CN114088784B - Electrochemical aptamer sensor for detecting staphylococcus aureus and preparation method and application method thereof - Google Patents

Electrochemical aptamer sensor for detecting staphylococcus aureus and preparation method and application method thereof Download PDF

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CN114088784B
CN114088784B CN202111334847.XA CN202111334847A CN114088784B CN 114088784 B CN114088784 B CN 114088784B CN 202111334847 A CN202111334847 A CN 202111334847A CN 114088784 B CN114088784 B CN 114088784B
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staphylococcus aureus
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catechol
aptamer
chitosan
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CN114088784A (en
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刘正春
陈伟
来庆腾
张燕科
龙孟秋
梁波
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Central South University
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Abstract

The invention provides an electrochemical aptamer sensor for detecting staphylococcus aureus and a preparation and application method thereof. The preparation method comprises the following steps: (1) Depositing chitosan on the glassy carbon electrode to obtain a chitosan film modified glassy carbon electrode; (2) Depositing catechol on the glass carbon electrode modified by the chitosan film to obtain a glass carbon electrode modified by the catechol-chitosan film; (3) And finally, combining the glassy carbon electrode with an aptamer of staphylococcus aureus to obtain the aptamer-catechol-chitosan membrane modified glassy carbon electrode. And measuring current by using the prepared sensor through a CV method, and obtaining the content of staphylococcus aureus in the sample to be measured according to a standard curve equation of the peak value of the Fc oxidation current and the concentration of the staphylococcus aureus. The electrochemical aptamer sensor has higher specificity, affinity and reproducibility, and has good selectivity and quick response characteristic; the method can overcome the interference of sulfur groups during the detection of the whole blood sample, has high detection precision, and can realize the direct detection of clinical samples.

Description

Electrochemical aptamer sensor for detecting staphylococcus aureus and preparation method and application method thereof
Technical Field
The invention relates to an electrochemical aptamer sensor for detecting staphylococcus aureus, a preparation method and an application method thereof, and belongs to the technical field of biosensors.
Background
Staphylococcus aureus is a common opportunistic pathogen that is commonly colonized on human skin and mucosal surfaces, particularly the anterior nares (about 30% of the general population). When the host's immunity is reduced or the skin and mucosal barrier is disrupted, it can enter any organ or enter the blood, mild causes skin and soft tissue infections (impetigo, folliculitis and scalded skin syndrome), severe causes severe systemic diseases such as bacteremia, endocarditis, osteomyelitis, hemolytic pneumonia and toxic shock syndrome. Although mild skin and mucosal infections are often self-limiting, severe systemic infections are often accompanied by high mortality (20% -50%), high recurrence (5-10%) and sustained injury (more than one third of survivors). Staphylococcus aureus is the primary pathogen responsible for hospital-acquired infections. Up to 2% of hospitalized patients are infected with staphylococcus aureus due to impaired immune system and frequent invasive surgery. Staphylococcus aureus is increasingly more toxic and resistant to antibiotics.
The rapid, effective and accurate diagnosis of staphylococcus aureus is of great importance for the rapid treatment of infected patients, prevention of infection transmission and reduction of the formation of resistant strains. The existing gold standard for detecting staphylococcus aureus is still a culture method, but is very time-consuming, usually requires 1-2 days to obtain a single colony, and then requires 1-2 days to obtain chemical identification and drug sensitivity results. In recent years, rapid and automated detection methods, such as enzyme-linked immunosorbent assay (ELISA), polymerase Chain Reaction (PCR), flow cytometry and mass spectrometry, have been developed, which require expensive instruments, complicated sample preparation and high professional requirements, and are not well suited for remote poor areas nor for bedside detection, although only 1-5 hours are required for the results. In addition, biosensor methods have been increasingly focused by researchers due to their rapid, simple, specific, and sensitive properties. However, conventional biosensors have generally proven to be poorly clinically practical, mainly due to unstable or unreliable sensor responses (such as protein adsorption, platelet adhesion, and clot formation) caused by biological contamination. Even without the target, a rapid decay of peak current can be observed when a sensor constructed with Au electrodes detects a whole blood sample, due to interference by biological thiols in the blood sample. Therefore, the biosensor for detecting staphylococcus aureus, which can overcome interference, is quick, simple, convenient, specific and sensitive, shortens the detection and diagnosis time of staphylococcus aureus and has more accurate clinical diagnosis, and has important practical significance.
Disclosure of Invention
The invention aims at solving the technical problems in the background technology and provides an electrochemical aptamer sensor for detecting staphylococcus aureus, and a preparation method and an application method thereof.
The scheme provided by the invention is as follows:
a method for preparing an electrochemical aptamer sensor for detecting staphylococcus aureus, comprising the following steps:
(1) Immersing a double-electrode system formed by a glassy carbon electrode and a platinum wire into chitosan solution, and depositing for 300-800 seconds by using an electrochemical deposition method to obtain a glassy carbon electrode modified by a chitosan film;
(2) Taking a glassy carbon electrode modified by a chitosan film as a working electrode, taking a platinum wire as a counter electrode, taking an Ag/AgCl electrode as a reference electrode to form a three-electrode system, immersing the three-electrode system into catechol solution, and grafting catechol to obtain the glassy carbon electrode modified by the catechol-chitosan film;
(3) Immersing the obtained catechol-chitosan membrane modified glassy carbon electrode into staphylococcus aureus aptamer solution for chemical combination to obtain the aptamer-catechol-chitosan membrane modified glassy carbon electrode.
