CN115011563A - Preparation method of aminated gold nanoparticle-phage T156 recognition probe and kit thereof - Google Patents
Preparation method of aminated gold nanoparticle-phage T156 recognition probe and kit thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of an aminated gold nanoparticle-phage T156 recognition probe and a kit thereof; the method comprises the steps of reducing chloroauric acid modified by mercaptoethylamine by using sodium borohydride to synthesize aminated gold nanoparticles; the aminated gold nanoparticles and the salmonella typhimurium bacteriophage T156 are coupled by amido bonds to form a stable recognition probe. The kit takes the identification probe as a core, induces the gold nanoparticles coupled with the salmonella to gather while specifically identifying and adsorbing the salmonella, so as to cause the characteristic of weakening or enhancing the intensity of absorption peaks at different wavelengths in an ultraviolet absorption spectrum, and further indirectly obtain the content of the salmonella in a sample to be detected through the change of the ultraviolet absorption spectrum. The kit has the characteristics of simplicity, convenience, low detection limit, high specificity, high detection speed and capability of being distinguished by naked eyes. The kit is applied to the detection of crayfish, and provides a theoretical basis for the rapid detection of salmonella pollution in food.
Description
Technical Field
The invention relates to the field of food safety detection, in particular to a preparation method of an aminated gold nanoparticle-phage T156 recognition probe and a kit thereof.
Background
Salmonella (Salmonella), a common food-borne pathogenic bacterium, is one of the most prominent pathogenic bacteria causing food-borne diseases worldwide. To ensure food safety and to maintain human health, early detection of salmonella is essential to minimize the risk of bacterial disease. The existing detection method has more or less defects, such as tedious workload, time consumption and labor consumption in the traditional culture method; immunological-based assays require high affinity antibodies; molecular biology-based assays are prone to false positive results; the detection technology based on the biosensor has high cost and insufficient stability, and cannot be used in a large range.
The use of bacteriophages as effective biorecognition elements has numerous advantages. The detection technology developed by the phage is continuously concerned at present, and the phage has the advantages of high specificity, stability, sensitivity, selectivity, rapidity, accessibility, cost effectiveness and the like, so that the phage can be used as an ideal biological recognition element. Meanwhile, a plurality of nanotechnologies are applied to an immunoassay method, and the nanotechnologies are widely applied to the detection fields of clinical medicine, food safety, environmental sanitation and the like, particularly the excellent optical characteristics and good biocompatibility of the gold nanoparticles, and provide a new thought for establishing an efficient detection method. Compared with various traditional detection methods, colorimetric detection utilizing the special optical properties of gold nanoparticles is very attractive due to its convenience, simplicity and high cost-effectiveness. In addition, the colorimetric method can be monitored by naked eyes without depending on any complicated instrument and equipment, so that the detection method is suitable for rapid detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of an aminated gold nanoparticle-phage T156 recognition probe and a kit thereof. The kit utilizes the specific recognition capability of the salmonella typhimurium bacteriophage T156 on salmonella and the unique optical characteristics of the gold nanoparticles to visually detect the salmonella, and the method has the characteristics of short detection time, simple detection, low cost, strong specificity and visual detection.
In order to achieve the purpose, the invention designs a preparation method of an aminated gold nanoparticle-phage T156 recognition probe, which comprises the following steps:
s1: reducing chloroauric acid modified by mercaptoethylamine by using sodium borohydride to synthesize aminated gold nanoparticles (Cys-AuNPs);
s2: the aminated gold nanoparticles and the salmonella typhimurium bacteriophage T156 are coupled by amide bonds to form a stable aminated gold nanoparticle-bacteriophage T156 recognition probe (T156@ Cys-AuNPs).
The above salmonella bacteriophage T156 has been sent to the chinese type culture collection under the classification designation: salmonella Typhimurium bacteriophage (Salmonella Typhimurium bacteriophage) T156, which is preserved in China center for type culture Collection 7/6 of 2020 at the preservation address of Wuhan university, Wuhan City, Hubei province with the preservation number: CCTCC NO: m2020288. Wherein the Salmonella typhimurium bacteriophage is disclosed in the Chinese patent with the application number of 202010712852.9 and the invention name of Salmonella typhimurium bacteriophage T156 and the application thereof.
Further, in the step S1, the aminated gold nanoparticles (Cys-AuNPs) are synthesized by the following steps:
1) weighing 6-8 parts of mercaptoethylamine (Cys), 20-22 parts of chloroauric acid (HAuCl4) and 2-4 parts of sodium borohydride (NaBH4) according to parts by weight;
2) dissolving sodium borohydride (NaBH4) in water to obtain a sodium borohydride (NaBH4) solution
3) Mixing mercaptoethylamine (Cys) and chloroauric acid (HAuCl4), and magnetically stirring at room temperature and in dark to obtain a mixture;
4) adding the prepared sodium borohydride (NaBH4) solution into the mixture, continuously stirring, reducing chloroauric acid through the sodium borohydride (NaBH4), stabilizing the formed gold particles, and finally forming a clear and transparent wine red solution, namely the solution containing the aminated gold nanoparticles (Cys-AuNPs).
