Recognition peptide mediated nano fluorescence enhancement probe for detecting food-borne pathogenic bacteria and preparation method of recognition peptide mediated nano fluorescence enhancement probe

Abstract

The invention discloses a recognition peptide mediated nano fluorescence enhancement probe for detecting food-borne pathogenic bacteria and a preparation scheme of the recognition peptide mediated nano fluorescence enhancement probe, and belongs to the field of rapid detection of biosensors. The invention provides an AuNC (at) CDs heterodimer fluorescence enhancement probe, which is generated through hybridization between a CDSs-ssDNA2-SA-recognition peptide and AuNCs-ssDNA1, and can effectively realize rapid specific detection of food-borne pathogenic bacteria such as pseudomonas aeruginosa and the like. The linear range of the biosensor probe for detecting lipopolysaccharide of food-borne pathogenic bacteria is 0.1-100 [mu] g / mL, and the limit of detection (LOD) can reach 0.04 [mu] g / mL.

Description

Technical Field

[0001] The invention relates to an identification peptide-mediated nano fluorescence enhancement probe for detecting food-borne pathogenic bacteria and a preparation method thereof, and belongs to the field of rapid detection of biosensors. Background Art

[0002] Foodborne pathogens pose a threat to public health and life. They not only have low nutritional requirements, but also easily cause people with weakened immunity to suffer from diseases such as meningitis, pneumonia, and sepsis. Pseudomonas aeruginosa, as a common foodborne pathogen, has relatively low nutritional requirements and is frequently detected in packaged drinking water. For special people with weakened immunity, exposure to the bacteria may lead to diseases such as meningitis, pneumonia, and sepsis. Therefore, accurate, sensitive, and rapid methods for detecting foodborne pathogens are urgently needed.

[0003] Traditional biological probes for detecting foodborne pathogens have problems such as unstable structure, single detection index, low sensitivity, and slow signal response. In recent years, peptides have been used as recognition biomolecules, fixed on the surface of biosensors, and bound to bacterial cell membranes through electrostatic interactions. Research in this field has received widespread attention. In addition, gold nanocrosses (AuNCs), as a fluorescent nanomaterial, have a strong surface enhanced fluorescence effect on their end faces, and have a large absorption coefficient and a wide absorption range in the ultraviolet visible region, and can be used as a quencher. Carbon dots (CDs), as an electron donor acceptor, have low toxicity and good biocompatibility, and their fluorescence can be quenched by a variety of fluorescent reagents. However, at this stage, there are few nano-enhanced probes using CDs as fluorescent labeling materials, and how to improve the sensitivity and response speed of detection is an urgent problem to be solved.

[0004] Based on this, the present invention aims to construct an AuNC@CDs “OFF-enhanced ON” fluorescence visualization enhanced probe based on FRET and SEF effects, in order to detect Pseudomonas aeruginosa. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and design a fluorescence visualization enhanced probe for the detection of foodborne pathogenic bacteria Pseudomonas aeruginosa.

[0006] The first technical solution provided by the present invention is a recognition peptide-mediated nano-fluorescence enhanced probe, the probe comprising AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptide, the AuNCs-ssDNA1 being formed by connecting AuNCs modified with polyethylene glycol and ssDNA1, and the CDs-ssDNA2-SA-recognition peptide being formed by connecting ssDNA2 modified with carboxylated carbon dots and avidin SA-modified Pseudomonas aeruginosa recognition peptide.

[0007] In certain embodiments, the nucleotide sequence of the ssDNA1 is shown as SEQ ID NO.1, the nucleotide sequence of the ssDNA2 is shown as SEQ ID NO.2, and the amino acid sequence of the recognition peptide is shown as SEQ ID NO.3.

[0008] SEQ ID NO.1: CCC-AAAAAAAAAGTCTTA;

[0009] SEQ ID NO.2: AAGAC-CCCCCC;

[0010] SEQ ID NO. 3: RGLRRLGRKIAHGVKKYGPTVLRIIRIAGC.

[0011] The second technical solution provided by the present invention is a preparation method for identifying peptide-mediated nano fluorescence enhancement probes, wherein the preparation method comprises connecting polyethylene glycol-modified AuNCs with ssDNA1 to form AuNCs-ssDNA1, and connecting carboxylated carbon dot-modified ssDNA2 with avidin SA-modified Pseudomonas aeruginosa recognition peptide to form CDs-ssDNA2-SA-recognition peptide;

[0012] The probe was synthesized by complementary hybridization of AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptide.

