CN110161100B - Preparation method of label-free electrochemical sensor for cardiac troponin I and detection method for cTnI - Google Patents

Preparation method of label-free electrochemical sensor for cardiac troponin I and detection method for cTnI Download PDF

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CN110161100B
CN110161100B CN201910434617.7A CN201910434617A CN110161100B CN 110161100 B CN110161100 B CN 110161100B CN 201910434617 A CN201910434617 A CN 201910434617A CN 110161100 B CN110161100 B CN 110161100B
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高凤
汪庆祥
褚亚茹
宋娟
吴杨仪
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Minnan Normal University
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Abstract

The experiment constructs novel label-free BSA/anti-cTnI/Cu-MOF-74/NH by combining field preparation of Cu-MOF-74 with electrochemical activity and biological immune reaction2The rGO/GCE sensor is used for carrying out specific reaction on a sensor and a target molecule (a cardiac marker cTnI) to obtain a cardiac marker antigen-antibody binding layer, so that the electrochemical active Cu-MOF-74 layer is subjected to electron transfer disturbance, and the output electrochemical signal is subjected to regular change. The electrochemical sensor can be used for rapid and sensitive detection of cardiac biomarkers, and provides possibility for early discovery, early diagnosis and early treatment of patients with cardiovascular diseases.

Description

Preparation method of label-free electrochemical sensor for cardiac troponin I and detection method for cTnI
Technical Field
The invention relates to the field of biomedical detection, in particular to a method for inducing Cu-MOF-74/NH based on immune reaction2-rGO composite layer electron transfer perturbation label-free cardiac marker cTnI electrochemical sensing detection technology.
Background
MOFs are porous nanocrystalline materials composed of metal ions and multidentate organic ligands. MOFs have many advantages such as large specific surface area, high porosity, good thermal stability, controllable physicochemical properties, etc. Accordingly, MOFs have been widely used in many fields such as catalysis, adsorption, optics, electricity, magnetism, and environment. In recent years, based on the unique advantages of the MOFs in structure and physicochemical properties, the research of the MOFs in the field of electrochemical analysis is more and more emphasized, and the application analysis objects are more and more extensive, relating to inorganic ions, organic molecules and biological macromolecules. At present, more and more MOFs materials are applied to the construction of electrochemical sensors, and researchers more utilize the characteristics of their surface area effect and open three-dimensional structure. However, the excellent electrochemical activity exhibited by MOFs through screening for suitable ligands and metal ions has not been sufficiently valued in sensor construction.
Cardiac troponin t (ctnt) and cardiac troponin i (ctni) have been accepted as standard biochemical indicators for diagnosing Acute Myocardial Infarction (AMI) and Acute Coronary Syndrome (ACS) as standard biochemical indicators for the diagnosis of AMI and ACS by the european society for cardiology, american society for cardiology, and american association for cardiology. cTnT is expressed to a lesser extent in skeletal muscle, but cTnI has not been found outside the myocardium. cTnI begins to rise rapidly in blood flow within 3-4h after the onset of acute myocardial infarction and remains as long as 4-10d, which allows diagnosis of AMI with a longer window. Because of the cardiac specificity of cTnI and the long-lasting features in circulation, cTnI can be a good biomarker for AMI. Therefore, we need to quickly and accurately assess cTnI elevation to ensure correct diagnosis in the early development, prognosis and monitoring of AMI. Serum cTnI levels reported in AMI-diagnosed patients were as high as 5-50 ng/mL. Several methods and sensors for more accurate detection of serum cTnI have been provided in recent literature, such as
[ first document ] Tang M, Zhou Z, Shangguan L, et al, electrochemiluminescence detection of cardiac troponin I by using soybean peroxidase labeled antibody as signal amplifier [ J ]. Talanta,2018,180:47-53 (Tangmin, Weekly, Shanguan L, etc. ], electrochemiluminescence detection of cardiac troponin I [ J ]. Talanta, Vol.180: pages 47-53.)
