CN110333277B

CN110333277B - Aptamer sensor and preparation method thereof

Info

Publication number: CN110333277B
Application number: CN201910642147.3A
Authority: CN
Inventors: 杜淼; 何领好; 贾巧娟; 李真真; 张治红; 王明花; 宋英攀
Original assignee: Zhengzhou University of Light Industry
Current assignee: Zhengzhou University of Light Industry
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2021-11-23
Anticipated expiration: 2039-07-16
Also published as: CN110333277A

Abstract

The invention relates to an aptamer sensor and a preparation method thereof, and belongs to the technical field of electrochemical sensors. The invention provides an aptamer sensor, which comprises an electrode, an electrode material for the aptamer sensor coated on the surface of the electrode, and a nucleic acid aptamer attached to the electrode material for the aptamer sensor; the electrode material for the aptamer sensor is a nickel cobalt Prussian blue analogue. The aptamer sensor has good selectivity, stability, reproducibility and applicability, and the detection limit is low. For example, aptamer sensors from bare gold electrodes, NiCo Prussian blue analogs, and carcinoembryonic antigen aptamers were at 1.0 fg. mL^‑1‑5.0ng·mL^‑1Has high sensitivity and selectivity for CEA detection, and lower detection limit of only 0.74 fg. mL^‑1(1.62fM), and broadens the application of the nickel cobalt Prussian blue analogue.

Description

Aptamer sensor and preparation method thereof

Technical Field

The invention relates to an aptamer sensor and a preparation method thereof, and belongs to the technical field of electrochemical sensors.

Background

Carcinoembryonic antigen (CEA) is considered as a tumor marker, plays an important role in the diagnosis and screening of malignant tumors including lung cancer, breast cancer, gastrointestinal cancer and ovarian cancer since the carcinoembryonic antigen (CEA) is first proved to be a tumor-associated antigen in 1965, and particularly plays a key role in colorectal cancer surgery because clinical research shows that the CEA level is increased to indicate the tumor recurrence. Therefore, it is important to develop a precise quantitative method for determining CEA. Various methods have been developed for the detection of CEA, including colorimetric immunoassays, electrochemical immunoassays, electrochemiluminescent immunoassays, fluorescent immunoassays, and chemiluminescent immunoassays. However, these methods generally have disadvantages of low sensitivity, complicated test procedures, and poor reproducibility.

The electrochemical biosensor has the advantages of low equipment price, easy construction, strong flexibility and high sensitivity, the key point for constructing the electrochemical biosensor is to fix the antigen on the nano material platform to capture the target analyte, and the design of the nano material platform aims to improve the antigen binding affinity to ensure the stability of the biosensor. Aptamers are artificial single-stranded DNA or RNA sequences, high affinity and specificity of binding of aptamers to targets, and high selectivity, a key step in the development of high performance adaptive sensors (aptamer sensors) is the immobilization of aptamers on the sensor surface. Therefore, finding suitable aptamer sensors with electrode materials as a support to increase aptamer loading is very important to couple aptamers to the electrodes.

Disclosure of Invention

A first object of the present invention is to provide an aptamer sensor having a strong electrochemical sensing performance.

The second purpose of the invention is to provide a preparation method of the aptamer sensor.

The technical scheme of the invention is as follows:

an aptamer sensor comprises an electrode, an electrode material for the aptamer sensor coated on the surface of the electrode, and a nucleic acid aptamer attached to the electrode material for the aptamer sensor;

the electrode material for the aptamer sensor is a nickel cobalt Prussian blue analogue.

It is understood that the NiCo Prussian blue analog is abbreviated NiCo PBA is of the chemical formula Ni₃[Co(CN)₆]₂Or cobalt nickel cyanate with water of crystallization, e.g. Ni₃[Co(CN)₆]₂·12H₂O。

The aptamer sensor comprises an electrode, an electrode material (nickel cobalt Prussian blue analogue) for the aptamer sensor and a nucleic acid aptamer, wherein the nickel cobalt Prussian blue analogue has stronger biological affinity, large specific surface area, good biocompatibility and higher stability, and an inherent cavity, a metal coordination center, a C [ ident ] N group and a pi-pi [ pi ] bond of the nickel cobalt Prussian blue analogue, so that the nucleic acid aptamer and the nickel cobalt Prussian blue analogue can be attached to a nanocube of the nickel cobalt Prussian blue analogue through strong interaction, and the aptamer sensor has good selectivity, stability, reproducibility and applicability and low detection limit. For example, aptamer sensors derived from bare gold electrodes, NiCo Prussian blue analogs, and carcinoembryonic antigen aptamers have good selectivity, stability, reproducibility, and applicability at 1.0 fg. multidot.mL^-1-5.0ng·mL^-1Has high sensitivity and selectivity for CEA detection, and lower detection limit of only 0.74 fg. mL^-1(1.62fM), and in addition, the detection of H460 cancer cells has good sensitivity and lower detection limit, which is only 47cell mL^-1. Aptamer sensors obtained from the nickel cobalt prussian blue analogues provide significant and promising potential in early diagnosis of tumors, and the application of the nickel cobalt prussian blue analogues is widened.

The nickel cobalt prussian blue analogue can be obtained by purchasing or preparing by a preparation method in the prior art, and preferably, the nickel cobalt prussian blue analogue is prepared by a method comprising the following steps: adding cobalt cyanate aqueous solution into aqueous solution of nickel salt and citrate, and reacting to obtain the nickel cobalt Prussian blue analogue.

The nickel cobalt Prussian blue analogue obtained by adding the cobalt cyanate aqueous solution into the aqueous solution of the nickel salt and the citrate for reaction has stronger biological affinity, large specific surface area, good biocompatibility and higher stability, and the nucleic acid aptamer and the nickel cobalt Prussian blue analogue can be attached to a nanocube of the nickel cobalt Prussian blue analogue through strong interaction due to an inherent cavity, a metal coordination center, a C [ identical to ] N group and a pi-pi bond of the nickel cobalt Prussian blue analogue.

