CN113670907B - CGC hybridized nano-composite and preparation method and application thereof - Google Patents
CGC hybridized nano-composite and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a CGC hybridized nano-composite, a preparation method and application thereof, wherein the CGC hybridized nano-composite is Con A-GOx-Ca 2 (PO 4 ) 3 . On the basis of the compound, the invention designs a hybridized nano-compound colorimetric detection system and method for the escherichia coli O157:H27, realizes the visual detection of the escherichia coli O157:H27, and has the advantages of simple operation, strong detection specificity, high sensitivity and the lowest detection limit of 10 CFU.mL ‑1 . In the detection of an actual sample, the detection recovery rate of the detection method is 93-102%, the detection method has higher accuracy, is suitable for the on-site rapid detection of the escherichia coli O157H 7, and has great application potential in the detection of other pathogenic bacteria or clinical diagnosis.
Description
Technical Field
The invention belongs to the technical field of food-borne pathogenic bacteria detection. More specifically, it relates to a Con A-GOx-Ca 2 (PO 4 ) 3 (CGC) hybrid nanocomposites, methods of preparation and use thereof.
Background
Coli O157H 7, one of the most common food-borne pathogens, is widely found in meat, eggs and vegetables, which can cause severe hemorrhagic diarrhea, hemolytic uremia, neonatal sepsis or acute renal failure, and even death in humans. If the method can detect the Escherichia coli O157H 7 timely and accurately, the damage caused by the method can be effectively reduced. Therefore, the construction of a simple, rapid and accurate method for detecting the escherichia coli O157:H27 has important significance.
In the detection method of food-borne pathogenic bacteria, colorimetric biosensors are often used for rapid detection of pathogenic microorganisms due to their simplicity and visual recognition. The test strip is used as one of the common colorimetric sensor output modes, is widely applied to the field of biological detection, and has the advantages of small volume, simple operation, easy reading, low cost and the like compared with other colorimetric sensor signal output modes. Ge et al found that organic substances such as proteins and phosphates formed a porous flower-like organic-inorganic nanocomposite with a large specific surface area. When the enzyme or antibody is used as an organic component of the organic-inorganic nanocomposite, the enzyme or antibody has excellent stability and enzyme activity as compared with the free enzyme and has excellent capturing ability as compared with the free antibody. In recent years, biosensors based on such organic-inorganic nanocomposites find application in disease diagnosis and detection. Chinese patent CN108300758A discloses a Hemin hybrid nanoflower and a preparation method and application thereof, but when the method is used for detecting escherichia coli O157:H27, an ultraviolet spectrophotometer and the like are required to be used for measuring the absorbance of a reaction solution, and the method is still not portable enough and is not suitable for on-site rapid detection.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art and provide a specific Con A-GOx-Ca 2 (PO 4 ) 3 Hybrid nanocomposites, methods of making and uses thereof. The Con A-GOx-Ca 2 (PO 4 ) 3 Hybrid nanocomposites capable of binding H 2 O 2 The test paper is combined, and the visual rapid detection of food-borne pathogenic bacteria, namely escherichia coli O157:H27, is realized through the color reaction.
The first object of the present invention is to provide a Con A-GOx-Ca 2 (PO 4 ) 3 Hybrid nanocomposites.
A second object of the present invention is to provide a Con A-GOx-Ca 2 (PO 4 ) 3 A method for preparing a hybrid nano-composite.
A third object of the invention is to provide a hybrid nanocomposite colorimetric detection system for detecting E.coli O157: H7.
The fourth object of the invention is to provide a method for detecting escherichia coli O157: H7 by utilizing the hybridization nano-complex colorimetric detection system.
The above object of the present invention is achieved by the following technical scheme:
the invention firstly provides a Con A-GOx-Ca 2 (PO 4 ) 3 Hybrid nanocomposite is prepared from concanavalin A (Con A), glucose oxidase (GOx) and Ca 2+ The ion is synthesized by a one-pot method after being dissolved in Phosphate Buffered Saline (PBS).