As a preferable mode of the technical scheme, the sequence of the staphylococcus aureus aptamer is 5'-GCAATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCTACG TCA AAA GTG CAC GCT ACT TTG CTAA-3' -CHO; staphylococcus aureus aptamer is first dissolved in buffer and then denatured by heating for a period of time and then rapidly cooled to allow proper folding of the aptamer to bind the target molecule.
As a preferable mode of the above-mentioned technical scheme, the concentration of the catechol solution in the step (2) is controlled to be 1-10mM.
Based on the same technical conception, the invention also provides an electrochemical aptamer sensor for detecting staphylococcus aureus, which is prepared by the preparation method.
Based on the same technical conception, the invention also provides a detection application method of the electrochemical aptamer sensor for detecting staphylococcus aureus, which comprises the following steps:
1) Establishing a standard curve equation according to a CV curve obtained by a cyclic voltammetry of a staphylococcus aureus series concentration standard solution;
2) Taking or preparing a sample solution to be measured;
3) Detecting an Fc oxidation current peak value of the sample solution to be detected by adopting a cyclic voltammetry based on the sample solution to be detected prepared in the step 2);
4) And calculating the concentration content of staphylococcus aureus in the sample solution to be detected according to a standard curve equation between the Fc oxidation current peak value and the concentration of staphylococcus aureus.
As a preferable mode of the technical scheme, the standard curve equation is Fc-Ru of the microelectrode sensor 3+ Adding staphylococcus aureus series concentration standard solution into the dual-electron medium solution to obtain the product; the staphylococcus aureus series concentration standard solution is a staphylococcus aureus series concentration solution in PBS solution or a staphylococcus aureus series concentration solution in whole blood sample.
As a preferred embodiment of the foregoing technical solution, when the series concentration solution of staphylococcus aureus in PBS solution is used as the series concentration standard solution of staphylococcus aureus, the standard curve equation in the step 1) is: Δp (μa) = -0.155log c (cfu/mL) +0.059 (R) 2 = 0.9928); the detection Limit (LOD) and the quantification Limit (LOQ) of the standard curve equation are 2cfu/mL and 10cfu/mL, respectively.
As a preferable aspect of the above technical solution, when the staphylococcus aureus series concentration solution in the whole blood sample is used as the staphylococcus aureus series concentration standard solution, the standard curve equation in the step 1) is: Δp (μa) = -0.182log c (cfu/mL) +0.092; the detection Limit (LOD) and the quantification Limit (LOQ) of the standard curve equation are 2cfu/mL and 10cfu/mL, respectively.
As a preferable mode of the above technical solution, the step 1) of establishing a standard curve equation includes:
a: fc-Ru in electrochemical aptamer sensor 3+ Adding staphylococcus aureus solution with known concentration into the double-electron medium solution; the scanning voltage of CV was measured from-0 using cyclic voltammetry on a solution of Staphylococcus aureus of known concentration.5V to +0.5V, the CV curve is recorded until the signal stabilizes; recording the peak Fc oxidation current of the staphylococcus aureus solution at that concentration at the time of signal stabilization;
and B, repeating the step A by adopting staphylococcus aureus solutions with different concentrations, and obtaining a linear regression equation according to the linear relation between each Fc oxidation current peak value and staphylococcus aureus with different concentrations.
As the optimization of the technical scheme, when the cyclic voltammetry is adopted for measurement in the step A, a stable signal can be obtained after 7.5 cycles are executed for each measurement, and the final cycle is the Fc oxidation current peak test result.
Compared with the prior art, the invention has the beneficial effects that:
by using a glassy carbon electrode, a catechol-chitosan film and Fc-Ru 3+ The double-electron medium forms a redox capacitor as an amplifying system, and the grafting process of the catechol-chitosan film is optimally selected, so that the detection sensitivity of the prepared electrochemical aptamer sensor is high. The electrochemical deposition time of chitosan is controlled, so that the thickness of the chitosan film of the prepared glassy carbon electrode modified by the chitosan film is proper, and the chitosan film can be ensured to cover the glassy carbon electrode matrix so as to ensure that catechol and staphylococcus aureus aptamer can be grafted around the glassy carbon electrode in a sufficient and uniform manner; and the chitosan film is not too thick to influence the redox effect of the electron absorption and discharge of the working electrode.
The invention designs and develops an electrochemical aptamer sensor which is sensitive, specific and simple and convenient and can directly detect staphylococcus aureus. The aptamer has higher specificity, affinity and reproducibility, can successfully capture staphylococcus aureus and distinguish the staphylococcus aureus from other bacteria, and has good selectivity; meanwhile, the interference of sulfur groups in the whole blood sample detection can be overcome, and the detection precision is higher. Meanwhile, the electrochemical aptamer sensor has good reproducibility, so that the sensor can be applied for multiple times, and resources are saved.
