Bacteria identification and detection method based on colorimetric / SERS (Surface Enhanced Raman Scattering) signal amplification

Abstract

The invention relates to the technical field of biology, in particular to a bacteria identification and detection method based on colorimetric / SERS (Surface Enhanced Raman Scattering) signal amplification, which comprises the following steps: S1, preparing colorimetric nano-enzyme: the colorimetric nano-enzyme comprises gold and silver core-shell nanoparticles (Au (at) Ag) and a metal organic framework, and the metal organic framework is an iron-based metal organic framework; s2, constructing a bacterium / SERS label / colorimetric nano-enzyme-based sandwich system: uniformly mixing a bacterium suspension and colorimetric nano-enzyme, wherein the surface of the bacterium is modified with 4-MPBA; and S3, detecting by using a colorimetric method. The bacterium detection method is high in accuracy and sensitivity, low in cost and simple to operate, and can be used for detecting various types of bacteria.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a bacteria identification and detection method based on colorimetry / SERS signal amplification. Background Art

[0002] In biological experiments, the identification and detection of bacteria play an important role in determining the types of bacteria, understanding their biological characteristics and ecological distribution. In traditional bacterial detection methods, such as surface enhanced Raman scattering (SERS) technology, 4-MPBA (4-mercaptophenylboronic acid) modified metal nanoparticles are often used as Raman tags (SERS tags) to detect bacteria. This method combines the advantages of directly obtaining bacterial Raman fingerprints and detecting bacteria after Raman tagging. Among them, the 4-MPBA molecule has two functional groups: a thiol group that can strongly bind to metal nanoparticles; the other is a boric acid group that can form a covalent bond with peptidoglycan or polysaccharides in the bacterial cell wall. The formation of this covalent bond is a key step in detecting bacteria. In order to allow gold nanoparticles to react quickly with bacteria, 4-MPBA (4-mercaptophenylboronic acid) modified metal nanoparticles are usually prepared first in bacterial detection, and then combined with polysaccharides on the surface of bacteria. However, when preparing 4-MPBA-AuNPs, the size and distribution of nanoparticles will vary, which will affect its binding efficiency with bacteria and detection sensitivity.

[0003] Prior art, such as the invention of patent application number 202310204351.3, a rapid detection method based on a solid-phase substrate SERS intelligent sensing platform, has constructed a method for detecting pathogenic bacteria based on an aptamer-functionalized Au@Ag NPs / PDMS substrate "sandwich" to achieve specific quantitative detection of bacteria. The specific binding of aptamers and target bacteria is used to construct a sandwich structure of "capture substrate-target-signal molecule probe". However, the sensitivity and accuracy of bacterial detection still need to be further improved. Moreover, its cost is high and the operation is complicated, which is not conducive to promotion and application. Summary of the invention

[0004] In order to solve the problems in the above-mentioned background technology, the present invention provides a bacterial identification and detection method based on colorimetry / SERS signal amplification, which has high accuracy and sensitivity, low cost, simple operation, and can be used to detect various types of bacteria.

[0005] The solution adopted by the present invention to solve the technical problem is: a method for bacterial identification and detection based on colorimetry / SERS signal amplification, the method comprising the following steps:

[0006] S1, preparing a colorimetric nanozyme: the colorimetric nanozyme comprises gold-silver core-shell nanoparticles (Au@Ag) and a metal-organic framework, wherein the metal-organic framework is an iron-based metal-organic framework;

[0007] S2, constructing a bacteria / SERS tag / colorimetric nanozyme-based sandwich system: mixing a bacterial suspension with the colorimetric nanozyme, wherein the bacterial surface is modified with 4-MPBA;

[0008] S3, detection using colorimetry.

[0009] Preferably, the colorimetric nanozyme is prepared by a preparation method comprising the following steps:

[0010] HAuC l 4 Pour it into an aqueous solution, add sodium citrate solution into the aqueous solution, stir evenly, and obtain gold nanoparticles (AuNPs). 3 The solution was added dropwise to a solution of gold nanoparticles (AuNPs) and stirred for 2 h to obtain Au@Ag; K3[Fe(CN)6] was added to Au@Ag and stirred evenly to obtain a colorimetric nanozyme.

