CN112630276A - Preparation method of electrochemical sensor for detecting sepsis marker procalcitonin - Google Patents

Preparation method of electrochemical sensor for detecting sepsis marker procalcitonin Download PDF

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CN112630276A
CN112630276A CN202011286938.6A CN202011286938A CN112630276A CN 112630276 A CN112630276 A CN 112630276A CN 202011286938 A CN202011286938 A CN 202011286938A CN 112630276 A CN112630276 A CN 112630276A
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electrochemical sensor
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CN112630276B (en
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刘雨双
包腊月
刘景海
海文峰
鲍文吉
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Inner Mongolia University for Nationlities
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Abstract

The invention discloses a preparation method of an electrochemical sensor for detecting a sepsis marker procalcitonin. According to the electrochemical sensor prepared by the invention, the specific surface area and the electron transfer capacity can be increased during detection by modifying the exfoliated graphite-phase carbon nitride on the surface of the glassy carbon electrode, and the polypeptide probe capable of specifically recognizing procalcitonin is modified on the glassy carbon electrode, so that the sensitivity, the accuracy and the characteristic selectivity of the sensor detection can be improved conveniently, and the detection sensitivity of the electrochemical sensor on the procalcitonin can reach 0.15 fg/mL.

Description

Preparation method of electrochemical sensor for detecting sepsis marker procalcitonin
Technical Field
The invention relates to the technical field of electrochemical sensors, in particular to a preparation method of an electrochemical sensor for detecting a sepsis marker procalcitonin.
Background
Sepsis is a potentially fatal or life-altering syndrome in which the body's response to an infection is a systemic immune response. Sepsis is considered by many to be three stages, starting with sepsis, progressing to severe sepsis and septic shock. Currently, approximately 30% to 70% of sepsis patients fail to survive multiple organ failure. Therefore, early, timely, specific treatment of sepsis is important and clinically necessary.
Procalcitonin (ProCT) is a sepsis-inducing protein, a hormone-inactive calcitonin propeptide material, which can be used as a marker of infectious inflammation to distinguish between bacterial and viral infections and to determine the severity of bacterial infections and prognostic tests. ProCT has been recognized as the most ideal indicator for diagnosing severe bacterial infection compared to conventional indicators for inflammation diagnosis, and has now been widely used in clinical applications such as monitoring of autoimmune diseases, monitoring of organ transplantation, monitoring of sepsis, monitoring of acute pancreatitis, and the like.
Currently, a number of ProCT detection methods based on conventional methods have been reported, including Blood Culture (BC), molecular diagnostic techniques, PCR-based methods, mass spectrometry, and protein microarrays. However, these methods all have the disadvantages of high instrument and equipment, high labor intensity, complex sample preparation, long time consumption and the like. Electrochemical detection methods are of great interest because of their high sensitivity, low cost, short time consumption, and the like. However, for the electrochemical sensor, designing a high-sensitivity and high-selectivity recognition system is one direction capable of effectively improving the detection efficiency and sensitivity.
In recent years, two-dimensional graphite phase carbon nitride (g-C)3N4) As a new class of carbon nanomaterials, they are gradually appearing in the line of sight of people due to their remarkable characteristics of large specific surface area, no metal, easy synthesis, thermal stability, low toxicity, low cost, and the like. g-C3N4In drug delivery, tumor imagingAnd the sensing process has attracted worldwide attention. However, g-C3N4The wide bandgap limits their use in electrochemical applications. For this reason, several methods such as doping impurities, coupling with a typical semiconductor, and nanostructure synthesis have been proposed.
And the exfoliated body g-C3N4g-C of NS than usual3N4Has better electrochemical activity and stability, so the exfoliant g-C is adopted3N4The use of NS for the preparation of electrochemical sensors for the detection of sepsis is a promising direction.
Disclosure of Invention
Accordingly, based on the above background, the present invention provides an exfoliated graphite phase-based carbon nitride (g-C)3N4NS) is used for detecting the sepsis marker procalcitonin, and has higher sensitivity and specificity selectivity.
The technical scheme of the invention is as follows: a preparation method of an electrochemical sensor for detecting a sepsis marker procalcitonin comprises the following steps:
s1: synthesizing urea serving as a raw material by using the urea at a high temperature to obtain carbon nitride powder;