[ document two ] Tong Z, Ning M, Asghar A, et al, electrochemical ultrasensitive detection of cardiac troponin I using a covalent framework for signal amplification [ J ] Biosensors and Bioelectronics,2018,119:176-
[ document III ] Lv H, Li Y, Zhang X, et al.A. ion in functionalized signal amplified double-module electrochemical analysis for sensitive detection of cardiac troponin I [ J ]. Biosensors and Bioelectronics,2019,133:72-78.(Lv H, Li Y, Zhang X, et al. thionine functionalized signal amplified marker derived bimodal electrochemical immunoassay for sensitive detection of cardiac troponin I [ J ]. Biosensors and Bioelectronics, Vol.133: 72-78.)
[ IV ] Chekin F, Vasilescu A, Jijie R, et al, sensitive electrochemical detection of cardiac troponin I in serum and saliva by nitro-produced graphene oxide electrode [ J ]. Sensors & activators B Chemical,2018,262:180-
However, the detection limit of the above documents is high and the detection range is narrow, and as the sensitivity of detecting troponin is increased, the concentration of cTnI in serum used for the evaluation of ACS and AMI is decreased, currently in the range of 0.01 to 0.1 ng/mL. Recent studies found that lower cut-off values and repeated tests after 1-3h could better identify patients at risk for AMI and improve survival of AMI. Therefore, obtaining a rapid and reliable diagnostic tool for serum levels of cTnI in the pg/mL to ng/mL range has become a common goal in the industry.
Disclosure of Invention
The invention aims to provide a rapid and reliable diagnostic tool for cTnI serum level in the range of pg/mL to ng/mL.
In order to achieve the above purpose, the solution of the invention is: the preparation method of the label-free electrochemical sensor of the cardiac troponin I comprises the following specific steps:
the method comprises the following steps: 1mg of NH2-rGO is dispersed in 1mL of ultrapure water and is subjected to ultrasonic treatment for 10min to obtain uniform black NH2-rGO dispersion;
step two: pretreating the bare glassy carbon electrode, and then pretreating the bare glassy carbon electrodePlacing in a mixed solution of 2.5% potassium dichromate and 10% nitric acid, and oxidizing by potentiostatic method to generate oxygen-containing active groups such as-COOH on the surface, wherein the potential in the potentiostatic method is +1.5V, and the time is 15 s; placing the oxidized bare glassy carbon electrode in 200 μ L of activating solution for activating treatment for 2h, naturally drying, and taking 10 μ L of 1.0mg/mL NH2-rGO is drop-coated onto the electrode surface and allowed to air dry naturally, thus obtaining NH2-rGO/GCE electrodes;
step three: Cu-MOF-74/NH2-preparation of rGO/GCE: preparation of 20mM Cu (NO)3)2·3H2Mixing O and 0.1M KCl solution as deposition solution, introducing nitrogen gas into the deposition solution for 20min to remove oxygen in the solution, and adding NH obtained in step two2Placing the-rGO/GCE electrode in the deoxidized deposition solution, electrodepositing for 70s under-0.4V by a potentiostatic method, introducing nitrogen into the deposition solution in the whole electrodeposition process to prevent oxygen from entering, leaching the electrode with water for the second time after electrodeposition, and drying to obtain Cu/NH2-rGO/GCE; preparing phosphate buffer solution containing 0.5mM 2, 5-dihydroxy terephthalic acid, introducing nitrogen to remove oxygen in the solution, and adding Cu/NH2Putting the-rGO/GCE in phosphate buffer salt solution of 2, 5-dihydroxyterephthalic acid, performing 20 cycles in a scanning potential interval of-1.0V to +1.0V by adopting cyclic voltammetry, then leaching with ultrapure water, and finally preparing Cu-MOF-74/NH2-rGO/GCE electrodes;
step four: preparation of a label-free electrochemical sensor of cardiac troponin I: the Cu-MOF-74/NH obtained in the third step2Putting rGO/GCE into 200 mu L of activation solution, soaking the rGO/GCE into 200 mu L of immunohistochemical phosphate buffered saline solution containing 4 mu g/mL of anti-cTnI after 2h of activation treatment for 4h, and leaching with water for the second time to obtain the anti-cTnI/Cu-MOF-74/NH2-rGO/GCE; finally, anti-cTnI/Cu-MOF-74/NH2Soaking the-rGO/GCE in 200 mu L of 1% BSA solution for 2h, leaching with water for the second time, and finally preparing BSA/anti-cTnI/Cu-MOF-74/NH2-rGO/GCE sensor, i.e. label-free electrochemical sensor of cardiac troponin I.