[0011] Preferably, the bacteria / SERS tag / colorimetric nanozyme-based sandwich system is prepared by a preparation method comprising the following steps: first, the bacterial suspension is evenly mixed with 4-MPBA, reacted at 37°C for 1 hour, and then centrifuged and washed to obtain a bacteria / 4-MPBA complex; then, the colorimetric nanozyme and the bacteria / 4-MPBA complex are evenly mixed to obtain a bacteria / SERS tag / colorimetric nanozyme-based sandwich system.

[0012] Preferably, the gold-silver core-shell nanoparticles (Au@Ag) are in a cubic shape.

[0013] The present application forms a bacteria / SERS tag / colorimetric nanozyme-based sandwich system by adding colorimetric nanozymes to bacteria modified with 4-MPBA. By using this system for colorimetric experiments, workers can quickly perform cell detection and greatly improve the accuracy and sensitivity of detection.

[0014] The colorimetric nanozyme of the present application has a MOF nanostructure doped with AuAgFe tri-metals. The colorimetric nanozyme has excellent enzyme catalytic properties and excellent surface sensitive response. It does not need to rely on specific labels and can quickly combine with 4-MPBA-modified bacteria to form a bacteria / SERS label / colorimetric nanozyme-based sandwich system to achieve rapid detection, improve the detection sensitivity and detection range of bacteria, and at the same time improve the stability of the system, ensuring the reliability of the detection results of the system on the intelligent platform.

[0015] In summary, the beneficial effects of the present invention are:

[0016] The sensitivity and accuracy of bacterial detection are improved by metal nanoparticles and metal organic frameworks grown in situ on metal nanoparticles; the bacterial identification and detection method based on colorimetric / SERS signal amplification in the present application can detect Staphylococcus aureus in the range of 6-10 8 CFU / mL,

[0017] The bacteria / SERS tag / colorimetric nanozyme-based sandwich system of the present application improves the specificity of bacterial detection and can accurately distinguish and detect different types of bacteria;

[0018] The cost of the detection materials required by this application is low. The colorimetric detection system integrated with the smartphone reading sensor platform is used. The bacterial identification and detection method based on colorimetric / SERS signal amplification in this application can detect Escherichia coli in the range of 4-10 9 CFU / mL, the detection range of Staphylococcus aureus can reach 2-10 8 CFU / mL, high accuracy and sensitivity, simple operation steps, more convenient for outdoor use.

[0019] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The synthesis diagram of the colorimetric nanozyme (Au@Ag-PB NPs) of this example;

[0021] Figure 2 are transmission electron microscope (TEM) bright field images of the nanoparticles of this embodiment, wherein B is a TEM bright field image of Au nanoparticles, C is a TEM bright field image of Au@Ag nanoparticles, and D is a TEM bright field image of Au@Ag-PB NPs;

[0022] Figure 3 is the EDS image of Au@Ag-PB NPs in this embodiment, where E is the element mapping image of Au@Ag-PB NPs, and F is the energy spectrum of Au@Ag-PB NPs;

[0023] Figure 4 This is the UV-visible absorption spectrum of the colorimetric nanozyme of this example;

[0024] Figure 5A is the XPS image of Au@Ag-PB NPs in this example, B is the full spectrum of Au@Ag-PB NPs, and C is the high-resolution spectrum of the Fe element in Au@Ag-PB NPs;

[0025] Figure 6 is the X-ray diffraction (XRD) pattern of Au@Ag-PB NPs in this example;

[0026] Figure 7 is the adsorption-desorption isotherm diagram of Au@Ag-PB NPs in this example;

[0027] Figure 8 TEM images of bacteria, wherein A is a TEM image of normal Escherichia coli, B is a TEM image of Escherichia coli adsorbed by the colorimetric nanozyme of this embodiment, C is a TEM image of normal Staphylococcus aureus, and D is a TEM image of Staphylococcus aureus adsorbed by the colorimetric nanozyme of this embodiment;

[0028] Fig. 9 The UV-visible absorption spectrum of Escherichia coli obtained by the bacteria / SERS tag / colorimetric nanozyme-based sandwich system of this embodiment, wherein the inset is the corresponding colorimetric image of different Escherichia coli concentrations;

