Bacterial biosensor as well as preparation method and application thereof

Abstract

The invention discloses a bacterial biosensor as well as a preparation method and application thereof. According to the bacterial biosensor, glass (BK7) with the surface covered with gold nanoparticles serves as a substrate, the surfaces of the gold nanoparticles are coupled and modified with specific antibodies, and the gold nanomaterial is prepared and applied to detection of bacterial endophytic ophthalmia. Gold nanoparticles are taken as a substrate, a high-sensitivity, selective and label-free method for simultaneously detecting gram-positive (G +) and gram-negative (G-) bacteria in a sample is constructed, small sample size requirements and short experiment operation time are met, and when the method is used for detecting the bacteria, the use of tachypleus amebocyte lysate can be avoided; the method has the advantages that the method is simple and convenient to operate, can effectively distinguish G < + > and G <-> bacteria, is more efficient to use, is a more humanized and ecologically sustainable bacteria detection method, plays an important role in protecting the horseshoe crabs, and is convenient to popularize and use.

Description

Technical Field

[0001] The present invention relates to the field of bacteria inspection technology, and in particular to a bacteria biosensor and a preparation method and application thereof. Background Art

[0002] Rapid detection of bacteria is of great significance to food safety, environmental monitoring, public health and other fields. Although traditional detection technologies such as culture and staining can detect bacteria, traditional methods have problems such as long detection time, complex detection process, and high detection environment requirements, which are not conducive to the promotion of bacterial detection technology. Currently, the commonly used bacterial detection methods mainly include colorimetry, flow cytometry, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). These methods have the advantages of high detection sensitivity, strong specificity and strong selectivity, but the detection technology is expensive and the detection process requires skilled technicians, which is not conducive to on-site application.

[0003] The horseshoe crab reagent is a sterile freeze-dried product made from the blood amebocyte lysate of the horseshoe crab, and the horseshoe crab reagent contains factor C, factor B, procoagulant, and prothrombin. Under suitable conditions, Gram-negative bacteria activate factor C, causing a series of enzymatic reactions, causing the horseshoe crab reagent to produce an agglutination reaction to form a gel. The gel method is used to perform limit detection or semi-quantitative detection of endotoxins based on the principle of agglutination reaction between the horseshoe crab reagent and endotoxins, but the method cannot perform quantitative detection, and is prone to false positives or false negatives due to the influence of interfering substances in the sample to be tested. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterial biosensor and a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0005] To achieve the above purpose, the present invention is implemented by the following technical means:

[0006] A bacterial biosensor comprises a plasma resonance substrate, which is covered with a gold nanomaterial structure layer. The gold nanomaterial structure layer forms a self-assembled gold nanoparticle optical resonance array embedded in a glass (BK7) substrate. The self-assembled gold nanoparticles are solid spherical nanoparticles.

[0007] The plasma resonance substrate is a nanoparticle structure array.

[0008] The bacterial biosensor is based on glass (BK7) covered with gold nanoparticles on the surface, and specific antibodies are coupled and modified on the surface of the gold nanoparticles.

[0009] The self-assembled gold nanoparticles have a diameter of 38-42 nm and are embedded in the glass (BK7) substrate to a depth of 8-10 nm.

[0010] The gold nanomaterial is first cleaned before being used for preparation. The cleaning method is: the glass chip covered with gold nanoparticles is ultrasonically cleaned twice with anhydrous ethanol for 5 minutes each time, and then the cleaned gold nanomaterial is obtained after drying.

[0011] The pretreatment method of the gold nanomaterial is as follows: washing the gold nanomaterial with anhydrous ethanol for 10 minutes, injecting MUA solution for treatment for 15 minutes, washing the unbound MUA on the surface of the gold nanomaterial with anhydrous ethanol for 10 minutes, injecting EDC / NHS mixed solution for reaction for 15 minutes, and then washing with PBS buffer for 10 minutes to obtain the pretreated gold nanomaterial.

[0012] The MUA solution is composed of: MUA 10mM, the solvent is anhydrous ethanol;

[0013] The EDC / NHS mixed solution is composed of: EDC 0.4M, NHS 0.15M, and the solvent is water;

[0014] The PBS buffer composition is: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.4.

