阅读说明：本技术 碳化硅@bsa-抗菌肽纳米探针的制备方法及其应用 (Preparation method and application of silicon carbide @ BSA-antibacterial peptide nanoprobe ) 是由 蔡继峰 李杏梅 丁艳君 孟凡明 李介男 于 2019-10-25 设计创作，主要内容包括：本发明提供一种碳化硅@BSA-抗菌肽纳米探针的制备方法及其应用,属于法医检测方法技术领域。所述的基于碳化硅@BSA-抗菌肽纳米探针的制备方法,包括以下步骤：(1)碳化硅@BSA纳米的制备；(2)碳化硅@BSA-抗菌肽纳米探针-抗菌肽的制备。所述碳化硅@BSA-抗菌肽纳米探针在检测口腔细菌及其在法医唾液鉴定中的应用方法包括：S1：标准溶液中口腔细菌的检测；S2：唾液样本中口腔细菌的检测；S3：法医唾液样本的鉴别。本发明基于碳化硅@BSA-抗菌肽纳米探针的荧光传感器,拥有灵敏度高、线性范围宽、成本低等优良分析性能,在法医微生物检测及法医唾液鉴定检测领域中具有很好的实用前景。(The invention provides a preparation method and application of a silicon carbide @ BSA-antibacterial peptide nanoprobe, and belongs to the technical field of forensic detection methods. The preparation method of the nano probe based on the silicon carbide @ BSA-antibacterial peptide comprises the following steps: (1) preparing silicon carbide @ BSA nano-particles; (2) and (3) preparation of silicon carbide @ BSA-antibacterial peptide nanoprobe-antibacterial peptide. The application method of the silicon carbide @ BSA-antibacterial peptide nanoprobe in detection of oral bacteria and identification of forensic saliva comprises the following steps: s1: detection of oral bacteria in a standard solution; s2: detecting oral bacteria in the saliva sample; s3: and (5) identifying the forensic saliva sample. The fluorescence sensor based on the silicon carbide @ BSA-antibacterial peptide nanoprobe has excellent analysis performances of high sensitivity, wide linear range, low cost and the like, and has good practical prospect in the fields of forensic microbial detection and forensic saliva identification detection.)

1. A preparation method of a silicon carbide @ BSA-antibacterial peptide nanoprobe is characterized by comprising the following steps:

(1) preparation of silicon carbide @ BSA nano: weighing silicon carbide powder, adding the silicon carbide powder into distilled water for ultrasonic treatment, and adjusting the pH value with ammonia water to obtain a silicon carbide solution; adding a proper amount of BSA (bovine serum albumin) into the silicon carbide solution to form a mixture, then putting the mixture into a polytetrafluoroethylene-lined reaction kettle, heating, and finally performing dialysis filtration to obtain a purified SiC @ BSA nanoprobe;

(2) preparation of silicon carbide @ BSA-antibacterial peptide nanoprobe-antibacterial peptide: coupling the silicon carbide @ BSA-antibacterial peptide nanoprobe prepared in the step (1) with antibacterial peptide through EDC and NHS, oscillating for 10-20 minutes, incubating for 2-4 hours, removing redundant antibacterial peptide through centrifugation to obtain the silicon carbide @ BSA-antibacterial peptide nanoprobe, and storing the prepared silicon carbide @ BSA-antibacterial peptide nanoprobe at 4 ℃ for later use.

2. The preparation method of the silicon carbide @ BSA-antibacterial peptide nanoprobe according to claim 1, wherein the mass ratio of the silicon carbide to the BSA in the step (1) is 1: 4-4: 1.

3. The method for preparing the silicon carbide @ BSA-antibacterial peptide nanoprobe according to claim 2, wherein the mass ratio of the silicon carbide to the BSA in the step (1) is 1: 1.

4. The method for preparing the silicon carbide @ BSA-antibacterial peptide nanoprobe according to claim 1, wherein the silicon carbide @ BSA-antibacterial peptide nanoprobe and the antibacterial peptide in the step (2) are coupled through EDC and NHS, shaken for 15 minutes and incubated for 3 hours.

5. The preparation method of the silicon carbide @ BSA-antibacterial peptide nanoprobe according to claim 1, wherein the concentration of the antibacterial peptide in the step (2) is 0.001-5 mg/mL.

6. The application of the silicon carbide @ BSA-antibacterial peptide nanoprobe is characterized in that the silicon carbide @ BSA-antibacterial peptide nanoprobe is used for detecting oral bacteria and the application of the silicon carbide @ BSA-antibacterial peptide nanoprobe in forensic saliva identification.

