CN111122675B - Strip-block-strip electrode and VEGF based on strip-block-strip electrode165Sensor, preparation method and detection method thereof - Google Patents
Strip-block-strip electrode and VEGF based on strip-block-strip electrode165Sensor, preparation method and detection method thereof Download PDFInfo
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Abstract
The invention discloses a strip-block strip electrode which comprises two opposite strip electrodes, wherein a plurality of block electrodes are distributed between the two strip electrodes. Also discloses VEGF based on the strip electrode165The sensor comprises the strip-block-strip electrode, and VEGF is fixedly loaded on the strip-block-strip electrode165An aptamer. Also discloses a preparation method of the sensor: 1) taking the strip electrodes, cleaning and drying; 2) VEGF165And (3) dripping the aptamer solution on the surfaces of the strip electrodes, and incubating to obtain the aptamer. Also discloses a detection method of the sensor: taking the VEGF based on the strip electrode165Sensor, dripping the solution to be tested on the surface of electrode, incubating, connecting with electrochemical analyzer or impedance measuring instrument to determine its impedance value under different frequencies, and calculating and judging whether the solution to be tested contains VEGF or not according to corresponding impedance diagram165And VEGF contained therein165The concentration of (c).
Description
Technical Field
The invention belongs to the technical field of electrochemical sensors, and particularly relates to a strip electrode,VEGF based on strip-block-strip electrodes165A sensor, a preparation method and a detection method thereof.
Background
The malignant tumor has high mortality rate and is a serious disease threatening the life health of human beings. Due to its invasiveness and destructiveness in the human body, the rapid growth of malignant tumors destroys the organ tissue structure of the human body, resulting in dysfunction. The report of 'analysis of the onset and death of malignant tumors in different regions in China' 2015 published by the national cancer center in 2019 shows that 392.9 ten thousands of new cases of malignant tumors in 2015 of China have the morbidity of 285.83/10 ten thousands, 233.8 thousands of cases of deaths and the mortality of 170.05/10 ten thousands. Malignant tumors become the 1 st cause of death of residents in China, and lung cancer, liver cancer, gastric cancer, colorectal cancer and esophageal cancer are main tumor causes of death. The world health organization has recently published data indicating that 880 million people die of cancer annually worldwide, accounting for nearly 1/6 total annual death worldwide, with the majority of deaths in mid-to-low income countries. There are over 1400 million new cancer cases per year, and this figure is expected to increase to over 2100 million by 2030.
As mitogens specific for endothelial cells, Vascular Endothelial Growth Factor (VEGF)165) Is a major regulator of tumor angiogenesis and stimulates the formation of new blood vessels. Since abnormally rapid growth and division of tumors in the vascular phase requires the formation of new blood vessels to supply nutrients and oxygen independently, VEGF is involved in the growth of tumors165Often over-expressed in tumor cells, affecting tumor angiogenesis and metastasis. Thus, one of the most effective methods for detecting malignant tumors is the detection of VEGF165And (4) horizontal. To date, several traditional methods for the detection of vascular endothelial growth factor have been reported in the literature, however, these techniques are expensive, laborious and time-consuming due to their expensive instrumentation and complex sample pre-treatment.
Printed circuit boards (also known as Printed circuit boards) are providers of electrical connections for electronic components. Aptamers (aptamers) are single-stranded DNA or RNA fragments which are obtained by screening from an artificially constructed random nucleic acid library and have efficient and specific binding with target ligands by using a systematic evolution of ligands by exponential enrichment (SELEX) technology. Compared with the antibody, the aptamer has the advantages of good stability, low cost, wide target action range, high specificity, affinity and the like. The electrochemical aptamer sensor constructed by combining the aptamer serving as a recognition element with an electrochemical sensing technology has the advantages of high sensitivity, quick response and low cost of electrochemical analysis, high selectivity, strong specificity and the like of the aptamer, and has wide application prospects in the aspects of disease diagnosis, environmental monitoring, drug analysis and the like. In order to further improve the detection sensitivity of the electrochemical aptamer sensor to meet the requirement of practical application, in recent years, a novel signal amplification technology is widely applied to the construction of the electrochemical aptamer sensor.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a strip-block strip electrode and VEGF based on the strip-block strip electrode165A sensor, a preparation method and a detection method thereof.