The Fc oxidation peak current signal gradually decreasing due to the blocking effect of staphylococcus aureus was recorded using CV method,the quantitative detection of staphylococcus aureus can be realized in 5 minutes without any pretreatment (incubation reaction or sample pre-concentration), and the potential application of the staphylococcus aureus in a POCT system is demonstrated. The aptamer electrochemical sensor showed good detection performance, as low as 2cfu/mL detection limit and linear range spanning seven orders of magnitude (10-10 7 cfu/mL), and can directly quantitatively detect staphylococcus aureus in whole blood. In addition, easy replacement of the aptamer can ensure that the sensor is easy to customize, so as to facilitate manufacturing of a practical high-performance biosensor suitable for bacteria measurement.
Drawings
FIG. 1 is a schematic diagram of catechol-chitosan film amplifying current signals in Fc, ru3+ media. Wherein: (a) is a result graph of current signals of unmodified glassy carbon electrodes in Fc and Ru < 3+ > media, (b) is a result graph of current signals of glassy carbon electrodes modified by catechol-chitosan films in Fc and Ru < 3+ > media, and (c) is a schematic diagram of current amplification principle.
FIG. 2 is a schematic diagram of the electrochemical aptamer sensor preparation and detection process for Staphylococcus aureus.
FIG. 3 is a graph showing the results of Fc oxidation peak currents of sensors for different times (300 s, 400s, 500s, 600s, 700s, 800 s) of electrodeposited chitosan.
FIG. 4 is a graph showing the results of Fc oxidation peak currents of sensors at different concentrations of catechol solutions (1 mM, 3mM, 5mM, 7mM, 9 mM).
FIG. 5 is a FT-IR spectrum of a glassy carbon electrode modified with a chitosan (Chi), catechol-chitosan (Cat-Chi), and aptamer-catechol-chitosan (Apt-Chi-Cat) film, respectively.
FIG. 6 is an X-ray photoelectron spectrum of a glassy carbon electrode modified with a chitosan (Chi), catechol-chitosan (Cat-Chi), and aptamer-catechol-chitosan (Apt-Chi-Cat) film, respectively.
FIG. 7 shows the glassy carbon electrode with 50. Mu.M Fc and Ru under different modification conditions 3+ Typical response CV curve in PBS.
FIG. 8 is a graph of the results of selective detection of different bacteria by an electrochemical aptamer sensor.
FIG. 9 is a diagram ofElectrochemical aptamer sensors at different concentrations (1-10 9 cfu/mL) CVs response plot of staphylococcus aureus PBS solution.
FIG. 10 is a graph of the logarithmic change of the Fc oxidation peak current value versus the concentration of Staphylococcus aureus of FIG. 9.
FIG. 11 shows Staphylococcus aureus (10) at different magnification 10 cfu/mL) and aptamer-catechol-chitosan membrane binding. Wherein (a) is 3000 times; (b) 6000 times; (c) 12000 times; (d) 24000 times.
FIG. 12 is a gram stain of Staphylococcus aureus (10 10 cfu/mL), the plot was captured electrochemically at different magnification; wherein: (a) 1000 times; (b) 3000 times.
FIG. 13 is a graph of the peak Fc oxidation current of an electrochemical aptamer sensor in a whole blood sample versus the logarithmic change of the concentration of Staphylococcus aureus in the blood.
FIG. 14 shows a sample of Staphylococcus aureus (10 6 cfu/mL), a CV curve for multiple cycles of the electrochemical aptamer sensor.
FIG. 15 shows a sample of Staphylococcus aureus (10 6 cfu/mL) input-output current profile for a plurality of cycles of the electrochemical aptamer sensor in the presence of cfu/mL).
Detailed Description
The present invention will be described more fully hereinafter for the purpose of facilitating understanding of the present invention, but the scope of protection of the present invention is not limited to the following specific examples.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Design and response mechanism of electrochemical aptamer sensor:
the glassy carbon electrode is immersed in a solution containing a double medium (F C And Ru (Rust) 3+ ) As shown in FIG. 1a, two pairs of redox current peaks, one F, can be observed for conventional cyclic voltammetry in PBS (0.1M, PH7.4) solution C Oxidation peak, another is Ru 3+ Reduction peak (peak current about 2. Mu.A). As shown in FIG. 1b, catechol-chitosan film modified glassy carbon electrode and dual media in solution (Fc and Ru 3+ ) Can form a redox capacitor and oxidize Fc and Ru 3+ The reduction current signal produces an amplifying effect (peak current increases to 17 mua). Principle of current amplification as shown in fig. 1c, briefly, when a forward voltage is applied to an electrode, the mediator Fc can be oxidized to Fc by transferring electrons to the electrode + Then, the catechol is reduced again to Fc by receiving electrons of the catechol in the membrane, and the catechol is converted to oxidized catechol (o-quinone). When a reverse voltage is applied to the electrode, medium Ru 3+ By receiving electrons from the electrode to reduce to Ru 2+ Ru is then oxidized by transferring the electrons to catechol (o-quinone) in the film 2+ Reoxidation to Ru 3+ . Meanwhile, oxidized catechol (o-quinone) is reduced to catechol structure. Thus, one electron will be sequentially taken from Fc, electrode, ru 3+ O-quinone, catechol, and Fc. Only when an electron is transferred from Fc to electrode will it be reacted by Ru 3+ Rapidly captured and transferred back to Fc. Due to Fc + Directly reduced by catechol rather than accepting electrons from the electrode, and Ru 2+ Is oxidized by the o-quinone in time rather than transferring electrons to the electrode, thus Fc + Reduction and Ru 2+ The oxidation current peak will decrease. Thus, catechol-chitosan film modified electrode can act as a redox capacitor and amplify Fc and Ru 3+ Current in the medium.