7. The use of silicon carbide @ BSA-antimicrobial peptide nanoprobes as claimed in claim 6, wherein the method for forensic saliva identification comprises:

s1: detection of oral bacteria in standard solutions: preparing oral bacteria solutions with different concentrations, adding the oral bacteria solutions into the silicon carbide @ BSA-antibacterial peptide nanoprobe, reacting for 0.1-6 h, measuring the fluorescence intensity of the oral bacteria solutions at 410nm, drawing a linear relation between the bacteria concentration and the fluorescence intensity, and preparing a standard solution curve so as to realize the detection of the oral bacteria in the standard solution;

s2: detection of oral bacteria in saliva samples: preparing saliva samples, adding oral bacteria with different concentrations into the saliva samples, respectively carrying out 3 parallel tests on each sample, adding the saliva samples containing the oral bacteria into the silicon carbide @ BSA-antibacterial peptide nanoprobe, measuring the fluorescence intensity of the saliva samples at 410nm after reacting for 0.1-6 h, and establishing a standard working curve for detection through comparison of the fluorescence intensity before and after the reaction, thereby realizing detection of the oral bacteria in the saliva samples;

s3: identification of forensic saliva samples: saliva, blood, urine and vaginal fluid samples in the actual forensic case are collected, pretreated, and then quantitatively detected through the silicon carbide @ BSA-antibacterial peptide nanoprobe for oral specific bacteria in different body fluid samples, and the oral specific bacteria can be detected only in the saliva, so that the saliva can be identified from the forensic body fluid samples.

Technical Field

The invention belongs to the technical field of forensic medicine, and particularly relates to a preparation method and application of a silicon carbide @ BSA-antibacterial peptide nanoprobe.

Background

Forensic saliva identification is an increasingly effective aid to criminal investigation, particularly in sexual crime, over the past few decades, various saliva identification techniques have evolved the most common method of identifying saliva is the salivary amylase test, although saliva α -amylase is an enzyme that is abundant in human saliva, but small amounts such as lip mucus, blood, urine, semen and peritoneal fluid are also present in other body fluids, as false positive results are produced to some extent (m.j. auvdel,% jjouranlof forensiicsciences, 1986,31, 426-431).

Through long-term development, the existing saliva identification methods, such as chemical methods and immunological methods, etc., and the analysis of cell-specific mRNA expression and protein level (t. akutsu, k.y. watanabeandk. sakura, international journanalof legalmedicine,2010,124,493 498.). However, these conventional methods require a long time and a large number of samples, which have not yet satisfied the appeal of high-efficiency forensic identification.

In recent years, oral bacteria have received increasing attention for the identification of saliva. Nakanishi et al, which performed preliminary work on saliva identification by oral bacterial detection, found that oral streptococcus (s.salivarius and s.mutans) could successfully detect saliva in mock forensic samples by Polymerase Chain Reaction (PCR) -based methods (h.nakanishi, a.kido, t.ohmori, a.takada, m.hara, n.adachi and k.j.f.s.i.saito,2009,183,20-23. J.). A recent study by the junggy jy team described that detection of oral specific bacteria (s.salivarius, s.sanguinis and n.bioflava) could be used to verify the presence of saliva in forensic samples (j.y.jung, h.k.yoon, s.an, j.w.lee, e.r.ahn, y.j.kim, h.c.park, k.lee, j.h.hwang and s.k.lim, scientific reports,2018,8,10852. R.). The above studies indicate that saliva identification based on oral bacterial detection is a prospect for future forensic studies.

The biosensing technology is widely applied in the fields of biochemistry, environmental protection, biomedicine and the like. Biosensor technology offers a more attractive alternative to other methods for target bacteria detection, requiring simple manipulations and fast reaction times, while not affecting specificity (o.r.miranda, l.xiaoning, g.g.limay, z.zheng-Jiang, y.bo, u.h.f.bunzand v.m.rotello, journal of american chemical society,2011,133,9650-9653. M.). Over the past two decades, various antimicrobial peptides have been reported as recognition units for detecting bacteria to construct biosensors (m.hoyos-nogue, f.j.gilandec.ma-Moruno, Molecules,2018,23,1683. S.). Antimicrobial peptides (AMPs) are anti-infective drugs from natural sources that have been recognized as potential next generation antibiotics. In addition, amps also exhibit several prominent features parallel to traditional antibodies, including small size, good chemical stability, and high sensitivity to bacteria (s. arctiaceno, p. pivarnik, c. m. melloanand a. senecal, Biosensors & bioelectronics,2008,23, 1721-. By virtue of the outstanding characteristics, AMP has been introduced into various microbial sensing applications, has higher selectivity and sensitivity, enables the detection of typhoid bacillus, E.coli and periodontal pathogenic bacteria to be reliable, and enables the effective detection of oral bacteria to be unexplored.

