CN113466303A - Preparation method of pathogenic bacteria detection electrode in water environment of construction site - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention discloses a preparation method and application of a pathogenic bacteria detection electrode in a water environment of a construction site, which comprises the following steps: s1 preparing a gold electrode; s2, treating the surface of the electrode; s3 surface modification single-walled carbon nanotubes (SWNTs); incubation of the S4 linker molecule; s5 blocking reaction, the electrode for detecting pathogenic bacteria in the water environment prepared by the process has long service life and high detection efficiency, and can accurately identify different types of bacteria.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a preparation method of a pathogenic bacteria detection electrode in a water environment of a construction site.
Background
Bacteria detection is of great significance to human health, and the current methods for detecting pathogenic bacteria mainly use antibodies or DNA. Standard methods are enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR). These methods have high sensitivity and specificity. However, they require trained personnel and long analysis times. These limitations prevent their use as a real-time, wide-range detection platform that requires detection and processing of local water sources, especially when working at remote outdoor construction sites, and the simplicity and rapidity of effective detection techniques is an ongoing goal.
The electrochemical method obtains map data of different bacteria through resistance data obtained by electrochemistry, so that the aim of identifying different types of bacteria is fulfilled, and the electrochemical method has the advantages of rapidness, stability, simplicity and low cost, but the problems that the service life of an electrode is short and the detection efficiency is low and different types of bacteria cannot be accurately identified due to the incomplete preparation process of the electrode for detecting pathogenic bacteria in a water environment are solved.
Disclosure of Invention
The invention aims to solve the problems that the electrode has short service life and low detection efficiency and can not accurately identify different types of bacteria due to the incomplete preparation process of the electrode for detecting pathogenic bacteria in the water environment in the prior art, and provides a preparation method of the electrode for detecting pathogenic bacteria in the water environment of a construction site.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a pathogenic bacteria detection electrode in a building site water environment comprises the following steps:
s1, preparing a gold electrode: photoetching gold interdigital electrodes on a Si/SiO2 substrate;
s2, electrode surface treatment: treating the surface of the electrode obtained in S1 with 1-Aminopropyltriethoxysilane (APTES) in acetone ultrasonic treatment for 10 minutes, nitrogen ultrasonic treatment for 30 minutes, oxygen plasma gas bath for 10 minutes and ultraviolet ozone for 30 minutes;
s3, electrode surface modification single-walled carbon nanotubes (SWNTs): incubating the carbon nanotubes to realize uniform assembly of the carbon nanotubes on the surface of the electrode, washing off redundant single-walled carbon nanotubes under the action of deionized water, and annealing in a Lindberg blue M tube furnace;
s4, incubation of linker molecules: the device obtained in S3 was incubated in a 3mg/mL solution of 1-pyrenebutyric acid succinimidyl ester (PBASE) in Dimethylformamide (DMF) for 1 hour, followed by washing with dimethylformamide and 10mM Phosphate Buffer (PB);
s5, blocking reaction: ethanolamine (0.1%) was used to block unreacted PBASE sites, tween 20 (0.1%) was used to block open SWNT regions, minimizing non-specific binding, incubation time was 10 minutes, and then washed with PB.
Preferred scheme in step S1:
s101, photoetching interdigital gold electrodes on Si/SiO2, wherein the shape structure of the interdigital gold electrodes is 20, 200 and mu m long, 5 and mu m wide and 3 and mu m spacing between fingers.
Preferred scheme in step S2:
s301, incubating the carbon nanotubes to realize uniform assembly of the carbon nanotubes on the surface of the electrode, washing off redundant single-walled carbon nanotubes under the action of deionized water, and annealing for 1 hour at 250 ℃ in a Lindberg blue M tube furnace.