Preferably, the aptamer is a carcinoembryonic antigen aptamer. The aptamer sensor obtained by attaching the carcinoembryonic antigen aptamer to the electrode material for the aptamer sensor can be used for detecting carcinoembryonic antigen and H460 cells.

Preferably, the coating amount of the electrode material for the aptamer sensor on the surface of the electrode is 0.07-6.37 mu g/mm². The coating amount of the electrode surface is 0.07-6.37 mu g/mm²The electrode material for the aptamer sensor is beneficial to fixing the nucleic acid aptamer and expanding the detection range of CEA.

Preferably, the electrode is a bare gold electrode or a glassy carbon electrode.

A method of making an aptamer sensor, comprising the steps of:

and coating the aptamer sensor on the surface of the electrode by using the electrode material suspension, drying to obtain a modified electrode, and then attaching the aptamer on the modified electrode to obtain the aptamer sensor.

According to the preparation method of the aptamer sensor, the aptamer sensor can be prepared only by coating the nickel cobalt Prussian blue analogue suspension on the surface of the electrode and then attaching the aptamer on the surface of the modified electrode, and the preparation method is high in preparation efficiency, simple and strong in operability.

Preferably, the concentration of the electrode material suspension for the aptamer sensor is 1-9 mg/mL. The concentration is 1-9mg/mL^-1The aptamer sensor obtained by coating the NiCo PBA suspension on the surface of AE has better performance, if the concentration of the NiCo PBA suspension is too large, the thickness of the NiCo PBA layer is too thick, and the result is that the aptamer sensor detects the delta R before and after CEA_ctThe value decreases due to exfoliation caused by too large a layer thickness of NiCo PBA. If the concentration of the NiCo PBA suspension is too low, the modification amount of the NiCo PBA on the AE surface is insufficient, so that enough nucleic acid aptamers cannot be immobilized, and sufficient nucleic acid aptamers cannot be provided when detecting CEAThe aptamer is fixed with CEA, so that the obtained aptamer sensor detects delta R before and after CEA_ctThe value decreases.

Preferably, the concentration of the electrode material suspension for the aptamer sensor is 1-2 mg/mL. By reasonably adjusting the concentration of the electrode material suspension for the aptamer sensor to be 1-2mg/mL, enough NiCo PBA can be modified on the surface of AE, and then a large amount of aptamers can be fixed, so that when the aptamer sensor detects CEA, enough aptamers are fixed with CEA, and the sensitivity, stability and reproducibility of the aptamer sensor are improved.

Preferably, the nucleic acid aptamer is attached to the modified electrode by a method comprising the steps of:

and (3) incubating the modified electrode in the aptamer solution, taking out and drying to obtain the aptamer sensor.

Preferably, the concentration of the aptamer solution is 5-200 nM. The aptamer solution with the concentration of 5-200nM can fix a sufficient amount of aptamer on the modified electrode and reduce the cost.

Preferably, the concentration of the aptamer solution is 50-100 nM. The aptamer solution with the concentration of 50-100nM can better enable the modified electrode to fix a sufficient amount of aptamer, and further control the cost. If the concentration of the aptamer solution is more than 100nM, although the overall performance of the aptamer sensor is not reduced, it is disadvantageous to reduce the cost, and if the concentration of the aptamer solution is less than 50nM, it is disadvantageous to attach a sufficient amount of aptamer to the surface of the modified electrode.

Preferably, the incubation temperature is 2-5 ℃ and the incubation time is 1.5-2.5 h. Through reasonable setting and optimization incubation temperature and time, the aptamer is more favorably attached to the modified electrode.

Drawings

FIG. 1 is an XRD pattern and FT-IR spectrum of NiCo PBA obtained in step (1) of example 1;

FIG. 2 is a high resolution XPS spectrum of C1s and N1s of NiCo PBA obtained in step (1) of example 1;

FIG. 3 is a surface topography map of NiCo PBA obtained in step (1) of example 1;

FIG. 4 shows the difference Δ R of the embodiment of the present invention_ctA visualization of (a); wherein a is the bare gold electrode before and after modification of the electrodes R of examples 1-6_ctDifference of value Δ R_ct(ii) a b is the electrode R of AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE of examples 1-5_ctDifference of value Δ R_ct(ii) a c front and rear electrodes R of Apt/NiCo PBA/AE for detecting CEA of example 1 and example 7-example 10_ctDifference of value Δ R_ct(ii) a d is the electrode R of AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE of example 1 and example 7-example 10_ctDifference of value Δ R_ct；

FIG. 5 is the electrochemical signal response of AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE of example 11; a is the electrochemical signal response of the EIS test method to the detection of AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE in example 11; b is the electrochemical signal response of the CV test method to the detection of AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE in example 11;

FIG. 6 is an EIS response of the aptamer sensor of example 11 to different concentrations of CEA;

FIG. 7 is a graph showing Δ R before and after the detection of CEA at different concentrations by the aptamer sensor obtained in example 11_ctValue versus CEA concentration;

FIG. 8 is the selectivity, reproducibility, stability and reproducibility of detection of CEA by the Apt/NiCo PBA/AE aptamer sensor of example 11; a is the selectivity of the Apt/NiCo PBA/AE aptamer sensor for detecting CEA; b is the reproducibility of detecting CEA by an Apt/NiCo PBA/AE aptamer sensor; c, detecting the stability of the CEA by using an Apt/NiCo PBA/AE aptamer sensor; d is the reproducibility of detecting CEA by an Apt/NiCo PBA/AE aptamer sensor;

FIG. 9 is an EIS curve of the procedure for detecting H460 live cells and L929 cells by the aptamer sensor of example 11; a is an EIS curve of the aptamer sensor of example 11 on the detection process of H460 living cells; b is the EIS curve of the aptamer sensor of example 11 on the L929 cell detection process;

FIG. 10 is an EIS plot of the aptamer sensor of example 11 against different concentrations of H460 cells;

FIG. 11 is a graph showing Δ R before and after detection of H460 cells at different concentrations by the aptamer sensor obtained in example 11_ctValue versus H460 cell concentration; a is Δ R_ctRelationship to H460 cell concentration; b is DeltaR_ctA linear fit to the logarithm of H460 cell concentration;

FIG. 12 is the reproducibility and stability of the Apt/NiCo PBA/AE aptamer sensor of example 11 for detecting H460 cells; a is the reproducibility of detecting H460 cells by an Apt/NiCo PBA/AE aptamer sensor; b is the stability of the Apt/NiCo PBA/AE aptamer sensor for detecting H460 cells.