Besides designing a ligand specifically bound to a target, the construction of the biosensor also provides a very important component for designing a fluorescent probe, and the detection efficiency of a substance is directly concerned. Quantum dots (quantumdots) are semiconductor nanocrystals (semiconductor-on-semiconductor nanoparticles), which are nanoparticles composed of ii-vi or iii-V elements, and their unique optical properties are of great interest in biological fluorescence labeling and imaging. As a novel nano material, the application of the nano material in the field of biosensing is more and more paid attention. Compared with the traditional fluorescent probe, the quantum dot has excellent optical effect, high luminous efficiency and difficult photobleaching. Silicon carbide quantum dots are quantum dots made of silicon carbide, are non-toxic, have good biocompatibility, are simple in preparation process, are gradually valued by researchers, and are initially applied to imaging research of living cells (y.cao, h.dong, s.punand x.zhang, NanoResearch,2018,1-8.C.) at present, however, the silicon carbide quantum dots are not applied to the field of forensic research.

Therefore, the development of a fluorescent bacterial sensor based on the silicon carbide nanoprobe and the antibacterial peptide and the application thereof in the field of forensic identification have very important significance, wherein the fluorescent bacterial sensor has excellent analysis performances such as simple operation, rapidness, high efficiency, higher sensitivity and the like.

Disclosure of Invention

The invention provides a preparation method and application of a silicon carbide @ BSA-antibacterial peptide nanoprobe, and aims to solve the problems that a forensic saliva identification operation method is complex, reaction time is long, and a complex sample is prepared in the prior art.

In order to achieve the purpose, the technical solution of the invention is as follows:

a preparation method of a silicon carbide @ BSA-antibacterial peptide nanoprobe comprises the following steps:

(1) preparation of silicon carbide @ BSA nano: weighing silicon carbide powder, adding the silicon carbide powder into distilled water for ultrasonic treatment, and adjusting the pH value with ammonia water to obtain a silicon carbide solution; adding a proper amount of BSA (bovine serum albumin) into the silicon carbide solution to form a mixture, then putting the mixture into a polytetrafluoroethylene-lined reaction kettle, heating, and finally performing dialysis filtration to obtain a purified SiC @ BSA nanoprobe;

(2) preparation of silicon carbide @ BSA-antibacterial peptide nanoprobe-antibacterial peptide: coupling the silicon carbide @ BSA-antibacterial peptide nanoprobe prepared in the step (1) with antibacterial peptide through EDC and NHS, oscillating for 10-20 minutes, incubating for 2-4 hours, removing redundant antibacterial peptide through centrifugation to obtain the silicon carbide @ BSA-antibacterial peptide nanoprobe, and storing the prepared silicon carbide @ BSA-antibacterial peptide nanoprobe at 4 ℃ for later use.

The silicon carbide @ BSA is a silicon carbide quantum dot with stable bovine protein; BSA is bovine serum albumin; EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS for N-hydroxy succinimide.

Preferably, the mass ratio of the silicon carbide to the BSA in the step (1) is 1: 4-4: 1.

Preferably, the mass ratio of the silicon carbide to the BSA in the step (1) is 1: 1.

Preferably, the silicon carbide @ BSA-antibacterial peptide nanoprobe and the antibacterial peptide in the step (2) are coupled through EDC and NHS, shaken for 15 minutes and incubated for 3 hours.

Preferably, the concentration of the antibacterial peptide in the step (2) is 0.001-5 mg/mL.

The invention also provides application of the silicon carbide @ BSA-antibacterial peptide nanoprobe in detection of oral bacteria and identification of forensic saliva.