Further, the bare glass is carbonized in the second stepThe pretreatment steps are as follows: first, 1.0 μm, 0.3 μm, 0.05 μm Al was used2O3Polishing the powder by the powder in sequence; then, carrying out ultrasonic treatment on the mixture for 5min by using ethanol and ultrapure water; finally using N2And drying the surface of the electrode for later use.
Further, the activating solution is 5.0 × 10-3mol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 8.0X 10-3A phosphate buffered saline solution mixed mol/LN-hydroxysuccinimide.
The detection method of the label-free electrochemical sensor of the cardiac troponin I on the cTnI comprises the following steps: soaking the unmarked electrochemical sensor of the cardiac troponin I in cTnI with different concentrations for 50 minutes, and then carrying out electrochemical detection on the result of identifying the unmarked electrochemical sensor of the cardiac troponin I with different concentrations by adopting a differential pulse voltammetry in a phosphate buffered saline solution with the pH value of 6.86, wherein the differential pulse voltammetry is used for testing parameters: the scanning potential is-0.4- +0.2V, the scanning speed is 0.1V/s, the potential increment is 0.004V, the potential amplitude is 0.05V, the pulse width is 0.05s, and the pulse period is 0.5 s.
Further, when the label-free electrochemical sensor of the cardiac troponin I is soaked in cTnI with different concentrations, the oxidation peak current of the label-free electrochemical sensor is gradually reduced along with the increase of the concentration of the cTnI, and the delta I of the label-free electrochemical sensor is within the range of 0.01pg/mL to 1.0ng/mL of the concentration of the cTnIpaHas good linear relation with the negative logarithm of the concentration of cTnI, and the linear equation is delta Ipa(μA)＝5.515-0.3649lgCcTnI(g/mL)，R＝0.9950。
Further, the detection limit of the method cTnI is 4.5 fg/mL.
After the scheme is adopted, the scheme combines the on-site preparation of the electrochemical sensor Cu-MOF-74 with electrochemical activity and the biological immune reaction to construct the novel label-free BSA/anti-cTnI/Cu-MOF-74/NH2-an rGO/GCE sensor (unmarked electrochemical sensor of cardiac troponin I) which reacts specifically with a target molecule (cardiac marker) to obtain a cardiac marker antigen-antibody binding layer, thereby causing an electrochemically active Cu-MOF-74 layer "electron transfer perturbation",the output electrochemical signal is changed regularly. Therefore, the electrochemical sensor can be used for quickly and sensitively detecting the cardiac biomarkers, and provides possibility for early discovery, early diagnosis and early treatment of patients with cardiovascular diseases.
Drawings
FIG. 1 shows BSA/anti-cTnI/Cu-MOF-74/NH2Preparation process of rGO/GCE sensor (unmarked electrochemical sensor of cardiac troponin I) and detection schematic diagram of cTnI by using the sensor;
FIG. 2 is NH2SEM picture (A) of rGO/GCE, Cu/NH2SEM picture (B) of rGO/GCE, Cu-MOF-74/NH2SEM image (C, D) of rGO/GCE: SEM images at low magnification (C), SEM images at high magnification (D);
in FIG. 3, (a) is Cu/NH2-rGO, (b) is Cu-MOF-74/NH2-ATR-FTIR profile of rGO/GCE;
FIG. 4 shows Cu-MOF-74/NH2-rGO/GCE(a)、anti-cTnI/Cu-MOF-74/NH2-rGO/GCE(b)、 BSA/anti-cTnI/Cu-MOF-74/NH2-rGO/GCE(c)、 cTnI/BSA/anti-cTnI/Cu-MOF-74/NH2-cyclic voltammogram of rGO/gce (d) in 0.01M phosphate buffered saline (pH 6.86);
FIG. 5 (A) Cu-MOF-74/NH2Cyclic voltammograms (CV maps) of different sweeps of rGO/GCE in phosphate buffered saline (PBS solution) (pH 6.86) (a → n): 0.01,0.02,0.04,0.06,0.08,0.10,0.15, 0.20,0.25,0.30,0.35,0.40,0.45,0.50V/s, (B) redox peak current (Ip) and number of roots of sweep speed (V)1/2) A linear relationship graph;
in FIG. 6 (A) BSA/anti-cTnI/Cu-MOF-74/NH2-rGO/GCE (label-free electrochemical sensor of cardiac troponin I) differential pulse voltammogram (DPV-graph) of the oxidation peaks in different concentrations of cTnI (red to black: 0, 0.01pg/mL, 0.1pg/mL, 1.0pg/mL, 10pg/mL, 100pg/mL, 1.0ng/mL, 10ng/mL, 100 ng/mL); (B) oxidation peak current values (Δ I) for the sensor after exposure to different concentrations of cTnIp) Negative logarithm of concentration (-lgC)cTnI) A linear relationship graph of (a);
