CN108152349A - Colorimetric and electrochemistry binary channels aptamer sensor based on nano silver catalysis - Google Patents

Abstract

The present invention provides the colorimetric and electrochemistry binary channels aptamer sensor being catalyzed based on nano silver, for the measure of PDGF BB.Nano silver has certain catalytic capability, can be catalyzed the reaction of substrate sodium borohydride and o-nitrophenol.By it is aptamer modified on nano silver after, the dual channel sensor of structure realizes the colorimetric to detecting target, and electrochemistry measures simultaneously.1 colorimetric detection of channel, method is easy, is adapted to live quick screening；2 Electrochemical Detection of channel utilizes the nano-complex with excellent conductive ability, obtains sensitive electrochemical detection signal.Method proposed by the present invention combines the high specific of aptamer screening albumen and the high sensitivity of nano silver catalysis, using Dual channel detection pattern, has simplicity, accuracy is high, the advantages of sensitivity is good, and detection limit is low, the detection for PDGF BB provide a kind of new method.

Description

Colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on nanosilver catalysis

technical field

The invention belongs to the technical field of biological detection and analysis instruments, and specifically relates to the construction of a colorimetric and electrochemical dual-channel nucleic acid aptamer sensor by utilizing the catalytic ability of nano silver to realize the measurement of platelet-derived growth factor (PDGF-BB).

Background technique

Due to the large specific surface area of nanomaterials, they can be highly efficient catalysts in biosensors. Nano-silver, as a special nano-material, has been verified as a high-efficiency catalytic material in various application fields, and can be used in the development of biosensors. Nano-silver also has a certain catalytic reduction ability for dye molecules, so that corresponding colorimetric sensors can be designed to achieve quantitative analysis of targets.

Electrochemical sensors have significant advantages in sensitivity, detection cost, simplicity, reliability, and miniaturization, and thus have gained extensive attention in medical diagnostic research. Printed electrodes are easy to prepare, low in cost, easy to mass produce, and easy to integrate portable on-site rapid detection instruments. They have become an important aspect of electrochemical sensors and have been widely used. In recent years, the emergence of nanomaterials has provided a new way for the development of novel electrochemical biosensors.

In nanomaterial-based bioassays, catalytically active nanomaterials are generally labeled with corresponding antibodies. Nucleic acid aptamer is also a single-stranded nucleic acid molecule with a specific three-dimensional structure that can specifically bind to proteins, and is a new protein recognition probe. Since aptamers are oligonucleotides with a specific structure and sequence, in addition to the stability of nucleic acids, they are also easy to synthesize and derive, so they are very suitable as recognition molecules for sensors, and can recognize many target molecules including proteins. , can largely replace the original antibody recognition task. In recent years, researchers at home and abroad have constructed a variety of highly efficient and sensitive nucleic acid aptamer sensors, realizing the sensitive determination of various proteins.

At present, polymers have attracted considerable attention because of their unique electronic and chemical properties, and have been widely used in many fields, such as energy storage, information memory devices, and electrochemical biosensors. In the development of biosensors, electrochemical polymerization methods are a powerful tool, but such methods mainly use the products of electropolymerization as substrate materials for immobilizing biomolecules and electrocatalysts. The direct electrochemical determination of electropolymerization products can also be the signal conduction mode of the sensor.

The biological assay method of PDGF-BB is usually an enzyme-linked immunosorbent assay kit detection method. Although the operation method is relatively simple, the detection method is single, the biological enzymes and antibodies used in the method are expensive, and the storage and operation are inconvenient. This brings certain limitations to the detection. In the present invention, nanomaterials with catalytic activity are used in color reaction or electrochemical reaction, combined with nucleic acid aptamers as capture probes, which can detect target proteins and obtain amplified signals. The proposed dual-channel detection platform of colorimetric method and electrochemical method can realize accurate express detection of PDGF-BB and make up for the shortcomings of traditional enzyme-labeled methods.