Detailed Description

The present invention will be further described with reference to the following embodiments.

According to the preparation method of the aptamer sensor, the electrode material for the aptamer sensor is dispersed in ethanol to obtain the electrode material suspension for the aptamer sensor.

Preferably, the concentration of the electrode material suspension for the aptamer sensor is 1 mg/mL.

In the method for preparing the aptamer sensor, the coating amount of the electrode material suspension for the aptamer sensor on the surface of the electrode is 5.0 mu L.

In the preparation method of the aptamer sensor, the drying time of the modified electrode is 3-8 h. The drying temperature was room temperature. The drying temperature may be 20-30 ℃.

The water was Milli-Q water (resistivity at 25 ℃ C. was 18.2. omega. cm).

Reagents and materials:

nickel nitrate (Ni (NO)₃)₂·6H₂O)、K₃[Co(CN)₆]Purchased from alatin chemicals, ltd (shanghai, china). Sodium citrate was purchased from national pharmaceutical group chemical agents, ltd.

Carcinoembryonic antigen (CEA), human epidermal growth factor receptor-2 (HER2), mouse immunoglobulin G (IgG), Bovine Serum Albumin (BSA), mucin-1 (MUC-1), Prostate Specific Antigen (PSA), VEGR and human serum were purchased from Solebao bioengineering, Inc. (Beijing, China).

The chemical reagents used were analytical reagent grade and were used without further purification.

Ultrapure water (resistivity at 25 ℃ C. was 18.2. omega. cm) was used throughout the experiment.

The carcinoembryonic antigen aptamer is derived from Biyunshi biotechnology, and has the following sequence: 5'-CCACGATACCAGCTTATTCAATTCGTGG-3' are provided.

Bare gold electrodes were purchased from Gaoss Union instruments and had a diameter of 3 mm.

Preparation of the solution: a nucleic acid aptamer solution was prepared using a phosphate buffer (pH 7.4, 0.01M). Nucleic acid aptamer (100. mu.M), human epidermal growth factor receptor-2 (HER2), mouse immunoglobulin G (IgG), Bovine Serum Albumin (BSA), mucin-1 (MUC-1), Prostate Specific Antigen (PSA), Vascular Endothelial Growth Factor (VEGF), and CEA (1 mg. mL)^-1) The stock solution of (2) was prepared with the above phosphate buffer solution and stored at 4 ℃. The solution was diluted with buffer to obtain the desired concentration. Human serum was diluted 100-fold with 0.01M phosphate buffer (pH 7.4) and spiked with different amounts of CEA for actual sample analysis.

The specific embodiment of the preparation method of the aptamer sensor is as follows:

example 1

The preparation method of the aptamer sensor comprises the following steps:

(1) preparation of nickel cobalt prussian blue analogue

0.6mM (174mg) nickel nitrate and 0.6mM (265mg) sodium citrate were dissolved in 20mL deionized water at room temperature to form solution A.

Potassium (iii) hexacyanocobaltate (134mg) was dissolved in 20mL of deionized water at room temperature to form solution B.

And dropwise adding the solution B into the solution A at 37 ℃ while stirring until a precipitate is formed, separating to obtain a precipitate, washing the precipitate with ethanol for three times, and drying in a vacuum oven at 60 ℃ for 10 hours to obtain the NiCo Prussian blue analogue, namely NiCo PBA.

(2) Pretreatment of bare gold electrodes

First, bare gold electrodes were polished with 0.05mm alumina slurry and rinsed with ultra-pure water.

Then, in the piranha solution (v/v, H)₂SO₄：H₂O₂7: 3) rinsed for 15min, rinsed with ultra pure water and dried under nitrogen.

Finally, H at 0.5M by potential cycling between-0.2V to 1.6V₂SO₄And (3) electrochemically washing the bare gold electrode, then rinsing with ultrapure water and drying under nitrogen to obtain the pretreated bare gold electrode, and recording as AE.

(3) Preparation of modified electrode

Dispersing NiCo PBA obtained in the step (1) in ethanol to form a solution with the concentration of 1mg/mL^-1A suspension of (a).

And (3) coating 5 mu L of NiCo PBA suspension on the surface of the AE obtained in the step (2), and drying at room temperature for 6h to obtain a modified electrode which is recorded as NiCo PBA/AE.

(4) Preparation of aptamer sensor

The carcinoembryonic antigen aptamer was dissolved in a phosphate buffer (pH 7.4, 0.01M) to obtain an aptamer solution having a carcinoembryonic antigen aptamer concentration of 100 nM.

And (4) incubating the NiCo PBA/AE obtained in the step (3) in 100nM aptamer solution for 2h at 4 ℃ to ensure that the NiCo PBA/AE is immobilized by the aptamer to obtain an aptamer sensor which is marked as Apt/NiCo PBA/AE.

Example 2 to example 6

The aptamer sensors of examples 2 to 6 were prepared in the same manner as in example 1 except that the concentration of NiCo PBA in step (3) was different from that in example 1, and the concentrations of NiCo PBA in the remaining steps are shown in table 1 in example 1 and examples 2 to 6.