[0013] In certain embodiments, the preparation method comprises the following steps:

[0014] (1) The carboxylated carbon dots and ssDNA2 were shaken at 4°C for 12 h, and the biotin-labeled Pseudomonas aeruginosa recognition peptide and avidin SA were also incubated at 4°C for 12 h. The two were then mixed and incubated at 4°C for another 12 h to synthesize CDs-ssDNA2-SA-recognition peptide;

[0015] (2) AuNCs were modified with polyethylene glycol and incubated in a metal bath at 30 °C for 12 h with shaking, and then ssDNA1 was added and incubated at 20-30 °C for 12 h to generate AuNCs-ssDNA1;

[0016] (3) The AuNC-ssDNA1 of step (2) and the CD-ssDNA2-SA-recognition peptide of step (1) are hybridized in a DNA hybridization solution at 37° C. for 12 h to obtain an AuNC@CDs heterodimer fluorescence enhanced probe.

[0017] In certain embodiments, 1 mg of carboxylated carbon dots in step (1) needs to be pre-activated with 8 mg of N-hydroxysuccinimide (NHS) and 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) before use to prepare a 1 mg / m LCDs sample.

[0018] In certain embodiments, AuNCs modified with polyethylene glycol-2000 in step (2) are more stable and more soluble in water, wherein the amount of polyethylene glycol (PEG) required for each 1 mL of AuNCs is 3 mmol.

[0019] In certain embodiments, the molar ratio of the biotin-labeled Pseudomonas aeruginosa recognition peptide to avidin SA is 1:(1-1.2), and the added amount of the recognition peptide is 100 μmol / L.

[0020] In certain embodiments, the added amount of ssDNA1 and ssDNA2 is 1 μmol / L, the mass volume ratio of CDs to ssDNA2 is 1 mg:10 μL, and the reaction molar ratio of the ssDNA1 to the AuNCs is 200:1.

[0021] In certain embodiments, the molar ratio of CD-ssDNA2-SA-recognition peptide to AuNC-ssDNA1 is 1:(1.5-2).

[0022] In some embodiments, the DNA hybridization solution is 1 mM Tris-HCl, 0.01% SDS, 20 mM MgCl 2 Mix the solution.

[0023] The third technical solution provided by the present invention is a detection kit, which contains the probe described in the first technical solution.

[0024] The fourth technical solution provided by the present invention is a method for detecting foodborne pathogens, which comprises adding the fluorescence enhancement probe described in the first technical solution to the sample to be tested, characterizing the LPS content in the bacteria by the intensity of the fluorescence signal, and then determining the concentration of the foodborne pathogens.

[0025] In certain embodiments, the foodborne pathogen is Pseudomonas aeruginosa.

[0026] The present invention also provides a method for extracting lipopolysaccharide (LPS) from a liquid and detecting bacteria, the method comprising the following steps:

[0027] (1) LPS is extracted from Pseudomonas aeruginosa, mainly by the hot phenol water method, which requires the cells to be collected by high-speed centrifugation;

[0028] (2) Add lysis buffer to the bacteria and vortex vigorously; then add chloroform and vortex, incubate at room temperature for 5 minutes; then centrifuge to extract LPS, take the supernatant and add purification buffer, incubate at 20°C for 10 minutes. Finally, centrifuge and purify, remove the upper layer to obtain crude LPS particles;

[0029] (3) Wash the LPS particles with 70% ethanol 2-3 times, centrifuge and dry the lower layer of LPS particles. Then add Tris-HCl buffer, resuspend with ultrapure water, and dry to obtain LPS purified sample;

[0030] (4) constructing a standard curve for detecting LPS samples of Pseudomonas aeruginosa, preparing 7 to 9 LPS standard concentration solutions with different concentrations in the range of LPS concentration of 0 to 100 μg / mL, and including a blank LPS solution, adding the fluorescence enhancement probe described in the first technical solution, and recording the change in the fluorescence intensity value of the fluorescence enhancement probe described in the first technical solution at 622 nm under an excitation wavelength of 560 nm, and obtaining a mapping relationship between LPS concentration and fluorescence signal intensity;

[0031] (5) Disperse the sample to be tested in pure water, with a bacterial concentration of 0 cfu / mL to 10 8 cfu / mL, the sample can be processed in sequence according to steps (1) to (3), the fluorescence enhancement probe described in the first technical solution is added, and the fluorescence intensity of the fluorescence enhancement probe at 622 nm under an excitation wavelength of 560 nm is recorded. The LPS concentration in the sample to be tested can be determined by the mapping relationship obtained in step (4), and the concentration of Pseudomonas aeruginosa can be further determined.

[0032] The above detection method has a low detection limit, high sensitivity and a good mapping function relationship.

[0033] In certain embodiments, the centrifugal speed in step (1) is preferably 13000 r / min, and the time is preferably 10 min.