Con A-GOx-Ca according to the invention 2 (PO 4 ) 3 Ca in hybrid nanocomposite (CGC for short) 3 (PO 4 ) 2 Con A and GOx are immobilized as inorganic frameworks, and Ca 2+ And the synergistic effect is generated by coexistence with GOx, so that the enzyme catalytic activity can be enhanced, the CGC has better adaptability and activity, low sensitivity to external conditions and strong stability.
The invention also provides the Con A-GOx-Ca 2 (PO 4 ) 3 A method of preparing a hybrid nanocomposite comprising the steps of:
s1, sequentially adding 500-1500 mu L of 10mM PBS buffer solution, 50-200 mu L of 0.1-1 mg/mL Con A, 50-200 mu L of 0.1-1 mg/mL glucose oxidase and 10-100 mu L of 100-500 mM CaCl into a centrifuge tube 2 Immediately shaking and uniformly mixing the solution, and standing for 16-24 hours at room temperature until white flocculent precipitate is generated;
s2, centrifuging the mixed solution at 8000-12 rpm for 3-5 min at room temperature, removing supernatant, adding 1-5 mL of 0.1-10 mM PBS buffer solution for washing and centrifuging for two times, removing supernatant, adding 100-500 mu L of 0.1-10 mM PBS buffer solution for suspending, and preserving at 4 ℃.
The invention utilizes the Con A-GOx-Ca 2 (PO 4 ) 3 The principle of the hybridized nano-composite (CGC) for carrying out visual quantitative detection on the escherichia coli O157:H27 is as follows: con A in CGC can bind to E.coli O157:H2 7 while the E.coli O157 is bound by magnetic beads carrying an antibody specific for E.coli O157:H2 7H7 is identified and enriched. Under the condition that target escherichia coli O157: H7 exists, a 'magnetic bead-target-CGC' sandwich structure is formed by the three, and redundant CGC hybridized nano-complex in the reaction is removed through rinsing with PBS buffer solution. Furthermore, glucose oxidase in CGC nanocomposites can catalyze β -D-glucose as H 2 O 2 And gluconic acid. Thus, the addition of beta-D-glucose solution to the sandwich structure allows for the catalytic production of H 2 O 2 I.e. by direct use of H 2 O 2 The test strip realizes the visual quantitative detection and analysis of the escherichia coli O157H 7 and is used for the rapid detection on site.
Thus, the present invention is described in the Con A-GOx-Ca 2 (PO 4 ) 3 On the basis of the hybridized nano-composite, a hybridized nano-composite colorimetric detection system for detecting the escherichia coli O157:H27 is also provided, which comprises escherichia coli O157:H2 antibody magnetic beads and Con A-GOx-Ca 2 (PO4) 3 Hybrid nanocomposites and H 2 O 2 Test strips.
Preferably, the beta-D-glucose solution is also included.
The invention also provides a method for utilizing the Con A-GOx-Ca 2 (PO4) 3 The method for detecting the escherichia coli O157:H27 by the hybridized nano-composite colorimetric detection system comprises the following steps:
s1, washing magnetic beads of an escherichia coli O157/H7 antibody with PBS buffer solution, and suspending for later use;
s2, suspending the antibody magnetic beads obtained in the step S1, and performing escherichia coli O157:H7 and Con A-GOx-Ca 2 (PO 4 ) 3 Mixing and incubating the hybridized nano-composites to form a sandwich structure combined with magnetic beads and targets and CGC, and washing with PBS buffer solution for later use;
s3, adding a proper amount of beta-D-glucose solution into the sandwich structure of the S2, uniformly mixing, adsorbing magnetic beads by a magnetic bead separator, sucking supernatant, and dripping the supernatant into H 2 O 2 And on the test strip, immediately shooting signals by shooting equipment after reacting for 10-60 s.
