| Pathogenicity aroused by food-borne pathogen is one of the most common problems in the world today. Microbial food poisoning can be caused by bacteria, viruses, fungi and protists, in which most of bacterial infections are caused by Salmonella. Currently, eggs’and meat products’ contaminations and food poisoning resulted from Salmonella have become a serious food safety issues that posed the adverse influence on economic development and human health. With the increasing attention to food safety problem, higher requirements were proposed for the development of food safety detective techniques that are fast, accurate, sensitive and efficient. However, the traditional detection methods have many limitations, such as time-consuming, expensive, complicated operation, low sensitivity and specificity, which can not satisfy the new requirement of rapid detection of Salmonella. Therefore, it is particularly important to establish simple, efficient, rapid and sensitive detection methods for Salmonella supervision. Carbon nanotubes have many application prospects in biosensors, drug delivery and other fields because of their unique chemical, electrical, and mechanical properties. Especially in recent years, they, as a broad-spectrum fluorescent quencher, have been gained more and more attention, becoming one of the most promising nano-materials. In this paper, upon studying their properties, biosensors were constructed for Salmonella detection based on the bio-functionalized carbon nanotubes. In addition, graphene, another carbon material similar to them, had been also applied in the detection of ATP in Salmonella. It was indicated that these sensors have high sensitivity, specificity, stability and other advantages. Finally, given the status that the Salmonella’s biofilm is difficult to remove and treat after its formation, an aptamer was well screened out for controlling the biofilm. Based on the above descriptions, the main contents list as follows:(1) Detection of S. typhimurium carrying SSeC gene located in pathogenicity island. A novel method has been fabricated to detect the bacteria carrying SSeC gene by using carbon nanotubes as a carrier and fluorescent quencher, molecular beacon as recognition element. In this detection, amino modified molecular beacons were convalently coupled to carboxyl modified carbon nanotubes through EDC/Sulfo-NHS chemistry, generating bio-functionalized carbon nanotubes. In the absence of target, the fluorescence of daunorubicin was fairly week as result of dual fluorescence quenching. On the contrary, the daunorubicin was competed from the beacon due to the target-induced formation of rigid structure between the loop structure of the MB and the target sequence, which resulted in a decrease in the effect of dual fluorescence quenching, thereby the fluorescence intensity increased substantially. The quantity of the target was achieved by fluorescence increment. However, the signal to noise ratio was much lower than the target’when the mismatches were added. These results clearly demonstrated a high selectivity and specificity for the biosensor. The experimental results showed that the recovery of fluorescence of daunorubicin is proportion to the concentration of the target DNA with the range0.2-0.7μM and the low detection limit is50nM.(2) The research for S. paratyphi A. In this work, the flagellin protein were obtained from S. paratyphi A by acid hydrolysis, and then single-stranded DNA aptamers that are highly specific to enterotoxigenic Salmonella paratyphi A were obtained from an enriched oligonucleotide pool using14rounds’SELEX to target the flagellin protein. DNAMAN software was used to analyze the similarity of their primary structure and secondary structure prediction, the selected aptamers can be divided into four families according to the similarity of secondary structure and repeatability of primary structure. By comparing the Kd values of typical aptamers picked out of the four families, an optimal aptamer was ultimately screened out. A probe containing the DNAzyme sequence and FITC-labeled truncated aptamer sequence wrapped onto carbon nanotubes was employed as a dual probe for S. paratyphi A detection.The results displayed that the milk spiked with S. paratyphi A could be quantified by fluorescence and absorbance increment, and the low detection limits increasing to105CFU/mL by fluorescence and106CFU/mL by spectrophotometry. The combination of fluorescence and spectrophotometry offers an accurate, reliable and non false-positive way for detecting Salmonella paratyphi A and has potential for detecting other pathogens and materials.(3) Label-free fluorescent aptasensor based on a graphene oxide self-assembled probe for the determination of adenosine triphosphate. The feasibility of this approach was first demonstrated that the fluorescence intensity of probe/graphene oxide did not increase when the cDNA was mixed only, while the fluorescence enhancement occurred only under ATP present. Therefore, the enhancement of the fluorescence was only related to the number of added ATP, making it can be used to detect ATP quantitatively. The fluorescence intensity considerably increased with the increasing concentration of ATP from0to800nM, which enabled ATP to be detected in a linear range of10-700nM with a low limit of lnM. In addition, GTP, CTP and UTP, analogues of ATP, were selected to verify the selectivity of this approach. The results indicated that only ATP can induce substantial fluorescence enhancement rather than the analogues, which implied that this aptasensor could detect ATP with high specificity. Compared to other conventional detection methods, the molecular recognition technology used for ATP detection was not only high sensitivity and specificity, but also simple, low cost and good stability, which paved the way for the development and use of other detection methods.(4) The study of controlling the biofilm formation by aptamer. Single-stranded DNA aptamers with high specificity to S. choleraesuis were obtained from an enriched oligonucleotide pool using14rounds’SELEX procedures. During the SELEX, S. paratyphi A and S. typhimurium were used as counter-selection targets at round10and11to improve its affinity. After the Kd was measured, an optimal aptamer was ultimately screened out, and the selected biotinylated aptamer-conjugated with streptavidin-modified magnetic beads, were then used to capture their binding targets on the bacteria. After mass spectrometry, the flagellin was ultimately identified as target captured by the aptamer. As motility and initial attachment mediated by flagella plays a critical role in biofilm formation, a functionalized aptamer was developed to control their movement, reducing the biofilm formation. Inhibition experiments, inverted microscope and atomic force microscope observation demonstrated that the selected aptamer was able to control the biofilm formation, more importantly, it could promote inhibitory effect of antibiotic against biofilm formation. Consequently, this method not only expanded the application of aptamer, but also reduced the use of antibiotics, which made it sense in decreasing drug resistance strains. |
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