[0034] In certain embodiments, in step (4), the lysis buffer is preferably 1-1.2 mL, and the chloroform is preferably 200-250 μL.

[0035] In certain embodiments, the centrifugal speed in step (4) is preferably 13000 r / min, the time is preferably 10 min and 15 min respectively, and the low temperature environment needs to be maintained at 4°C.

[0036] In certain embodiments, in step (4), the ratio of supernatant to purification buffer is preferably 1:(2-2.5), and the supernatant is preferably 400 μL to 500 μL.

[0037] In certain embodiments, in step (5), 1 mL of ethanol is preferably used for each wash, the concentration of Tris-HCl is preferably 10 mM and the pH is maintained at 8.0, preferably in an amount of 30 to 50 μL, and the amount of ultrapure water used for resuspension is preferably 1 mL.

[0038] In certain embodiments, the centrifugal speed in step (5) is preferably 13000 r / min, the time is preferably 3 min, and it is preferably carried out at 4°C.

[0039] The technical effects of the present invention are as follows:

[0040] The present invention provides a recognition peptide-mediated nano fluorescence enhancement technology for detecting foodborne pathogens and a preparation scheme thereof. The present invention is used for a specific set of foodborne pathogens and AuNC@CDs heterodimer fluorescence enhancement probes. When used in a system of foodborne pathogens and detection probes in water bodies, as the LPS content in a unit volume increases, the overall fluorescence intensity value at 622nm at an excitation wavelength of 560nm gradually increases, and a certain mapping function relationship is formed. In the presence of the target strain Pseudomonas aeruginosa, the pathogen recognition peptide as a biological recognition element preferentially binds to the bacterial LPS, causing AuNCs-ssDNA1 to dissociate from the constructed nanodimer AuNC@CDs and release CDs-ssDNA2-SA-recognition peptides. The CDs fluorescence intensity is continuously enhanced at a fixed excitation wavelength, and the pathogen content in the liquid to be tested can be further determined by detecting LPS. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The synthetic route of the AuNC@CDs heterodimer fluorescence enhancement probe in the present invention is shown in FIG.

[0042] Figure 2 The UV absorption graphs of AuNC, AuNC-PEG, and AuNC-ssDNA1 compare the changes before and after AuNC surface modification.

[0043] Figure 3 Comparison of the UV absorption patterns of AuNC-ssDNA1, CDs-ssDNA2-SA-recognition peptide and AuNC@CDs composite probe.

[0044] Figure 4 The optimization test results of the probe effect generated by AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptide under different ratios; (A) the vertical axis △F represents the fluorescence intensity quenching value, which represents the fluorescence quenching effect after the synthesis of the probe; (B) the vertical axis FF 0 is the fluorescence enhancement value, indicating the fluorescence enhancement effect after the probe recognizes LPS.

[0045] Figure 5The fluorescence quenching effect after probe generation was optimized for AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptides under different hybridization time incubations.

[0046] Figure 6 The test results were optimized for AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptides under different hybridization time incubations, and the fluorescence enhancement effect after the generated probes recognized LPS.

[0047] Figure 7 This is the transmission electron microscopy characterization image of AuNC@CDs fluorescent probe.

[0048] Figure 8 Dynamic light scattering (DLS) characterization before and after coupling of AuNC-ssDNA1 and CDs-ssDNA2-SA-recognition peptides

[0049] Fig. 9 is the relative fluorescence intensity (FF 0 ) and the logarithm of Pseudomonas aeruginosa LPS concentration.

[0050] Fig.10 This is the correlation diagram between bacterial solution concentration and fluorescence intensity.

[0051] Fig.11 This is a graph showing the relationship between the actual water samples with different bacterial solution concentrations and the fluorescence intensity. DETAILED DESCRIPTION

[0052] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0053] The present invention provides a nano fluorescence enhancement probe mediated by recognition peptides, and the principle of the probe is described as follows.

[0054] Based on fluorescence resonance energy transfer (FRET) and surface enhanced fluorescence effect (SEF), an "OFF-enhanced ON" type fluorescence enhanced probe was constructed for rapid separation and detection of Pseudomonas aeruginosa. The conformational change of ssDNA was used to regulate the distance between the gold nanocross and the fluorescent carbon dots, thereby causing a change in the fluorescence value. When the distance between AuNC and CDs is less than 10nm, a FRET effect is exhibited; when the distance between AuNC and CDs is 10nm, AuNC has an obvious SEF effect on CDs, and an obvious fluorescence amplification signal is generated by the change of fluorescence intensity from "OFF" to "enhanced ON". When the target strain Pseudomonas aeruginosa lipopolysaccharide is present, the pathogen recognition peptide of the above probe preferentially binds to the bacterial lipopolysaccharide and separates from AuNCs-ssDNA1, resulting in enhanced fluorescence.