s2: dissolving and stripping the carbon nitride powder obtained in the step S1 by using an ultrasonic melting mode to prepare graphite-phase carbon nitride;
s3: modifying the graphite-phase carbon nitride prepared in the step S2 to the surface of the electrode in a pi-pi stacking mode;
s4: and (3) modifying the Polypeptide Probe (PP) capable of specifically recognizing procalcitonin on the surface of the glassy carbon electrode modified in the step S3, and then sealing the surface by using bovine serum albumin to prepare the electrochemical sensor for detecting sepsis.
Further, the modified electrode of steps S3 and S4 is a working electrode.
Furthermore, the system of the electrochemical sensor takes glassy carbon as a working electrode, Ag/AgCl as a reference electrode and platinum as a counter electrode.
Further, the preparation step of the carbon nitride powder in the step S1 is:
1) firstly, weighing a certain amount of urea, placing the urea in a quartz ceramic crucible, placing the quartz ceramic crucible in a tube furnace, heating the urea to 550 ℃ at the speed of 5 ℃/min,
2) keeping the temperature of a quartz ceramic crucible at 550 ℃ for 3 hours under normal pressure;
3) and cooling the quartz ceramic crucible to room temperature, and collecting the product in the quartz ceramic crucible to prepare the carbon nitride powder.
Further, the preparation step of the graphite phase carbon nitride in the step S2 is:
1) dispersing the carbon nitride powder prepared in the step S1 in ethanol, and carrying out ultrasonic treatment on the mixed solution for 1 h;
2) standing the mixture subjected to ultrasonic treatment in the step 1), and collecting uniformly dispersed and stripped graphite-phase carbon nitride dispersion liquid after large-particle graphite-phase carbon nitride is completely precipitated at the bottom of the ethanol solution.
Further, the step of modifying the glassy carbon electrode with graphite-phase carbon nitride in step S3 is:
1) firstly, polishing a glassy carbon electrode by using alumina powder, and then continuously washing for a plurality of times by using distilled water;
2) and (4) dripping the stripped graphite-phase nitrogen carbide dispersion liquid collected in the step (S2) on the surface of the glassy carbon electrode, and drying at room temperature for 30 min.
Further, the preparation step of step S4 is:
1) and (3) dissolving the peptide powder in Tris-HCl, dripping synthetic peptide modified by proline on the surface of the glassy carbon electrode modified by graphite-phase carbon nitride in the step S3, culturing at room temperature for 1h, and washing the surface of the glassy carbon electrode by Tris-HCl and deionized water in sequence to remove unbound synthetic peptide.
By adopting the technical scheme, the beneficial effects are as follows:
according to the electrochemical sensor prepared by the invention, the vitreous graphite phase carbon nitride is modified on the surface of the glassy carbon electrode, the specific surface area and the electron transfer capacity can be increased during detection, and the polypeptide probe capable of specifically recognizing procalcitonin is modified on the glassy carbon electrode, so that the sensitivity, the accuracy and the specific selectivity of the sensor detection can be improved conveniently, and the detection sensitivity of the electrochemical sensor to the procalcitonin can reach 0.15 fg/mL.
Drawings
FIG. 1 is a schematic diagram of a preparation process of the present invention;
FIG. 2 is a graph of atomic force microscope pairs g-C according to the present invention3N4And PP/g-C3N4Characterizing the morphology of the electrode modification;
FIG. 3 shows a graph of the present invention through g-C3N4And PP/g-C3N4The electrochemical performance characterization result of the modified glassy carbon electrode;
FIG. 4 is a graph showing the results of the detection of procalcitonin at various concentrations using the electrochemical sensor of the example;
fig. 5 is a graph showing the verification result of the specific selection of the electrochemical sensor according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: a method for preparing an electrochemical sensor for detecting sepsis comprises the following steps (the preparation flow is shown in figure 1):
s1: preparing carbon nitride powder:
1) firstly, 0.25g of urea is weighed and placed in a quartz ceramic crucible, then the quartz ceramic crucible is placed in a tube furnace, the temperature is raised to 550 ℃ at the speed of 5 ℃/min,
2) keeping the temperature of a quartz ceramic crucible at 550 ℃ for 3 hours under normal pressure;
3) and cooling the quartz ceramic crucible to room temperature, and collecting the product in the quartz ceramic crucible to prepare the carbon nitride powder.
S2: preparation of graphite phase carbon nitride
1) Weighing 0.001g of the carbon nitride powder prepared in the step S1, dispersing the carbon nitride powder in 2ml of ethanol (HPLC grade), and carrying out ultrasonic treatment on the mixed solution for 1 h;
2) standing the mixture subjected to ultrasonic treatment in the step 1), and collecting uniformly dispersed and stripped graphite-phase carbon nitride dispersion liquid after large-particle graphite-phase carbon nitride is completely precipitated at the bottom of the ethanol solution.