FIG. 7 shows BSA/anti-cTnI/Cu-MOF-74/NH2rGO/GCE (Label-free electrochemical transduction of cardiac troponin I)Sensor) for a, blank solution; hb (1.0 ng/mL); myoglobin (Myo) (1.0 ng/mL); d is cTnI (1.0 ng/mL); and e, electrochemical response of the mixed solution of Hb, myoglobin (Myo) and cTnI with the solubility of 1.0 ng/mL.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments.
The preparation method of the label-free electrochemical sensor of the cardiac troponin I comprises the following specific steps:
the method comprises the following steps: 1mg of NH2-rGO is dispersed in 1mL of ultrapure water and is subjected to ultrasonic treatment for 10min to obtain uniform black NH2-rGO dispersion;
step two: pretreating the bare glassy carbon electrode: first, 1.0 μm, 0.3 μm, 0.05 μm Al was used2O3Polishing the powder by the powder in sequence; then, carrying out ultrasonic treatment on the mixture for 5min by using ethanol and ultrapure water; finally, drying the surface of the electrode by using N2 for later use; placing the pretreated bare Glassy Carbon Electrode (GCE) in a mixed solution of 2.5% potassium dichromate and 10% nitric acid, and oxidizing the electrode by a potentiostatic method to generate oxygen-containing active groups such as-COOH on the surface of the electrode, wherein the potential in the potentiostatic method is +1.5V and the time is 15 s; the oxidized bare glassy carbon electrode was placed in 200. mu.L of activating solution (5.0X 10)-3mol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 8.0X 10-3Phosphate buffer solution mixed by mol/LN-hydroxysuccinimide) for 2 hours, and taking 10 mu L of 1.0mg/mL NH after naturally airing2-rGO is drop-coated onto the electrode surface and allowed to air dry naturally, thus obtaining NH2-rGO/GCE electrodes;
step three: Cu-MOF-74/NH2-preparation of rGO/GCE: preparation of 20mM Cu (NO)3)2·3H2Mixing O and 0.1M KCl solution as deposition solution, introducing nitrogen gas into the deposition solution for 20min to remove oxygen in the solution, and adding NH obtained in step two2Placing the rGO/GCE electrode in the deoxidized deposition solution, electrodepositing for 70s under-0.4V by a potentiostatic method, introducing nitrogen into the deposition solution in the whole process of electrodeposition to prevent oxygen from entering, rinsing the electrode with water for the second time after electrodeposition, and airingDrying to obtain Cu/NH2-rGO/GCE; preparing phosphate buffer solution containing 0.5mM 2, 5-dihydroxy terephthalic acid, introducing nitrogen to remove oxygen in the solution, and adding Cu/NH2Putting the-rGO/GCE in phosphate buffer salt solution of 2, 5-dihydroxyterephthalic acid, performing 20 cycles in a scanning potential interval of-1.0V to +1.0V by adopting Cyclic Voltammetry (CV), then leaching with ultrapure water, and finally preparing Cu-MOF-74/NH2-rGO/GCE electrodes;
step four: preparation of a label-free electrochemical sensor of cardiac troponin I: the Cu-MOF-74/NH obtained in the third step2Putting rGO/GCE into 200 mu L of activation solution, soaking the rGO/GCE into 200 mu L of immunohistochemical phosphate buffered saline solution containing 4 mu g/mL of anti-cTnI after 2h of activation treatment for 4h, and leaching with water for the second time to obtain the anti-cTnI/Cu-MOF-74/NH2-rGO/GCE; finally, anti-cTnI/Cu-MOF-74/NH2Soaking the-rGO/GCE in 200 mu L of 1% BSA solution for 2h, leaching with water for the second time, and finally preparing BSA/anti-cTnI/Cu-MOF-74/NH2-rGO/GCE sensor, i.e. label-free electrochemical sensor of cardiac troponin I;
step five: the detection method of the cTnI by taking the label-free electrochemical sensor of the cardiac troponin I as a sensor comprises the following steps: the label-free electrochemical sensor of the cardiac troponin I is respectively soaked in cTnI with different concentrations for 50 minutes, and then the result of the label-free electrochemical sensor of the cardiac troponin I for identifying the cTnI with different concentrations is electrochemically detected in phosphate buffered saline (PBS solution) with pH of 6.86 by adopting a Differential Pulse Voltammetry (DPV), and the DPV testing parameters are as follows: the scanning potential is-0.4- +0.2V, the scanning speed is 0.1V/s, the potential increment is 0.004V, the potential amplitude is 0.05V, the pulse width is 0.05s, and the pulse period is 0.5 s.