Preferred scheme in step S4:
incubation of linker molecules the device obtained in S3 was incubated for 1 hour in a solution of 1-Pyrene Butyric Acid Succinimidyl Ester (PBASE) Dimethylformamide (DMF) at a concentration of 3 mg/mL. Then washed with dimethylformamide and 10mM Phosphate Buffer (PB), I-V measurements were taken before and after each incubation step using the CHI 1202A electrochemical workstation, and the resistance was calculated from the ramp, in order to monitor the effectiveness and size of each subsequent functionalization.
The method has the advantages of simple process, convenient operation and high yield; the electrode modified by the method can be applied to the electrochemical identification of bacterial species.
Drawings
Fig. 1 is an SEM image of a carbon nanotube functionalized interdigitated electrode.
FIG. 2 is an interdigitated gold electrode on Si/SiO2, 20 electrodes 200 μm long, 5 μm wide, 3 μm apart.
FIG. 3 shows the results of the principal component analysis in the experiment: 6 receptors (3 saccharides and 3 lectins) and 4 receptors (3 saccharides and ConA lectins) have better discrimination on the clustering of the bacterial types, and effectively distinguish the four bacteria.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
A preparation method of a pathogenic bacteria detection electrode in a building site water environment comprises the following steps:
s1, preparing a gold electrode: photoetching gold interdigital electrodes on a Si/SiO2 substrate; the shape structure of the material is 20 with the length of 200 mu m, the width of 5 mu m and the inter-finger interval of 3 mu m.
S2, electrode surface treatment: treating the surface of the electrode obtained in S1 with 1-Aminopropyltriethoxysilane (APTES) in acetone ultrasonic treatment for 10 minutes, nitrogen ultrasonic treatment for 30 minutes, oxygen plasma gas bath for 10 minutes and ultraviolet ozone for 30 minutes;
s3, electrode surface modification single-walled carbon nanotubes (SWNTs): incubating the carbon nanotubes to achieve uniform assembly of the carbon nanotubes on the electrode surface, washing off excess single-walled carbon nanotubes under the action of deionized water, and annealing at 250 ℃ for 1 hour in a lindenberg blue M tube furnace.
S4, incubation of linker molecules: the device obtained in S3 was incubated in a 3mg/mL solution of 1-pyrenebutyric acid succinimidyl ester (PBASE) in Dimethylformamide (DMF) for 1 hour, followed by washing with dimethylformamide and 10mM Phosphate Buffer (PB); to monitor the effectiveness and size of each subsequent functionalization, I-V measurements were taken before and after each incubation step using the CHI 1202A electrochemical workstation and the resistance was calculated from the ramp.
S5, blocking reaction: ethanolamine (0.1%) was used to block unreacted PBASE sites, tween 20 (0.1%) was used to block open SWNT regions, minimizing non-specific binding, incubation time was 10 minutes, and then washed with PB.
Referring to fig. 1 to 3, the electrodes prepared by the above-described method through experiments have excellent reproducibility in initial resistance, performance, and stability.
The ratio between the on-chip resistances (55 electrodes) is within 19%. An average of 25 electrodes maintained 12% of their initial resistance over a one month period. In addition to showing the effect of functionalization on device resistance (slope of the I-V curve), the response of the sensor to E.coli at concentrations of 1x103 and 1x107 cfu/mL is shown, with the signal increasing with increasing bacterial cell concentration. The lectins used in these studies, which have high or no specificity for p-aminosugars, were initially tested to demonstrate that the device is capable of binding bacteria via its naturally occurring surface lectins. The literature reports that ConA has high affinity for glucose and mannose and peanuts have high affinity for galactose and WGA for n-acetylglucosamine. 16, 17 lectin stocks were freshly prepared for each test, formulated to 1mM enriched PB 3mg/mL Ca2+ and Mn2+ ions. These ions are used in specific lectins, called c-lectins, which have metal binding sites that need to be occupied in order to achieve the correct conformation for binding to carbohydrates. Low concentrations were achieved by serial dilution. Each concentration was incubated for 1h, rinsed with PB and the resistance determined.