[0055] The preparation method of the above probe includes the following steps: CDs-ssDNA2-recognition peptide and AuNCs-ssDNA1 are synthesized separately, and then the corresponding probe is synthesized by complementary hybridization of ssDNA1 and ssDNA2, and then separated, purified and used. The specific synthesis process is as follows Figure 1 shown.

[0056] The raw materials used in the examples are:

[0057] All ssDNA used in the present invention were purchased from Jiangsu Saisuofei Biotechnology Co., Ltd.; chloroauric acid (HAuCl 4 , 99%), L-ascorbic acid (VC), silver nitrate (AgNO 3 , >99%), sodium salicylate (>98%), sodium borohydride (NaBH 4 , 99%), hexadecyltrimethylammonium bromide (CTAB), chloroform (CHCl 3 ), ethanol (70%), Tris-HCl buffer (pH 8.0), and plate count agar medium were purchased from Shanghai Sinopharm Group; tris(2-carboxy)-methanephosphine hydrochloride (TCEP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), and Pseudomonas aeruginosa LPS standards were purchased from Sigma-Aldrich; avidin (SA) was purchased from Beijing Solebao Technology Co., Ltd.; thiol-polyethylene glycol 5000 (PEG-SH) was purchased from Shanghai Tuochang Biotechnology Co., Ltd.; carboxylated CDs was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; and lipopolysaccharide extraction kit (LPS extraction kit) was purchased from iNtRON Biotechnology; Nutrient broth (NB) was purchased from Shanghai Bio-Microbiology Technology Co., Ltd.; The SMAPs peptide sequence of Pseudomonas aeruginosa was Biotin-RGLRRLGRKIAHGVKKYGPTVLRIIRIAGC (N-terminus-C-terminus), which was synthesized by GenScript Biotech Co., Ltd.; Pseudomonas aeruginosa (P. aeruginosa) ATCC9027 was a commercial strain derived from a laboratory-preserved strain (Wuxi, Jiangsu).

[0058] Example 1: Overall synthesis route of AuNC@CDs heterodimer fluorescence enhanced probe

[0059] As an example of the present invention, a recognition peptide-mediated nano fluorescence enhancement probe for detecting foodborne pathogens, the synthesis route of the probe is as follows: Figure 1 , CDs-ssDNA2-SA-recognition peptide and AuNCs-ssDNA1 need to be synthesized separately and then hybridized to generate probes.

[0060] Example 2: Construction of CDs-ssDNA2-SA-recognition peptide

[0061] (1) First, CDs were connected to ssDNA2. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to 1 mg / mL carboxylated carbon dots (CDs) and stirred at 4°C for 4-5 hours. Then, the activated CDs and ssDNA2 were fully mixed and shaken at 4°C for 12 hours. The amount of CDs used was 1 mL, the amount of 1-Ethyl-(3-dimethylaminopropyl)carbodiimide used was 8 mg, the amount of N-hydroxysuccinimide used was 8 mg, and the amount of ssDNA2 added was 100 μmol / L.

[0062] (2) Secondly, biotin-labeled Pseudomonas aeruginosa SMAPs (recognition peptides) were linked to avidin SA. Biotin-labeled Pseudomonas aeruginosa SMAPs (recognition peptides) were incubated with 0.5 mg / mL avidin SA at 4°C for 12 hours and purified by dialyzing with an ultrafiltration centrifuge tube. The amount of biotin-labeled Pseudomonas aeruginosa SMAPs was 100 μmol, and the amount of avidin SA was 100 μmol.

[0063] (3) Finally, CDs-ssDNA2 was added to the dialyzed SA-recognition peptide and incubated for 12 hours. Uncoupled substances were dialyzed using an ultrafiltration centrifuge tube to obtain CDs-ssDNA2-SA-recognition peptide. The entire process was performed at 4°C.

[0064] Example 3: Construction of AuNCs-ssDNA1

[0065] (1) First, prepare the gold seed solution: add 0.1 M HAuCl 4 0.2 M hexadecyltrimethylammonium bromide (CTAB) solution, ultrapure water and 0.01 M NaBH were added to the solution in sequence. 4 The solution was stirred rapidly for 2 minutes and then allowed to stand for 30 minutes to obtain a gold seed solution. The entire process of preparing gold seeds was carried out in a 30°C water bath with stirring. 4 The solution dosage is 5mL, the CTAB solution dosage is 5mL, the ultrapure water dosage is 0.4mL, and the NaBH 4 The dosage is 0.6mL.