TABLE 1 concentration of NiCo PBA in example 1 and examples 2-6

Example 7 example 10

Example 7-example 10 the aptamer sensor was prepared in a manner different from example 1 in the concentration of the aptamer solution in step (4), and the concentrations of the aptamer solutions in the other steps, which are the same as those in example 1, example 1 and example 7-example 10, are shown in table 2.

Table 2 concentration of aptamer solutions in example 1 and example 7-example 10

	Concentration of aptamer solution (nM)
		Example 1	100
Example 7	10
		Example 8	25
Example 9	50
		Example 10	200

Example 11

The preparation method of the aptamer sensor comprises the following steps:

(1) preparation of nickel cobalt prussian blue analogue

(2) Pretreatment of bare gold electrodes

(3) Preparation of modified electrode

NiCo PBA was dispersed in ethanol to a concentration of 1 mg. mL^-1NiCo PBA suspension.

mu.L of NiCo PBA suspension was coated on the AE surface and dried at room temperature for 6h to give a modified electrode, designated NiCo PBA/AE.

(4) Preparation of aptamer sensor

The carcinoembryonic antigen aptamer was dissolved in a phosphate buffer (pH 7.4, 0.01M) to obtain an aptamer solution having a carcinoembryonic antigen aptamer concentration of 50 nM.

And (2) incubating the NiCo PBA/AE obtained in the step (1) in a 50nM aptamer solution at 4 ℃ for 2h to ensure that the NiCo PBA/AE is immobilized by the aptamer to obtain an aptamer sensor which is marked as Apt/NiCo PBA/AE.

Example 12

The preparation method of the aptamer sensor comprises the following steps:

(1) preparation of modified electrode

NiCo PBA was dispersed in ethanol to a concentration of 2 mg. mL^-1NiCo PBA suspension.

(2) Preparation of aptamer sensor

Second, the embodiments of the aptamer sensor of the present invention correspond to the end products of the methods for manufacturing the aptamer sensors, examples 1 to 12, respectively.

Third, related test example

Basic characteristics of test example 1NiCo PBA

The crystal and chemical structure of NiCo PBA obtained in step (1) of example 1 were characterized by XRD, FT-IR and XPS, and the obtained results are shown in fig. 1 and fig. 2, and the surface morphology of NiCo PBA obtained in step (1) of example 1 was characterized by TEM, HR-TEM and FE-SEM, and the obtained results are shown in fig. 3.

1、XRD

In fig. 1, (a) is an XRD pattern of NiCo PBA, and diffraction peaks at 2 θ ═ 15.07 °, 17.50 °, 24.78 °, 35.28 °, 39.66 °, 43.60 °, 50.82 °, 54.10 °, 57.27 °, and 66.31 ° correspond to crystal planes of (111), (200), (220), (400), (420), (422), (440), (600), (620), and (640) NiCo PBA, respectively.

2、FT-IR

In FIG. 1, (b) is a FT-IR spectrum of NiCo PBA, which is at 2160cm as shown in FIG. 1(b)^-1And 2146cm^-1The characteristic peak at (a) is due to the bridging C ≡ N group. 3420cm^-1And 1610cm^-1The broad band at (a) is related to v (-OH) stretching of the hydroxide groups of the crystal water in the layer and interlayer regions, respectively. 1615cm^-1Assignment of nearby absorption peaksAsymmetric stretching vibration at carbonyl.

3、XPS

FIG. 2(a) is a high resolution XPS spectrum of C1s for NiCo PBA and FIG. 2(b) is a high resolution XPS spectrum of N1s for NiCo PBA. As shown in fig. 2, in the high resolution C1s XPS spectra, peaks located at 285.2, 286.3 and 288.5eV correspond to CN, CO and COO groups, respectively. For XPS spectra of high resolution N1s, only one major peak with Binding Energy (BEs) of 398.4eV was observed, due to CN.

The results of XRD, FT-IR and XPS indicate the successful synthesis of NiCo PBA, which contains Co³⁺、Ni²⁺And a CN-containing functional group.

4. TEM, HR-TEM and FE-SEM

In FIG. 3, (a) is a low-magnification FE-SEM image of NiCo PBA, (b) is a high-magnification FE-SEM image of NiCo PBA, (c) is a low-magnification TEM image of NiCo PBA, (d) is a high-magnification TEM image of NiCo PBA, and (e) is a HR TEM image of NiCo PBA.

Fig. 3 shows that NiCo PBA has a typical nano-cubic shape with uniform distribution (fig. 3a), while high magnification SEM images (fig. 3b) show that NiCo PBA consists of monodisperse cubic particles with uniform size, about 100 nm. TEM images show that NiCo PBA is in the form of nanocubes, which can be dispersed separately (fig. 3c and 3 d). In addition, NiCo PBA nanocubes show sharp edges and corners. From the HR-TEM image of the NiCo PBA nanocubes (FIG. 3e), lattice spacings of 0.207 and 0.227nm were observed, corresponding to the (422) and (420) crystallographic planes of the NiCo PBA, respectively.

Test example 2 electrochemical sensing Performance

First, electrochemical measurement experiment conditions

All electrochemical data were tested using a CHI 760E electrochemical workstation (Chenghua instruments, Inc., Shanghai, China) equipped with a conventional three-electrode system, 5mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆](K₃[Fe(CN)₆]And K₄[Fe(CN)₆]In a molar ratio of 1: 1) as electrolyte solution, platinum wire as auxiliary electrode, silver/silver chloride (Ag/AgCl) electrode as reference electrode, AE or modified AE asIs the working electrode. Cyclic Voltammetry (CV) at-0.2V to 0.8V at 100 mV. multidot.s^-1Is performed at the scanning rate of (1). Electrochemical Impedance Spectroscopy (EIS) was recorded at a frequency range of 0.01Hz to 100kHz with an amplitude of 5 mV. EIS data were analyzed by nyquist equivalent circuit using Zview2 software.