S3: modified glassy carbon electrode of graphite phase carbon nitride
1) Polishing a glassy carbon electrode with the diameter of 3mm by using alumina powder polishing cloth, and then continuously washing for a plurality of times by using distilled water;
2) dripping 10 μ L of the graphite phase nitrogen carbide dispersion liquid collected in step S2 after peeling on the surface of a glassy carbon electrode, and drying at room temperature for 30 min.
S4: probe modified glassy carbon electrode
1) Dissolving 25mg of peptide powder in 1ml of Tris-HCl to prepare a solution with the final concentration of 500um, dripping 50 mu L of synthetic peptide with the final concentration of 500mM and modified by proline on the surface of the glassy carbon electrode modified by graphite-phase carbon nitride in the step S3, culturing for 1h at room temperature, and washing the surface of the glassy carbon electrode by sequentially using 1 xTris-HCl (pH 7.4) and deionized water to remove unbound synthetic peptide to prepare the electrochemical sensor for detecting sepsis.
In the electrochemical sensor system in this embodiment, glassy carbon is used as a working electrode, Ag/AgCl is used as a reference electrode, and platinum is used as a counter electrode.
The urea and NaCl in this example were purchased from Solarbio, Tris-HCl, deionized water, and Bovine Serum Albumin (BSA) were purchased from Solarbio (Beijing), and procalcitonin was purchased from cusag (China, Wuhan.).
In this embodiment, a series of characterization and verification are performed on the preparation process and the detection result of the sensor, and the characterization result is as follows:
1. using atomic force microscope to g-C3N4And PP/g-C3N4The characterization result of the surface morphology of the glassy carbon electrode in the modification process is shown in fig. 2, as can be seen from the characterization picture of the glassy carbon electrode before being unmodified in fig. 2(a), the surface of the glassy carbon electrode before being unmodified presents a relatively uniform flat structure, and as can be seen from the characterization result in fig. 2(b) and fig. 2(C) after passing through g-C3N4And PP/g-C3N4Modified glassy carbon electrode due to g-C3N4And PP/g-C3N4The pi-pi stacking effect of the glass carbon electrode obviously changes the shape of the surface of the glass carbon electrode, and the g-C process can be seen from figure 2(b)3N4The surface of the modified glassy carbon electrode is structured like a pancake filled with spherical pores, and thus the g-C can be determined3N4Has been successfully decorated on a glassy carbon electrode; as can be seen from FIG. 2(C), the PP solution was added dropwise to g-C3N4After the modified glassy carbon electrode surface is modified, the electrode surface is obviously changed, and bright spots and high increase in the figure show that PP is firmly combined on the electrode.
2. Adopting three modes of Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS) to adopt g-C3N4And PP/g-C3N4The electrochemical performance of the modified glassy carbon electrode was characterized and the results are shown in figure 3. FIG. 3(A) shows the characterization results of Cyclic Voltammetry (CV), in which the scan rate during characterization is 100mv/s, and it can be seen from FIG. 3(A) that the background current follows the g-C3N4And PP, thereby indicating g-C3N4Formation of/GCE and PP/g-C3N 4/GCE; while the DPV technique was used to identify the modification step, it is evident from FIG. 3(B) that the peak current shows a decreasing trend with depth of modification, which shows the g-C3N4Successfully coupled with PP, the impedance change in the glassy carbon electrode stepwise modification process is shown in FIG. 3(C), which shows the unmodified glassy carbon electrode, gC3N4(ii)/GCE and PP/gC3N4/The impedance of the GCE is almost a straight line on the Nyquist plotThe charge transfer resistance at the time of post-modification of the electrode indicates gC3N4And PP are gradually fixed on the surface of the glassy carbon electrode to form an additional barrier layer, so that the electronic exchange between the electrode and the redox probe can be prevented.
3. The electrochemical sensor is adopted to detect and verify procalcitonin with different concentrations, wherein the concentration preparation modes of the different procalcitonins are as follows: firstly, preparing a ProCT solution with the final concentration of 1.5pg/mL, and gradually diluting to reach the concentration required by detection; the results of the test are shown in FIG. 4, and it is apparent from FIG. 4(A) that the peak current exhibited by DPV is significantly reduced as the concentration of procalcitonin is increased, and FIG. 4(B) shows I/I0The change in the value of procalcitonin concentration from 0.15fg/mL to 11.7fg/mL indicates that the detection sensitivity of the sensor reaches 0.15 fg/mL.
4. The sensitivity of the electrochemical sensor of the present invention was compared with that of the conventional sensor, and the comparison results are shown in table 1.