Further, the parts by weight of mercaptoethylamine (Cys), chloroauric acid (HAuCl4) and sodium borohydride (NaBH4) are respectively as follows: 6.57 parts, 19.03 parts and 3.78 parts.
Still further, in the solution containing the aminated gold nanoparticles (Cys-AuNPs), the particle size of the aminated gold nanoparticles (Cys-AuNPs) is 25-27 nm, and the potential is 38.6 mV.
Still further, in the step S2, the aminated gold nanoparticle-phage T156 recognition probe (T156@ Cys-AuNPs) is formed by coupling the following steps:
1) performing activated culture on the bacteriophage T156, performing centrifugal resuspension, and filtering by using a microporous filter membrane to obtain a high-titer bacteriophage stock solution; wherein the content of the bacteriophage T156 in the bacteriophage stock solution is 1010 PFU/mL.
2) Preparing a tert-butyl carbamate (BOC) solution by taking Tetrahydrofuran (THF) as a solvent; wherein the concentration of the tert-butyl carbamate (BOC) solution is 1-3 mM;
3) adding a tert-butyl carbamate (BOC) solution into a phage stock solution according to a volume ratio of 1:100 for reaction (in order to avoid the phenomenon that phage is self-coupled under subsequent coupling conditions); obtaining a reaction bacterial liquid;
4) simultaneously adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy thiosuccinimide sodium salt (NHS) into the reaction bacterial liquid according to the volume ratio of 1:1:100 to activate (activate-COOH group of bacteriophage T156) to obtain activated bacterial liquid; wherein the concentration of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) is 2-4 mM and the concentration of N-hydroxythiosuccinimide sodium salt (NHS) is 2-4 mM.
5) And (2) mixing the phage stock solution with activated carboxyl and a solution containing aminated gold nanoparticles (Cys-AuNPs) at a ratio of 1: 300-100, coupling overnight (stable covalent bond connection of amide bonds is formed between the phage and the aminated gold nanoparticles), finally adding trifluoroacetic acid to remove tert-butyl carbamate (BOC), and centrifuging to obtain the aminated gold nanoparticles-phage T156 identification probe (T156@ Cys-AuNPs).
Still further, the volume of the phage stock solution activated with carboxyl groups and the solution containing aminated gold nanoparticles was 1: 150.
Still further, the particle size of the aminated gold nanoparticle-phage T156 recognition probe is 190-210 nm, and the coupling amount of the phage is 0.78 multiplied by 10 8 ～0.95×10 8 PFU/mL, coupling ratio 65% -75%.
The invention also provides a kit for detecting salmonella ATCC 13311, which comprises the aminated gold nanoparticle-phage T156 recognition probe prepared by the method.
The colorimetric method for rapidly detecting the salmonella typhimurium ATCC 13311 by using the kit comprises the following steps:
1) salmonella typhimurium ATCC 13311 was activated and then treated at 0 CFU/mL-1.2X 10 6 Preparing Salmonella typhimurium ATCC 13311 solutions with different concentrations in the CFU/mL range;
2) respectively adding the aminated gold nanoparticles and phage T156 recognition probes (T156@ Cys-AuNPs) into Salmonella typhimurium ATCC 13311 solutions with different concentrations, mixing and incubating for 25-35 min, placing the mixture into an ultraviolet spectrophotometer for detection, respectively recording light absorption values at 526nm and 680nm, and analyzing the light absorption A 680 /A 526 The ratio is changed, and the concentration of the bacteria liquid is used as the abscissa and the absorbance A is established 680 /A 526 The ratio is a standard curve of a vertical coordinate;
3) mixing a sample to be detected with an aminated gold nanoparticle-phage T156 recognition probe (T156@ Cys-AuNPs), detecting and recording absorbance values at 526nm and 680nm, and analyzing the absorbance A 660 /A 520 And obtaining the content of the salmonella typhimurium ATCC 13311 in the sample to be detected according to the standard curve.
The invention also provides application of the kit in detection of artificially polluted crayfishes.