Preferably, the method for forensic saliva identification comprises:

s1: detection of oral bacteria in standard solutions: preparing oral bacteria solutions with different concentrations, adding the oral bacteria solutions into the silicon carbide @ BSA-antibacterial peptide nanoprobe, reacting for 0.1-6 h, measuring the fluorescence intensity of the oral bacteria solutions at 410nm, drawing a linear relation between the bacteria concentration and the fluorescence intensity, and preparing a standard solution curve so as to realize the detection of the oral bacteria in the standard solution;

s2: detection of oral bacteria in saliva samples: preparing saliva samples, adding oral bacteria with different concentrations into the saliva samples, respectively carrying out 3 parallel tests on each sample, adding the saliva samples containing the oral bacteria into the silicon carbide @ BSA-antibacterial peptide nanoprobe, measuring the fluorescence intensity of the saliva samples at 410nm after reacting for 0.1-6 h, and establishing a standard working curve for detection through comparison of the fluorescence intensity before and after the reaction, thereby realizing detection of the oral bacteria in the saliva samples;

s3: identification of forensic saliva samples: saliva, blood, urine and vaginal fluid samples in the actual forensic case are collected, pretreated, and then qualitatively and quantitatively detected by the silicon carbide @ BSA-antibacterial peptide nanoprobe for oral specific bacteria in different body fluid samples, and the oral specific bacteria can be detected only in the saliva, so that the saliva can be identified from the forensic body fluid samples.

The invention has the beneficial effects that:

1. the fluorescence sensor based on the silicon carbide @ BSA-antibacterial peptide nanoprobe has the advantages of simple preparation, good biocompatibility and good light stability, the antibacterial peptide is a polypeptide with a specific binding effect on bacteria, and the coupling silicon carbide nanoprobe realizes specific response on oral bacteria, so that the method has excellent analysis performances such as high sensitivity, wide linear range, low cost and the like, and has good practical prospects in the fields of forensic microbial detection and forensic saliva identification detection.

2. The invention adopts fluorescence resonance energy transfer FRET technology to realize quantitative detection of streptococcus salivarius, and when bacteria enter an oral cavity, because fluorescence resonance energy transfer FRET effect exists between the silicon carbide @ BSA-antibacterial peptide nanoprobe coupled with the antibacterial peptide and the oral bacteria, the luminescence peak position of the silicon carbide @ BSA-antibacterial peptide nanoprobe is quenched, when the binding capacity of the oral bacteria and the antibacterial peptide is stronger, the FRET effect between the silicon carbide @ BSA-antibacterial peptide nanoprobe and the combination of the oral bacteria is destroyed, and the fluorescence of the silicon carbide @ BSA-antibacterial peptide nanoprobe is quenched, thereby realizing the rapid quantitative detection of the oral bacteria.

3. The silicon carbide @ BSA-antibacterial peptide nanoprobe fluorescence sensor constructed by the invention can be quickly and simply used for quantitative analysis of the content of microorganisms in forensic samples, and the fluorescence signal can be 1.0 multiplied by 10²cfu/mL-1.0×10⁷The method realizes quantitative response to oral bacteria within the concentration range of cfu/mL, has excellent analysis performances of high sensitivity, wide linear range, no need of sample pretreatment, high detection speed, low cost and the like, and well overcomes the defects of complex operation, long reaction time, low sensitivity and the like of the traditional method.

4. The reagent for preparing the silicon carbide @ BSA nano fluorescent probe is a common chemical reagent, is safe and nontoxic, is environment-friendly and pollution-free, and can be widely applied.

5. The method for determining the bacterial concentration can rapidly and accurately determine the bacterial concentration, applies the fluorescence technology to forensic saliva identification for the first time, and provides a method for forensic workers to accurately infer the death reason of the dead.

6. Compared with the saliva amylase test method in the prior forensic identification, the method adopts the saliva qualitative result analysis to be superior to the saliva amylase result, and realizes the forensic saliva identification.

Drawings

FIG. 1 is a schematic flow diagram of the present invention.

FIG. 2 shows the change in fluorescence intensity of the system in the presence of different concentrations of Streptococcus salivarius (10)²-10⁷cfu mL^-1)。

FIG. 3 is the relationship between the fluorescence intensity of the target Streptococcus salivarius and the system obtained from FIG. 2.

FIG. 4 shows the change in fluorescence intensity of the system in the presence of different concentrations of Streptococcus salivarius (10)³-10⁸cfu mL^-1)。

FIG. 5 is the relationship between the fluorescence intensity of the target Streptococcus salivarius and the system obtained from FIG. 4.

FIG. 6 shows the change in fluorescence intensity of the system in the presence of different concentrations of Streptococcus sanguis (10)²-10⁷cfu mL^-1)。

FIG. 7 is the relationship between the fluorescence intensity of the target Streptococcus sanguis and the system obtained from FIG. 6.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of those skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination of technical solutions should not be considered to exist, and is not within the protection scope of the present invention.