The Application Of Carbon Nano-materials In Detection Of Pathogens

In recent years, the applications of carbon materials have been wildly dispersed into every aspect of biology, in which they displayed brilliant advantages such as biocompatibility, low cost, nontoxic and stabilization. These nano-carbon materials include mainly carbon nanotubes, graphene, carbon nano-particles. They can be separated into two parts based on the property of fluorescence. In this article, we fabricated a molecule beacon, which contains three parts:carbon nano-particles (CNPs), black hole quencher1(BHQ1) and a specific sequence of DNA. Using such molecule beacon, we have developed two methods to detect two kinds of pathogens(Salmonella enteritidis and Salmonella paratyphi A). Major works are as following.1. Detecting the16S rRNA of S. enteritidis. Ribosomal ribonucleic acid (rRNA) molecules have been used to improve the detecting ability of sensors because they allow the design of taxon-specific oligonucleotides. Hybridization using oligonucleotides directly as sensor for16S rRNA could be an interesting alternative to identify S. enteritidis much more rapidly than current methods. In this section, we have developed a biosensor based on oligonucleotide-specific hybridization and fluorescence resonance energy transfer (FRET). It is well known that SWNTs can interact with ssDNA non-covalently through "π-π" stacking interactions between the nucleotide bases and them. The binding of the complementary DNA sequence of16S rRNA and target unzipped the connection between the CNT-DNA and SWNTs, because of the probe was energetically more stable when hybridized with16S rRNA, which is a substrate of Nb.BbvC I. Then the nicking enzyme quickly binds to and cleaves the molecule beacons. The molecule beacon is then too short to maintain its conformation, so when the CNPs and BHQ1disconnected, a fluorescence signal appears. In the absence of the target, molecule beacons will not be hybridized and cleave the3’end of the CNT-DNA sequence is anchored onto the SWNTs through a covalent bond, which prevents the release of the sequence from the SWNTs. The biosensor exhibited a linear relationship with the concentration of S. enteritidis ranging from102to3×103CFU/mL in water and from1.5×102to3×103CFU/mL in milk. The present results indicated that our FRET-based detection system can be widely employed for the effective detection of pathogens.2. Detection of Salmonella paratyphi A using aptamer. This method was based on FRET, specification of EXOIII and the affinity between aptamer and target. In this detecting system, the aptamer locating on the DNA probe can twine around the pathogen, while other two parts of probe (located on3’ end and5’ end) will still dislocate. This complex structure can be regard as "Ω". Ultimately, these two parts and molecule beacon fabricated a dsDNA, which is a substrate of EXOⅢ. Continually, the enzyme quickly bound to and cleaved the molecule beacon. The latter is then too short to maintain its conformation, resulted in producing fluorescence signals when the CNPs and BHQ1disconnect. We validated that this method can be a specific way to detect Salmonella paratyphi A, and that the method can be wildly used to detect other bio-molecules when replacing the specific sequence of the probe.