The principle of the detection method is as follows: con A-GOx-Ca 2 (PO4) 3 Con A in the hybridized nano-composite is used as a biological recognition element and is used for O on the surface of escherichia coli O157:H7 - The antigen has high affinity, and can be combined with Escherichia coli O157:H27; the glucose oxidase is used as a signal output element to catalyze beta-D-glucose into H 2 O 2 And gluconic acid. When target escherichia coli O157: H7 exists, adding magnetic beads with escherichia coli O157: H7 specific antibodies and CGC, wherein the magnetic beads, the target-CGC and the CGC can form a sandwich structure compound of the magnetic beads and the target; after the incubation is finished, the excess CGC is washed off by PBS buffer solution, and then beta-D-glucose is added to catalyze the beta-D-glucose to H by glucose oxidase 2 O 2 And gluconic acid, H 2 O 2 Test paper pair H 2 O 2 Color development is carried out, so that the visual detection of the escherichia coli O157:H27 is realized; further, the imaging device such as a smart phone can be used for recording the color development result, and the Image J software can be used for H 2 O 2 And (5) analyzing the color development result of the test strip. On the contrary, when the Escherichia coli O157H 7 target is not present, a sandwich structure cannot be formed, and beta-D-glucose cannot be enzymatically decomposed to generate H without glucose oxidase 2 O 2 ，H 2 O 2 The test strip has no color change.
The invention uses the Escherichia coli O157 to H7 concentration change and H 2 O 2 The correlation of the color change of the test strip can be used for quantitatively analyzing the concentration of the escherichia coli O157 to H7.
Preferably, the amount of the antibody bead suspension in step S2 is 1 to 6. Mu.L, the amount of E.coli O157:H2 7 is 10 to 100. Mu.L, con A-GOx-Ca 2 (PO 4 ) 3 The amount of hybrid nanocomposite used was 1 to 10. Mu.L, see example 5.
Preferably, the incubation temperature of the antibody beads, E.coli O157: H7 and nanocomposite CGC in step S2 is 25℃to 55℃as described in example 5.
Preferably, in step S2, the incubation time of the antibody beads, E.coli O157:H7 and nanocomposite CGC is 20-60 min, see example 5.
More preferably, in step S2, the incubation time of the antibody beads, E.coli O157:H7 and nanocomposite CGC is 40-60 min, see example 5.
Preferably, in step S2, the pH of the PBS buffer is between 4.5 and 8.5, see example 5.
More preferably, in step S2, the pH of the PBS buffer is between 5.5 and 7.5, see example 5.
Preferably, in step S3, the concentration of the beta-D-glucose solution is 10-60 mM, see example 5.
More preferably, in step S3, the concentration of the beta-D-glucose solution is 40-60 mM, see example 5.
The invention has the following beneficial effects:
the invention is based on Con A-GOx-Ca 2 (PO 4 ) 3 Hybridization nanocomposite signal amplification technique for determining food-borne pathogenic bacteria Escherichia coli O157:H7, con A-GOx-Ca 2 (PO 4 ) 3 The hybrid nanocomposite has glucose oxidase activity, which has the following advantages:
(1) The invention synthesizes Con A-GOx-Ca by a one-pot method 2 (PO 4 ) 3 Ca in hybrid nanocomposites 3 (PO 4 ) 2 Con A and GOx are immobilized as inorganic frameworks, and Ca 2+ The catalyst has synergistic effect with GOx, can enhance the catalytic activity of enzyme, has better adaptability and activity, low sensitivity to external conditions, strong stability and simple operation;
(2) The invention forms a sandwich structure compound of magnetic bead-target-CGC by a one-step method, wherein ConA is taken as a recognition element to be recognized and combined with an E.coli O157-H7 cell membrane, and glucose oxidase in the nano compound is caused by Ca 2+ The ion function enhances the catalytic activity and stability, and catalyzes the conversion of beta-D-glucose into gluconic acid and H 2 O 2 And H is 2 O 2 The specific chromaticity change intensity of the test strip is used as a signal output end detection result, so that the visual detection of the escherichia coli O157:H27 is realized;
(3) The invention is based on CGC and H 2 O 2 The test paper strip is large for food-borne pathogenic bacteriaThe enterobacteria O157H 7 has strong specificity and high sensitivity, and the lowest detection limit is 10 CFU.mL -1 The linear range of detection is 10-10 6 CFU·mL -1 。
(4) In the detection of the actual sample, the detection recovery rate is 93-102%, and the detection method has higher accuracy.
(5) The detection method is simple and convenient to operate, is suitable for on-site detection of pathogenic bacteria such as escherichia coli O157:H27, and has great application potential in detection of other pathogenic bacteria or clinical diagnosis.