[0015] The coupling method of the specific antibody is as follows: placing the surface pretreated gold nanomaterial substrate in an antibody buffer, carrying out the coupling reaction at room temperature for 30 minutes, washing with a PBS buffer for 10 minutes to wash away the unbound antibody, injecting an EA / BSA blocking solution to block the nonspecific binding sites, and washing with a PBS buffer for 10 minutes to wash away the blocking solution, thereby obtaining a bacterial detection biosensor;

[0016] The coupling reaction system is composed of: 1ug / ml antibody diluted in PBS; the PBS buffer is composed of: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.4.

[0017] The composition of the EA / BSA blocking solution is: EA 0.1M, BSA 2% (W / V).

[0018] The bacterial biosensor has a three-unit structure (A, B, C), which can be coupled with anti-LPS (specific to G- bacteria) antibodies and anti-LTA (specific to G+ bacteria) antibodies respectively to form a G- bacteria detection unit (A unit), a G+ bacteria detection unit (B unit), and a negative control detection unit (C unit).

[0019] A method for preparing a bacterial biosensor comprises the following steps:

[0020] The glass chip covered with gold nanoparticles was ultrasonically cleaned twice with anhydrous ethanol for 5 minutes each time, and then dried to obtain the cleaned gold nanomaterial;

[0021] The gold nanomaterials were rinsed with anhydrous ethanol for 10 minutes, injected with MUA solution for 15 minutes, the unbound MUA on the surface of the gold nanomaterials was rinsed with anhydrous ethanol for 10 minutes, injected with EDC / NHS mixed solution for reaction for 15 minutes, and then rinsed with PBS buffer for 10 minutes to obtain pretreated gold nanoparticles; the composition of the MUA solution was: MUA 10mM, the solvent was anhydrous ethanol; the composition of the EDC / NHS mixed solution was: EDC 0.4M, NHS 0.15M, the solvent was water; the composition of the PBS buffer was: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent was water, pH 7.4;

[0022] The surface pretreated gold nanomaterial substrate was placed in an antibody buffer, and the coupling reaction was carried out at room temperature for 30 minutes. The unbound antibody was washed away with a PBS buffer for 10 minutes, and an EA / BSA blocking solution was injected to block the nonspecific binding sites. The blocking solution was washed away with a PBS buffer for 10 minutes to obtain a bacterial detection biosensor.

[0023] The coupling reaction system is composed of: 1ug / ml antibody diluted in PBS; the PBS is composed of: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.4; the EA / BSA blocking solution is composed of: EA 0.1M, BSA 2% (W / V), the solvent is water.

[0024] Application of a bacterial biosensor in detecting bacterial endophthalmitis and hospital-acquired pneumonia.

[0025] Furthermore, the specific application method is as follows: turn on the spectrometer, perform spectral measurement, rinse with washing buffer for 5 minutes, inject the sample to be tested into the bacterial biosensor, the flow rate is 15μl / min, react at room temperature for 10-15 minutes, wash away the unbound sample with washing buffer, and perform spectral measurement again; according to the data of the two previous and subsequent spectral measurements, calculate the phase difference of the light of the three detection units A, B, and C respectively, wherein the phase difference value of the C detection unit is used as a negative control; according to the LPS and LTA standard curves, obtain the bacterial content in the sample to be tested, and judge the bacterial properties in the sample to be tested; the standard curve is made under the same conditions for the detection of the sample to be tested, with the logarithm of the LPS or LTA concentration as the horizontal axis and the phase difference value as the vertical axis; the washing buffer is a PBS buffer with a pH of 7.4.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention uses gold nanoparticles as a substrate to construct a highly sensitive, selective and label-free method for simultaneously detecting Gram-positive (G+) and Gram-negative (G-) bacteria in a sample, as well as a small sample size requirement and a short experimental running time. By using this method to detect bacteria, the use of horseshoe crab reagents can be avoided, while G+ and G- bacteria can be effectively distinguished, making it more efficient to use. In addition, this method is a more humane and ecologically sustainable bacterial detection method, which plays an important role in the protection of horseshoe crabs and is easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of gradient alcohol of bacterial biosensor in an embodiment of the present invention;