Table 1: sensitivity comparison table of electrochemical sensor and other sensors
Figure BDA0002782682140000041
5. The specificity of the electrochemical sensor is verified by adopting C-reactive protein, and an electrolyte blank sample, a C-reactive protein sample containing procalcitonin and a procalcitonin sample are respectively prepared, wherein the preparation method of the pure procalcitonin comprises the following steps: firstly, ProCT solution with the final concentration of 1.5pg/mL is prepared and gradually diluted to reach the concentration required by detection, then the detection and verification result of the sample is shown in figure 5, and as is obvious from figure 5, compared with a procalcitonin sample, the rest samples have very weak peak current, and the electrochemical sensor disclosed by the invention has very excellent specific selectivity on the procalcitonin.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A preparation method of an electrochemical sensor for detecting a sepsis marker procalcitonin is characterized by comprising the following steps:
s1: synthesizing urea serving as a raw material by using the urea at a high temperature to obtain carbon nitride powder;
s2: dissolving and stripping the carbon nitride powder obtained in the step S1 by using an ultrasonic melting mode to prepare graphite-phase carbon nitride;
s3: modifying the graphite-phase carbon nitride prepared in the step S2 to the surface of the electrode in a pi-pi stacking mode;
s4: and (3) modifying the Polypeptide Probe (PP) capable of specifically recognizing procalcitonin on the surface of the glassy carbon electrode modified in the step S3, and then sealing the surface by using bovine serum albumin to prepare the electrochemical sensor for detecting sepsis.
2. The method for preparing an electrochemical sensor for detecting the sepsis marker procalcitonin according to claim 1, wherein the electrochemical sensor system uses glassy carbon as a working electrode, Ag/AgCl as a reference electrode, and platinum as a counter electrode.
3. The method for preparing an electrochemical sensor for detecting the sepsis marker procalcitonin according to claim 1, wherein the carbon nitride powder in the step S1 is prepared by the steps of:
1) firstly, weighing a certain amount of urea, placing the urea in a quartz ceramic crucible, placing the quartz ceramic crucible in a tube furnace, heating the urea to 550 ℃ at the speed of 5 ℃/min,
2) keeping the temperature of a quartz ceramic crucible at 550 ℃ for 3 hours under normal pressure;
3) and cooling the quartz ceramic crucible to room temperature, and collecting the product in the quartz ceramic crucible to prepare the carbon nitride powder.
4. The method for preparing an electrochemical sensor for detecting the sepsis marker procalcitonin according to claim 1, wherein the step of preparing the graphite-phase carbon nitride in the step S2 comprises the following steps:
1) dispersing the carbon nitride powder prepared in the step S1 in ethanol, and carrying out ultrasonic treatment on the mixed solution for 1 h;
2) standing the mixture subjected to ultrasonic treatment in the step 1), and collecting uniformly dispersed and stripped graphite-phase carbon nitride dispersion liquid after large-particle graphite-phase carbon nitride is completely precipitated at the bottom of the ethanol solution.
5. The preparation method of the electrochemical sensor for detecting the sepsis marker procalcitonin according to claim 4, wherein the step of modifying the glassy carbon electrode by the graphite-phase carbon nitride in the step S3 is as follows:
1) firstly, polishing a glassy carbon electrode by using alumina powder, and then continuously washing for a plurality of times by using distilled water;
2) and (4) dripping the stripped graphite-phase nitrogen carbide dispersion liquid collected in the step (S2) on the surface of the glassy carbon electrode, and drying at room temperature for 30 min.
6. The method for preparing an electrochemical sensor for detecting the sepsis marker procalcitonin according to claim 1, wherein the step S4 is carried out by the following steps:
1) and (3) dissolving the peptide powder in Tris-HCl, dripping synthetic peptide modified by proline on the surface of the glassy carbon electrode modified by graphite-phase carbon nitride in the step S3, culturing at room temperature for 1h, and washing the surface of the glassy carbon electrode by Tris-HCl and deionized water in sequence to remove unbound synthetic peptide.
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Citations (4)

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CN108802015A (en) * 2018-06-13 2018-11-13 福建省妇幼保健院 A kind of preparation of the nontoxic electrochemical luminescence sensor of zearalenone based on peptide sensor
CN110687177A (en) * 2019-11-26 2020-01-14 济南大学 Based on g-C3N4Preparation and application of electrochemical luminescence sensor for detecting procalcitonin through energy transfer between CuO and electrochemical luminescence sensor

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Title
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