The principle of the invention is as follows:
a colorimetric method for detecting salmonella by using a kit based on an aminated gold nanoparticle-bacteriophage T156(T156@ Cys-AuNPs) identification probe comprises the steps of firstly modifying gold nanoparticles by mercaptoethylamine, preparing aminated gold nanoparticles under the reduction action of sodium borohydride, activating carboxyl on bacteriophage, and carrying out stable amido bond covalent bonding with amino groups of the aminated gold nanoparticles to form the aminated gold nanoparticle-bacteriophage T156 identification probe (T156@ Cys-AuNPs) serving as a biological identification probe, and simultaneously adding BOC to prevent the bacteriophage from undergoing a self-coupling phenomenon. The biological recognition element (T156@ Cys-AuNPs) is used for visually detecting the salmonella. Phage T156 in the T156@ Cys-AuNPs biological recognition probe can specifically recognize and adsorb to the surface of host bacteria, so that a large amount of T156@ Cys-AuNPs are gathered on the surface of the host bacteria. Due to the optical characteristics of the gold nanoparticles, namely the color change caused by a large amount of aggregation, the change of a solution from red to purple can be observed by naked eyes, and the phenomenon that the highest ultraviolet absorption peak is reduced and red shift occurs can be found by representing an ultraviolet absorption spectrogram through an ultraviolet spectrophotometer. The more the bacteria to be detected, the larger the aggregation degree, the more obvious the color change of the gold nanoparticle solution, and the larger the change degree of the ultraviolet absorption spectrogram. The qualitative and quantitative detection of the salmonella can be rapidly carried out through the change of the colorimetric color and the ultraviolet absorption peak of the naked eye.
The invention has the beneficial effects that:
1. the aminated gold nanoparticles (Cys-AuNPs) are synthesized by reducing chloroauric acid modified by mercaptoethylamine through sodium borohydride, the synthesized Cys-AuNPs are transparent wine red solution and uniform in size and are dispersed, amino groups are modified on the surfaces of the gold nanoparticles, functional modification of the gold nanoparticles is facilitated, the particle size is 25-27 nm, and the potential is 38.6 mV.
2. The amino group modified on the aminated gold nanoparticle and the carboxyl group on the salmonella typhimurium bacteriophage T156 are utilized, amide bond coupling is carried out between the amino group modified on the aminated gold nanoparticle and the carboxyl group on the bacteriophage T156 through activating the carboxyl group on the bacteriophage, the addition amount of the bacteriophage T156 is optimized simultaneously to form a stable aminated gold nanoparticle-bacteriophage T156 identification probe (T156@ Cys-AuNPs), the prepared T156@ Cys-AuNPs are dispersed and stable, the particle size is 190-210 nm, and the particle size is 0.78 multiplied by 10 8 ～0.95×10 8 PFU/mL, coupling ratio of about 65% -75%.
3. A simple, rapid and sensitive colorimetric method for detecting salmonella by using a kit based on an aminated gold nanoparticle-phage T156 recognition probe is established on the basis of T156@ Cys-AuNPs, the detection time of the detection method is about 40min, compared with the traditional culture method of 5-7d, the detection time is greatly shortened, the detection efficiency is effectively improved, the detection limit is 12CFU/mL, and the linear range is 1.2 multiplied by 10 1 CFU/mL～1.2×10 6 CFU/mL, detection specificity of 100%.
Drawings
FIG. 1 shows the transmission electron microscope patterns (A) and (B) of bacteriophage T156
FIG. 2 is the UV absorption spectrum of Cys-AuNPs
FIG. 3 is a graph showing the particle size distribution of Cys-AuNPs
FIG. 4 shows Zeta potential of Cys-AuNPs
FIG. 5 shows the transmission electron microscope patterns (A) and (B) of Cys-AuNPs
FIG. 6 shows the particle size distribution of the T156@ Cys-AuNPs recognition probe
FIG. 7 is a graph showing the activity change of Salmonella in aminated gold nanoparticles/PBS at different times
FIG. 8 is a graph showing UV absorption spectra after coupling various amounts of phage
FIG. 9 shows A after coupling of various amounts of phage 680 /A 526 Variations in
FIG. 10 shows the change in the amount of coupling of phage
FIG. 11 shows the change in coupling ratio of phage
FIG. 12 shows the preparation process and detection principle of T156@ Cys-AuNPs
FIG. 13 is a graph of the UV absorption spectra of T156@ Cys-AuNPs incubated with Salmonella for different periods of time
FIG. 14 is A of T156@ Cys-AuNPs incubated with Salmonella for various periods of time 680 /A 526 Variations in
FIG. 15 is an ultraviolet absorption spectrum of a colorimetry for detecting Salmonella of different concentrations
FIG. 16 is a standard curve of a colorimetric method
FIG. 17 shows TEM images of T156@ Cys-AuNPs adsorbed on the surface of Salmonella (A) and (B)
FIG. 18 is a graph of UV absorption spectra for detection of different bacteria
FIG. 19 shows A for detection of different bacteria 680 /A 520 Comparison of
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Strains and numbers: salmonella Typhimurium (Salmonella Typhimurium ATCC 13311, ATCC 14028, ATCC 13076), Listeria monocytogenes (Listeria monocytogenes ATCC 19114), Vibrio parahaemolyticus (Vibrio parahaemolyticus ATCC 33846), Staphylococcus aureus (Staphylococcus aureus ATCC 25923) were derived from the American ATCC biological Standard resource center (ATCC). Escherichia coli (Escherichia coli NCTC 12900) was derived from the national Standard cell Bank (NCTC).