[0029] Figure 2 It is a linear schematic diagram between the bacterial biosensor and the logarithm of the concentration of the LTA aqueous solution in an embodiment of the present invention;

[0030] Figure 3 It is a linear schematic diagram between the bacterial biosensor and the logarithm of the concentration of the LPS aqueous solution in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The following is a detailed description of the embodiments of the technical solution of the present application in conjunction with the accompanying drawings. The following embodiments and drawings are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application. The accompanying drawings only schematically show the parts related to the technical solution of the present application, and they do not represent the actual structure of the product.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0033] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present application, the term "multiple" refers to more than two (including two), similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple pieces" refers to more than two pieces (including two pieces).

[0034] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0035] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0036] In the description of the embodiments of the present application, the orientations or positional relationships indicated by technical terms such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", and "circumferential" are based on the orientations or positional relationships shown in the accompanying drawings and are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the embodiments of the present application.

[0037] In the description of the embodiments of the present application, unless otherwise clearly specified and limited, technical terms such as "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0038] In this embodiment, a bacterial biosensor includes a plasma resonance substrate, on which is covered a gold nanomaterial structure layer, wherein the gold nanomaterial structure layer forms a self-assembled gold nanoparticle optical resonance array embedded in a glass (BK7) substrate, wherein the self-assembled gold nanoparticles are solid spherical nanoparticles.

[0039] The plasma resonance substrate is a nanoparticle structure array.

[0040] The bacterial biosensor is based on glass (BK7) covered with gold nanoparticles on the surface, and specific antibodies are coupled and modified on the surface of the gold nanoparticles.

[0041] The self-assembled gold nanoparticles have a diameter of 38-42 nm and are embedded in the glass (BK7) substrate to a depth of 8-10 nm.

[0042] The gold nanomaterial is first cleaned before being used for preparation. The cleaning method is: the glass chip covered with gold nanoparticles is ultrasonically cleaned twice with anhydrous ethanol for 5 minutes each time, and then the cleaned gold nanomaterial is obtained after drying.

[0043] The pretreatment method of the gold nanomaterial is as follows: washing the gold nanomaterial with anhydrous ethanol for 10 minutes, injecting MUA solution for treatment for 15 minutes, washing the unbound MUA on the surface of the gold nanomaterial with anhydrous ethanol for 10 minutes, injecting EDC / NHS mixed solution for reaction for 15 minutes, and then washing with PBS buffer for 10 minutes to obtain the pretreated gold nanomaterial.

[0044] The MUA solution is composed of: MUA 10mM, the solvent is anhydrous ethanol;

[0045] The EDC / NHS mixed solution is composed of: EDC 0.4M, NHS 0.15M, and the solvent is water;

[0046] The PBS buffer composition is: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.4.

[0047] The coupling method of the specific antibody is as follows: placing the surface pretreated gold nanomaterial substrate in an antibody buffer, carrying out the coupling reaction at room temperature for 30 minutes, washing with a PBS buffer for 10 minutes to wash away the unbound antibody, injecting an EA / BSA blocking solution to block the nonspecific binding sites, and washing with a PBS buffer for 10 minutes to wash away the blocking solution, thereby obtaining a bacterial detection biosensor;

[0048] The coupling reaction system is composed of: 1ug / ml antibody diluted in PBS; the PBS buffer is composed of: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.4.

[0049] The composition of the EA / BSA blocking solution is: EA 0.1M, BSA 2% (W / V).

[0050] The bacterial biosensor has a three-unit structure (A, B, C), which can be coupled with anti-LPS (specific to G- bacteria) antibodies and anti-LTA (specific to G+ bacteria) antibodies respectively to form a G- bacteria detection unit (A unit), a G+ bacteria detection unit (B unit), and a negative control detection unit (C unit).