Basic experimental appliance:
1. reagents and bacteria
Chi (Chi, deacetylation. Gtoreq.75%) catechol (Cat, gtoreq.99), 1' -ferrocenedimethanol (Fc, 97%) and ruthenium hexamine (III) chloride (Ru) 3+ ，98％)。
Staphylococcus aureus (Staphylococcus aureus, s. Aureus), staphylococcus epidermidis (Staphylococcus epidermidis, s. Epidrmidis), klebsiella pneumoniae (Klebsiella pneumoniae, k. Pneumaloiae), acinetobacter baumannii (Acinetobacter baumannii, a. Baumannii), pseudomonas aeruginosa (Pseudomonas aeruginosa, p. Aeromonas), escherichia Coli (Escherichia Coli, e.coli), enterococcus faecium (Enterococcus faecium, e.faecium), proteus mirabilis (Proteus mirabilis, p. Mirabilis), streptococcus pyogenes (Streptococcus pyogenes, s. Pyogenes), streptococcus pneumoniae (Streptococcus pneumoniae, s. Pneumaloiae). Firstly, separating and culturing, selecting single colony for enrichment, and preparing staphylococcus aureus with different concentrations by using PBS. Staphylococcus aureus was counted using standard plate counting methods.
2. Aptamer
The aldehyde group modified staphylococcus aureus aptamer was synthesized by Shanghai bioengineering technology services limited. The sequence was 5'-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCTACG TCAAAA GTG CAC GCT ACT TTG CTA A-3' -CHO. Staphylococcus aureus aptamer was dissolved in buffer (10 mM Tris-HCl, with 150mM NaCl,5mM KCl,2mM MgCl) 2 ,1mM CaCl 2 pH 7.4) and heated at 95℃for 5 minutes, and then rapidly cooled at 4℃for 15 minutes to allow the aptamer to properly fold to bind the target molecule.
The determination method used by the invention comprises the following steps:
1) Fourier transform Infrared Spectroscopy (FTIR)
FTIR (Thermo Scientific Nicolet iS 5) was used to analyze the chemical functional groups on the glassy carbon electrode membrane to determine if chitosan, catechol, and aptamer were successfully deposited on the glassy carbon electrode. After chitosan, catechol and aptamer were bound to the glassy carbon electrode, respectively, the membrane was scraped off the glassy carbon electrode and dried for FTIR analysis. A sample of 2mg of the powder scraped from the glassy carbon electrode and 200mg of pure potassium bromide were taken and the mixture was ground to a fine powder with a mortar and pestle. The mixture was then pressed into a sheet for measuring FTIR spectra (4000-400 cm -1 Wavelength, 32 scans, 4cm -1 Resolution).
2) X-ray photoelectron spectroscopy (XPS)
XPS is used for analyzing chemical components and states of chemical elements in chitosan, catechol-chitosan and aptamer-catechol-chitosan films. XPS measurements were obtained on a Thermo Scientific K-Alpha instrument with a monochromatic A1K Alpha (1486.6 eV) X-ray source. The spectrum was recorded at 50eV pass energy under ultra-high vacuum using the C1s peak at 284.8 as a reference.
3) Freezing electron microscope
The specific binding of staphylococcus aureus to the aptamer-catechol-chitosan membrane was confirmed using Cryo SEM (FEI Quanta 450). An aptamer-catechol-chitosan film combined with staphylococcus aureus is stuck on conductive carbon gel on a sample table. The sample stage with the sample was then placed in liquid nitrogen for 30 seconds and flash frozen. The sample was sublimated at-90℃for 10 minutes, then sputtered with a 10mA current and gold plated for 60 seconds. Finally, the sample is sent to a sample chamber of a scanning electron microscope for observation. The freezing temperature is-140 ℃ and the accelerating voltage is 5kV.
4) Gram staining
Gram staining was used to confirm that staphylococcus aureus specifically bound to the aptamer-catechol-chitosan membrane. Staphylococcus aureus-aptamer-catechol-chitosan membranes were printed directly onto clean glass slides and air dried. Gram staining was then performed and examined under an oil microscope (×1000 times) to find membrane-bound gram-positive cocci (staphylococcus aureus).
Example 1
An electrochemical aptamer sensor for detecting staphylococcus aureus and a preparation method thereof, as shown in fig. 2, comprises the following steps:
(1) Immersing a double-electrode system formed by the glassy carbon electrode and the platinum wire into chitosan solution, and depositing by an electrochemical deposition method to obtain the glassy carbon electrode modified by the chitosan film.