Example 1 characterization of bacteriophage T156
1. Multiplication of phage titer
Performing phage solid proliferation by adopting a double-layer plate method, continuously diluting a phage stock solution by 10 times of gradient, uniformly mixing 100 mu L of diluent with proper gradient (ensuring that plaques grow over the surface of the whole culture medium), 100 mu L of host bacterium solution and 3.8mL of semisolid culture medium (45-55 ℃), pouring the mixture into a plate containing LA solid culture medium, and putting the plate into a constant-temperature incubator at 37 ℃ for inverted culture for 8-12 h after solidification; gently scraping three upper-layer culture media by using a sterilized medical cotton stick, adding 20mL of LB culture medium and 200 mu L of freshly activated host bacterium suspension, and carrying out shake culture in a constant-temperature incubator at 37 ℃ and 160r/min for 3-5 h; centrifuging the proliferated culture solution at 4 deg.C and 8500r/min for 15min, sucking supernatant, filtering with 0.22 μm filter membrane to remove host bacteria to obtain bacteriophage stock solution, and placing in 4 deg.C refrigerator for use.
The titer of the phage is determined by a double-layer plate method, the phage stock solution is continuously diluted by 10 times of gradient, 100 mu L of diluent with proper gradient, 100 mu L of host bacterium solution and 3.8mL of semisolid culture medium (45-55 ℃) are uniformly mixed and poured into a plate containing LA solid culture medium, and the plate is placed into a constant temperature incubator at 37 ℃ for inverted culture for 8-12 h after solidification. The titer of the phage was calculated by the number of plaques. Phage titer (PFU/mL) equals plaque number × dilution multiple × 10. The titer of the phage can reach 10 after being measured 10 PFU/mL。
2. Characterization of phage Transmission Electron microscopy
The morphology of the phage can be clearly observed by a transmission electron microscope and classified and identified. Before the observation of the phage by a transmission electron microscope, the phage is dyed by a phosphotungstic acid negative dyeing method.
The method comprises the following specific steps: the phage suspension was ultracentrifuged at 40000r/min at 4 ℃ for 1h, and then the phage precipitated at the bottom of the centrifuge tube was resuspended with 0.1mol/L ammonium acetate. Sucking 20 mu L of heavy suspension liquid to be dripped on a sealing film, taking a copper net, placing the copper net in phage suspension liquid, soaking for 10min, sucking excess liquid by using filter paper, dyeing the copper net by using phosphotungstic acid dye for 10min, sucking excess liquid at the edge of the copper net by using the filter paper, naturally airing until the copper net is completely dried, observing the shape of the phage by using a transmission electron microscope, measuring the size of the prepared copper net by using software Digital Micrograph Demo 3.9.1, and taking a picture for recording.
The appearance of the phage was observed by transmission electron microscopy as shown in FIG. 1, in which phage T156 had an icosahedral head with a longer tail that was curved and non-shrinkable. The size of the sample is measured: the diameter of the head part is 57.0 +/-1.0 nm, the length of the tail part is 150.0 +/-3.0 nm, and the diameter of the tail part is 9.50 +/-0.20 nm. Phage T156 belongs to the Long-tailed phage family (Siphonviridae) according to the standard for morphological classification of phages.
Example 2 preparation of gold nanoparticles
1. Cleaning of glassware
In order to avoid the impure prepared gold nanoparticles, all glassware required by the experiment needs to be cleaned by a detergent before the experiment begins, and then the glassware is put into an oven for drying. Then soaking the mixture for more than 72 hours by using a freshly prepared chromic acid washing solution, taking out the mixture, washing the mixture for three times by using ultrapure water, and putting the mixture into an oven for drying for use.