Drawings
FIG. 1 is a diagram of Con A-GOx-Ca 2 (PO 4 ) 3 Scanning electron microscopy images of the hybrid nanocomposite, with magnification of 10000× for panel a and 25000× for panel B.
FIG. 2 is a diagram of Con A-GOx-Ca 2 (PO 4 ) 3 Hybrid nanocomposite binding H 2 O 2 The schematic diagram of the visual detection of the test strip on the escherichia coli O157 to H7.
FIG. 3 shows the results of optimizing the detection conditions, wherein FIG. A corresponds to the incubation time of the magnetic bead-target-CGC, FIG. B corresponds to the incubation temperature of the magnetic bead-target-CGC, FIG. C corresponds to the pH of the PBS buffer, and FIG. D corresponds to the concentration of β -D-glucose.
FIG. 4 shows the result of a sensitivity analysis of the visual detection of E.coli O157H 7, wherein FIG. A is H 2 O 2 The detection results of the test strips on the escherichia coli O157:H7 with different concentrations are shown in the graph B, wherein the graph B shows the specific chromaticity change intensity of the escherichia coli O157:H7 with different concentrations, and the graph C shows H 2 O 2 The delta intensity% of the test strip is linearly related to the E.coli O157: H7 concentration.
FIG. 5 shows the result of a specific analysis of the visual inspection, wherein FIG. A is H 2 O 2 And the test strip is used for detecting and developing the results of different strains, and the graph B is the delta intensity% statistical result of the different strains.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Food-borne pathogenic bacteria strains related to the invention, including Escherichia coli O157: H7 (CICC 21530), escherichia coli (CICC 10389), salmonella typhimurium (CCICC 21482), staphylococcus aureus (CICC 21600), listeria monocytogenes (CICC 21529) and Vibrio parahaemolyticus (CICC 21617) are all provided by the China center for type culture Collection of microorganisms. H 2 O 2 Test strips were purchased from Merck, germany.
Example 1 Con A-GOx-Ca 2 (PO 4 ) 3 Synthesis and analysis of hybrid nanocomposites
Con A-GOx-Ca 2 (PO 4 ) 3 Synthesis of hybrid nanocomposites
(1) 780. Mu.L of PBS buffer at a concentration of 10mM, 100. Mu.L of concanavalin (Con A) at a concentration of 0.1mg/mL, 100. Mu.L of glucose oxidase (GOx) at a concentration of 0.1mg/mL, and 20. Mu.L of CaCl at a concentration of 200mM were sequentially added to a 1.5mL centrifuge tube 2 Immediately shaking and uniformly mixing the solution, and standing at room temperature for 16 hours to generate white flocculent precipitate;
(2) Centrifuging the mixed solution obtained in the step (1) at room temperature at 10000rpm for 5min, removing supernatant, washing with 1mL of 1mM PBS buffer, centrifuging twice, and adding 100 μl of 1mM PBS buffer to obtain Con A-GOx-Ca 2 (PO 4 ) 3 And (3) taking a proper amount of suspension of the organic-inorganic nano-composite (CGC), displaying the appearance characteristics of the synthesized nano-composite by using a scanning electron microscope, and storing the rest in a refrigerator at 4 ℃ for later use.
Con A-GOx-Ca 2 (PO 4 ) 3 The scanning electron micrograph of the hybrid nanocomposite is shown in FIG. 1, wherein the magnification of panel A is 10000X and the magnification of panel B is 25000X. As shown in fig. 1A, CGC is a flower-like nanostructure of about 6 μm. Further analysis of its structure, it can be seen from fig. 1B that CGC has a nanoscale layered petal structure, which allows the CGC to have a higher specific surface area, contributing to increased target capture of recognition proteins and enhanced enzymatic activity.