[0029] Fig.10 is the linear relationship between the absorbance at 652 nm of the bacteria / SERS tag / colorimetric nanozyme-based sandwich system of this example and the number of E. coli;

[0030] Fig.11 The UV-visible absorption spectrum of Staphylococcus aureus obtained by the bacteria / SERS tag / colorimetric nanozyme-based sandwich system of this embodiment, wherein the inset is the corresponding colorimetric image of different Staphylococcus aureus;

[0031] Fig.12 is the linear relationship between the bacteria / SERS tag / colorimetric nanozyme-based sandwich system of this example at 652 nm absorbance and the number of Staphylococcus aureus;

[0032] Fig.13 This is a flow chart of the bacteria / SERS tag / colorimetric nanozyme-based sandwich system of this embodiment, which integrates saturation analysis in a smartphone for bacteria detection;

[0033] Fig.14 Colorimetric images showing the gradual increase in the number of bacteria of Escherichia coli (E.co li) and Staphylococcus aureus (S.aureus);

[0034] Fig.15 The linear relationship between the colorimetric image saturation analysis and different Escherichia coli (E. co li) concentrations;

[0035] Fig.16 Linear relationship between colorimetric image saturation analysis and different Staphylococcus aureus (S. aureus) concentrations;

[0036] Fig.17 This is the pore size distribution diagram of Au@Ag-PB NPs in this example. DETAILED DESCRIPTION

[0037] The present application is a method for bacterial identification and detection based on colorimetric / SERS signal amplification, the method comprising the following steps:

[0038] S1, preparing a colorimetric nanozyme: the colorimetric nanozyme comprises gold-silver core-shell nanoparticles (Au@Ag) and a metal-organic framework, wherein the metal-organic framework is an iron-based metal-organic framework;

[0039] S2, constructing a bacteria / SERS tag / colorimetric nanozyme-based sandwich system: mixing a bacterial suspension with the colorimetric nanozyme, wherein the bacterial surface is modified with 4-MPBA;

[0040] S3, detection using colorimetry.

[0041] Preferably, the metal nanoparticles are in a cubic shape.

[0042] This application improves the sensitivity and accuracy of bacterial detection by developing a multifunctional colorimetric nanozyme, with low detection cost, simple operation steps, and rapid detection. At the same time, the technical solution of this application can also detect different types of bacteria, and does not rely on specific nanomaterials or labels, which improves the specificity of bacterial detection and can accurately distinguish and detect different types of bacteria.

[0043] In addition, the detection method of the present invention can be applied not only to the detection of bacteria, but also to the detection of other microorganisms. Compared with the existing technology, it has a wider application range, can meet more detection needs, and has broad application prospects.

[0044] In order to make the content of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the accompanying drawings.

[0045] Example

[0046] S1, preparation of multifunctional colorimetric nanozymes:

[0047] Add 250uL of 0.1M HAuCl 4 Pour into 100 mL of aqueous solution, add 1.5 mL of 1% sodium citrate solution into the aqueous solution, stir evenly, and obtain gold nanoparticles (AuNPs) with a particle size of 20 nm. Subsequently, add 6.5 mL of 1 mM AgNO3 The solution was added dropwise to the solution containing the above-mentioned gold nanoparticles (AuNPs) and stirred for 2 h to obtain Au@Ag;

[0048] Add 1 ml of K3[Fe(CN)6] to Au@Ag and stir evenly to grow Fe-based MOF in situ to obtain colorimetric nanozymes (Au@Ag-PB NPs).

[0049] S2, construction of bacteria / SERS tag / colorimetric nanozyme-based sandwich system:

[0050] First, 0.5 mL of bacterial suspension was mixed evenly with 0.5 mL of 4-MPBA, reacted at 37°C for 1 h, and then centrifuged and washed with PBS at pH 7.4 to remove unbound 4-MPBA and bacteria to obtain bacteria / 4-MPBA complex;

[0051] Subsequently, 0.5 mL of Au@Ag-PB NPs was evenly mixed with 0.5 mL of bacteria / 4-MPBA complex to obtain the bacteria / SERS tag / colorimetric nanozyme-based sandwich system.