Contents of the invention

The invention overcomes the deficiencies of the prior art, and proposes a colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on nano-silver catalysis to realize the determination of PDGF-BB.

For realizing above-mentioned purpose of the present invention, the present invention adopts following technical scheme:

A colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on nano-silver catalysis to realize the determination of PDGF-BB. The PDGF-BB nucleic acid aptamer is immobilized in the wells of the multi-well plate, and the target analyte PDGF-BB is added to form a complex between the aptamer and the protein, and then the nano-silver probe modified by the PDGF-BB nucleic acid aptamer is added. Add the probe reaction and then add the catalytic reaction substrate. After the catalytic reaction is completed, dual-channel detection is realized. Channel 1 adopts visible spectrophotometry, detects the absorbance of the substrate system at the maximum absorption wavelength, and uses the colorimetric method to read the absorbance value to determine the content of the sample. Channel 2 adopts the electrochemical detection method, which can directly add reduced graphene oxide into the hole as the electrolyte, insert the printed electrode into it for electrodeposition, and place the electrode in the electrolyte solution after completion to detect the amplification of the polymer formed on the surface of the electrode. Electrochemical signals to achieve quantitative determination of targets.

The multi-well plate is a commercially available 96-well multi-well plate.

The method of immobilizing the PDGF-BB nucleic acid aptamer in the wells of a multi-well plate is as follows: dilute the streptavidin with a carbonate buffer solution, add it to the wells, let stand at 4°C for 12 hours, wash and dry, and the biotin-modified The PDGF-BB nucleic acid aptamer was added, reacted at 37°C for 30 minutes, washed and dried.

The complex formation reaction between the aptamer and the protein adopts the following steps: add 1% BSA to the wells immobilized with PDGF-BB nucleic acid aptamers, block at 37°C for 1 hour, wash and dry in the air after the blocking is completed. Add the PDGF-BB standard or sample into the wells of the multi-well plate, react at 37°C for 30 minutes, wash and dry in the air. During this process, aptamer-protein complexes are finally formed on the well plate.

The preparation method of the PDGF-BB nucleic acid aptamer-modified nano-silver probe is as follows: silver nitrate and sodium borohydride are reacted in an ice bath to prepare nano-silver. The streptavidin and nano silver were mixed and reacted at 37°C for 2 hours, centrifuged and then biotin-modified PDGF-BB nucleic acid aptamer was added, reacted at 37°C for 30 minutes and then centrifuged. The obtained PDGF-BB nucleic acid aptamer-modified nano-silver probe can form an aptamer-protein complex with PDGF-BB.

The reaction of adding the probe is as follows: add the nano-silver probe modified by the PDGF-BB nucleic acid aptamer into the well, react at 37° C. for 30 minutes, wash and dry in the air.

The reaction substrates are as follows: sodium borohydride and o-nitrophenol.

The detection method of channel 1 is as follows: 10 minutes after adding the reaction substrate, the reaction solution is placed in a cuvette, and the absorbance value is measured at a wavelength of 423 nm, and the quantitative method is a colorimetric control method. Use a visible spectrophotometer to read the absorbance value (423 nm) of the faded substrate system in the PDGF-BB standard and sample reaction wells, and calculate the difference ⊿ between the absorbance values of the standard well and the blank sample well A The difference between the absorbance values of _{the standard} and sample wells and the blank sample well ⊿A _sample . The concentration of PDGF-BB in the sample can be directly calculated by using the method of direct comparison.

The detection method of channel 2 is as follows: after adding the reaction substrate for 10 minutes, add 1 mg/mL reduced graphene oxide into the hole and mix well, insert the printed electrode, perform electrodeposition, and wash and dry the electrode. 0.1 mol/L hydrochloric acid was added dropwise to the electrode surface, and the electrochemical signal value was read by differential pulse voltammetry, and the detection signal was the peak current value. The quantitative method is the standard curve method. The standard curve is drawn with the concentration of the PDGF-BB standard series standard solution as the abscissa and the detected peak current value as the ordinate, and the standard curve equation can be obtained. By measuring the peak current value of the sample, the concentration of PDGF-BB in the sample can be obtained by using the standard curve equation.