The chitosan was dissolved using 1M HCl solution and then its pH was adjusted to 5-6 with 1M NaOH solution. The chitosan solution was stirred overnight with a magnetic stirrer and filtered to remove undissolved particles. With particle size ofAlumina powder of 1.0 μm,0.3 μm,0.05 μm was used to polish the glassy carbon electrode three minutes at a time. Soaking glassy carbon electrode in distilled water, ultrasonic treating for 10 min, and adding aqua regia (H) 2 SO 4 /30％H 2 O 2 7:3, V/V) for 10 minutes, and finally rinsed 3 times with water and ethanol, respectively. Immersing a glassy carbon electrode into chitosan solution, and electrodepositing chitosan on the surface of the glassy carbon electrode by using a timing amperometric method (-0.5 to-3V, wherein the glassy carbon electrode is used as a working electrode and a platinum wire is used as a counter electrode). After electrodeposition for 500 seconds, the chitosan modified working electrode was removed from the chitosan solution, rinsed with distilled water and dried with nitrogen.
(2) And the glassy carbon electrode modified by the chitosan film is used as a working electrode, a platinum wire is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode to form a three-electrode system, the three-electrode system is immersed into catechol solution, and catechol is grafted to obtain the glassy carbon electrode modified by the catechol-chitosan film.
Immersing the working electrode obtained in the step (1) into catechol solution (0.1M PBS, pH 7.4) with the concentration of 5mM, and grafting catechol onto chitosan by adopting a timing voltage method (the voltage is 0.6V, the timing is 500 seconds, a glassy carbon electrode is used as the working electrode, a platinum wire is used as a counter electrode, and an Ag/AgCl electrode is used as a reference electrode). And taking out the pyrocatechol-chitosan membrane modified glassy carbon electrode from the pyrocatechol solution after the reaction, washing with distilled water, and drying with nitrogen.
(3) Immersing the obtained catechol-chitosan membrane modified glassy carbon electrode into staphylococcus aureus aptamer solution for chemical combination to obtain the aptamer-catechol-chitosan membrane modified glassy carbon electrode.
The resulting catechol-chitosan film-modified glassy carbon electrode was immersed in a solution containing 2. Mu.M Staphylococcus aureus aptamer (0.1 MPBS, PH7.4), the temperature was maintained at 4 ℃, and after 12 hours, the aptamer-catechol-chitosan film-modified glassy carbon electrode was washed with distilled water and dried with nitrogen.
Optimal design of electrodeposited chitosan time and catechol concentration:
the thickness of the chitosan affects the amplified current signal effect of the catechol-chitosan film. If the chitosan film is too thick, it can affect the electron exchange between the GCE, catechol-chitosan film and the redox mediator. However, insufficient chitosan film thickness can limit the amount of catechol and aptamer grafted on the electrode, which can reduce the performance of the modified electrode.
Therefore, in order to explore the optimal performance of the chitosan modified electrode, the CV method is adopted to optimally select the electrodeposition time of the chitosan. As shown in fig. 3, the electrodeposition time of chitosan was selected to be 300s, 400s, 500s, 600s, 700s, 800s; from 300 seconds to 800 seconds, the peak Fc oxidation current reached a maximum at 500 seconds of deposition. Thus, electrodeposited chitosan 500s was chosen to modify the electrode optimally.
The concentration of catechol also affects the electrochemical activity of the modified electrode and thus the amplification of the peak redox current. In order to obtain the catechol-chitosan membrane modified electrode with better performance, the invention performs an optimization selection test on the concentration of the catechol solution. In this test, the electrodeposition time of chitosan was 500s, and catechol-chitosan membrane modified electrodes were prepared with catechol solutions of different concentrations (1 mM, 3mM, 5mM, 7mM, 10 mM). As shown in FIG. 4, the peak value of Fc oxidation current of the modified electrode increases with an increase in catechol concentration, but the growth rate is slow when the catechol concentration is more than 5 mM. Therefore, it is optimal to modify the electrode with a 5mM catechol solution.
Structural detection and characterization of the electrochemical aptamer sensor for detecting staphylococcus aureus prepared in example 1:
1) Modification and characterization of the electrode:
modification of the electrodes was characterized by FTIR and XPS. As shown in FIG. 5, the main characteristic absorption peak of the infrared absorption spectrum of the chitosan film was 3310cm -1 (O-H and N-H stretching vibration), 2935 and 2880cm -1 (stretching vibration of C-H), 1617cm -1 (C=O stretching vibration), 1509cm -1 (C-H and N-H deformation vibration), 1380cm -1 (-bending vibration of CH 3), 1309cm -1 (stretching vibration of C-O), 1020cm -1 (C-O-C stretching vibration). When chitosan is graftedAfter the catechol has been branched, a new characteristic absorption peak 1640cm is observed -1 、1580cm -1 、1500cm -1 And 1450cm -1 These are mainly caused by vibrations of the benzene ring skeleton. As hydroxyl is introduced into catechol, the characteristic adsorption peak is 3310cm -1 Become deeper and wider. Furthermore, 1735cm -1 The newly added adsorption peak indicates that Schiff base reaction occurs between catechol and chitosan, which confirms the formation of catechol-chitosan film. 1090cm due to the presence of P-O-C as an aptamer -1 The decrease in transmittance of (c) indicates the formation of an aptamer-catechol-chitosan film. Furthermore, as shown in fig. 6, XPS spectra showed the presence of N-c=o, indicating that an amide bond is formed between the aldehyde group modified aptamer and the amino group of chitosan by schiff base reaction, which further confirms the formation of aptamer-catechol-chitosan film. Furthermore, XPS spectra showed the presence of elemental phosphorus in the aptamer-catechol-chitosan film only in the aptamer, further confirming the successful formation of the aptamer-catechol-chitosan film.