Example 2 Con A-GOx-Ca 2 (PO 4 ) 3 Synthesis and analysis of hybrid nanocomposites
Con A-GOx-Ca 2 (PO 4 ) 3 Synthesis of hybrid nanocomposites
(1) Into a 1.5mL centrifuge tube, 500. Mu.L of PBS buffer at a concentration of 10mM, 50. Mu.L of concanavalin (Con A) at a concentration of 1mg/mL, 50. Mu.L of glucose oxidase (GOx) at a concentration of 1mg/mL, and 10. Mu.L of CaCl at a concentration of 500mM were sequentially added 2 Immediately shaking and uniformly mixing the solution, and standing at room temperature for 20 hours to generate white flocculent precipitate;
(2) Centrifuging the mixed solution obtained in the step (1) at room temperature at 12000rpm for 3min, removing supernatant, washing with 5mL of PBS buffer with concentration of 0.1mM, centrifuging twice, and adding 500 μl of PBS buffer with concentration of 0.5mM to obtain Con A-GOx-Ca 2 (PO 4 ) 3 And (3) taking a proper amount of suspension of the organic-inorganic nano-composite (CGC), displaying the appearance characteristics of the synthesized nano-composite by using a scanning electron microscope, and storing the rest in a refrigerator at 4 ℃ for later use.
Con A-GOx-Ca prepared in this example 2 (PO 4 ) 3 The scanning electron micrograph of the hybrid nanocomposite is similar to that of fig. 1, with a nanoscale layered petal structure.
Example 3 Con A-GOx-Ca 2 (PO 4 ) 3 Synthesis and analysis of hybrid nanocomposites
Con A-GOx-Ca 2 (PO 4 ) 3 Synthesis of hybrid nanocomposites
(1) 1500. Mu.L of 10mM PBS buffer and 200. Mu.L of concentrated solution were sequentially added to a 1.5mL centrifuge tubeConcanavalin (Con A) at a concentration of 0.1mg/mL, glucose oxidase (GOx) at a concentration of 200. Mu.L at a concentration of 0.1mg/mL, and CaCl at a concentration of 300mM at 100. Mu.L 2 Immediately shaking and uniformly mixing the solution, and standing at room temperature for 24 hours to generate white flocculent precipitate;
(2) Centrifuging the mixed solution obtained in the step (1) at room temperature at 8000rpm for 5min, removing supernatant, washing with 1mL of 10mM PBS buffer, centrifuging twice, and adding 100 μl of 10mM PBS buffer to obtain Con A-GOx-Ca 2 (PO 4 ) 3 And (3) taking a proper amount of suspension of the organic-inorganic nano-composite (CGC), displaying the appearance characteristics of the synthesized nano-composite by using a scanning electron microscope, and storing the rest in a refrigerator at 4 ℃ for later use.
Con A-GOx-Ca prepared in this example 2 (PO 4 ) 3 The scanning electron micrograph of the hybrid nanocomposite is similar to that of fig. 1, with a nanoscale layered petal structure.
Example 4 visualized detection of E.coli O157: H7
The invention is based on CGC nano-composite and H 2 O 2 The principle of colorimetric detection of the escherichia coli O157-H7 by the test strip is shown in figure 2. The invention uses the one-pot method to prepare concanavalin A (Con A), glucose oxidase (GOx) and Ca at room temperature 2+ Ion synthesis of Con A-GOx-Ca 2 (PO 4 ) 3 Organic-inorganic nanocomposite (CGC). Wherein Ca is 2 (PO 4 ) 3 Con A and GOx are immobilized as inorganic frameworks while Ca 2+ Ions can coexist with GOx to produce synergistic effect and enhance the catalytic activity of enzyme. Con A is used as a biological recognition element and has high affinity to O-antigen on the surface of escherichia coli O157H 7, and glucose oxidase is used as a signal output element and can catalyze beta-D-glucose into H 2 O 2 And gluconic acid. When the target escherichia coli O157: H7 exists, the antibody magnetic bead, the target and the CGC nano-composite can form a sandwich structure of 'magnetic bead-target-CGC', so that beta-D-glucose is catalyzed to be H 2 O 2 And gluconic acid, H 2 O 2 Make H 2 O 2 The test strip changes color, which shows that the sample contains colibacillus O157:H27, the color result can be recorded by using shooting equipment, and the color result can be recorded by using imageJ software 2 O 2 And (5) quantitatively analyzing the color development result of the test strip. On the contrary, when no target exists, a sandwich structure cannot be formed, and beta-D-glucose cannot be enzymatically decomposed to generate H without glucose oxidase 2 O 2 ，H 2 O 2 The test strip has no color change.