2. Preparation of gold nanoparticles
1) Weighing 6.57mg of mercaptoethylamine (Cys) and 19.03mg of chloroauric acid (HAuCl) according to parts by weight 4 ) 3.78mg of sodium borohydride (NaBH) 4 )；
2) Reacting sodium borohydride (NaBH) 4 ) Dissolving in water to obtain sodium borohydride (NaBH) 4 ) Solutions of
3) Reacting mercaptoethylamine (Cys) with chloroauric acid (HAuCl) 4 ) Mixing, and stirring with a magnetic stirrer at room temperature in dark environment for 20min to obtain a mixture;
4) to the mixture was added the sodium borohydride (NaBH) prepared above 4 ) The solution was stirred further through sodium borohydride (NaBH) 4 ) Reducing chloroauric acid to stabilize the formed gold particles, finally forming a clear and transparent wine red solution, namely a solution containing the aminated gold nanoparticles (Cys-AuNPs), and storing the solution at 4 ℃ in a dark environment for later use.
Example 3 Cys-AuNPs Effect on Salmonella Activity assay
Because the activity influence of gold nanoparticles on salmonella can directly influence the detection result, in order to verify the activity influence, fresh activated salmonella ATCC 13311 is added into the prepared aminated gold nanoparticle (Cys-AuNPs) solution, the aminated gold nanoparticle solution is placed into a constant-temperature shaking table at 37 ℃ and 160r/min for incubation for different time (5-50 min), the activity change of salmonella is determined by a dilution coating plate method, and meanwhile, a PBS solution is used as a blank control group.
The results are shown in fig. 7, by comparing the activities of salmonella in the gold nanoparticle solution and PBS, the activity of host bacteria is substantially not greatly changed from 5min to 50min, so that the environment of the gold nanoparticle solution has no great influence on the activity of the host bacteria, and the detection result is not greatly influenced.
EXAMPLE 4 optimization of Synthesis conditions for T156(T156@ Cys-AuNPs)
1. Propagation and pretreatment of phages
In order to improve the stable binding effect of the phage and the gold nanoparticles, the titer of the phage stock solution needs to be increased, meanwhile, the LB culture solution is removed, and sterile water is used as a resuspension of the phage stock solution. Therefore, the solid proliferated phage stock solution is ultracentrifuged at 4 ℃ and 40000r/min for 1h, the phage are precipitated at the bottom of a centrifuge tube after centrifugation, resuspended by 1mL of sterile water, filtered by a 0.22 μm microporous membrane to obtain a high-titer phage stock solution (the titer of the phage can be determined by a double-layer plate method), and the phage stock solution is placed in a refrigerator at 4 ℃ for standby.
2. Optimization of phage addition
A2 mM solution of tert-butyl carbamate (BOC) in THF was added to the stock solution of phage T156 (10) 10 PFU/mL) in order to avoid the phenomenon of phage self-coupling in subsequent coupling conditions. 3mM EDC and 3mM NHS were used to activate the-COOH group of phage T156, and different amounts (1-8. mu.L) of activated phage liquid (10. mu.L) were added 10 PFU/mL) to freshly prepared 300 μ L of aminated gold nanoparticle solution, coupled overnight at 4 ℃ in the dark, and finally stripped of BOC with trifluoroacetic acid, and the by-products were separated by centrifugation at 12000r/min for 15min at 4 ℃. And (3) characterizing an ultraviolet absorption spectrogram by using an ultraviolet spectrophotometer, and selecting and preparing the most stable T156@ Cys-AuNPs recognition probe according to the color change of the solution and the ultraviolet absorption spectrum. And simultaneously measuring the coupling amount and coupling success rate of the phage.
As shown in FIGS. 8, 9, 10 and 11, the coupling activity of phage increased with the addition of phage, but decreased when the amount of phage was increasedBecause excessive phage causes the instability and aggregation of the aminated gold nanoparticles, the coupling success rate of the phage also shows the trend of increasing first and then decreasing; when the phage amount is 4 mu L, the coupling activity and coupling success rate of the phage are the highest and are respectively 0.937 multiplied by 10 8 PFU/mL and 70.21%. According to analysis of the color, the ultraviolet absorption spectrogram, the phage coupling amount and the coupling rate of the prepared T156@ Cys-AuNPs, when 4 mu L of phage T156 is added, the phage is coupled on the gold nanoparticles most and the coupling rate is highest, the prepared identification probe is most stable; namely, the volume ratio of the phage stock solution to the aminated gold nanoparticles is 1: 75.
the coupling synthesis method of the aminated gold nanoparticle-phage T156 recognition probe (T156@ Cys-AuNPs) obtained by optimization is as follows:
1) performing activated culture on the bacteriophage T156, performing centrifugal resuspension, and filtering by using a microporous filter membrane to obtain a high-titer bacteriophage stock solution; wherein the content of bacteriophage T156 in the bacteriophage stock solution is 10 10 PFU/mL。
2) Preparing a tert-butyl carbamate (BOC) solution by taking Tetrahydrofuran (THF) as a solvent; wherein the concentration of the tert-butyl carbamate (BOC) solution is 1-3 mM;
3) according to the volume ratio of 1:100, adding a tert-butyl carbamate (BOC) solution into a phage stock solution for reaction; obtaining a reaction bacterial liquid;
4) according to the volume ratio of 1:1:100, simultaneously adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy thiosuccinimide sodium salt (NHS) into the reaction bacterial liquid for activation to obtain activated bacterial liquid; wherein the concentration of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) is 2-4 mM and the concentration of N-hydroxythiosuccinimide sodium salt (NHS) is 2-4 mM.