[0052] S3, detection using colorimetry.

[0053] Colorimetric detection of bacteria

[0054] 50 μL of 2.0 mM TMB substrate and 50 μL of 250 mM H2O were added to the bacteria / SERS tag / colorimetric nanozyme-based sandwich system containing different bacterial concentrations. 2 O 2 , the signal is amplified through peroxidase-catalyzed reaction, and then the colorimetric image of the reaction solution is recorded to perform quantitative colorimetric detection of the solution.

[0055] The colorimetric detection results are transmitted to an integrated intelligent platform, and the built-in saturation calculation algorithm of the intelligent platform is used to analyze and obtain the bacterial detection results.

[0056] Figure 2 FIG. 1 is a transmission electron microscope (TEM) bright field image of the nanoparticles of this embodiment. Figure 2 In B, it can be seen that the gold nanoparticles (AuNPs) are spherical in structure, and their average particle size is 10nm; Figure 2 It can be seen from C that Au@Ag is a spherical structure with an average particle size of 20nm. Figure 2 As can be seen in D, the colorimetric nanozyme (Au@Ag-PB NPs) has a cubic structure and its average particle size is 100 nm.

[0057] from Figure 3It can be observed in Figure E that Au, Ag, Fe, C, and N are distributed throughout the nanosheet, with the Ag and Fe components mainly overlapping on the surface of the sheet, while the Au component is mainly confined to the core, forming an AuAgFe tri-metal doped PB MOF nanosheet. Figure 3 The analysis in Figure F showed that the ratio of Au / Ag / Fe in the Au@Ag-PB NPs was 1:7:96, indicating a high Fe loading and a moderate Ag doping level.

[0058] Figure 4 The UV-visible absorption spectra of the colorimetric nanozymes of Au@Ag, Au@Ag-PB1, Au@Ag-PB2, Au@Ag-PB3, Au@Ag-PB4, Au@Ag-PB5 and Au@Ag-PB6, where the concentrations of K added in step S1 of Au@Ag, Au@Ag-PB1, Au@Ag-PB2, Au@Ag-PB3, Au@Ag-PB4, Au@Ag-PB5 and Au@Ag-PB6 were 0, 0.6, 1.0, 1.4, 1.8, 2.2 and 2.6 mM, respectively. 3 [Fe(CN)6] prepared colorimetric nanozyme, in addition Figure 4 In the upper left corner of the figure, the colorimetric images of the colorimetric nanozymes corresponding to Au@Ag, Au@Ag-PB1, Au@Ag-PB2, Au@Ag-PB3, Au@Ag-PB4, Au@Ag-PB5 and Au@Ag-PB6 are inserted. It can be analyzed from the image that as K 3 [Fe(CN) 6 ] concentration increased, the absorbance of the colorimetric nanozyme at 420nm decreased significantly, accompanied by a red shift of the peak signal, confirming that the Ag shell in Au@Ag was etched. The results showed that Au@Ag-PB NPs have adjustable local surface plasmon resonance (LSPR) properties, which can quickly interact with specific molecules on the surface of bacteria and generate detectable signals in a shorter time to achieve specific recognition. There is no need for additional labeling of bacteria, which simplifies the detection process and makes the detection faster and more direct.

[0059] Figure 5 B shows that Au@Ag-PB NPs contain Au, Cl, O, Ag, and Fe elements. Figure 5 C indicates the presence of Fe in Au@Ag-PB NPs 3+ and Fe 2+ .

[0060] Figure 6 The heterogeneous structure in Au@Ag-PB NPs was further confirmed.

[0061] Figure 7The nitrogen adsorption-desorption experiment proves that Au@Ag-PB NPs are typical mesoporous materials. The width of their mesopore size distribution is very consistent with the MOF structure. From the data in the figure, it can be calculated that the Brunauer-Emmett-Teller specific surface area of ​​Au@Ag-PB NPs is 1532 m 2 / g.