Con A-GOx-Ca 2 (PO 4 ) 3 Hybrid nanocomposite binding H 2 O 2 The test strip carries out visual detection on the escherichia coli O157:H27, and the steps are as follows:
(1) Pretreatment of E.coli O157H 7 antibody magnetic beads: washing the antibody magnetic beads for 2-5 times by using an equal volume of 1mM PBS buffer solution before the test, and suspending the antibody magnetic beads for later use by using the equal volume of PBS buffer solution;
(2) Adding 3 mu L of treated antibody magnetic beads, 50 mu L of target (E.coli O157: H7) and 6 mu L of CGC hybridized nano-composite into a centrifuge tube, incubating for 40min at 25 ℃ to form a sandwich structure combined with magnetic beads and target and CGC, and washing three times with 50 mu L of PBS buffer solution with the concentration of 1mM for later use;
(3) Adding 10 mu L of beta-D-glucose with the concentration of 40mM into the sandwich structure obtained in the step (2), uniformly mixing by a pipetting gun, adsorbing magnetic beads in an RCR tube by using a magnetic bead separator, sucking the supernatant reaction solution, and dripping the supernatant reaction solution into H 2 O 2 Immediately after reaction for 30 seconds on the test strip, shooting a reaction signal by using shooting equipment, judging whether the escherichia coli O157:H7 exists or not through the color change of the test strip, and performing quantitative analysis by using imageJ software.
As a result, it was found that H 2 O 2 The color of the test strip changes, which indicates that a rapid visual detection method of the escherichia coli O157: H7 is successfully constructed.
Example 5 optimization of detection conditions
The invention further researches that the incubation time, the incubation temperature and the pH value of the washing buffer solution of the sandwich structure compound of the magnetic bead-target-CGC are combined with the three in the compound systemThe efficiency is greatly affected. The amount of beta-D-glucose affects the amount of glucose oxidase in the complex that catalyzes it as gluconic acid, and has an effect on the chromogenic signal of the test strip. The present invention is therefore based on glucose-induced H 2 O 2 The specific chromaticity change intensity of the test strip optimizes the detection condition. H 2 O 2 The specific colorimetric change intensity (Δintensity%) of the test strip was calculated as follows: Δintensity% = [ (In) Positive and negative –In Negative of )/In Positive and negative ]100% and the results were repeated 3 times. The optimized results of the detection conditions are shown in fig. 3, wherein graph a corresponds to the incubation time of the magnetic bead-target-CGC, graph B corresponds to the incubation temperature of the magnetic bead-target-CGC, graph C corresponds to the pH of the PBS buffer solution, and graph D corresponds to the concentration of β -D-glucose.
1. Optimization of incubation time of magnetic bead-target-CGC sandwich structure complex
Incubation times were set to 20, 30, 40, 50 and 60min, respectively, using the same colorimetric detection procedure as in example 4. The optimized result of the incubation time is shown in FIG. 3A, and it can be seen from FIG. 3A that as the incubation time increases, H 2 O 2 The delta intensity% of the test strip also increases. The detection purpose can be realized when the incubation time is 20-60 min, but when the incubation time is 50min, the delta intensity% reaches the maximum value and tends to be stable.
2. Optimization of incubation temperature of magnetic bead-target-CGC sandwich structure complex
The incubation temperatures were set to 4 ℃,25 ℃, 37 ℃ and 55 ℃ respectively using the same colorimetric detection procedure as in example 4. The optimal result of the incubation temperature is shown in FIG. 3B, and as can be seen from FIG. 3B, H as the incubation temperature increases 2 O 2 The delta intensity% of the test strip is increased and then decreased, and when the incubation temperature reaches 37 ℃, H 2 O 2 As can be seen from the result, the too high or too low incubation temperature can affect the combination efficiency of the magnetic bead-target-CGC, has an influence on the delta intensity%, and is ideal in the range of 25-55 ℃.