5) The phage stock solution with activated carboxyl groups and the solution containing aminated gold nanoparticles (Cys-AuNPs) were mixed in a 1:100 are mixed and coupled overnight, and finally trifluoroacetic acid is added to remove tert-butyl carbamate (BOC), and the mixture is centrifuged to obtain an aminated gold nanoparticle-phage T156 recognition probe (T156@ Cys-AuNPs).
Example 5
The kit for detecting salmonella ATCC 13311 comprises the T156@ Cys-AuNPs prepared by the method.
The colorimetric conditions for detecting salmonella with the kit for detecting salmonella ATCC 13311 were established as follows:
optimization of incubation time of T156@ Cys-AuNPs and host bacteria
Adding the freshly activated host bacteria ATCC 13311 into the prepared T156@ Cys-AuNPs solution, respectively incubating the solution in a constant-temperature shaking table at 37 ℃ and 160r/min for different times (4-36 min), observing color change, representing an ultraviolet absorption spectrogram by using an ultraviolet spectrophotometer, recording absorbance values at 526nm and 680nm, and obtaining the absorbance value according to the absorbance A 680 /A 526 The change of the ratio explores the optimal time for the T156@ Cys-AuNPs to be adsorbed to the host bacteria.
The results are shown in FIGS. 13 and 14, as the adsorption time of T156@ Cys-AuNPs and host bacteria is increased, the highest absorption peak of the ultraviolet absorption spectrum is continuously reduced, and the absorbance A is continuously reduced 680 /A 526 The ratio continuously rises, which indicates that the recognition probe is continuously adsorbed to the surface of the host bacteria, so that the gold nanoparticles are aggregated; when the adsorption time is 32min later, the highest absorption peak changes slowly, and the absorbance A 680 /A 526 The ratio is slightly reduced even if the change is small, which indicates that the recognition probes are completely adsorbed to the surface of the host bacteria at the moment, so that the gold nanoparticles are completely gathered on the host bacteria; if incubation continues, the phage will lyse the host bacteria, which in turn affects the assay results. Therefore, the incubation time of the T156@ Cys-AuNPs and the host bacteria is 32 min.
2. Detection of Salmonella at different concentrations
In order to detect the sensitivity of the visual detection, a series of concentrations of Salmonella typhimurium ATCC 13311(0 CFU/mL-1.2X 10) were detected by the method under the optimal experimental conditions after condition optimization 6 CFU/mL). Observing color change, representing its ultraviolet absorption spectrogram by ultraviolet spectrophotometer, recording absorbance values at 526nm and 680nm, and analyzing absorbance A 680 /A 526 The ratio is changed.
The result is shown in fig. 15, when the concentration of the bacterial liquid is less than 12CFU/mL, the highest absorption peak of the ultraviolet absorption spectrogram at 526nm has no great change, so the lowest detection limit is 12 CFU/mL; when the concentration is more than 12CFU/mL, the highest absorption peak at 526nm of the ultraviolet absorption spectrogram is continuously reduced along with the increase of the concentration of the bacterial liquid, the absorption peak gradually appears at 680nm, and the color of the T156@ Cys-AuNPs solution is continuously deepened, so that the wine red color is changed into purple red and deep purple; the presence of Salmonella can be determined by colorimetric methods.
3. Establishment of standard curve of colorimetric method
After salmonella with different concentrations are respectively detected by a colorimetric method based on an aminated gold nanoparticle-phage T156 identification probe, the concentration of a bacteria liquid is established as a horizontal coordinate and the absorbance A is established 680 /A 526 The ratio is a standard curve of a vertical coordinate, so that the qualitative and quantitative detection of the salmonella is achieved.
As a result, as shown in FIG. 16, the bacterial liquid concentration was 1.2X 10 1 CFU/mL～1.2×10 6 CFU/mL range, and absorbance A 680 /A 526 The ratio shows a good linear relationship, and the standard equation is that y is 0.1198x +0.1209, R 2 ＝0.9580。
4. Colorimetric specificity assessment
In order to detect the specificity of the visual detection, the method is used for detecting salmonella typhimurium ATCC 13311, salmonella typhimurium ATCC 14028, salmonella typhimurium ATCC 13076, vibrio parahaemolyticus ATCC 33846, escherichia coli NCTC 12900, staphylococcus aureus ATCC 25923, listeria monocytogenes ATCC 19114 and a blank control group under the optimal experimental condition after condition optimization. Observing color change, representing its ultraviolet absorption spectrogram by ultraviolet spectrophotometer, recording absorbance values at 526nm and 680nm, and determining absorbance A 680 /A 526 The ratio is changed.