The technical scheme adopted by the invention is as follows:
a strip and block electrode comprises a base material and electrodes, wherein the electrodes comprise two opposite strip electrodes, and a plurality of block electrodes are distributed between the two strip electrodes.
In the technical scheme, one strip electrode is T-shaped, the other strip electrode is U-shaped, the T-shaped strip electrode comprises a transverse strip and a vertical strip, the top end of the vertical strip is connected with the middle of the transverse strip, and the vertical strip faces to the concave part of the U-shaped strip electrode.
The distances between the strip electrodes and the block electrodes and between the block electrodes are 0.2 mm.
VEGF based on strip electrode165The sensor comprises the strip-block-strip electrode, and VEGF is fixedly loaded on the strip-block-strip electrode165An aptamer.
The VEGF165The sequence of the aptamer is 5 '-SH-TTTCCCGTCTTCCAGACAAGAGTGCAGGG-3'.
VEGF based on strip electrode165The preparation method of the sensor comprises the following steps:
1) taking the bar block and bar electrodes, cleaning and drying;
2) VEGF165And (3) dropwise coating the aptamer solution on the surfaces of the cleaned and dried strip electrodes, and incubating to make the aptamer immobilized on the surfaces of the electrodes, thereby obtaining the aptamer-coated electrode.
The cleaning of the strip electrodes in the step 1) adopts sodium hydroxide, hydrochloric acid and NH3、H2O2、H2The mixed solution of O is washed sequentially and then rinsed with water.
In the above-mentioned embodiment, VEGF165Aptamer solution concentration was 500nM and incubation at 4 ℃ for 24 h.
VEGF based on strip electrode165The detection method of the sensor comprises the following steps: taking the VEGF based on the strip electrode165Sensor, dripping the solution to be tested on the surface of the electrode, incubating, connecting with electrochemical analyzer or impedance measuring instrument to measure its impedance value under different frequencies, and calculating and judging whether the solution to be tested contains VEGF or not according to the corresponding impedance diagram165And VEGF contained therein165The concentration of (c).
In the technical scheme, the solution to be detected is dripped on the surface of the electrode and reacts for 60min at the incubation temperature of 37 ℃.
The invention has the beneficial effects that: (1) the strip-block-strip electrode is a novel electrode manufactured based on a printed circuit board, and has the characteristics of simple preparation, low cost, easiness in mass production, high sensitivity and the like. (2) Detection of VEGF of the invention165The impedance sensor has high affinity and specificity between the aptamer and a target molecule, high specificity and detection sensitivity, and the aptamer has no immunogenicity, low cost and easy storage.
Drawings
Fig. 1 is a schematic diagram of the structure of a bar-block bar electrode of the present invention.
FIG. 2 is a microscope image and a physical image of the strip electrode of the present invention, wherein A is a microscope image and B is a physical image.
FIG. 3 is an electrochemical impedance characterization and fitted equivalent circuit diagram.
Fig. 4 is a comparison of the analytical performance of the gold interdigitated electrode and the striped block electrode of the present invention, wherein a is the striped block electrode and B is the gold interdigitated electrode.
FIG. 5 is a diagram of the detection of VEGF according to the invention165Working principle diagram of the impedance sensor.
FIG. 6 shows the results of optimization of aptamer concentration and reaction time, wherein A is the result of concentration optimization and B is the result of time optimization.
FIG. 7 is the antigen VEGF165The reaction time of (c) optimizes the results.
FIG. 8 is the antigen VEGF165The linear range of (2) detection results.
FIG. 9 is the antigen VEGF165The result of the specific detection of (1).
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The experimental procedures in the following examples are conventional unless otherwise specified.
First embodiment, a bar electrode is manufactured
The strip-block strip electrode (LPLE) is manufactured by taking FR-4 epoxy glass cloth as a base material and carrying out gold immersion on a printed circuit board. The electrode is a capacitor electrode and mainly comprises two parts of capacitors, namely a strip-to-block capacitor and a block-to-block capacitor.