3. Optimization of PBS wash buffer pH
The pH of the PBS wash buffer was selected to be 4.5, 5.5, 6.5, 7.5 and 8.5, respectively. As shown in FIG. 3C, the result of the optimization of the pH of the PBS wash buffer is shown in FIG. 3C, and as the pH of the PBS wash buffer increases, H 2 O 2 The delta intensity% of the test strip increases and then decreases, and reaches a maximum value when the pH value of the washing buffer solution reaches 7.5. From the results, it can be seen that too high or too low a pH of the PBS wash buffer affects the binding efficiency of the "bead-target-CGC" and has an effect on the Δintensity% value.
4. Optimization of beta-D-glucose solution concentration
beta-D-glucose solutions at concentrations of 10, 20, 30, 40, 50 and 60mM, respectively, were selected for optimization experiments. The results of optimizing the concentration of the beta-D-glucose solution are shown in FIG. 3D, and it is understood from FIG. 3D that H increases with the increase of the beta-D-glucose concentration 2 O 2 The delta intensity% of the test strip also increases. The detection can be achieved at a concentration of 10 to 60mM of beta-D-glucose, but when the concentration of beta-D-glucose reaches 40mM, the delta intensity% reaches a maximum value and becomes stable.
If an optimal detection effect is desired, the "magnetic bead-target-CGC" sandwich complex may be incubated at 37℃for 50min, washed with PBS wash buffer at pH 7.5, and detected with beta-D-glucose at a concentration of 40 mM.
While optimizing the conditions, the invention simultaneously optimizes the antibody magnetic bead suspension, the escherichia coli O157:H7 and the Con A-GOx-Ca 2 (PO 4 ) 3 The amount of hybridized nanocomposite and beta-D-glucose used was optimized. As a result, it was found that the visual detection of E.coli O157H 7 was achieved when the amount of the treated E.coli O157H 7 antibody beads was 1 to 6. Mu.L, the amount of E.coli O157H 7 was 10 to 100. Mu.L, and the amount of the CGC hybrid nanocomposite was 1 to 10. Mu.L, and the amount of beta-D-glucose was 10 to 50. Mu.L, which was not a key factor for the detection effect.
Example 6 detection sensitivity and specificity analysis
1. Sensitivity analysis
The sensitivity analysis of the visual detection of the escherichia coli O157: H7 is carried out under the optimal reaction condition described in the example 4, the sensitivity of the detection method is analyzed, a linear equation and a detection range are determined, the detection target is the lowest detection limit, and the result data are repeated for 3 times or more. Calculating bacterial liquid concentration by nucleic acid protein analyzer, and sequentially diluting to 10 and 10 by 10-fold gradient dilution method 2 、10 3 、10 4 、10 5 、10 6 、10 7 CFU·mL -1 And (5) standby application.
The sensitivity analysis results are shown in FIG. 4, where A is H 2 O 2 The detection results of the test strips on the escherichia coli O157:H7 with different concentrations are shown in the graph B, wherein the graph B shows the specific chromaticity change intensity of the escherichia coli O157:H7 with different concentrations, and the graph C shows H 2 O 2 The delta intensity% of the test strip is linearly related to the E.coli O157: H7 concentration. As can be seen from FIG. 4A, H increases with increasing E.coli O157: H7 concentration 2 O 2 The color of the test strip gradually deepens. And as can be seen in FIG. 4B, H 2 O 2 The delta intensity% of the test strip increases with increasing E.coli O157: H7 concentration. As can be seen from FIG. 4C, H 2 O 2 The delta intensity% of the test strip and the concentration of the escherichia coli O157:H7 are 10-10 6 CFU·mL -1 Has good linear relation in the range, the linear equation is Y=11.22X+20.69 (wherein Y represents the specific chromaticity change intensity, X represents the logarithm of the concentration of the escherichia coli O157: H7), and the correlation coefficient R 2 Can reach 0.9878, and the lowest detection limit is 10 CFU.mL -1 。
2. Specificity analysis
The specificity analysis of the detection method is also carried out under the optimal experimental condition, and the concentration is 10 respectively based on a sandwich structure formed by magnetic bead-target-CGC 7 CFU·mL -1 The specificity of the visual detection was investigated for E.coli O157: H7 (E.coli O157: H7), E.coli (E.coli), sal. Typhimurium (Sal. T), staphylococcus aureus (S.aur), listeria monocytogenes (List. M) and Vibrio parahaemolyticus (Vibiro. P), and the results were repeated 3 times or more. Specific analysis of visual detectionThe results are shown in FIG. 5, where A is H 2 O 2 And the test strip is used for detecting and developing the results of different strains, and the graph B is the delta intensity% statistical result of the different strains. From the figure, H 2 O 2 The test strip was developed only when E.coli O157: H7 was detected, and H of the control strains E.coli, sal. T, S.aur, list. M and Vibiro. P 2 O 2 The delta intensity% value of the test strip is lower than 20% and lower than the minimum detection limit of the target bacteria, and the target escherichia coli O157:H27 triggers stronger H 2 O 2 As can be seen from the delta intensity of the test strip, the CGC-based nanocomposite and H of the invention 2 O 2 The test strip has strong specificity for the visual detection of the escherichia coli O157-H7.