The method for making the printed electrode is as follows: using PET as the substrate, printing silver layer, carbon layer, silver chloride layer and insulating layer layer by layer on a printing machine. The electrode is a three-electrode system, and the electrode size is 0.4 mm × 5 mm × 26 mm.

The electrodeposition reaction was scanned by cyclic voltammetry in the range of -0.3 V to 1.3 V for 20 cycles as follows.

The detection method of the electrochemical signal value is as follows: use 0.1 mol/L hydrochloric acid solution as the electrolyte, record the differential pulse voltammetry curve in the range of -0.3V to 0.4V, and the detection signal is the peak current value.

The sample processing method is as follows: for plasma or serum sample processing, it is diluted 100 times with PBS, and the standard product is not diluted.

The colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on nano-silver catalysis proposed by the present invention is suitable for rapid and portable determination of PDGF-BB. PDGF-BB is measured using the dual-channel method proposed by the present invention, and the detection limits are respectively: 100 pg/mL (channel 1) and 3 pg/mL (channel 2). Channel 1 has fast colorimetric measurement and is suitable for on-site detection, while channel 2 has high electrochemical detection sensitivity and is suitable for low-concentration sample analysis. There is no cross-reaction between the detection results and PDGF-AA, PDGF-AB, and thrombin. At the same time, the CV value of the detection between wells is lower than 10%. The method has high precision, and the recovery rate is between 97% and 105%. The method has high accuracy.

Beneficial effects of the present invention: the present invention provides a colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on the catalytic ability of nano-silver for the determination of PDGF-BB. Nano silver can catalyze the substrates sodium borohydride and o-nitrophenol. After the nucleic acid aptamer is modified on the nano-silver, a dual-channel sensor is constructed, which realizes the colorimetric and electrochemical simultaneous determination of the detection target. Channel 1 colorimetric detection is simple and suitable for on-site rapid screening; channel 2 electrochemical detection uses nanocomposites with excellent conductivity to obtain sensitive electrochemical detection signals. The method proposed by the invention combines the high specificity of nucleic acid aptamer screening proteins and the high sensitivity of nano-silver catalysis, adopts a dual-channel detection mode, and has the advantages of simple method, high accuracy, good sensitivity and low detection limit. The invention provides a new method for detecting PDGF-BB.

Description of drawings

Fig. 1 Schematic diagram of colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on nano-silver catalysis.

Fig. 2 UV-Vis absorption spectrum curve of colorimetric method detection channel.

Figure 3 Standard curve in the electrochemical detection channel.

Fig. 4 The influence of interfering substances in the electrochemical detection channel on the measurement results of PDGF-BB.

Detailed ways

The sequences used in the following examples are as follows:

Biotin-modified PDFG-BB nucleic acid aptamer (Bio-APT):

5'-AAAAAAAAATACTCAGGGCACTTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAT, the 5' end of which is modified with biotin.

Biotin-modified DNA strands (Bio-DNA):

5'-AAAAAAAAAAAAAAAA whose 5' end is modified with biotin.

In order to better understand the present invention, the technical solution of the present invention will be described in detail below with specific examples, but the present invention is not limited thereto.