The structure of the strip-block strip electrode is shown in figure 1: the capacitive touch screen mainly comprises a substrate 1 and electrodes, wherein the electrodes are capacitance electrodes, the input electrodes are two opposite strip-shaped electrodes 2, and a plurality of block-shaped electrodes 3 are uniformly distributed between the two strip-shaped electrodes 2. The electrode is formed by dipping gold on a substrate 1, one strip-shaped electrode 2 is T-shaped, the other strip-shaped electrode 2 is U-shaped, the T-shaped strip-shaped electrode 2 comprises a transverse strip 2a and a vertical strip 2b, the top end of the vertical strip 2b is connected with the middle part of the transverse strip 2a, and the vertical strip 2b faces to the concave part of the U-shaped strip-shaped electrode 2. The strip-shaped electrodes 2 may also be of other shapes, such as arc-shaped; two strip electrodes 2 enclose a gold square frame of a measuring area, a plurality of block electrodes 3 are distributed in the gold square frame, the block electrodes 3 can be square or round,
FIG. 2 is a microscope image and representation of a bar-and-block bar electrode of the inventionIn the figure, the coin used in fig. 2 has a diameter of 19 mm. The Electrochemical Impedance Spectroscopy (EIS) detection is carried out by connecting the strip-block strip electrodes of the invention with CHI 604E electrochemical workstation (Shanghai Chenghua apparatus, China). The electrochemical detection uses a two-electrode system. The processing parameters of the strip electrodes in this embodiment are as follows: the total size of the base material 1 is 20mm multiplied by 9.7mm multiplied by 0.6mm, and the length and the width of the measuring area of a gold frame surrounded by the two strip electrodes 2 are both 4 mm; the distances between the strip-shaped electrodes 2 and the block-shaped electrodes 3 and between the block-shaped electrodes 3 and the block-shaped electrodes 3 are 0.2 mm. The two strip electrodes 2 are respectively connected with the two leads 4, the two leads 4 are respectively welded with the two copper wires, one copper wire is connected with a green chuck of the electrochemical workstation, and the other copper wire is simultaneously connected with a red chuck and a white chuck of the electrochemical workstation. The amplitude of the alternating voltage is 5mV, and the frequency range of the alternating impedance spectrum (Bode diagram) is 1Hz-105Hz. The dc bias is set to 0V. The strip electrodes are placed in 0.1M PBS buffer solution (PH 7.0) for electrochemical impedance measurement, and in the electrochemical impedance characterization, the naked electrode, aptamer fixation and VEGF are obtained by detecting gradual modification165Graph of the corresponding Z' -Z "(real-imaginary) relationship of the incubations, as shown in fig. 3. The detection principle is shown in a fitting equivalent circuit of an inset in fig. 3, the interface of the strip electrode and the solution forms an electric double layer capacitor structure, and different modifications of the electrode can promote or obstruct the detection of the electrode interface property, so that the impedance detection of the electrode is carried out.
Compared with gold interdigital electrodes, the target detection analysis performance of the strip-block strip electrodes at 8.25Hz is remarkably improved (as shown in FIG. 4). Electrode and VEGF with modified impedance difference165After reaction, the impedance amplitude changes at a single frequency point. The strip-block strip electrode manufactured by the embodiment sensitively generates signals according to dielectric characteristics after combining an analyte by utilizing a non-Faraday impedance spectrum principle; due to its geometrical properties, the signal output can be amplified without multiple labeling or DNA intercalating agents and additional amplification steps, and thus can be used to design many impedance biosensors for detecting various chemical and biological samples.
Example two, the application of the vascular endothelial cell growth factor 165 detection
The strip and strip electrodes of the first embodiment are used for preparing VEGF (vascular endothelial growth factor) detection165The impedance sensor (the working principle of the sensor is shown in fig. 5) operates according to the following steps:
step one, processing the strip electrodes
Taking the bar and strip electrodes manufactured in the first embodiment, and sequentially cleaning: 1M sodium hydroxide for 5min, 1M hydrochloric acid for 3min, NH3:H2O2:H2Cleaning with O (1:1:5, v/v/v) mixed solution for 2min, then rinsing with ultrapure water (18.25M omega cm), and blowing with nitrogen for later use.