FIG. 1 is a schematic diagram of the preparation process of the label-free electrochemical sensor for cardiac troponin I and the detection of cTnI by using the sensor. From the figure, the whole principle process of the electrode in the preparation process can be seen, and the novel label-free BSA/anti-cTnI/Cu-MOF-74/NH is constructed by combining the in-situ preparation of the Cu-MOF-74 with electrochemical activity and the biological immune reaction2-rGO/GCE sensor, with sensorAnd the specific reaction is carried out with a target molecule (cardiac marker) to obtain a cardiac marker antigen-antibody binding layer, so that the Cu-MOF-74 layer with electrochemical activity causes 'electron transfer disturbance', and the output electrochemical signal is regularly changed.
Fig. 2 is a morphology characterization diagram of different modified electrodes through SEM (scanning electron microscope), and fig. 2A is a SEM characterization diagram of NH2-rGO/GCE, from which it can be observed that the flaky aminated graphene is uniformly distributed on the electrode surface. FIG. 2B is Cu/NH2SEM representation of rGO/GCE, and many uniform particles attached to the surface of the electrode can be seen, which indicates that elemental copper is successfully deposited on the surface of the electrode. FIG. 2C is Cu-MOF-74/NH2SEM image of rGO/GCE, and it can be observed that the electrode surface is distributed with many clusters formed by rod-shaped stacks. FIG. 2D shows Cu-MOF-74/NH at high position rate2SEM image of rGO/GCE, from which it can be observed that clusters formed by stacking rods grow on the plate-like aminated graphene, which indicates that Cu-MOF-74 is successfully synthesized on the surface of NH 2-rGO/GCE.
FIG. 3 is Cu/NH2-rGO (a) and Cu-MOF-74/NH2IR spectrum of rGO/GCE (b), shown in the figure, Cu-MOF-74/NH2Spectra of rGO/GCE (b) and Cu/NH2-rGO (a) at a ratio of 600-1700 cm-1A series of new bands appeared in the range, which are mainly attributed to vibrational modes of the organic ligands. 1640 and 1423cm-1The strong band appeared here is due to the stretching vibration of-COOH in the organic ligand, confirming the presence of a dicarboxylic acid linker, 700cm, in the frame-1The nearby wavelength band is attributable to the stretching and plane bending vibration of the benzene ring. 842 and 1045cm-1At corresponds to M2 (C)8H2O6) Bending vibration of hydroxyl groups on a common hexagon, 1172cm-1The strong peak at (b) is due to stretching vibration of the hydroxyl group. The spectrum also shows 3400-3600 cm-1Very broad wavelength band, which is the adsorbed H in the material2O is caused by. In conclusion, the characterization results show that Cu-MOF-74 is in NH2-successfully synthesized on rGO/GCE.