[0062] Figure 8 Gram-negative Escherichia coli (E.co li) and Gram-positive Staphylococcus aureus (S.aureus) were selected as analytes. After TEM image analysis of normal Gram-negative Escherichia coli (E.co li) and normal Gram-positive Staphylococcus aureus (S.aureus), 4-MPBA was added to Escherichia coli and Staphylococcus aureus, respectively, and the reaction was carried out according to step S2 to obtain a bacteria / 4-MPBA complex. Among them, the 4-MPBA molecules on the bacteria / 4-MPBA complex serve as bifunctional molecules, serving as both Raman tags and bacterial recognition units. Nanocolorimetric enzymes were then added to capture the target bacteria, respectively, and a bacteria / SERS tag / colorimetric nanozyme-based sandwich system was established. TEM image analysis of Escherichia coli (E.co li) and Staphylococcus aureus (S.aureus) based on the bacteria / SERS tag / colorimetric nanozyme-based sandwich system was performed. By Figure 8 A and Figure 8 The comparison of B clearly confirms that the colorimetric nanozymes aggregate on the surface of E. coli; Figure 8 C and Figure 8 The comparison of D clearly confirmed the aggregation of colorimetric nanozymes on the surface of Staphylococcus aureus, verifying the successful capture of bacteria by the bacteria / SERS tag / colorimetric nanozyme-based sandwich system.

[0063] These results indicate that the detection based on the bacteria / SERS tag / colorimetric nanozyme-based sandwich system can quantitatively detect the bacterial concentration with high detection sensitivity.

[0064] Colorimetric detection of bacteria based on bacteria / SERS tag / colorimetric nanozyme-based sandwich system, e.g. Fig. 9 The values ​​are 0, 10, and 10 respectively. 3 , 10 5 , 10 7 and 10 9 UV-visible absorption spectra of Escherichia coli at CFU / mL detection concentrations, with the illustration in the upper right corner showing 10, 10 3 , 10 5 , 10 7 and 10 9 Colorimetric image of CFU / mL of E. coli; Fig.10Under the light of 652nm wavelength, the absorbance is 0, 10, 10 3 , 10 5 , 10 7 and 10 9 The linear relationship between CFU / mL concentration of Escherichia coli. Note that the Escherichia coli used in the above UV-visible absorption spectrum detection, colorimetric image, and linear relationship detection are all detected based on the bacteria / SERS tag / colorimetric nanozyme-based sandwich system.

[0065] pass Fig. 9 It can be observed that the absorbance at 652 nm increases with the increase of E. coli concentration. Fig.10 It can be seen that 10-10 9 The linear relationship between absorbance and the number of E. coli was obtained within the range of CFU / mL, and the corresponding calibration curve was fitted as y=0.0939x+0.00635, R 2 =0.997.

[0066] Colorimetric detection of bacteria based on bacteria / SERS tag / colorimetric nanozyme-based sandwich system, e.g. Fig.11 The values ​​are 0, 10, and 10 respectively. 2 , 10 4 , 10 6 and 10 8 UV-visible absorption spectra of Staphylococcus aureus at CFU / mL test concentrations, with the illustration in the upper right corner showing 10, 10 2 , 10 4 , 10 6 and 10 8 Colorimetric image of Staphylococcus aureus at the CFU / mL test concentration; Fig.12 Under the light of 652nm wavelength, the absorbance and concentration are 0, 10, 10 2 , 10 4 , 10 6 and 10 8 Note that the Staphylococcus aureus used in the above UV-visible absorption spectrum detection, colorimetric image, and linear relationship detection are all detected based on the bacteria / SERS tag / colorimetric nanozyme-based sandwich system.

[0067] pass Fig.11 It can be observed that the absorbance at 652 nm increases with the increase of Staphylococcus aureus concentration. Fig.12 It can be seen that 10-10 8 The linear relationship between absorbance and the number of Staphylococcus aureus was obtained in the range of CFU / mL, and the corresponding calibration curve was fitted as y=0.137x-0.127, R2 =0.996. Based on 3σ / S, the detection limit (LOD) of bacteria was calculated to be 6 CFU / mL.