Step two, preparation of aptamer
Thiol-modified aptamer (5 '-SH-TTTCCCGTCTTCCAGACAAGAGTGCAG-GG-3') was selected and 1. mu.M of this aptamer solution was reacted with 10mM TCEP at room temperature for 1h to activate DNA and open disulfide bonds. And (3) dropwise coating the aptamer solution on the surface of a cleaned bar electrode, incubating for 24h at 4 ℃, and well fixing the aptamer on the surface of the electrode through a gold-sulfur bond. The plates were washed in 0.1M PBS buffer (pH 7.0), then rinsed with ultra-pure water and blown dry with nitrogen for further use. And then sealing in 0.25% (w/v) bovine serum albumin solution for 30min, washing after the reaction is finished, and drying.
Step three, antigen VEGF165Capture of
Adding 1ng/ml VEGF165And (3) dropwise coating the antigen solution on the surface of the bar electrode treated in the step two, incubating for 1h at 37 ℃, and capturing the antigen through the specific binding of the aptamer and the antigen.
Step four, determining the optimal fixed concentration of the probe
On the electrode washed clean in the first step, by performing reaction with aptamer (100nM,300nM,500nM,700nM,1 μ M) in different concentrations for 24h at the incubation temperature of 4 ℃, the optimal fixed concentration of the probe is obtained by the late calculation of the corresponding impedance diagram, and the result is shown in FIG. 6A, and the optimal fixed concentration of the probe is 500 nM. Step five, determining the optimal reaction time of the probe
On the electrode washed cleanly in the first step, by carrying out reactions with 500nM aptamer at different times (12h,16h,20h,24h,28h) at an incubation temperature of 4 ℃, the optimal reaction time of the probe is obtained by post calculation, and the result is shown in FIG. 6B, and the optimal reaction time is 24 h.
Step six, determining antigen VEGF165Optimum reaction time of
The aptamer with the optimal concentration (500nM) is fixed on the electrode with the optimal reaction time (24h), after 30min of blocking treatment, 1ng/ml antigen is used for reaction at different times (40min,50min, 60min,70min) at the incubation temperature of 37 ℃, and the optimal reaction time of the antigen is obtained through later stage calculation, and the result is shown in figure 7, and the optimal reaction time is 60 min.
Step seven, linear range and detection limit of experiment
Under the optimized conditions and the proper temperature, the antigen with different concentrations (1fg/ml,100fg/ml, 200fg/ml,600fg/ml,1pg/ml,1.2pg/ml and 1.4 pg/ml) is reacted for 1h, and the linear range and the detection limit of the antigen are obtained through later calculation, so that the current response value and the antigen are in good linear relation in the range of 1fg/ml to 1.2pg/ml, the concentration is in good linear relation in the numerical range, and the response curve is smooth in 1.4pg/ml, as shown in FIG. 8. Method for obtaining resistance variable delta Z and VEGF by using least square method165The linear relationship of the concentration C is: Δ Z ═ 1.422C +2411.01 (linear relationship R)20.9950), i.e. C ═ Δ Z-2411.01)/1.422, VEGF can be calculated from the measured impedance change Δ Z165The concentration of (2). The detection limit LOD is 3 δ/S (where δ is the remaining standard deviation of the standard curve and S is the slope of the standard curve), and the detection limit is 0.64 fg/ml.