[0066] (2) Synchronously prepare the growth solution of the gold nanocross: Dissolve CTAB and sodium salicylate in ultrapure water at 55°C, cool to 30°C, add 4mM silver nitrate solution and 1% chloroauric acid solution, then add ultrapure water to dilute and stir for 15 minutes, finally add 0.1M ascorbic acid solution, and stir vigorously until the solution is colorless and transparent. The amount of CTAB is 0.9g, the amount of sodium salicylate is 0.08g, the amount of ultrapure water is 25mL and 23.9712mL respectively, the amount of silver nitrate solution is 0.6mL, the amount of chloroauric acid solution is 1028.8μL, and the amount of ascorbic acid solution is 700μL.

[0067] (3) Preparation of AuNCs: Add the prepared gold seed to the growth solution and stir rapidly for 30 seconds, then let it stand for 12 hours. Centrifuge the AuNCs solution and discard the supernatant. Take the precipitate and dissolve it in 5mM CTAB, which can be stored in a refrigerator at 4°C. During the synthesis of AuNCs, the preparation temperature must be maintained at 30°C, the amount of gold seed solution is 80μL, the amount of growth solution is 50mL, the amount of CTAB is 35mL, and the centrifugation must be stabilized at 10,000-12,000r / min and maintained for 15-25min.

[0068] (4) AuNCs surface functionalization: 5 mM thiolated PEG was covalently modified on the surface of AuNCs, and ultrapure water was added for further dilution and incubated in a metal bath for 12 to 15 hours. Excess PEG was then centrifuged out and resuspended with 5 mM CTAB. The reaction molar ratio of PEG to AuNCs was 30:1, the amount of AuNCs was 1 mL, the amount of PEG solution was 600 μL, the amount of ultrapure water was 400 μL, and the amount of CTAB solution was 1 mL. In addition, the temperature of the modification and centrifugation process was kept stable at 30 °C, and the centrifugal speed was 10,000 r / min and maintained for 10 minutes.

[0069] (5) Reduction of thiolated ssDNA1 and binding to AuNCs epitope: ssDNA1 was added to 100 μM tri(2-carboxyethyl)phosphine (TCEP) and incubated in the dark for 4 to 5 h. Unreacted substances were filtered using a G25 extraction column. The functionalized ssDNA1 and AuNCs solution were mixed and incubated for 12 h. The supernatant was removed by centrifugation and dissolved in a buffer to obtain AuNCs-ssDNA1. The amount of TCEP was 100 μL, ssDNA1 and TCEP were combined in equimolar amounts, the reaction molar ratio of ssDNA1 and AuNCs solution was 200:1, the ssDNA1 activation incubation temperature was 4 °C, the AuNCs and ssDNA1 binding incubation temperature was 30 °C, the centrifugal speed was 10,000 r / min and maintained for 20 min, and the AuNCs in each step were characterized by UV absorption ( Figure 2 ).

[0070] The base sequences of ssDNA1 and ssDNA2 are shown in Table 1.

[0071] Table 1 ssDNA1 and ssDNA2 base sequences

[0072] name Base sequence (5'-3') ssDNA1 HS-C3-AAAAAAAAAGTCTTA ssDNA2 Biotin-TAAGAC-C6-NH2

[0073] Figure 2 It can be seen that after AuNC was modified with PEG, the peak shape did not change significantly, and the local surface plasmon resonance absorption peak (LSPR) had a slight blue shift from 630nm to 626nm, indicating that the end face of AuNC was covalently coupled with thiolated PEG. After further modification with ssDNA1, the LPSR shifted to 623nm, indicating that the thiolated ssDNA1 was covalently bound to the end face of AuNCs.

[0074] Example 4: Construction of AuNC@CDs heterodimer fluorescence enhanced probe

[0075] The specific steps are as follows:

[0076] (1) Prepare DNA hybridization solution: 1 mM Tris-HCl, 0.01% SDS, 20 mM MgCl 2 As raw material.

[0077] (2) CD-ssDNA2-SA-recognition peptide was hybridized and connected with AuNC-ssDNA1. Based on the head-to-tail parallel self-assembly effect, CD-ssDNA2-SA-recognition peptide and AuNC-ssDNA1 were self-assembled in the DNA hybridization solution, and then dialyzed to obtain the corresponding AuNC@CDs heterodimer fluorescence enhancement probe, which can measure the fluorescence emission peak at 622nm under 560nm wavelength excitation, and the quenching effect of the probe under different synthesis conditions and the enhancement effect after recognizing LPS are used as optimization indicators ( Figure 3 CD-ssDNA2-SA-recognition peptide and AuNC-ssDNA1 were self-assembled at 37°C in a ratio of 1:1, 1:2, and 2:1, respectively ( Figure 4 ), and the fluorescence quenching and enhancement effects of the probes under different incubation time conditions were also compared ( Figure 5 , Figure 6 ).

