The method can be used for measuring the A beta oligomer in human serum, has important significance for the auxiliary diagnosis and treatment of AD, is an electrochemical biosensor with high sensitivity, high selectivity and high stability, and has good development and application prospects.

The DNA sequence of the aptamer-T30 of the Abeta oligomer and the amino acid sequence of the prion protein are shown in a sequence table.

Drawings

FIG. 1 is a technical scheme of the present invention.

FIG. 2 is a fluorescence spectrum (a) and a transmission electron microscope (b) of the copper nanocluster electrochemical probe prepared in the present invention, wherein (1) to (2) in a represent fluorescence spectrum curves before and after the aptamer-poly T template is added, respectively.

FIG. 3 is a scanning electron microscope before and after gold nanoparticle modification of the vertical graphene/carbon cloth film of the present invention.

Fig. 4 is a representation diagram of impedance and cyclic voltammetry in the preparation process of the sensor of the present invention, wherein (1) - (5) represent a vertical graphene/carbon cloth electrode, a gold nanoparticle-modified vertical graphene/carbon cloth electrode, an electrode after prion protein fixation, an electrode for identifying a β oligomers, and an electrode after copper nanoclusters are introduced, respectively.

Fig. 5 is the response of the sensor of the invention to a β oligomers: a is a measured differential pulse voltammogram; b is a standard curve of the current change value along with the change of concentration; c is a selectivity chart of the electrochemical sensor of the invention, and (1) to (7) respectively represent A beta oligomer, blank, A beta fiber, A beta monomer, insulin, human tumor necrosis factor and C-reactive protein; d is the stability of the electrochemical sensor of the invention: (1) - (2) represent responses before and after two weeks of standing, respectively.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples, which are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention in any way.

Example 1 preparation method of aptamer electrochemical sensor for detecting A beta oligomer based on Au-VG/CC substrate and poly T template CuNPs dual signal amplification strategy

(1) Deposition of gold nanoparticles on a vertical graphene/carbon cloth electrode: soaking the clean vertical graphene/carbon cloth membrane in 5mL of 20 mmol.L^-1And in a chloroauric acid solution, performing electrodeposition for 100s under a constant potential of-0.1V to obtain the gold nanoparticle modified vertical graphene/carbon cloth electrode.

(2) Immobilization of prion protein and recognition of a β oligomers: gold nanoparticle modified vertical graphene/carbon cloth electrode and 10 mu L of 4 mu mol/L^-1HS-PrP^CThe solution was incubated for 6h and then with 4 mmol. multidot.L^-1And sealing the surface of the electrode for 20min by using a 1-hexanethiol solution to prepare the prion protein modified sensor. The modified sensor was incubated with a solution containing different concentrations of a β oligomers for 4h, and then the electrodes were rinsed with pure water.

(3) Preparation and introduction of probe molecules: at room temperature, 25. mu.L of 100. mu. mol. L^-1Aptamer of (4) -T30 and 25. mu.L of 300 mmol.L^-1After fully mixing ascorbic acid, 2.5. mu.L of 500 mmol. multidot.L was added^-1CuSO₄·5H₂And (4) O solution. Stirring for 5min in the dark to obtain the aptamer-poly T-copper nano cluster biological conjugate. The prepared solution was diluted 40-fold in MOPS buffer solution at pH 7.6 and its fluorescence spectrum was measured with Cary Eclipse fluorescence spectrophotometer from Agilent Technologies. The optical path length, excitation wavelength, emission spectrum recording range, excitation slit and emission slit of the quartz colorimeter were set to 5nm, 1.0cm, 345nm, 550nm and 5nm, respectively. And (3) incubating the solution and the electrode after the A beta oligomer is identified in the step (2) together to prepare the A beta oligomer electrochemical sensor.