Example 7 detection of actual samples
To evaluate the CGC-based hybrid nanocomposites and H according to the present invention 2 O 2 The method for visually detecting the escherichia coli O157 to H7 of the test strip is applicable to detection of actual samples, and recovery tests are carried out by using skimmed milk powder samples. Based on the sandwich structure combined by antibody-target-CGC, respectively adding escherichia coli O157 to H7 into sterilized skimmed milk powder to reach the concentration of 10 4 、10 5 And 10 6 CFU·mL -1 To explore the recovery rate and accuracy of the detection method, the result data were repeated 3 times and more.
The result is shown in table 1, the recovery rate of actual detection is from 93+/-0.71% to 102+/-1.32%, and the recovery result shows that the detection method can provide feasible and reliable pathogen measurement for preliminary practical application, and the detection result is accurate and reliable.
TABLE 1 detection of E.coli O157: H7 in skimmed milk powder samples
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. Con A-GOx-Ca 2 （PO 4 ） 3 A hybrid nanocomposite, characterized in that the nanocomposite is concanavalin A, glucose oxidase and Ca 2+ The ions are dissolved in PBS buffer solution and then synthesized by a one-pot method; the preparation method comprises the following steps:
s1, 500 to 1500 mu L of 10mM PBS buffer solution, 50 to 200 mu L of 0.1mg/mL concanavalin A, 50 to 200 mu L of 0.1mg/mL glucose oxidase and 10 to 100 mu L of 100 to 500mM CaCl 2 Mixing the solution evenly, standing at room temperature for 16-24 h until white flocculent precipitate is generated;
s2, centrifuging the mixed solution at 8000-12 rpm for 3-5 min at room temperature, removing supernatant, adding 1-5 mL of 0.1-10 mM PBS buffer solution for washing, and centrifuging to obtain the composite material.
2. A method for detecting escherichia coli O157:H27 is characterized by comprising the following steps of:
s1, washing magnetic beads of an escherichia coli O157/H7 antibody with PBS buffer solution, and suspending for later use;
s2, suspending the antibody magnetic beads obtained in the step S1, a sample to be tested and Con A-GOx-Ca as set forth in claim 1 2 （PO 4 ） 3 Mixing and incubating the hybridized nano-composites to form a sandwich structure combined with magnetic beads and targets and CGC, and washing with PBS buffer solution for later use; the incubation temperature of the antibody magnetic beads and the escherichia coli O157:H7 and the nanocomposite CGC is 25-55 ℃, and the incubation time is 20-60 min; the pH value of the PBS buffer solution is 4.5-8.5;
s3, adding a proper amount of beta-D-glucose solution into the sandwich structure of the S2, uniformly mixing and reacting, adsorbing magnetic beads after the reaction, sucking supernatant fluid and dripping the supernatant fluid into H 2 O 2 And (3) carrying out reaction on the test strip for 10-60 s, and then judging the result.
3. The method according to claim 2, wherein the amount of the antibody bead suspension in step S2 is 1 to 6. Mu.L, the amount of the sample to be measured is 10 to 100. Mu.L, and Con A-GOx-Ca 2 （PO 4 ） 3 The dosage of the hybridized nano-composite is 1-10 mu L.
4. The method according to claim 2, wherein in step S3, the concentration of the beta-D-glucose solution is 10 to 60 mM.
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