[0078] (3) Characterization of the probe, including transmission electron microscopy characterization of the AuNC@CDs fluorescent probe ( Figure 7 ) and dynamic light scattering (DLS) characterization before and after coupling ( Figure 8 ).

[0079] Depend on Figure 3It can be seen that AuNC-ssDNA1 and AuNC@CDs composite probes have obvious absorption peaks at 623nm and 623nm, respectively, and the UV spectra of AuNC@CDs composite probe and CDs-ssDNA2-SA-recognition peptide are basically consistent before 400nm, which proves that AuNC-ssDNA1 chain and CDs-ssDNA2-SA-recognition peptide chain have been successfully coupled. Among them, as the hybridization time increases, the fluorescence intensity of CDs is gradually quenched by AuNCs, and the fluorescence intensity of the system continues to decrease. The fluorescence intensity drops to the minimum at 12h and remains basically unchanged. Therefore, the hybridization time of CDs-ssDNA2-SA-recognition peptide and AuNCs-ssDNA1 is usually selected as 12h.

[0080] Depend on Figure 4 It can be seen that as the hybridization time increases, the fluorescence intensity of CDs is gradually quenched by AuNCs, and the fluorescence intensity of the system continues to decrease. The fluorescence intensity drops to the lowest at 12h and remains basically unchanged. Therefore, the hybridization time of CDs-ssDNA2-SA-recognition peptide and AuNCs-ssDNA1 is usually selected as 12h.

[0081] Figure 5 , Figure 6 It shows that when the target bacterial LPS is added, the fluorescence intensity of the system increases with the incubation time. The fluorescence intensity returns to the maximum value at 5h and remains basically stable. Therefore, in the subsequent experiments, the incubation time of AuNC@CDs heterodimer fluorescence enhancement probe and the bacterial LPS to be tested is selected as 5h.

[0082] Figure 7 Illustration, transmission electron microscopy characterization of AuNC@CDs fluorescent probe, where the gray part is CDs-ssDNA2-recognition peptide, the dark part is AuNCs-ssDNA1, and the electron microscope image of AuNC shows four tips in a cross structure. It can be seen that CDs and AuNCs are successfully coupled, and there is no obvious change in size and morphology between the two.

[0083] Figure 8 It can be seen from the dynamic light scattering results that the original particle size of AuNC is 28nm, and the original particle size of CDs is 5.6nm. The particle size after coupling is 43nm, which is consistent with the characterization under transmission electron microscopy. This further shows that AuNCs and CDs have completed surface functionalization and synthesized AuNC@CDs fluorescent probe.

[0084] Example 5: Extraction of LPS from bacteria in the test solution

[0085] A method for extracting bacterial LPS in a liquid comprises the following steps:

[0086] (1) The Pseudomonas aeruginosa strain was inoculated into sterilized nutrient broth and incubated overnight at 37°C with shaking at 150 rpm. The bacterial cell count was quantified by plate colony counting method. 2 , 10 3 , 10 4 Three dilution gradients of CFU / mL were coated on three plates respectively, and the plates were cultured in a 37°C constant temperature biological incubator for 18 h. The bacterial concentration was then calculated based on the plate count results.

[0087] (2) Meanwhile, 2 mL of bacterial cells were centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the cells were washed three times with PBS buffer to obtain clean bacterial cells.

[0088] (3) Add 1 mL of lysis buffer and vortex vigorously until the cell clumps disappear. Add 200 μL of chloroform, vortex for 10-20 seconds, and incubate at room temperature for 5 minutes. Then centrifuge at 13,000 r / min at 4°C for 10 minutes, transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube, add 800 μL of purification buffer, stir well, and incubate at 20°C for 10 minutes. Then centrifuge at 13,000 r / min at 4°C for 15 minutes, remove the upper layer to obtain LPS particles.

[0089] (4) Rinse the LPS particles 2-3 times with 1 mL of 70% ethanol and centrifuge at 13,000 r / min for 3 min at 4°C. Discard the upper layer and air-dry the remaining LPS particles. Add 30-50 μL of 10 mM Tris-HCl buffer (pH 8.0) to the LPS particles, vortex and boil for 2 min to completely dissolve the LPS, and finally resuspend with 1 mL of ultrapure water and freeze-dry in vacuum for 24 hours to obtain a purified LPS sample.