The respective sugar is used for detecting the free agglutinin, and the experimental scheme and the incubation method are verified, so that the method has good correlation with the literature report. For each lectin tested, although each lectin had a carbohydrate to which it bound most strongly, its binding strength differed, as evidenced by the affinity binding constant. Detection of WGA with affinity for n-acetylglucosamine, by capturing the glycoside with lower affinity for n-acetylglucosamine, produces a small signal. For these reasons, the signals for detecting lectins by their respective sugars are not very large.
As described above, PBASE can be reacted with amine-modified saccharides. Its goal is to capture lectins on the surface of bacteria. The aminophenyl sugar functionalized devices were incubated with one of the four different bacteria used in this work at different concentrations for 1h, followed by PB wash and resistance measurement for quantitative detection, sensitivity, dynamic range and limit of detection. Normalized biosensor response (R R0)/R0, where resistance of R0 device versus gradual functionalization and blocked empty website 20R after resistance versus bacteria culture +0.2 to 0.2V plot from the inverse of the slope of the V curve as a function of bacteria concentration. The response is linear over the detection range and different biological receptors are used differently. In the concentration range of 2x103 to 2x108 cfu/mL, the regression equation of escherichia coli for galactose is 0.92x0.90(R2 is 0.95), the regression equation for mannoside is 0.61x2.2(R2 is 0.92), and the regression equation for glucoside is 0.31x-1.1(R2 is 0.62), where y is the relative change of resistance ((R R0)/R0) and x is the logarithm of the bacterial concentration. In addition, PBASE can react with amines on lectins. This in turn targets carbohydrates on the bacterial surface for binding. In this case, a stock solution of lectin was prepared in enriched PB, incubated on PBASE equipment for 2 hours, and then the bacteria were incubated. 4 bacteria, 1 phage and 1 virus were detected in phosphate buffered saline using the same 3 lectins and 3 sugars. The results showed that phage had no affinity for any of the 6 receptors, whereas the H1N1 virus had a slight affinity for each receptor, most notably for lectins. This was confirmed by previous studies which reported isolation of the virus from the matrix by lectin agglutination. However, the detection of bacteria by these 6 receptors results in a greater response and unique characteristics for each bacterium. Because gram-negative bacteria have a LPS layer, which varies widely in structure and composition, the patterns of response produced by these bacteria vary most and are easily distinguished. Gram-positive bacteria, on the other hand, are similar in their external structure and consist mainly of peptidoglycans. Because of the similarity, the two gram-positive bacteria tested in this study gave rise to a similar response pattern. It is important to note that the response of the sugar-functionalized device produces a higher response than the lectin, which provides a higher sensitivity. In addition, because not all bacteria have lectins on their surfaces, higher specificity can be achieved using sugars as bioreceptors. From these results, one array can be used to distinguish between bacterial and viral infections and has the potential to distinguish gram types, in the case of gram positive bacteria, to further identify species. Several challenges are faced when detecting bacteria in the relevant artificial matrix. Detection of E.coli in artificial urine inhibited the presence of urea by a factor of 10. It is well known that urea can unfold proteins at sufficiently high concentrations. For the detection protocol employed here, unfolding the protein means that the binding sites are lost, and thus detectability is lost. In the case of S.mutans, mucin in the artificial saliva provides some background signal. To minimize the adhesion of mucins to the device, the CNT device was treated with 0.1% Mercaptohexanol (MCH) prior to other functionalization steps. The contribution of mucin following MCH treatment was determined to be 20% of the signal, a result similar to previously reported work.