Second, electrochemical measurement results

1. Electrochemical performance of different NiCo PBA concentrations and different carcinoembryonic antigen aptamer concentrations

The NiCo PBA nanocubes in the aptamer sensor Apt/NiCo PBA/AE are biomembrane materials for fixing the CEA aptamer, and have excellent sensing capability. The electrochemical signal when detecting CEA for NiCo PBA based aptamer sensors was detected by Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV).

CEA(1mg·mL^-1) The concentration of the buffer solution was 1mg/mL using a phosphate buffer solution (pH 7.4, 0.01M) as a solvent^-1The CEA solution of (1).

Examples 1 to 6 differ only in the concentration of NiCo PBA in the NiCo PBA suspension, and the AE of examples 1 to 6 and NiCo PBA/AE were tested under the same conditions for EIS to obtain the R of AE_ctThe value is denoted as R_ct，AER of NiCo PBA/AE_ctThe value is denoted as R_{ct，material}Calculating the front and rear electrodes R of the NiCo PBA coated suspension_ctDifference of value Δ R_ct(△R_ct＝R_{ct，material}-R_ct，AE) FIG. 4a shows the results obtained, and FIG. 4a shows electrodes R before and after bare gold electrode modification in examples 1 to 6_ctDifference of value Δ R_ct. As can be seen from FIG. 4a, the concentration of NiCo PBA in the NiCo PBA suspension was 1-9 mg. multidot.mL^-1When in range, Δ R_ctThe value increases with increasing concentration, the concentration being 5.0 mg. multidot.mL^-1Substantially reaching saturation.

For the AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE of examples 1-5, respectively (Apt/NiCo PBA/AE vs. 1 mg. mL. mu.L)^-1CEA of (E) is detected and is marked as CEA/Apt/NiCo PBA/AE) is tested by EIS under the same condition, and R of the obtained AE is_ctThe value is denoted as R_ct，AER of NiCo PBA/AE_ctThe value is denoted as R_{ct，material}R of Apt/NiCo PBA/AE_ctThe value is denoted as R_ct，AptR of CEA/Apt/NiCo PBA/AE_ctThe value is denoted as R_ct，_CEACalculating the front and rear electrodes R of the modified NiCo PBA_ctDifference of value Δ R_ct(△R_ct＝R_{ct，material}-R _ct，AE) Front and rear electrodes R of modified electrode fixed aptamer (Apt)_ctDifference of value Δ R_ct(△R_ct＝R_ct，Apt-R_{ct，material}) Front and rear electrodes R for detecting CEA_ctDifference of value Δ R_ct(△R_ct＝R_ct，CEA-R_ct，Apt) The results obtained are shown in FIG. 4b, when the suspension concentration reached 1 mg. multidot.mL^-1Then, Δ R before and after CEA detection_ctTo the maximum, the concentration of NiCo PBA is further increased, which in turn leads to Δ R before and after CEA detection_ctThe value decreases because the NiCo PBA layer thickness is too large to cause exfoliation. Therefore, the concentration was 1 mg. multidot.mL^-1Coating of the NiCo PBA suspension on the AE surface is preferred.

The preparation methods of the aptamer sensors of example 1 and example 7 to example 10 are different only in the concentration of the aptamer in the aptamer solution, the concentration range of the aptamer solution is 10 nM to 200nM, the aptamer sensor (Apt/NiCo PBA/AE) of example 7 to example 10 is used for detecting CEA, which is recorded as CEA/Apt/NiCo PBA/AE, and Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE of example 7 to example 10 are tested by EIS under the same conditions to obtain R of Apt/NiCo PBA/AE_ctThe value is denoted as R_ct，AptR of CEA/Apt/NiCo PBA/AE_ctThe value is denoted as R_ct，CEACalculating front and rear electrodes R for detecting CEA_ctDifference of value Δ R_ct(△R_ct＝R_ct，CEA-R_ct，Apt) The results obtained are shown in FIG. 4c, Δ R before and after CEA detection when the aptamer solution concentration is in the range of 10-50nM_ctThe value increases with increasing concentration of aptamer solution, whereas with further increasing concentration of aptamer solution, Δ R_ctThe value remains substantially constant.

The AE of example 1 and example 7 to example 10, respectively, was tested by EIS under the same conditions to obtain R of AE_ctThe value is denoted as R_ct，AER of NiCo PBA/AE_ctThe value is denoted as R_{ct，material}R of Apt/NiCo PBA/AE_ctThe value is denoted as R_ct，AptR of CEA/Apt/NiCo PBA/AE_ctThe value is denoted as R_ct，CEACalculating the front and rear electrodes R of the modified NiCo PBA_ctDifference of value Δ R_ct(△R_ct＝R_{ct，material}-R _ct，AE) Front and rear electrodes R of modified electrode fixed aptamer (Apt)_ctDifference of value Δ R_ct(△R_ct＝R_ct，Apt-R_{ct，material}) Front and rear electrodes R for detecting CEA_ctDifference of value Δ R_ct(△R_ct＝R_ct，CEA-R_ct，Apt) The results obtained are shown in FIG. 4d, Δ R induced by each step when the concentration of aptamer solution reached 50nM_ctThe change in value was essentially saturated, indicating that the immobilization of the aptamer solution at a concentration of 50nM on the modified electrode was saturated.

From the electrochemical sensing performance of example 1-example 10, when the concentration of the aptamer solution is 50nM and the concentration of the NiCo PBA suspension is 1 mg-mL^-1In the case of the aptamer sensor, the electrochemical performance is better, so that the aptamer solution concentration is 50nM, and the NiCo PBA suspension concentration is 1mg/mL^-1The aptamer sensor of example 11 as a subject for subsequent testing of the electrochemical portion for detection of CEA, including selectivity, stability, reproducibility, and applicability tests.