[0090] Example 6: Construction of standard curve

[0091] The construction of the standard curve for detecting Pseudomonas aeruginosa based on AuNC@CDs heterodimer fluorescence enhanced probe includes the following steps:

[0092] (1) The LPS standard of Pseudomonas aeruginosa was prepared with ultrapure water into standard solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, and 100 μg / mL for the detection of AuNC@CDs heterodimer fluorescence enhanced probe.

[0093] (2) The AuNC@CDs heterodimer fluorescence enhancement probe described in Example 4 was used as a fluorescent probe, 10 μL of the standard solution and 100 μL of the fluorescent probe were mixed in a 1.5 mL centrifuge tube, and incubated at 37° C. and 300 rpm for 5 hours to obtain a test solution.

[0094] (3) Then, 100 μL of the solution to be tested was added to the microplate reader using a pipette, and the fluorescence intensity at 622 nm under an excitation wavelength of 560 nm was measured. Three parallels were measured for each concentration. The fluorescence recovery value was used as the ordinate and recorded as Y (FF 0 ), the logarithm of LPS concentration is the horizontal axis and marked as X(log 10 C).

[0095] (4) The fluorescence intensity value at 622 nm under an excitation wavelength of 560 nm was measured to obtain the standard curve of the AuNC@CDs heterodimer fluorescence enhanced probe for the detection of Pseudomonas aeruginosa LPS, as shown in Fig. 9 shown.

[0096] (5) In addition, the correlation between bacterial solution concentration and fluorescence intensity was established. The total number of colonies was calculated by plate counting method, and the extraction of LPS by (1) to (5) of Example 5 was repeated. The fluorescence intensity was detected after dilution. The results are as follows: Fig.10 shown.

[0097] The standard curve between LPS concentration and fluorescence value is as follows Fig. 9 ,from Fig. 9 It can be seen that: with the gradual increase of LPS concentration, the fluorescence recovery degree of the detection system gradually increases and is enhanced to a certain extent; when the LPS concentration is in the range of 0.1-100 μg / mL, the logarithm of the LPS concentration and the recovered fluorescence intensity show a good linear relationship, and the linear equation is Y=4637X+23501, where Y is the fluorescence recovery degree FF 0 , X is the logarithmic value of LPS concentration; R 2 =0.9909, and the detection limit LOD can reach 0.04μg / mL.

[0098] The relationship between bacterial concentration and fluorescence intensity is as follows: Fig.10 ,from Fig.10 It can be seen that: as the bacterial solution concentration increases, the fluorescence intensity value shows an upward trend; the y-axis is the measured fluorescence intensity value, the x-axis represents the logarithm of the Pseudomonas aeruginosa bacterial solution concentration, and a relationship diagram between the fluorescence intensity and the bacterial solution concentration is obtained.

[0099] Example 7: Actual sample testing

[0100] Purified water was selected as the actual sample analysis material to evaluate the accuracy of AuNC@CDs heterodimer fluorescence enhanced probe for Pseudomonas aeruginosa in actual sample detection. The purchased purified water was diluted 10 times and 10 2 , 10 4 , 10 8CFU / mL of Pseudomonas aeruginosa was added to the sample as the food sample to be tested, and compared with the standard culture medium with different bacterial concentrations. The results are as follows Fig.11 And as shown in Table 2.

[0101] The results are as follows Fig.11 and Table 2, from Fig.11 As can be seen from Table 2, the fluorescence intensity measured in the actual water sample is basically consistent with that in the culture medium, and the relative standard deviation ranges from 0.5% to 2%. The probe can be used to quickly and accurately detect the concentration of foodborne pathogens in the reagent sample, and will not change with the viscosity, composition and interference of the liquid to be tested for the same concentration gradient of the bacterial solution.

[0102] Table 2 Experimental results of AuNC@CDs heterodimer fluorescence enhanced probe for detecting Pseudomonas aeruginosa in actual samples

[0103]

[0104] In addition, the detection accuracy of other methods (electrochemical detection, nuclear magnetic resonance) was also compared. The electrochemical detection method was referenced to "A novel impedimetric biosensor based on the antimicrobial activity of the peptide nisin for the detection of Salmonella spp.", and the nuclear magnetic resonance detection method was referenced to "A Low-Field Magnetic Resonance Imaging Aptasensor for the Rapid and Visual Sensing of Pseudomonas aeruginosa in Food, Juice, and Water." The results are shown in Table 3.

[0105] Table 3 Comparison results of AuNC@CDs heterodimer fluorescence enhanced probe detection with other AMPs-based detection methods

[0106]

[0107] The results are shown in Table 3. It can be seen from Table 3 that this method is also applicable to the detection of Pseudomonas aeruginosa in actual samples. Compared with the existing methods for detecting foodborne pathogens based on recognition peptides, it has higher sensitivity and is simpler and convenient for rapid on-site detection.