As shown in FIG. 4, Cu-MOF-74/NH2-rGO/GCE (a) has a scanning interval of-0.8- +0.4VThe obvious redox peak indicates that the Cu-MOF-74 has good redox electrical activity on the surface of the electrode. Activation of Cu-MOF-74/NH with an activating liquid2-rGO/GCE 2h, activating-COOH on the Cu-MOF-74 material to be-COO-, and continuing to activate the activated Cu-MOF-74/NH2Soaking rGO/GCE in immunohistochemical phosphate buffered saline (PBS solution) containing 4 mu g/mL anti-cTnI for 4h, and utilizing-NH possessed by protein2The immobilization of the antibody on the electrode is realized by the condensation reaction with-COO-on Cu-MOF-74 to generate an amide bond, and as a result, as shown in a curve b, compared with a curve a, the peak current signal is obviously reduced because the immobilization of the antibody on the surface of the electrode forms an insulating layer, which hinders the electron conduction rate, and thus the electric conduction capability of the electrode is reduced. As a result of blocking the excess-COO-active sites with BSA, the conductivity of the electrode was further reduced and the peak current signal was further reduced due to the BSA attachment to the electrode surface, as shown in curve c. The prepared label-free electrochemical sensor for cardiac troponin I is used for detecting cTnI of 1.0ng/mL, as shown by a curve d, due to specific binding immunoreaction of an antigen-antibody, an antigen is also bound to the surface of an electrode, an insulating layer is thickened so as to further obstruct the transmission of electrons, and a peak current signal is continuously reduced, which indicates that the sensor has good recognition capability on the cTnI.
Further examine different scan speeds for Cu-MOF-74/NH2-effect of rGO/GCE electrochemical response in phosphate buffered saline (PBS solution). As can be seen from fig. 5A, in the process of increasing the scanning speed, the redox peak current signal also increases; from FIG. 5B, the redox peak current signal and v1/2Presents excellent linear relation, and the linear regression equation of the oxidation reduction peak is respectively as follows:
Ipa(μA)＝-4.700+391.8ν1/2(V1/2 ·s-1/2)，R＝0.9962；
Ipc(μA)＝17.17-207.5ν1/2(V1/2 ·s-1/2)，R＝0.9951。
the results show that Cu-MOF-74/NH2Electrochemical behavior of-rGO/GCE surfaces is mainly influenced by diffusion control。
In order to explore the detection performance of the sensor as a sensor on a target object cTnI, when the unmarked electrochemical sensor of the troponin I in the five central muscle in the step is soaked in cTnI with different concentrations, the result of DPV detection is shown in figure 6A, the oxidation peak current of the electrochemical sensor is gradually reduced along with the increase of the concentration of the cTnI, and the delta I of the electrochemical sensor is shown in figure 6B, wherein the concentration of the cTnI is in the range of 0.01pg/mL to 1.0ng/mLpaAnd concentration (C)cTnI) The negative logarithm of (d) is in good linear relationship, as shown in the data in table I, the obtained linear equation is Δ Ipa(μA)＝5.515- 0.3649lgCcTnI(g/mL), R-0.9950, limit of detection of cTnI is 4.5 fg/mL.
TABLE-different concentrations of cTnI (C)cTnI) And corresponding negative logarithm of concentration (lgC)cTnI) Lower sensor peak current value
Concentration (g/mL) 1×10-9 1×10-10 1×10-11 1×10-12 1×10-13 1×10-14 0
Negative logarithm of concentration 9 10 11 12 13 14 －
Ip(μA) -3.926 -4.159 -4.605 -4.849 -5.320 -5.746 -6.088
ΔIp(μA) 2.162 1.929 1.483 1.239 0.768 0.342 －
As shown in fig. 6A, the DPV detection result shows that the oxidation peak current gradually decreases with the increase of the cTnI concentration, because the antibody immobilized on the surface of the electrode specifically binds to the cTnI antigen to cause an immune reaction, so that the insulating layer gradually becomes thicker due to the increase of the cTnI concentration, thereby blocking the electron conduction rate and causing the electrochemical behavior of the electrode to change.
And the second table is the comparison of the performance of the sensor constructed in the text with that of other recently reported documents, and the comparison result shows that the sensor constructed in the text has lower detection limit, wide detection range and excellent performance.