Test example 3 detection of CEA by aptamer sensor

1. Feasibility of aptamer sensor for detecting CEA

The electrochemical signal responses of AE, NiCo PBA/AE, Apt/NiCo PBA/AE and CEA/Apt/NiCo PBA/AE of example 11 were detected by using EIS and CV test methods, and CEA/Apt/NiCo PBA/AE was Apt/NiCo PBA/AE to detect CEA (0.5 ng. mL. for^-1) The results obtained are shown in fig. 5a and 5b, respectively.

FIG. 5a shows that AE appears as an approximate straight line, TableThe electron transfer between the AE electrode and the electrolyte solution is faster, and after the NiCo PBA modified AE electrode is used, the R of the modified electrode NiCo PBA/AE is obtained_ctThe value increased to 0.370k Ω, indicating successful modification of NiCo PBA to the AE surface, and the low conductivity of NiCo PBA prevented electron transfer between the electrode surface and the electrolyte solution. R of Apt/NiCo PBA/AE obtained after the aptamer was immobilized on NiCo PBA/AE_ctThe value increased to 0.706 k.OMEGA.due to the electronegative phosphate group and the negatively charged [ Fe (CN) ]of the nucleic acid aptamer₆]^3-/4-There is a repulsive force between the redox pairs that further hinders the redox probe from contacting the electrode surface. When NiCo PBA-based aptamer sensor is used to detect CEA (0.5ng mL)^-1) R of CEA/Apt/NiCo PBA/AE_ctThe value reached 1.031k Ω, because the G-quadruplex formed between CEA and the nucleic acid aptamer formed an insulating layer on the outer layer of the electrode, which in turn hindered the electron transfer around the aptamer sensor and the solution, resulting in a continuous decrease in the electrochemical activity of the constructed aptamer sensor.

Fig. 5b shows. The redox peak current is significantly reduced as the material is gradually immobilized on the electrode. The CV curve of the naked AE has a pair of clear redox peaks, and the peak current of NiCo PBA/AE is reduced after NiCo PBA is modified. After the aptamer was immobilized on the NiCo PBA/AE surface, the peak current was further reduced, indicating that successful binding of the aptamer prevented electron transfer by the redox probe. A continuous decrease in peak current during the CEA detection phase was observed, indicating successful interaction of CEA with the nucleic acid aptamer.

All results indicate that the constructed NiCo PBA based aptamer sensor can be used to detect CEA.

2. Sensitivity and minimum detection Limit

The high sensitivity detection of cancer markers is very important for the early diagnosis and effective treatment of cancer, so the sensitivity of the aptamer sensor obtained in example 11 for detecting CEA is further determined by EIS, and the aptamer sensor obtained in example 11 is used for carrying out gradient test on CEA with different concentrations, wherein the concentration range is 0.001-5000 pg.mL^-1The concentration is 0.001, 0.01, 0.1, 1.0, 10, 100 in sequence，1000，2000，5000pg·mL^-1. FIG. 6 shows the EIS response results of the aptamer sensor of example 11 to CEA of different concentrations, and Δ R before and after the aptamer sensor of example 11 detects CEA of different concentrations_ctThe relationship between the value and the CEA concentration is shown in FIG. 7, and Δ R is shown in FIG. 7a_ctThe relationship with CEA concentration; FIG. 7b shows Δ R_ctA linear fit to the logarithm of CEA concentration; Δ R with CEA concentration_ctThe value increased greatly from 0.25 kOmega to 1.750 kOmega, which resulted in Δ R due to the inhibition of electron transfer by the binding of CEA at various concentrations to the aptamer_ctIs increased. Then, Δ R_ctValues nearly reach equilibrium, indicating that binding of CEA to the aptamer is saturated. The sensitivity of the aptamer sensor of example 11 to detect CEA was evaluated by the limit of detection (LOD), and the lowest detection concentration was determined, resulting in Δ R_ctThe linear regression equation of the linear relation with the logarithm of the CEA concentration is as follows: delta R_ct(kΩ)＝0.23LogC_CEA-0.84, LOD calculated to be 0.74 fg. mL^-1. Compared to the prior art method for detecting CEA, the aptamer sensor of example 11 has an ultra-low LOD, a wider linear range and a high sensitivity, as shown in table 3.

Table 3 LOD of aptamer sensor of example 11 compared to prior art

Document 1: wu T, Y.Zhang, D.Wei, X.Wang, T.Yan, B.Du, Q.Wei, Label-free photochemical electrochemical analyzer for carbonic organic detection based on g-C3N4nanosheets hybridized with Zn0.1Cd0.9S nanocrystals, Sensors and activators B Chemical 2018,256, 812-.

Document 2: miao H., L.Wang, Y.ZHuo, Z.Zhou, X.Yang, Label-free fluorine detection of CEA using carbon dots derived from a tomato juice, Biosensors and Bioelectronics 2016,86,83-89.

Document 3: jie G, J.Ge, X.Gao, C.Li, Amplified electrochemiluminescence detection of CEA based on magnetic Fe3O4@ Au nano particles-assembled Ru@SiO₂nanocomposites combined with multiple cycling amplification strategy,Biosensors and Bioelectronics 2018,118,115-121.

Example 11 the superior sensing ability of the aptamer sensor is mainly attributed to the following factors: (1) the NiCo PBA nanocube has an inherent cavity structure, a large specific surface area and excellent biocompatibility, so that aptamer immobilization can be promoted, and the stability of an aptamer sensor can be enhanced; (2) the coordination center of the transition metals Ni and Co can further increase the fixed amount of the biological molecules; (3) the coexistence of the C ≡ N group with the pi-bonds in NiCo PBA leads to strong interactions with the CEA aptamer chain, resulting in efficient immobilization of the aptamer chain.

Test example 4 aptamer sensor for detecting selectivity, stability and reproducibility of CEA

1. Selectivity is

Bovine Serum Albumin (BSA), an interferent solution prepared using phosphate buffered saline (pH 7.4, 0.01M), human epidermal growth factor receptor-2 (HER2), immunoglobulin g (igg), immunoglobulin (IgE), MUC1, Prostate Specific Antigen (PSA), and Vascular Endothelial Growth Factor (VEGF) at a concentration of 1.0pg mL^-1As a test interferent, a solution of (100 times greater than CEA concentration).