[0051] A method for preparing a bacterial biosensor comprises the following steps:

[0052] The glass chip covered with gold nanoparticles was ultrasonically cleaned twice with anhydrous ethanol for 5 minutes each time, and then dried to obtain the cleaned gold nanomaterial;

[0053] The gold nanomaterials were rinsed with anhydrous ethanol for 10 minutes, injected with MUA solution for 15 minutes, the unbound MUA on the surface of the gold nanomaterials was rinsed with anhydrous ethanol for 10 minutes, injected with EDC / NHS mixed solution for reaction for 15 minutes, and then rinsed with PBS buffer for 10 minutes to obtain pretreated gold nanoparticles; the composition of the MUA solution was: MUA 10mM, the solvent was anhydrous ethanol; the composition of the EDC / NHS mixed solution was: EDC 0.4M, NHS 0.15M, the solvent was water; the composition of the PBS buffer was: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent was water, pH 7.4;

[0054] The surface pretreated gold nanomaterial substrate was placed in an antibody buffer, and the coupling reaction was carried out at room temperature for 30 minutes. The unbound antibody was washed away with a PBS buffer for 10 minutes, and an EA / BSA blocking solution was injected to block the nonspecific binding sites. The blocking solution was washed away with a PBS buffer for 10 minutes to obtain a bacterial detection biosensor.

[0055] The coupling reaction system is composed of: 1ug / ml antibody diluted in PBS; the PBS is composed of: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.4; the EA / BSA blocking solution is composed of: EA 0.1M, BSA 2% (W / V), the solvent is water.

[0056] Application of a bacterial biosensor in detecting bacterial endophthalmitis and hospital-acquired pneumonia.

[0057] Furthermore, the specific application method is as follows: turn on the spectrometer, perform spectral measurement, rinse with washing buffer for 5 minutes, inject the sample to be tested into the bacterial biosensor, the flow rate is 15μl / min, react at room temperature for 10-15 minutes, wash away the unbound sample with washing buffer, and perform spectral measurement again; according to the data of the two previous and subsequent spectral measurements, calculate the phase difference of the light of the three detection units A, B, and C respectively, wherein the phase difference value of the C detection unit is used as a negative control; according to the LPS and LTA standard curves, obtain the bacterial content in the sample to be tested, and judge the bacterial properties in the sample to be tested; the standard curve is made under the same conditions for the detection of the sample to be tested, with the logarithm of the LPS or LTA concentration as the horizontal axis and the phase difference value as the vertical axis; the washing buffer is a PBS buffer with a pH of 7.4.

[0058] The LPS standard curve was prepared as follows: (1) Inject 10 -3 EU / mL, 10 -2 EU / mL, 10 -1 EU / mL and 1EU / mL G-bacterial LPS standard solutions were reacted at room temperature for 10 to 15 minutes, and unbound LPS was washed away with a washing buffer. Then, spectral characterization was performed, with the logarithmic value of LPS concentration as the abscissa and the optical phase difference obtained by characterizing different concentrations of LPS as the ordinate to obtain an LPS standard curve; the washing buffer was a PBS buffer with a pH of 7.4.

[0059] The LTA standard curve is prepared as follows: (1) the bacterial biosensor is placed in G+ bacterial LTA standard solutions of 100ug / mL, 10ug / mL, 1ug / mL, 0.5ug / mL, and 0.1ug / mL, respectively, and reacted at room temperature for 10 to 15 minutes. After washing away the unbound LTA with a washing buffer, spectral characterization is performed, with the logarithmic value of the LTA concentration as the horizontal axis and the optical phase difference obtained by the characterization of different concentrations of LTA as the vertical axis to obtain the LTA standard curve; the washing buffer is a PBS buffer with a pH of 7.4.

[0060]

[0061] The present invention uses gold nanoparticles as a substrate to construct a highly sensitive, selective and label-free method for simultaneously detecting Gram-positive (G+) and Gram-negative (G-) bacteria in a sample, as well as a small sample size requirement and a short experimental running time. By using this method to detect bacteria, the use of horseshoe crab reagents can be avoided, while G+ and G- bacteria can be effectively distinguished, making it more efficient to use. In addition, this method is a more humane and ecologically sustainable bacterial detection method, which plays an important role in the protection of horseshoe crabs and is easy to promote and use.

[0062] The specific embodiments disclosed in the present invention fall within the protection scope of the claims of the present invention, and are the specific lower-level implementation scope of the characteristic part of the present invention. The protection content of the specific embodiments is merely an explanation of the protection scope of the claims of the present invention. The protection scope of the present invention is not limited to the protection content of the specific embodiments, and the protection content of the specific embodiments should not be understood as a limitation on the protection scope of the claims of the present invention.