2) Electrochemical property detection of electrochemical aptamer sensor:
in the presence of 50 μm Fc and Ru 3+ In PBS (0.1M, PH7.4) solution, the electrochemical properties of the modified electrode were measured and studied using CVs. As shown in FIG. 7, bare GCE shows that Bare glassy carbon electrode contains 50 μm Fc and Ru 3+ Typical response in PBS (0.1M, PH7.4) solution, chi-GCE showed typical response of chitosan film modified glassy carbon electrode. It can be seen that the chitosan film modified glassy carbon electrode has no significant oxidation and reduction current due to low redox properties compared to the bare glassy carbon electrode. After catechol is grafted, a catechol-chitosan modified glassy carbon electrode (Cat-Chi-GCE) is used for carrying out oxidation current peak signal on Fc and Ru 3+ The reduction current peak signal shows an amplification characteristic, which is a remarkable characteristic of the redox capacitor electrode. Compared with the catechol-chitosan film modified glassy carbon electrode (Cat-Chi-GCE), the aptamer-catechol-chitosan film modified glassy carbon electrode (Apt-Cat-Chi-GCE) has no difference in oxidation and reduction peak current, which means that electron exchange between the catechol-chitosan film and the redox medium is not affectedInfluence of the aptamer.
3) Selective and reproducible detection of electrochemical aptamer sensor:
to explore the specificity of the electrochemical aptamer sensor, the prepared electrochemical aptamer sensor was used to detect 10 6 cfu/mL of Staphylococcus epidermidis, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, enterococcus faecium, bacillus mirabilis, streptococcus pyogenes, and Streptococcus pneumoniae. As shown in fig. 8, the peak Fc oxidation current signal change of staphylococcus aureus was much greater than that of other bacteria. The prepared catechol-chitosan membrane modified glassy carbon electrode is used for detecting staphylococcus aureus to investigate whether nonspecific binding exists between the catechol-chitosan membrane and the staphylococcus aureus, and the result shows that the Fc oxidation current signal peak current of the catechol-chitosan membrane modified GCE is not greatly changed before and after the catechol-chitosan membrane modified GCE reacts with the staphylococcus aureus, so that nonspecific binding does not exist between the catechol-chitosan membrane and the staphylococcus aureus. The results show that the electrochemical aptamer sensor has good specificity due to the high specificity of the aptamer.
The reproducibility is also an important feature of electrochemical sensors in practical applications. The prepared electrochemical aptamer sensor detects primary staphylococcus aureus (10 5 cfu/mL), the electrochemical aptamer sensor was thoroughly washed with sterile water, then the secondary structure formed by the aptamer was destroyed using 2.0M NaCl solution and the bound staphylococcus aureus was released for regeneration. The regenerated electrochemical aptamer sensor was washed three times with PBS to remove staphylococcus aureus and then the staphylococcus aureus solution was detected. Detection 10 using regenerated electrochemical aptamer sensor 5 The peak change in current of cfu/mL staphylococcus aureus was 0.91 μA, approaching the initial electrochemical aptamer sensor (0.94 μA), indicating good regeneration performance.
Example 2
An application method of an electrochemical aptamer sensor for detecting staphylococcus aureus in PBS solution. Detection using the electrochemical aptamer sensor prepared in example 1 includes the following steps:
1) Establishing a standard curve equation according to a CV curve obtained by cyclic voltammetry of staphylococcus aureus series concentration standard solution:
a: fc-Ru in electrochemical aptamer sensor 3+ Adding a known concentration of Staphylococcus aureus PBS solution into a two-electron medium solution (50. Mu.M each in 0.1M PBS, pH 7.4); measuring staphylococcus aureus solution with known concentration by cyclic voltammetry, and recording CV curve until signal is stable, wherein CV scanning voltage is from-0.5V to +0.5V; recording the peak Fc oxidation current of the staphylococcus aureus solution at that concentration at the time of signal stabilization;
b, the concentration is in the range of 1-10 9 Repeating the step A by cfu/mL of different concentration staphylococcus aureus PBS solutions, and obtaining a linear regression equation according to the linear relation between each Fc oxidation current peak value and different concentration staphylococcus aureus. As shown in fig. 9, the peak Fc oxidation current decreases in sequence with increasing concentration of staphylococcus aureus. The peak change in Fc oxidation current has a good linear relationship with the logarithmic value of staphylococcus aureus concentration. As shown in FIG. 10, the linear range is 10-10 8 cfu/mL, the linear regression equation can be expressed as ΔP (μA) = -0.155log C (cfu/mL) +0.059 (R) 2 = 0.9928). The detection Limit (LOD) and the quantification Limit (LOQ) are calculated using the equations: lod=3σ/k and loq=10σ/k, where σ=0.0149 (containing 50 μm Fc and Ru 3+ K=0.155 (slope of calibration curve). LOD and LOQ were calculated as 2cfu/mL and 10cfu/mL, respectively, with the better sensitivity due to the amplified current effect of catechol-chitosan redox capacitors and the high affinity of the aptamer to Staphylococcus aureus.