Testing the selectivity of the aptamer sensor of example 11, the results of the detection of CEA and interfering analogs by the aptamer sensor of example 11 are shown in FIG. 8a, and the resulting electrochemical signal for detecting interfering analogs is negligible, even at low concentrations of CEA (0.01 pg. mL) compared to detecting CEA^-1) Significant Δ R may also result due to specific recognition between CEA and aptamer chains_ctSignificant changes in value.

In addition, the aptamer sensor of example 11 was also used to test a mixed solution containing both CEA and interferent, producing a Δ R compared to the CEA solution_ctThere was no significant difference in the values, indicating that the presence of interferents had a negligible effect on CEA detection. These results demonstrate the specific recognition between carcinoembryonic antigen aptamer and CEA, with excellent selectivity for NiCo PBA-based aptamer sensors.

2. Reproducibility of

CEA (concentration of 0.01pg/mL) is detected by the Apt/NiCo PBA/AE obtained in example 11 to obtain CEA/Apt/NiCo PBA/AE, the reproducibility of the constructed NiCo PBA-based aptamer sensor Apt/NiCo PBA/AE is further evaluated by five times of parallel EIS tests, and the obtained result is shown in FIG. 8b, wherein Δ R caused by five electrodes_ctThe values varied almost identically with a Relative Standard Deviation (RSD) of 1.10% (n ═ 5), and the NiCo PBA-based aptamer sensors had good reproducibility.

3. Stability of

The stability of the aptamer sensor of example 11 was tested and evaluated by testing continuously at 4 ℃ for a period of 15 days, with the results shown in FIG. 8c, and Δ R of FIG. 8c, measured for 15 days_ctValue, Δ R on day fifteen_ctThe value was about 105% of the initial value, indicating that the aptamer sensor had excellent stability over a long-term test period of 15 days.

4. Reproducibility of

The reproducibility of the aptamer sensor of example 11 was tested by using 0.5 mol.L^-1H of (A) to (B)₂SO₄The aptamer sensor of example 11 was tested for regenerability by treating the CEA/Apt/NiCo PBA/AE in solution for 5 minutes and washing with distilled water to avoid contamination. Subsequently, CEA was detected using the aptamer sensor again. Repeat the same procedure 10 times for each measured R_ctThe change in value is summarized in fig. 8 d. The aptamer sensor detects the reappearance of the high-intensity electrochemical signal change of the CEA with the same concentration, and shows excellent reproducibility.

All the results demonstrate that the aptamer sensor of example 11 has ultrasensitiveness, good reproducibility and stability, and excellent reproducibility.

Test example 5 aptamer sensor for detecting CEA over-expressed in live cancer cells

1. Preparation of cell solutions

H460 cancer cells were obtained from the first subsidiary Hospital of Zhengzhou university and cultured in Dulbecco's Modified Eagle Medium (DMEM) (available from Beijing Soilebao Tech., Ltd.)The medium contained 10% heat-inactivated fetal bovine serum and antibiotics (50 units. mL)^-1Penicillin and 50 units. mL^-1Streptomycin). Cells were incubated at 37 ℃ with 5% CO₂Until use. In use, H460 viable cells were centrifuged and collected at 800rpm to separate the medium, and then carefully dispersed into PBS to give H460 solutions of varying concentrations, which were 1X 10 in turn H460 cell solutions²cell·mL^-1、5×10²cell·mL^-1、1×10³cell·mL^-1、5×10³cell·mL^-1、1×10⁴cell·mL^-1、5×10⁴cell·mL^-1、1×10⁵cell·mL^-1、5×10⁵cell·mL^-1、1×10⁶cell·mL^-1And 5X 10⁶cell·mL^-1。

The concentration of the prepared product was 5000cell mL using PBS^-1The solution of L929 cells of (1), wherein the L929 cells are derived from a syzigar organism.

2. Selectivity is

H460 cancer cells and L929 cells were detected using the aptamer sensor obtained in example 11. And (3) adopting EIS to characterize the electrochemical behavior and detection efficiency of the aptamer sensor. For this purpose, the electrodes were first incubated with a cell solution at 37 ℃ for 40min to remove non-specifically bound cells. Impedance spectra of NiCo PBA-modified electrodes were recorded after each step of CEA detection, and EIS data were fitted by an equivalent circuit.

In view of the characteristics of high sensitivity, excellent biocompatibility, capability of absorbing NiCo PBA by cancer cells, CEA over-expression in live cancer cells, and the like, the aptamer sensor of embodiment 11 is further applied to detect live cancer cells, i.e., H460 cells, the aptamer sensor Apt/NiCo PBA/AE detects H460 cells and is recorded as H460/Apt/NiCo PBA/AE, and the EIS of the aptamer sensor of embodiment 11 detects live H460 cells is shown in fig. 9 a. In H460/Apt/NiCo PBA/AE, R_ctThe value increased gradually from 0.747 kOmega to 1.119 kOmega for Apt/NiCo PBA/AE, indicating the formation of a specific recognition interaction between the aptamer and the H460 cell surface.

In addition, detection of other interfering cells by EIS(Normal L929 cells, concentration of 5000 cells. multidot.mL)^-1) The aptamer sensor of example 11 was tested for selectivity in detecting H460 cells (FIG. 9b), and the aptamer sensor Apt/NiCo PBA/AE for L929 cells was L929/Apt/NiCo PBA/AE, since L929 cells are normal cells and the CEA content is very low, even though the concentration of L929 cells is very high (5000 cells mL. L)^-1) Δ R produced by L929/Apt/NiCo PBA/AE_ctThe value is also relatively small (Δ R)_ct＝R_ct，L929-R_ct，Apt) And is about 80 omega. This indicates that the constructed NiCo PBA-based aptamer sensor has high selectivity for live cancer cells.