Step eight, specificity of experiment
A linear range of one concentration 0.026pM (i.e. 1pg/ml) was selected as antigen VEGF165The other four interfering proteins (thrombin), immunoglobulin G (IgG), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor 121 (VEGF) were used at the same concentrations121) The reaction is carried out under the same experimental conditions, and the specificity analysis of the antigen is obtained through later stage calculation, and the result is shown in figure 9, the sensor has good characteristicsHeterosexual, sensitive detection of VEGF165。
Claims (5)
1. VEGF based on strip electrode165The sensor is characterized in that VEGF is immobilized on the electrodes of the strip electrodes165Obtaining an aptamer, wherein the strip and strip electrode comprises a base material (1) and an electrode, the electrode comprises two opposite strip electrodes (2), and a plurality of block electrodes (3) are distributed between the two strip electrodes (2); one strip electrode (2) is T-shaped, the other strip electrode (2) is U-shaped, the T-shaped strip electrode (2) comprises a transverse strip (2 a) and a vertical strip (2 b), the top end of the vertical strip (2 b) is connected with the middle part of the transverse strip (2 a), and the vertical strip (2 b) faces to the concave part of the U-shaped strip electrode (2); the distances between the strip-shaped electrodes (2) and the block-shaped electrodes (3) and between the block-shaped electrodes (3) and the block-shaped electrodes (3) are all 0.2 mm;
the VEGF based on the strip electrode165The preparation method of the sensor comprises the following steps:
1) taking the strip electrodes, cleaning and drying;
2) VEGF165And (3) dropwise coating the aptamer solution on the surfaces of the cleaned and dried strip electrodes, and incubating to make the aptamer immobilized on the surfaces of the electrodes, thereby obtaining the aptamer-coated electrode.
2. The strip and strip electrode based VEGF of claim 1165A sensor, characterized by: the VEGF165The sequence of the aptamer is
5’-SH-TTTCCCGTCTTCCAGACAAGAGTGCAGGG-3’。
3. The strip and strip electrode based VEGF of claim 1165The sensor is characterized in that sodium hydroxide, hydrochloric acid and NH are adopted to clean the strip electrodes in the step 1)3、H2O2、H2The mixed solution of O is washed sequentially and then rinsed with water.
4. VE based on a bar-and-block bar electrode as claimed in claim 1GF165A sensor, characterized by: VEGF in step 2)165Aptamer solution concentration was 500nM and incubation at 4 ℃ for 24 h.
5. VEGF based on strip electrode165The detection method of the sensor is characterized by comprising the following steps: VEGF based on a striped electrode according to claim 1165Sensor, dripping solution to be detected on the surface of the electrode, reacting for 60min at 37 ℃ incubation temperature, connecting with an electrochemical analyzer or an impedance measuring instrument to measure impedance values of the electrode at different frequencies, and calculating and judging whether the solution to be detected contains VEGF or not through corresponding impedance diagram165And VEGF contained therein165The concentration of (2).
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| CN202010003065.7A CN111122675B (en) | 2020-01-02 | 2020-01-02 | Strip-block-strip electrode and VEGF based on strip-block-strip electrode165Sensor, preparation method and detection method thereof |
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| US6417574B1 (en) * | 1999-08-11 | 2002-07-09 | Fujitsu Media Devices Limted | Surface-acoustic-wave device for flip-chip mounting |
| WO2010104479A1 (en) * | 2009-03-11 | 2010-09-16 | Agency For Science, Technology And Research | Electrical sensor for ultrasensitive nucleic acid detection |
| CN107478690A (en) * | 2017-07-01 | 2017-12-15 | 华中科技大学 | Shearing orientation measuring method in a kind of macromolecule layer based on interdigital electrode |
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| US6417574B1 (en) * | 1999-08-11 | 2002-07-09 | Fujitsu Media Devices Limted | Surface-acoustic-wave device for flip-chip mounting |
| WO2010104479A1 (en) * | 2009-03-11 | 2010-09-16 | Agency For Science, Technology And Research | Electrical sensor for ultrasensitive nucleic acid detection |
| CN107478690A (en) * | 2017-07-01 | 2017-12-15 | 华中科技大学 | Shearing orientation measuring method in a kind of macromolecule layer based on interdigital electrode |
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| Enhanced performance of piezoelectric nanogenerator based on aligned nanofibers and three-dimensional interdigital electrodes;Lingling Zhang;《Nano Energy》;ELSEVIER;20191130;第65卷;第1-10页 * |
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