2) Taking or preparing a sample solution to be measured;
3) Detecting an Fc oxidation current peak value of the to-be-detected solution by adopting a cyclic voltammetry based on the to-be-detected sample solution prepared in the step 2); when the solution to be detected is detected, the detection is carried out at room temperature, and the solution to be detected before the detection is degassed by nitrogen so as to remove air in the solution; during the test, the solution to be tested was continuously filled with nitrogen.
4) And calculating the concentration content of staphylococcus aureus in the sample solution to be detected according to a standard curve equation between the Fc oxidation current peak value and the concentration of staphylococcus aureus.
In this embodiment, when the cyclic voltammetry is adopted for measurement in step a, a stable signal can be obtained after 7.5 cycles are performed for each measurement, and the last cycle is the Fc oxidation current peak test result.
The staphylococcus aureus bound to the aptamer-catechol-chitosan membrane was verified using a cryoscanning electron microscope and gram stain. FIG. 11 shows an aptamer-catechol-chitosan membrane modified electrode and 10 10 After cfu/mL staphylococcus aureus underwent CV reaction, staphylococcus aureus at different magnifications was bound on an aptamer-catechol-chitosan membrane. As shown in FIG. 11, the aptamer-catechol-chitosan membrane was covered with approximately 0.8 μm diameter, nearly spherical grape cluster bacteria. Meanwhile, the aptamer-catechol-chitosan membrane was gram-stained, and as shown in fig. 12, gram-stained positive spherical grape cluster bacteria appeared on the aptamer-catechol-chitosan membrane. The results indicate that staphylococcus aureus prevents Fc migration after being specifically captured by the aptamer and decreases the charge transfer rate, resulting in a gradual decrease in Fc oxidation current peak signal. The magnitude of the decrease in peak Fc oxidation current increases with increasing concentration of staphylococcus aureus.
Example 3
An application method of an electrochemical aptamer sensor for detecting staphylococcus aureus in a whole blood sample. Detection using the electrochemical aptamer sensor prepared in example 1 includes the following steps:
1) Establishing a standard curve equation according to a CV curve obtained by cyclic voltammetry of staphylococcus aureus series concentration standard solution:
a: fc-Ru in electrochemical aptamer sensor 3+ A whole blood sample of Staphylococcus aureus solution of known concentration was added to a duplex medium solution (50. Mu.M each in 0.1M PBS, pH 7.4); by cyclic voltaicsAn ampere method is used for measuring staphylococcus aureus solution of a whole blood sample with known concentration, the scanning voltage of CV is from-0.5V to +0.5V, and a CV curve is recorded until the signal is stable; recording the peak Fc oxidation current of the staphylococcus aureus solution at that concentration at the time of signal stabilization;
b, the concentration is in the range of 1-10 8 Repeating the step A by cfu/mL of staphylococcus aureus solutions with different concentrations, and obtaining a linear regression equation according to the linear relation between each Fc oxidation current peak value and staphylococcus aureus with different concentrations. The result is shown in fig. 13, and the linear regression equation is: Δp (μa) = -0.182log c (cfu/mL) +0.092 (R) 2 = 0.9951), linear range of 10-10 7 cfu/mL, detection Limit (LOD) and quantification Limit (LOQ) are 2cfu/mL and 10cfu/mL, respectively.
2) Taking or preparing a sample solution to be measured;
3) Detecting an Fc oxidation current peak value of the to-be-detected solution by adopting a cyclic voltammetry based on the to-be-detected sample solution prepared in the step 2); when the solution to be detected is detected, the detection is carried out at room temperature, and the solution to be detected before the detection is degassed by nitrogen so as to remove air in the solution; during the test, the solution to be tested was continuously filled with nitrogen.
4) And calculating the concentration content of staphylococcus aureus in the sample solution to be detected according to a standard curve equation between the Fc oxidation current peak value and the concentration of staphylococcus aureus.