[0108] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A peptide-mediated nano-fluorescence enhancement probe, characterized in that: The probe includes AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptide, wherein the AuNCs-ssDNA1 is formed by connecting polyethylene glycol-modified AuNCs and ssDNA1, and the CDs-ssDNA2-SA-recognition peptide is formed by connecting carboxylated carbon dot-modified ssDNA2 and avidin SA-modified Pseudomonas aeruginosa recognition peptide.

2. The nano fluorescence enhanced probe according to claim 1, characterized in that: The nucleotide sequence of the ssDNA1 is shown in SEQ ID NO.1, the nucleotide sequence of the ssDNA2 is shown in SEQ ID NO.2, and the amino acid sequence of the recognition peptide is shown in SEQ ID NO.

3.

3. A method for preparing a peptide-mediated nano-fluorescence enhancement probe, characterized in that: The preparation method comprises the following steps: AuNCs modified with polyethylene glycol are connected with ssDNA1 to form AuNCs-ssDNA1, and ssDNA2 modified with carboxylated carbon dots is connected with a Pseudomonas aeruginosa recognition peptide modified with avidin SA to form CDs-ssDNA2-SA-recognition peptide; The probe was synthesized by complementary hybridization of ssDNA1 and ssDNA2 using AuNCs-ssDNA1 and CDs-ssDNA2-SA-recognition peptide.

4. The method according to claim 3, characterized in that: The preparation method comprises the following steps: (1) The carboxylated carbon dots and ssDNA2 were shaken and reacted at 4°C for 10-14 h, and the biotin-labeled Pseudomonas aeruginosa recognition peptide and avidin SA were also incubated at 4°C for 10-14 h, and then the two were mixed and incubated at 4°C for another 10-14 h to synthesize CDs-ssDNA2-SA-recognition peptide; (2) AuNCs were modified with polyethylene glycol and incubated in a metal bath at 30°C for 10-14 h with shaking, and then ssDNA1 was added and incubated at 20-30°C for 10-14 h to generate AuNCs-ssDNA1; (3) The AuNC-ssDNA1 of step (2) and the CD-ssDNA2-SA-recognition peptide of step (1) are hybridized in a DNA hybridization solution at 37° C. for 10-14 h to obtain an AuNC@CDs heterodimer fluorescence enhanced probe.

5. The method according to claim 4, characterized in that In step (1), each 1 mg of carboxylated carbon dots needs to be pre-activated with 6-10 mg of N-hydroxysuccinimide and 6-10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide before use to prepare a 1 mg / mL CDs sample; The molar ratio of biotin-labeled Pseudomonas aeruginosa recognition peptide to avidin SA is 1:(1-1.2), and the added amount of the recognition peptide is 80-120 μmol / L; The added amount of ssDNA2 was 1 μmol / L, and the mass volume ratio of CDs to ssDNA2 was 1 mg:(8-12) μL.

6. The method according to claim 4, characterized in that In step (2), the amount of polyethylene glycol required per 1 mL of AuNCs is 2-4 mmol; The added amount of ssDNA1 was 1 μmol / L, and the reaction molar ratio of the ssDNA1 to the AuNCs was 200:

1.

7. The method according to claim 4, characterized in that In step (3), the molar ratio of CD-ssDNA2-SA-recognition peptide to AuNC-ssDNA1 is 1:(1.5-2).

8. A detection kit, characterized in that: The kit contains the probe according to claim 1 or 2.

9. A method for detecting Pseudomonas aeruginosa, characterized in that: The method comprises adding the fluorescence enhancement probe according to claim 1 or 2 to the sample to be tested, characterizing the content of LPS in the bacteria by the intensity of the fluorescence signal, and then determining the concentration of Pseudomonas aeruginosa.

10. The method according to claim 9, characterized in that The method comprises the following steps: (A) constructing a standard curve for detecting LPS samples of Pseudomonas aeruginosa, preparing 7 to 9 LPS standard concentration solutions with different concentrations in the range of LPS concentration of 0 to 100 μg / mL, and including a blank LPS solution, adding the fluorescence enhancement probe described in claim 1 or 2 and recording the change in the fluorescence intensity value of the fluorescence enhancement probe at 622 nm under an excitation wavelength of 560 nm, and obtaining a mapping relationship between LPS concentration and fluorescence signal intensity; (B) The sample to be tested is dispersed in pure water, the fluorescence enhancement probe is added, and the fluorescence intensity of the fluorescence enhancement probe at 622 nm under an excitation wavelength of 560 nm is measured. The LPS concentration in the sample to be tested can be determined through the mapping relationship obtained in step (1), and the concentration of Pseudomonas aeruginosa can be further determined.

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