Application example 1 assay of Abeta oligomers with the aptamer electrochemical sensor of the present invention

The potential test adopts a three-electrode system, the modified electrode of the invention is used as a working electrode, a platinum wire is used as a counter electrode, and Ag/AgCl is used as a reference electrode. At 0.1 mol. L^-1In a phosphate (pH 7.4) buffer solution, recording a voltammetric response signal of a sensor by using a differential pulse voltammetry method in a range of 0.4V to-0.2V under the conditions of a pulse amplitude of 0.004V, a pulse width of 0.1 mus and a sampling width of 0.0167 mus, measuring standard solutions of Abeta oligomers with different concentrations, calculating the change values of the peak current of a probe molecule copper nano cluster and the blank solution current after the Abeta oligomers are added, and drawing a standard curve by using the current change value-concentration. The current change value (. DELTA.I) is such that the concentration (C) of A.beta.oligomers is 10 to 2200 pmol. L^-1Is proportional to the correlation coefficient R in the range of²0.998, detection limit of 3.5 pmol. multidot.L^-1. The electrochemical sensor is proved to have high sensitivity when being used for determining the A beta oligomer.

And during actual serum sample measurement, determining the concentration of the A beta oligomer in the human serum according to the current change value and based on the linear equation, thereby realizing quantitative detection.

Application example 2 Performance examination of electrochemical sensor

To investigate the stability of the sensor of the invention, 2.5 nmol.L of the same sensor pair was used^-1The a β oligomer of (a) was measured in 7 replicates, and the relative standard deviation of the current response was 4.8%. The measurement of a β oligomers with 6 independent sensors was performed in a reproducibility test with a relative standard deviation of 2.6%. To investigate the long-term stability of the electrochemical assay, the voltammetric response maintained 91.8% of the initial signal response by measuring the change in voltammetric response after 2 weeks storage in a refrigerator at 4 ℃. The results show that the method has better stability due to the stability of the substrate electrode and the microenvironment of biocompatibility. In addition, an unstable fluorescence emission signal is replaced by an electrochemical signal of the copper nanocluster, and long-time and stable detection application is guaranteed.

To evaluate the specificity of the sensor to A beta oligomers, the potential interfering substances include A beta monomers, A beta fibersAnd three representative interfering substances in serum, tumor necrosis factor-alpha, insulin and C-reactive protein, and in the presence of the interfering substances, the electrochemical signals of other interfering substances are very low compared with that of the target A beta oligomer, indicating PrP^CThere was no significant binding to the other substrates mentioned above. This good specificity may be due to the A.beta.oligomers in combination with PrP^CAnd a specific recognition property between aptamers.

Application example 3 assay of A beta oligomers in serum samples

To evaluate the utility of electrochemical sensors for the detection of a β oligomers, human serum samples were tested and recovery rates were determined by standard addition methods on serum samples from healthy persons and AD patients. As shown in Table 1, the concentrations of A.beta.oligomers in the sera of 2 healthy persons (samples 1 and 2) and 2 AD patients (samples 3 and 4) were 72, 68, 97 and 90 pmol. multidot.L^-1Consistent with the levels of human serum a β oligomers in the literature. When the amount of the A beta oligomer added was 100 pmol. multidot.L, 500 pmol. multidot.L, 1000 pmol. multidot.L, respectively^-1And the recovery rate is between 93 and 108 percent, and the relative standard deviation is less than 7 percent. These results indicate that the electrochemical sensor has good accuracy and practical application potential in clinical diagnosis.

TABLE 1 assay of A.beta.oligomers in human serum samples

Sequence listing

< 110> Shangqiu college of education

<120> beta-amyloid oligomer sensor using copper nanocluster as electrochemical signal probe

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<210>1

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<212>DNA

<213> Artificial sequence

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<221> aptamer-T30

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gcctgtggtg ttggggcggg tgcgtttttt tttttttttt tttttttttt tttt 54

<210>2

<211>16

<212>PRT

<213> Artificial sequence

<220>

<221> prion protein

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Thr His Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys

1 5 10 15