The results are shown in FIGS. 18 and 19, the T156@ Cys-AuNPs of three groups of Salmonella typhimurium experimental groups are aggregated and subjected to color change, the highest absorption peak of the ultraviolet absorption spectrum at 526nm is obviously changed, and the absorbance A 680 /A 526 The ratio of (A) to (B) is significantly increased; t156@ Cys-AuNPs of the remaining experimental groupsAll have obvious aggregation and color change, the ultraviolet absorption spectrum has no great change, and the absorbance A 680 /A 526 The ratio does not change much. The result shows that the result has significant difference, and the colorimetric method based on the aminated gold nanoparticle-phage T156 recognition probe has stronger specificity.
The colorimetric method for rapidly detecting the salmonella typhimurium ATCC 13311 by using the kit comprises the following steps:
1) salmonella typhimurium ATCC 13311 was activated and then treated at 0 CFU/mL-1.2X 10 6 Preparing Salmonella typhimurium ATCC 13311 solutions with different concentrations in the CFU/mL range;
2) respectively adding the T156@ Cys-AuNPs into Salmonella typhimurium ATCC 13311 solutions with different concentrations, mixing and incubating for 25-35 min, placing the mixture into an ultraviolet spectrophotometer for detection, respectively recording light absorption values at 526nm and 680nm, and analyzing the light absorption A 680 /A 526 The ratio is changed, and the concentration of the bacteria liquid is used as the abscissa and the absorbance A is established 680 /A 526 The ratio is a standard curve of a vertical coordinate;
3) mixing a sample to be detected with T156@ Cys-AuNPs, detecting and recording light absorption values at 526nm and 680nm, and analyzing the light absorption A 660 /A 520 And obtaining the content of the salmonella typhimurium ATCC 13311 in the sample to be detected according to the standard curve.
Example 6 application of kit in artificially contaminated crayfish
1. Pretreatment of crayfish samples
Sterilizing crayfish with an autoclave at 121 deg.C for 15min, collecting 19 crayfish samples, respectively coating bacterial liquid on the tail of crayfish, adsorbing the crayfish samples in the crayfish, and simulating crayfish samples contaminated to different degrees. The 19 labeled crayfish samples were mashed with a sterilized grinding rod in a clean bench and then mixed with sterile water. And after the crayfish and the sterile water are fully and uniformly mixed, taking a sample liquid for later use.
2. Detection of artificially contaminated crayfishes
Adding sample solution of 19 crayfish samples into the kit T156@ Cys-AuNPs solution is put into a constant temperature shaking table with the temperature of 37 ℃ and the speed of 160r/min for incubation for 30min respectively, an ultraviolet absorption spectrogram is represented by an ultraviolet spectrophotometer, the absorbance values at 526nm and 680nm are recorded, and the absorbance A is determined 680 /A 526 The ratio is changed.
TABLE 1 detection results of Salmonella in crayfish samples
Note: the bacteria liquid concentration of the detected object has a linear relation within the range of 1.2 × 101-1.2 × 106CFU/mL, and the measured A 680 /A 526 The ratio of (A) is within the range of 0.2519-0.8492 to have reliability.
The result of the colorimetric method of the kit is not obviously different from the detection result of a flat plate counting method, the recovery rate is 85.71-117.86%, and the average recovery rate is 103.68%. The colorimetric method of the kit is proved to be applicable to detection of different food sample matrixes.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A preparation method of an aminated gold nanoparticle-phage T156 recognition probe is characterized by comprising the following steps: the method comprises the following steps:
s1: reducing chloroauric acid modified by mercaptoethylamine by using sodium borohydride to synthesize aminated gold nanoparticles;
s2: the aminated gold nanoparticles and the salmonella typhimurium bacteriophage T156 are coupled by amido bonds to form a stable aminated gold nanoparticle-bacteriophage T156 recognition probe.
2. The method for preparing the aminated gold nanoparticle-phage T156 recognition probe of claim 1, wherein: in step S1, the aminated gold nanoparticles are synthesized by the following steps:
1) weighing 6-8 parts of mercaptoethylamine, 20-22 parts of chloroauric acid and 2-4 parts of sodium borohydride according to parts by weight;
2) dissolving sodium borohydride in water to obtain sodium borohydride solution
3) Mixing mercaptoethylamine and chloroauric acid, and magnetically stirring uniformly at room temperature and in a dark environment to obtain a mixture;
4) adding the prepared sodium borohydride solution into the mixture, continuously stirring, reducing chloroauric acid through sodium borohydride, stably forming gold particles, and finally forming a clear and transparent wine red solution, namely the solution containing the aminated gold nanoparticles.