Example 1: The construction process of the colorimetric and electrochemical dual-channel nucleic acid aptamer sensor based on nano-silver catalysis:

(1) Immobilize the PDGF-BB nucleic acid aptamer on the well plate: prepare a 2 mg/mL streptavidin solution with carbonate coating solution (0.05 mol/L carbonate buffer solution, pH=9.6). Add 100 μL of streptavidin solution to each well, and place the plate in the refrigerator at 4°C for 12 hours. Using PBST solution (0.05% Tween-20 in PBS) as the washing solution, wash the plate wells coated with streptavidin three times, and pat dry for later use. A 200 nmol/L biotin-modified PDFG-BB nucleic acid aptamer (Bio-APT) and a 200 nmol/L biotin-modified DNA strand (Bio-DNA) were prepared with 0.1 mol/L PBS buffer solution. Mix Bio-APT and Bio-DNA (1:3) and add a total of 100 μL to the well, incubate in a water bath at 37 °C for 30 minutes, wash with PBST solution three times and pat dry, add 1 mg/mL BSA to block After blocking, the cells were incubated in a water bath at 37°C for 1 hour, washed three times with PBST solution and patted dry.

(2) Preparation of PDGF-BB nucleic acid aptamer-modified nano-silver probe: Place a round-bottomed flask containing 0.2 mmol/L silver nitrate solution in an ice bath, add 0.3 mmol/L sodium borohydride dropwise, and stir . The volume ratio of silver nitrate and sodium borohydride is 1:2. When the temperature of the reaction system returns to room temperature, the stirring is stopped to obtain a nano-silver solution. Add 200 μL of streptavidin solution (dissolved in PBS buffer solution) into 2 mL of nano-silver solution, mix and shake at 37 °C for 2 hours, centrifuge and wash 3 times with PBST washing solution, and redisperse to 2 mL. Then add 100 μL of 10 μmol/L biotin-modified PDFG-BB nucleic acid aptamer (Bio-APT) and 300 μL of 10 μmol/L biotin-modified DNA strand (Bio-DNA), mix at 37 °C After shaking for 1 hour, wash with PBST three times and redisperse to 2 mL.

(3) PDGF-BB detection process by the sensor: add PDGF-BB standard or sample to the well plate immobilized with PDGF-BB nucleic acid aptamer, 50 μL per well, incubate at 37°C for 30 minutes and wash with PBST solution 3 times and pat dry. Add 50 μL of the above-mentioned PDGF-BB nucleic acid aptamer-modified nano-silver probe to each well, incubate at 37°C for 30 minutes, wash with PBST solution 3 times and pat dry. Add the above-mentioned PDGF-BB nucleic acid aptamer-modified nano-silver probe to 50 μL of each well, incubate at 37 °C for 30 min, wash with PBST solution 3 times and pat dry. At this time, a sandwich-type nanocomposite of "aptamer-protein-aptamer-modified nanosilver" is formed in the hole.

(4) Catalytic reaction: The reaction substrate sodium borohydride (50 mmol/L) and o-nitrophenol (200 mmol/L) were mixed at a volume ratio of 1:1, added to the wells, 50 μL per well, and mixed well , and incubate for 10 minutes at 37°C in a humid chamber.

Embodiment 2: Passage 1 colorimetric method detects:

The above-mentioned substrate system is yellow, and it is added to the orifice plate, and under the catalysis of nano-silver, a fading reaction occurs, which can be used for quantitative determination. The results are shown in Figure 2, wherein curve a is the detection absorption curve of the blank sample hole, and curve b is Contain PDGF-BB standard sample to detect absorption curve. Use a visible spectrophotometer to read the absorbance value (423 nm) of the faded substrate system in the PDGF-BB standard and sample reaction wells, and calculate the difference ⊿ between the absorbance values of the standard well and the blank sample well A The difference between the absorbance values of _{the standard} and sample wells and the blank sample well ⊿A _sample . The concentration of PDGF-BB in the sample can be directly calculated by using the method of direct comparison. Standards and samples were averaged three times.

Embodiment 3: Channel 2 electrochemical detection:

(1) Printing of printed electrodes: Printed electrodes use PET as the substrate, silver, carbon, silver/silver chloride and insulating paste as materials, and print according to the design drawings. Silver is the conductive wire, carbon is the working electrode and auxiliary electrode, and silver/silver chloride is used as the reference electrode. After production, put it in an oven and cure it at 130°C for 120 minutes. Finally, print the insulating paste, and wash it after 10 minutes of light curing.