To statistically determine that the response of each bacterium is unique or distinguishable, Principal Component Analysis (PCA) was performed. PCA is a chemometric tool used to analyze multivariate data. The PCA scores of the results are typically plotted to produce clusters. The proximity of these clusters can be interpreted as the relevance or similarity, i.e. differentiability or indistinguishability, of the data, which is the target of performing the analysis. From the principal component analysis and subsequent figures we can extract which variables contribute the most or most to the uniqueness of the analyte, in this case bacteria. This is useful for designing an array of the strongest or most important receptors to identify bacteria. Thus, an optimized array with minimal elements can be designed with high selectivity. The individual delta R/R0 data for 6 receptors were arranged into 18 cell rows. Each row corresponds to a complete array. Four bacteria produce three rows each. 95% of the total variance in the array was captured by the first two principal component analysis factors. Plotting the PCA scores for these two factors shows 4 separate clusters, one for each bacterium, indicating that each cluster is distinguishable regardless of type. This analysis was repeated using different combinations of 6 receptors to determine which receptor contributed most uniquely to the response profile. The data for 3 saccharides and ConA were the most closely clustered of all combinations. This means that sugars and ConA lectins are the four major factors of uniqueness of the response spectrum, with 4 receptors being sufficient to keep clusters separated (i.e. bacterial identification). This approach helps to optimize the array to a minimum number, thereby improving the cost-effectiveness and overall efficiency of the device. Through the interaction of lectin and carbohydrate, an effective platform is provided for capturing bacteria. The results demonstrate the possibility of using such sensors to distinguish between viral and bacterial infections, which can be used for clinically relevant bacterial concentrations.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A preparation method of a pathogenic bacteria detection electrode in a building site water environment is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing a gold electrode: in Si/SiO2Photoetching gold interdigital electrodes on a substrate;
s2, electrode surface treatment: treating the surface of the electrode obtained in S1 with 1-Aminopropyltriethoxysilane (APTES) in acetone ultrasonic treatment for 10 minutes, nitrogen ultrasonic treatment for 30 minutes, oxygen plasma gas bath for 10 minutes and ultraviolet ozone for 30 minutes;
s3, electrode surface modification single-walled carbon nanotubes (SWNTs): incubating the carbon nanotubes to realize uniform assembly of the carbon nanotubes on the surface of the electrode, washing off redundant single-walled carbon nanotubes under the action of deionized water, and annealing in a Lindberg blue M tube furnace;
s4, incubation of linker molecules: the device obtained in S3 was incubated in a 3mg/mL solution of 1-pyrenebutyric acid succinimidyl ester (PBASE) in Dimethylformamide (DMF) for 1 hour, followed by washing with dimethylformamide and 10mM Phosphate Buffer (PB);
s5, blocking reaction: ethanolamine (0.1%) was used to block unreacted PBASE sites, tween 20 (0.1%) was used to block open SWNT regions, minimizing non-specific binding, incubation time was 10 minutes, and then washed with PB.
2. The method for preparing the pathogenic bacteria detection electrode in the water environment of the construction site according to claim 1, characterized in that: the step of S1 includes:
s101, photoetching interdigital gold electrodes on Si/SiO2, wherein the shape structure of the interdigital gold electrodes is 20, 200 and mu m long, 5 and mu m wide and 3 and mu m spacing between fingers.
3. The method for preparing the pathogenic bacteria detection electrode in the water environment of the construction site according to claim 1, characterized in that: the step of S2 includes:
s201, carrying out ultrasonic treatment on the electrode for 10 minutes in acetone, 30 minutes in nitrogen, 10 minutes in an oxygen plasma gas bath, and 30 minutes in ultraviolet ozone in strict sequence, and treating the surface with 1-Aminopropyltriethoxysilane (APTES).
4. The method for preparing the pathogenic bacteria detection electrode in the water environment of the construction site according to claim 1, characterized in that: the step of S4 includes:
incubation of linker molecules the device obtained in S3 was incubated for 1 hour in a solution of 1-Pyrene Butyric Acid Succinimidyl Ester (PBASE) Dimethylformamide (DMF) at a concentration of 3 mg/mL. Then washed with dimethylformamide and 10mM Phosphate Buffer (PB), I-V measurements were taken before and after each incubation step using the CHI 1202A electrochemical workstation, and the resistance was calculated from the ramp, in order to monitor the effectiveness and size of each subsequent functionalization.
5. The application of the electrode for detecting pathogenic bacteria in the water environment is characterized in that: for detecting different kinds of bacteria.
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