[0063] The above only describes some exemplary embodiments of the present invention by way of illustration. It is undoubted that for those of ordinary skill in the art, the described embodiments can be modified in various ways without departing from the scope of the present invention. Therefore, the above description is illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention. Unless otherwise defined, all academic and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art to which the present invention belongs. In the event of a conflict, the definitions in this specification shall prevail. Unless otherwise stated, all percentages, parts, ratios, etc. are by weight. When a numerical value or a numerical range, a preferred range, or a series of lower preferred values ​​and upper preferred values ​​are given, it should be understood that any range formed by any pair of numerical values ​​of any smaller range limit or preferred value and any larger range limit or preferred value is specifically disclosed, regardless of whether the range is disclosed separately. Unless otherwise stated, where the numerical range is described in this specification, the range is intended to include all integers and fractions within the range end value and range. When the term "about" or "about" is used to describe the end value of a numerical value or a range, the disclosed content should include the specific numerical value or the end value involved. The use of "a" and "an / kind" to describe elements of the present invention is only for convenience and to give a general description of the present invention. Unless otherwise clearly stated, the description should be understood to include one / kind or at least one / kind.

Claims

1. A bacterial biosensor, characterized in that: The invention comprises a plasma resonance substrate, on which a gold nano material structure layer is covered. The gold nano material forms a self-assembled gold nano particle optical resonance array embedded in a glass (BK7) substrate in the substrate. The self-assembled gold nano particles are solid spherical nano particles.

2. A bacterial biosensor according to claim 1, characterized in that: The gold nanomaterial is composed of self-assembled gold nanoparticles, the diameter of the self-assembled gold nanoparticles is 38-42nm, and the depth of embedding in the glass (BK7) substrate is 8-10nm.

3. A bacterial biosensor according to claim 1, characterized in that: The bacterial biosensor includes a three-unit structure, namely unit A, unit B, and unit C, wherein unit A is a G-bacteria detection unit, unit B is a G+bacteria detection unit, and unit C is a negative control detection unit, which can be coupled with anti-LPS (G-bacteria-specific) antibodies and anti-LTA (G+bacteria-specific) antibodies respectively.

4. The method for preparing a bacterial biosensor according to claims 1-3, characterized in that: The method comprises the following steps: using anhydrous ethanol to ultrasonically clean a glass chip covered with gold nanoparticles twice, each time for 5 minutes, and drying the glass chip to obtain the cleaned gold nanomaterial; The gold nanomaterials were rinsed with anhydrous ethanol for 10 minutes, injected with MUA solution for treatment for 15 minutes, unbound MUA on the surface of the gold nanomaterials was rinsed with anhydrous ethanol for 10 minutes, injected with EDC / NHS mixed solution for reaction for 15 minutes, and then rinsed with PBS buffer for 10 minutes to obtain pretreated gold nanomaterials; the surface pretreated gold nanomaterials were placed in antibody buffer, and the coupling reaction was carried out at room temperature for 30 minutes, and the unbound antibodies were washed away by rinsing with PBS buffer for 10 minutes, and EA / BSA blocking solution was injected to block nonspecific binding sites, and the blocking solution was washed away by rinsing with PBS buffer for 10 minutes to obtain a bacterial detection biosensor.

5. The method for preparing a bacterial biosensor according to claim 4, characterized in that: The MUA solution comprises: MUA 10 mM, and the solvent is anhydrous ethanol.

6. The method for preparing a bacterial biosensor according to claim 4, characterized in that: The EDC / NHS mixed solution consists of: EDC 0.4M, NHS 0.15M, and the solvent is water.

7. The method for preparing a bacterial biosensor according to claim 4, characterized in that: The PBS buffer composition is: NaCl 137mM, Na2HPO4 8mM, K2HPO4 1.2mM, KCl 2.7mM, the solvent is water, pH 7.

4.

8. The method for preparing a bacterial biosensor according to claim 4, characterized in that: The composition of the EA / BSA blocking solution is: EA 0.1M, BSA 2% (W / V), and the solvent is water.

9. Application of a bacterial biosensor in detecting bacterial endophthalmitis and hospital-acquired pneumonia.

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