In this embodiment, when the cyclic voltammetry is adopted for measurement in step a, a stable signal can be obtained after 7.5 cycles are performed for each measurement, and the last cycle is the Fc oxidation current peak test result. The principle is as follows: staphylococcus aureus in Fc-Ru 3+ Negatively charged in the medium (pH 7.4); because the isoelectric point is pH 2-3, the negatively charged Staphylococcus aureus will be continually bound to the aptamer under the drive of the circulating voltage, but it will tend to equilibrate at some point. To obtain the equilibration time, the present invention tested and observed a single tested CV graph and voltage input output graph. As shown in fig. 14, 15, decay of Fc oxidation current was observed during the first 12 cycles (0-8 minutes), but after 7.5 cycles (300 seconds total) of scanning, staphylococcus aureusThe interaction between the bacteria and the aptamer has reached equilibrium and a stable current signal is generated. Thus, the subsequent CV measurements will be scanned for 7.5 cycles to obtain a stable signal, and the last cycle scan is recorded as the test result. Therefore, the electrochemical aptamer sensor only passes 7.5 cycles (300 seconds in total) when in CV measurement detection, and the interaction between staphylococcus aureus and the aptamer can reach equilibrium, so that a stable current signal is generated; compared with the method that the sensor in the prior art needs to be incubated in a sample solution for more than 30 minutes to balance the reaction between the probe and the target, the detection method can realize timely reaction without sample pretreatment, has short detection time, and can obtain a detection result only by 5 minutes.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modification, equivalent variation and modification of the above embodiments according to the technical substance of the present invention shall fall within the scope of the technical solution of the present invention.

Claims (10)

1. A method for preparing an electrochemical aptamer sensor for detecting staphylococcus aureus, comprising the following steps:
(1) Immersing a double-electrode system formed by a glassy carbon electrode and a platinum wire into chitosan solution, and depositing for 300-800 seconds by using an electrochemical deposition method to obtain a glassy carbon electrode modified by a chitosan film;
(2) Taking a glassy carbon electrode modified by a chitosan film as a working electrode, taking a platinum wire as a counter electrode, taking an Ag/AgCl electrode as a reference electrode to form a three-electrode system, immersing the three-electrode system into catechol solution, and grafting catechol to obtain the glassy carbon electrode modified by the catechol-chitosan film;
(3) Immersing the obtained catechol-chitosan membrane modified glassy carbon electrode into staphylococcus aureus aptamer solution for chemical combination to obtain an aptamer-catechol-chitosan membrane modified glassy carbon electrode; the sequence of the staphylococcus aureus aptamer is 5'-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3' -CHO.
2. The method of claim 1, wherein the staphylococcus aureus aptamer is first dissolved in a buffer and denatured by heating for a period of time and then rapidly cooled to allow the aptamer to fold properly to bind the target molecule.
3. The method according to claim 1, wherein the concentration of the catechol solution in the step (2) is controlled to be 1 to 10mM.
4. An electrochemical aptamer sensor for detecting staphylococcus aureus, characterized in that the sensor is prepared by the preparation method of any one of claims 1-3.
5. A detection application method based on the electrochemical aptamer sensor for detecting staphylococcus aureus as claimed in claim 4, comprising the following steps:
1) Establishing a standard curve equation according to a CV curve obtained by a cyclic voltammetry of a staphylococcus aureus series concentration standard solution;
2) Taking or preparing a sample solution to be measured;
3) Detecting an Fc oxidation current peak value of the sample solution to be detected by adopting a cyclic voltammetry based on the sample solution to be detected prepared in the step 2);
4) And calculating the concentration content of staphylococcus aureus in the sample solution to be detected according to a standard curve equation between the Fc oxidation current peak value and the concentration of staphylococcus aureus.
6. The method of claim 5, wherein the standard curve equation is Fc-Ru at a microelectrode sensor 3+ Adding staphylococcus aureus series concentration standard solution into the dual-electron medium solution to obtain the product; the staphylococcus aureus series concentration standard solution is PA series concentration solution of staphylococcus aureus in BS solution or a series concentration solution of staphylococcus aureus in whole blood sample.
7. The method according to claim 6, wherein when the series concentration solution of staphylococcus aureus in PBS solution is used as the series concentration standard solution of staphylococcus aureus, the standard curve equation in the step 1) is: Δp (μa) = -0.155log c (cfu/mL) +0.059 (R) 2 = 0.9928); the detection limit and the quantification limit of the standard curve equation are 2cfu/mL and 10cfu/mL, respectively.
8. The method according to claim 6, wherein when the staphylococcus aureus concentration series solution in the whole blood sample is used as the staphylococcus aureus concentration series standard solution, the standard curve equation in the step 1) is: Δp (μa) = -0.182log c (cfu/mL) +0.092; the detection limit and the quantification limit of the standard curve equation are 2cfu/mL and 10cfu/mL, respectively.
9. The method according to claim 5, wherein the step 1) of establishing a standard curve equation includes:
a: fc-Ru in electrochemical aptamer sensor 3+ Adding staphylococcus aureus solution with known concentration into the double-electron medium solution; measuring staphylococcus aureus solution with known concentration by cyclic voltammetry, and recording CV curve until signal is stable, wherein CV scanning voltage is from-0.5V to +0.5V; recording the peak Fc oxidation current of the staphylococcus aureus solution at that concentration at the time of signal stabilization;
and B, repeating the step A by adopting staphylococcus aureus solutions with different concentrations, and obtaining a linear regression equation according to the linear relation between each Fc oxidation current peak value and staphylococcus aureus with different concentrations.
10. The method according to claim 9, wherein when the cyclic voltammetry is adopted in the step a, a stable signal can be obtained after 7.5 cycles are performed for each measurement, and the last cycle is the Fc oxidation current peak test result.
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