[0068] The above results indicate the unique advantages of the bacteria / SERS tag / colorimetric nanozyme-based sandwich system, which has strong nanozyme activity and excellent detection sensitivity of the colorimetric method because the formation of the MOF-encapsulated nanolayer significantly enhances the reduction of SERS activity.

[0069] The bacteria / SERS tag / colorimetric nanozyme-based sandwich system was used for quantitative detection of bacteria using a colorimetric method that was integrated with a smartphone readout sensing platform for ultrasensitive detection of bacteria.

[0070] like Fig.13 As shown, the bacteria / SERS tag / colorimetric nanozyme-based sandwich system is used to integrate saturation analysis in a smartphone for the detection of Escherichia coli and Staphylococcus aureus. The detection concentrations of Escherichia coli in the figure are 10 and 10, respectively. 3 , 10 5 , 10 7 and 10 9 The detection concentrations of Staphylococcus aureus were 10 and 10 2 , 10 4 , 10 6 and 10 8 ,Depend on Fig.14 It can be observed that the color change becomes more obvious as the bacterial concentration increases.

[0071] Fig.15 The bacterial concentrations of E. coli were 10 and 10 3 , 10 5 , 10 7 , 10 9 Corresponding to the detected colorimetric image, through analysis, it was found that there was a good linear relationship between the saturation value and the logarithm of the Escherichia coli concentration, which was fitted to y = 0.0971x-0.0407 (R2 = 0.995), and the detection limit was calculated to be 4 CFU / mL based on 3σ / S.

[0072] Fig.16 The bacterial concentrations of Staphylococcus aureus were shown to be 10, 10 2 , 10 4 , 10 6 , 10 8 Corresponding to the detected colorimetric image, through analysis, it was found that there was a good linear relationship between the saturation value and the logarithm of the Staphylococcus aureus concentration, which was fitted as y=0.0485x-0.0287 (R 2=0.996), and the detection limit was calculated to be 2 CFU / mL based on 3σ / S.

[0073] Combined with a smartphone-readout sensing platform, this colorimetric method based on the bacteria / SERS tag / colorimetric nanozyme-based sandwich system showed ultrasensitive detection and enhanced portability.

[0074] The embodiments described above are only preferred implementation modes of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention shall fall within the protection scope of the present invention.

Claims

1. A method for bacterial identification and detection based on colorimetry / SERS signal amplification, characterized in that: The method comprises the following steps: S1, preparing a colorimetric nanozyme: the colorimetric nanozyme comprises gold-silver core-shell nanoparticles (Au@Ag) and metal-organic frameworks (MOFs), wherein the metal-organic framework is an iron-based metal-organic framework; S2, constructing a bacteria / SERS tag / colorimetric nanozyme-based sandwich system: mixing a bacterial suspension with the colorimetric nanozyme, wherein the bacterial surface is modified with 4-MPBA; S3, detection using colorimetry.

2. A method for bacterial identification and detection based on colorimetry / SERS signal amplification according to claim 1, characterized in that: The colorimetric nanozyme is prepared by a preparation method comprising the following steps: HAuCl4 was poured into an aqueous solution, and then sodium citrate solution was added to the aqueous solution and stirred evenly to obtain gold nanoparticles (AuNPs). Subsequently, AgNO3 solution was added dropwise to the solution of gold nanoparticles (AuNPs) and stirred for 2 h to obtain Au@Ag. Add K3[Fe(CN)6] to Au@Ag and stir evenly to obtain colorimetric nanozyme.

3. A method for bacterial identification and detection based on colorimetry / SERS signal amplification according to claim 2, characterized in that: The bacteria / SERS tag / colorimetric nanozyme-based sandwich system is prepared by a preparation method comprising the following steps: First, the bacterial suspension was mixed evenly with 4-MPBA, reacted at 37°C for 1 h, and then centrifuged and washed to obtain a bacteria / 4-MPBA complex; Subsequently, the colorimetric nanozyme was evenly mixed with the bacteria / 4-MPBA complex to obtain a bacteria / SERS tag / colorimetric nanozyme-based sandwich system.

4. The method for bacterial identification and detection based on colorimetry / SERS signal amplification according to claim 1, characterized in that: The gold-silver core-shell nanoparticles (Au@Ag) are in a cubic shape.

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