(2) Electrochemical detection: After reacting the above substrates for 10 minutes, add 2 mg/mL reduced graphene oxide, 10 μL per well, and mix well. Insert and connect the printed electrodes to the electrochemical workstation. Electrodeposition was performed by scanning 20 cycles in the range of -0.3 V to 1.3 V by cyclic voltammetry at a rate of 100 mV/S. After deposition, a layer of conductive nanocomposites can be obtained on the electrode surface and washed with distilled water and dried. Insert the deposited electrode into 0.1 mol/L hydrochloric acid solution, perform electrochemical detection by differential pulse voltammetry, and record the peak current value from -0.3 V to 0.4 V. The standards and samples of each concentration were measured in parallel three times. Draw the standard curve with the concentration of PDGF-BB standard solution as the abscissa and the detected peak current value as the ordinate (Figure 3). The standard curve equation can be obtained, and the measured value of the sample can be used to obtain the PDGF-BB in the sample. content.

Embodiment 4: intersection test:

This example examines the specificity of the present invention to detecting PDGF-BB, because PDGF-AA, PDGF-AB, thrombin may cross-react, and interfere with the determination of PDGF-BB, so the above-mentioned interfering substances and PDGF- The BB standard was diluted to a concentration of 30 ng/mL, and measured according to the methods of Example 1 and Example 2. The results showed that the other three substances had no interference with the determination of PDGF-BB (Figure 4).

Claims (4)

1. Colorimetric and electrochemical dual-channel nucleic acid aptamer sensors based on nano-silver catalysis are used for the determination of PDGF-BB, characterized in that: the PDGF-BB nucleic acid aptamer is fixed in the well of a multi-well enzyme plate, Add target PDGF-BB to form a complex of aptamer and protein, then add PDGF-BB nucleic acid aptamer-modified nano-silver probe; add the reaction substrate after adding the probe reaction, and realize dual-channel after the nano-silver catalyzed reaction is completed Detection; channel 1 adopts spectrophotometry, detects the absorbance of the substrate system at the maximum absorption wavelength, and uses colorimetry to read the absorbance value to determine the content of the sample; channel 2 adopts electrochemical detection method, which can directly add reduced oxidation state to the hole Graphene is used as the electrolyte, and the printed electrode is inserted into it for electrodeposition. After completion, the electrode is placed in the electrolyte solution, and the amplified electrochemical signal of the nanocomposite formed on the electrode surface is detected to realize the quantitative determination of the target. 2. The nano-silver catalytic reaction as claimed in claim 1, characterized in that: nano-silver catalyzes the reaction between sodium borohydride (50 mmol/L) and o-nitrophenol (200 mmol/L) (volume ratio 1:1) Under the catalysis of nano-silver, sodium borohydride reduces yellow o-nitrophenol to colorless o-aminophenol. 3. Spectrophotometry as claimed in claim 1, is characterized in that: read respectively the absorbance value (measurement wavelength is 423 nm) of the solution in PDGF-BB standard substance and sample reaction well, and calculate standard substance hole respectively The difference between the absorbance value of the blank sample hole and the blank sample _{hole⊿A standard} , the difference between the sample hole and the blank sample hole absorbance value⊿A _sample ; the content of PDGF-BB in the sample can be calculated by using the direct comparison method . 4. The electrochemical detection method according to claim 1, characterized in that: directly add 2 mg/mL reduced graphene oxide as electrolyte to each hole after the catalytic reaction is completed, and insert the printed electrode into it for electrodeposition, Use cyclic voltammetry to scan 20 cycles in the range of -0.3 V to 1.3 V; after completion, detect the amplified electrochemical signal of the nanocomposite formed on the electrode surface, use 0.1 mol/L hydrochloric acid solution as the electrolyte, and record -0.3 V The differential pulse voltammetry curve within the range of 0.4 V, the detection signal is the peak current value, and the PDGF-BB is quantitatively determined by the standard curve method.