TABLE comparison of Performance of two different sensors to detect cTnI
Figure RE-GDA0002124204990000091
The selectivity, reproducibility of the present sensor was further investigated as follows:
1. hb, myoglobin (Myo) and cTnI with the concentration of 1.0ng/mL and a mixed solution of the Hb, the myoglobin (Myo) and the cTnI with the cardiac troponin I through a DPV differential pulse method are subjected to interference analysis, and the results are shown in figure 7, wherein Hb (b), Myo (c) and the signal are basically unchanged compared with the blank liquid (a), which indicates that the sensor does not recognize the Hb and the Myo. compared with the blank liquid (a), signals of the cTnI (c), (d) and the mixed liquid (e) are obviously reduced, and the signals of the cTnI (d) and the mixed liquid (e) are basically consistent, and experimental results show that Hb (b) and Myo (c) basically have no interference to the sensor, and the sensor has good selectivity to the cTnI.
2. The reproducibility is also an important index for inspecting the performance of the sensor, and in order to inspect the reproducibility of the sensor, the linear equation is used for measurement, and the solution with the concentration of 1.0ng/mL cTnI is subjected to parallel modification for 5 times by using a same branch electrode to obtain the actual measurement concentrations of 0.94, 0.99, 1.01, 1.03 and 1.08ng/mL, and the Relative Standard Deviation (RSD) is 5.1 percent; the assay was repeated 5 times with a 1.0ng/mL solution of cTnI using the homopolar modified electrode with a Relative Standard Deviation (RSD) of 5.2%. The experimental sensor has good reproducibility. The label-free electrochemical sensor of the cardiac troponin I is placed in a refrigerator to be stored for 10 days, DPV response signals are measured in a blank solution every 2 days, finally, the signals are attenuated to 91.2% of the original signals, the sensor is used for detecting the solution of 1.0ng/mL cTnI on the tenth day, the obtained signal current is brought into a linear equation, the measured concentration is 0.94ng/mL, and the sensor has good stability.
3. Determination of cTnI in actual serum samples by sensor: in order to further examine the analysis capability of the label-free electrochemical sensor for cardiac troponin I on actual samples, the serum labeling method is adopted for further research, the experimental results are shown in table three, and the recovery rate of cTnI in serum is between 95% and 104% by adding a known amount of cTnI standard solution into a blank serum sample, so that the sensor is feasible.
Detection of cTnI in serum samples
Figure RE-GDA0002124204990000101
In the experiment, a novel cTnI sensor combining electrochemical activity Cu-MOF-74 with biological immune reaction is successfully synthesized on the surface of a glassy carbon electrode through electrodeposition. The experimental result shows that under the optimal condition, the detection range of the cTnI is 10 fg/mL-1 ng/mL, and the detection limit is 5.4 fg/mL. In addition, the sensor has good selectivity on cTnI, and can be used for detecting the cTnI in a human serum sample. Thus, the sensor has considerable potential in aiding diagnosis of myocardial damage, particularly myocardial infarction.
The above description is only an embodiment of the present invention, and is not intended to limit the design of the present invention, and all equivalent changes made according to the design key of the present invention fall within the protection scope of the present invention.