3. The method for preparing the aminated gold nanoparticle-phage T156 recognition probe of claim 1, wherein: the mercaptoethylamine, the chloroauric acid and the sodium borohydride respectively comprise the following components in parts by weight: 6.57 parts, 19.03 parts and 3.78 parts.
4. The method for preparing the aminated gold nanoparticle-phage T156 recognition probe of claim 2, wherein: in the solution containing the aminated gold nanoparticles, the particle size of the aminated gold nanoparticles is 25-27 nm, and the potential is 38.6 mV.
5. The method for preparing the aminated gold nanoparticle-phage T156 recognition probe of claim 1, wherein: in the step S2, the aminated gold nanoparticle-phage T156 recognition probe is formed by coupling the following steps:
1) performing activated culture on the bacteriophage T156, performing centrifugal resuspension, and filtering by using a microporous filter membrane to obtain a high-titer bacteriophage stock solution; wherein the content of the bacteriophage T156 in the bacteriophage stock solution is 1010 PFU/mL.
2) Preparing a tert-butyl carbamate solution by taking tetrahydrofuran as a solvent; wherein the concentration of the tert-butyl carbamate solution is 1-3 mM;
3) adding the tert-butyl carbamate solution into the phage stock solution according to the volume ratio of 1:100 for reaction; obtaining a reaction bacterial liquid;
4) adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide sodium salt into the reaction bacterial liquid at the same time according to the volume ratio of 1:1:100 for activation to obtain activated bacterial liquid; wherein the concentration of 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 2-4 mM and the concentration of N-hydroxy thiosuccinimide sodium salt is 2-4 mM;
5) and mixing the phage stock solution with activated carboxyl and a solution containing the aminated gold nanoparticles at a ratio of 1: 300-100, coupling overnight, finally adding trifluoroacetic acid to remove tert-butyl carbamate, and centrifuging to obtain the aminated gold nanoparticles-phage T156 recognition probe.
6. The method for preparing the aminated gold nanoparticle-phage T156 recognition probe of claim 5, wherein: the volume of the phage stock solution with activated carboxyl and the solution containing the aminated gold nanoparticles was 1: 150.
7. The method for preparing the aminated gold nanoparticle-phage T156 recognition probe according to claim 5, wherein: the particle size of the aminated gold nanoparticle-phage T156 recognition probe is 190-210 nm, and the coupling amount of the phage is 0.78 multiplied by 10 8 ～0.95×10 8 PFU/mL, coupling ratio 65% -75%.
8. A kit for detecting salmonella ATCC 13311, comprising: the kit comprises an aminated gold nanoparticle-phage T156 recognition probe prepared by the method of claim 1.
9. A colorimetric method for rapidly detecting Salmonella typhimurium ATCC 13311 by using a kit is characterized in that: the method comprises the following steps:
1) salmonella typhimurium ATCC 13311 was activated and then treated at 0 CFU/mL-1.2X 10 6 Preparation of different concentrations of typhimurium in the CFU/mL rangeSalmonella ATCC 13311 solution;
2) respectively adding the aminated gold nanoparticles and the bacteriophage T156 recognition probe into salmonella typhimurium ATCC 13311 solutions with different concentrations, mixing and incubating for 25-35 min, placing the mixture into an ultraviolet spectrophotometer for detection, respectively recording light absorption values at 526nm and 680nm, and analyzing the light absorption A 680 /A 526 The ratio is changed, and the concentration of the bacteria liquid is used as the abscissa and the absorbance A is established 680 /A 526 The ratio is a standard curve of a vertical coordinate;
3) mixing a sample to be detected with an aminated gold nanoparticle-phage T156 recognition probe, detecting and recording light absorption values at 526nm and 680nm, and analyzing the light absorption A 660 /A 520 And obtaining the content of the salmonella typhimurium ATCC 13311 in the sample to be detected according to the standard curve.
10. Use of the kit of claim 9 for the detection of artificially contaminated crayfish.
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| CN113999820A (en) * | 2021-10-14 | 2022-02-01 | 华中农业大学 | Salmonella enteritidis phage SEP37 and electrochemical impedance spectroscopy sensor and detection method thereof |
| US20220112469A1 (en) * | 2018-10-02 | 2022-04-14 | The Regents Of The University Of California | Targeted Phage for Bacterial Detection and Destruction |
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| CN111944766A (en) * | 2020-07-22 | 2020-11-17 | 华中农业大学 | Salmonella typhimurium bacteriophage T156 and application thereof |
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