3. Sensitivity and detection Limit

Concentration gradient experiments were performed on H460 cells using the aptamer sensor of example 11. EIS results of Apt/NiCo PBA/AE detection of H460 cells at different concentrations are shown in FIG. 10 (0, 100, 500, 1000, 5000, 1X 10)⁴，5×10⁴，10⁵，10⁶And 5X 10⁶cell·mL^-1) R of Apt/NiCo PBA/AE with increasing H460 cell concentration_ctThe values are increased in proportion, and Δ R before and after detection of H460 cells at different concentrations by the aptamer sensor obtained in example 11_ctThe relationship between the value and H460 cell concentration is shown in FIG. 11a, Δ R_ctThe value is in linear relation with logarithm of H460 cell concentration, and the linear test range is 100-5 multiplied by 10⁶cell·mL^-1(FIG. 11b inset), fitting to obtain regression equation Δ R_ct(kΩ)＝0.71logC_cell-1.23. Finally, the LOD of the constructed NiCo PBA-based aptamer sensor is calculated to be 47cell mL^-1。

2. Reproducibility of

The Apt/NiCo PBA/AE obtained in example 11 was added to H460 cells (the concentration was 500 cells. multidot.mL in this order)^-1、5000cell·mL^-1And 100000 cell. mL^-1) The detection is carried out to obtain H460/Apt/NiCo PBA/AE, the reproducibility of the constructed aptamer sensor Apt/NiCo PBA/AE based on NiCo PBA is evaluated through five parallel EIS tests of 3 groups of H460 cells with different concentrations, and the obtained result is shown in FIG. 12a, wherein the Delta R caused by five electrodes in the H460 cells with the same concentration_ctChange of phase almostMeanwhile, the aptamer sensor based on NiCo PBA has good reproducibility in detecting H460 cells.

3. Stability of

The stability of the aptamer sensor of example 11 was tested by continuously testing H460 cells (at a concentration of 500cells/mL) for a period of 15 days at 4 ℃ under storage conditions, and the results are shown in FIG. 12b, where Δ R is measured for 15 days in FIG. 12b_ctValue of DeltaR_ctThe values were almost unchanged, indicating that the aptamer sensor has excellent stability for detecting H460 cells during a long-term test of 15 days.

Test example 6 detection of live cancer cells by aptamer sensor

Human serum samples with different CEA concentrations were tested using the electrochemical aptamer sensor of example 11 and the theoretical CEA concentrations were calculated according to the regression equation and the results are shown in table 4.

Table 4 test results of the aptamer sensor of example 11 for detecting CEA in human serum samples

Compared with the actual addition amount of CEA, the Relative Standard Deviation (RSD) of the detection concentration of the electrochemical aptamer sensor in the embodiment 11 is low and is only 1.7% -4.1%, and the recovery rate is high and is 98.6% -103.6%, which shows that the aptamer sensor based on NiCo PBA has a good application prospect.

The experiment result shows that NiCo PBA has stronger biological affinity, so that NiCo PBA has outstanding fixing capacity on carcinoembryonic antigen nucleic acid aptamers, and further promotes the specificity recognition of CEA by forming a stable G-quadruplex between the carcinoembryonic antigen nucleic acid aptamers and the CEA. Whereas live H460 cells are CEA-overexpressing cancer cells and can release CEA biomarkers, carcinoembryonic antigen nucleic acid aptamers can form stable complexes with H460 cancer cells, which can also cause significant changes in electrochemical signals when live cancer cells are detected. Thus, the aptamer sensor Apt/NiCo PBA/AE can serve as a bifunctional sensor for detecting cancer markers and live cancer cells.

Claims

1. An aptamer sensor is characterized by comprising an electrode, an electrode material for the aptamer sensor coated on the surface of the electrode, and a nucleic acid aptamer attached to the electrode material for the aptamer sensor; the aptamer is a carcinoembryonic antigen aptamer;

the electrode material for the aptamer sensor is a nickel cobalt Prussian blue analogue; the NiCo PBA is the NiCo Prussian blue analogue for short, and the chemical structural formula is Ni₃[Co(CN)₆]₂Or Ni₃[Co(CN)₆]₂·12H₂O。

2. The aptamer sensor of claim 1, wherein the nickel cobalt prussian blue analogue is prepared by a method comprising the steps of:

adding cobalt cyanate aqueous solution into aqueous solution of nickel salt and citrate, and reacting to obtain the nickel cobalt Prussian blue analogue.

3. The aptamer sensor according to claim 1, wherein the aptamer sensor is coated with the electrode material on the surface of the electrode in an amount of 0.07-6.37 μ g/mm²。

4. The aptamer sensor of claim 1, wherein the electrode is a bare gold electrode or a glassy carbon electrode.

5. A method for preparing an aptamer sensor according to claim 1, comprising the steps of:

6. The method for producing an aptamer sensor according to claim 5, wherein the concentration of the electrode material suspension for the aptamer sensor is 1 to 9 mg/mL.

7. The method for producing an aptamer sensor according to claim 6, wherein the concentration of the electrode material suspension for the aptamer sensor is 1 to 2 mg/mL.

8. The method for producing an aptamer sensor according to claim 5, wherein the aptamer is attached to the modified electrode by a method comprising the steps of:

and (3) immersing the modified electrode into the aptamer solution, taking out and drying to obtain the aptamer sensor.

9. The method for producing an aptamer sensor according to claim 8, wherein the concentration of the aptamer solution is 5 to 200 nM.

10. The method for producing an aptamer sensor according to claim 9, wherein the concentration of the aptamer solution is 50 to 100 nM.

11. The method for preparing the aptamer sensor according to claim 8, wherein the temperature of the modified electrode immersed in the aptamer solution is 2-5 ℃ and the time is 1.5-2.5 h.