Claims (6)

1. The preparation method of the label-free electrochemical sensor of the cardiac troponin I comprises the following specific steps:
the method comprises the following steps: 1mg of NH2-rGO is dispersed in 1mL of ultrapure water and is subjected to ultrasonic treatment for 10min to obtain uniform black NH2-rGO dispersion;
step two: pretreating a bare glassy carbon electrode, and placing the pretreated bare glassy carbon electrode in a mixed solution of 2.5% of potassium dichromate and 10% of nitric acid to carry out oxidation treatment by adopting a potentiostatic method, so that a-COOH oxygen-containing active group is generated on the surface of the bare glassy carbon electrode, wherein the potential in the potentiostatic method is +1.5V, and the time is 15 s; placing the oxidized bare glassy carbon electrode in 200 μ L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) activation solution for activation treatment for 2h, and naturally airingAfter drying, 10. mu.L of 1.0mg/mL NH was taken2-rGO is drop-coated onto the electrode surface and allowed to air dry naturally, thus obtaining NH2-rGO/GCE electrodes;
step three: Cu-MOF-74/NH2-preparation of rGO/GCE: preparation of 20mM Cu (NO)3)2·3H2Mixing O and 0.1M KCl solution as deposition solution, introducing nitrogen gas into the deposition solution for 20min to remove oxygen in the solution, and adding NH obtained in step two2Placing the-rGO/GCE electrode in the deoxidized deposition solution, electrodepositing for 70s under-0.4V by a potentiostatic method, introducing nitrogen into the deposition solution in the whole electrodeposition process to prevent oxygen from entering, leaching the electrode with water for the second time after electrodeposition, and drying to obtain Cu/NH2-rGO/GCE; preparing phosphate buffer solution containing 0.5mM 2, 5-dihydroxy terephthalic acid, introducing nitrogen to remove oxygen in the solution, and adding Cu/NH2Putting the-rGO/GCE in phosphate buffer salt solution of 2, 5-dihydroxyterephthalic acid, performing 20 cycles in a scanning potential interval of-1.0V to +1.0V by adopting cyclic voltammetry, then leaching with ultrapure water, and finally preparing Cu-MOF-74/NH2-rGO/GCE electrodes;
step four: preparation of a label-free electrochemical sensor of cardiac troponin I: the Cu-MOF-74/NH obtained in the third step2Putting rGO/GCE into 200 mu L of activation solution, soaking the rGO/GCE into 200 mu L of immunohistochemical phosphate buffered saline solution containing 4 mu g/mL of anti-cTnI after 2h of activation treatment for 4h, and leaching with water for the second time to obtain the anti-cTnI/Cu-MOF-74/NH2-rGO/GCE; finally, anti-cTnI/Cu-MOF-74/NH2Soaking the-rGO/GCE in 200 mu L of 1% BSA solution for 2h, leaching with water for the second time, and finally preparing BSA/anti-cTnI/Cu-MOF-74/NH2-rGO/GCE sensor, i.e. label-free electrochemical sensor of cardiac troponin I.
2. The method for preparing a label-free electrochemical sensor of cardiac troponin I according to claim 1, wherein: the step two of pretreating the bare glassy carbon electrode comprises the following steps: first, 1.0 μm, 0.3 μm, 0.05 μm Al was used2O3Polishing the powder by the powder in sequence; then using ethanol to carry out ultrafiltrationUltrasonic treating with pure water for 5 min; finally using N2And drying the surface of the electrode for later use.
3. The method for preparing a label-free electrochemical sensor of cardiac troponin I according to claim 1, wherein: the activating solution is 5.0 x 10-3mol/L EDC and 8.0X 10-3mol/L NHS mixed phosphate buffered saline.
4. A method for detecting cTnI by using a label-free electrochemical sensor for cardiac troponin I produced by the production method according to claim 1, comprising: respectively soaking the unmarked electrochemical sensor of the cardiac troponin I in cTnI with different concentrations for 50 minutes, and then carrying out electrochemical detection on the result of identifying the unmarked electrochemical sensor of the cardiac troponin I with the cTnI with different concentrations by adopting a differential pulse voltammetry in a phosphate buffered saline solution with the pH value of 6.86, wherein the differential pulse voltammetry is used for testing parameters: the scanning potential is-0.4- +0.2V, the scanning speed is 0.1V/s, the potential increment is 0.004V, the potential amplitude is 0.05V, the pulse width is 0.05s, and the pulse period is 0.5 s.
5. The method for detecting cTnI by using a label-free electrochemical sensor for detecting cardiac troponin I according to claim 4, wherein: in the method, when the label-free electrochemical sensor of the cardiac troponin I is soaked in cTnI with different concentrations, the oxidation peak current of the label-free electrochemical sensor is gradually reduced along with the increase of the concentration of the cTnI, and the delta I of the label-free electrochemical sensor is within the range of 0.01pg/mL to 1.0ng/mL of the concentration of the cTnIpaHas good linear relation with the negative logarithm of the concentration of cTnI, and the linear equation is delta Ipa(μA)＝5.515-0.3649lgCcTnI(g/mL)，R＝0.9950。
6. The method for detecting cTnI by using a label-free electrochemical sensor for detecting cardiac troponin I according to claim 5, wherein: the detection limit of cTnI in the above method was 4.5 fg/mL.
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