Ultrasensitive Detection Of Gene Expression Based On Molecular Beacons And Ultrasmall DNA & PH Sensors

The investigation range of life science had reached the sca1e of nanometer and the leve1 ofsing1e cell, singe molecue. How to obtain the useful biochdrical informaton on this scale isthe new tren in the research fie1d of analytical chehascy Therefore, single molecule detection,sing1e cell detection, DNA ~ and the shaPle DNA analySis were one of the main researchdireeons ofanalytcal chendscy Nove1 molecular probe and ultrasmalI biosensor for real tiIneand in vivo detection has been the focuses in the research field of analytical chendstryAccording tO the above mentioned advanced direetions, tWo pnd of inveshgations hasbeen pdrirmed in thes thesis. Ih ped one, a nove1 mo1ecuar probe onolecuar beason (MB)was develoPed tO establish a stwle and PreCise method for the quanitative analySis of genticmaterials such as ING1 InRN frOm differen cells and RNA fonn Tobacco Mosaic Virus. TheMilization method fOr MB was Studied fOr the fabrication of DNA biosensor and DNAchiP. Ih pall tWo, a series of inunobilization method such as sol-gel method and lighpolererization method were investigaed fOr the otilization of bio-molecues andfluOrescence dye. A series of ulthesmall OPtical fiber biosensors such as pH and DNA weredeveloPed. The pH ultra-smal1 biosensor was successfuly used to detect pH value in singie cell.Wr biosensors fOr proten and glucose were also PrOposed.Ped one: In chaPter l, three ams were synthsized to find a suitable probe to hybridize theexPression of ING1 gene, a recentiy identified tUmor SUPPressor gene. The looP sequence ofMB1 and MB2 were the anti sense and sense sequence ofING1, reSPectively The sequence ofMB3 was a piece of ssRNA sequenCe in Tobacco Mosaic Virus, which had no analogical tohUman gene. MBl was the moSt suitable Probe because MBl had the highest fluorescenceenhancemen after hybridizing wtth RNA extrated frOIn normal cell. A stwle and Pngisemethod was proPosed for the qUantitative analysis of mGl twA from nasoPharyngralcarcinoma (NPC) cell lines and normal ce11s. The exPerimen reSUits shOWed that theconcenthaon of ING1 InRNA in normal cells was higher than that in tUmr cells. ms resultwas consistwt tO the result of RT-PCR detection.Ih chaPter 2, the selected MBl in chaPter l was dellvered into ltheg hUman N'PC cells wunliPosome as a cAner. Ffuorescence was observed in the cytoPlasm and the region near thenucleolus of the cell. ms shOwed that MB hybridized wth the expression of INGl. The resultindcated this method had a pototial tO monitor the eXPression of ther suPPressor gene atsing1e cell level. INGl Plasndd was a1so prePared and delivered tO the NPC cell. Subsequenly,VMBl was also de1ivered to the cell and intensity fluorescence was observed bY nuorescenceconfOcal ndcroscope. The degree of flUorscence enhancemen can be wttatively obtaindthrough Anscence sPectr8. ms method had a potenial to develop a novel diagnse methodfor drior cell.lh mpter 3, the dCtection of plam virus was developed by the use of MB. An antisensemolecular beacon (MBl) was designed fOr the dCtonninon of the genome RNA of TobaccoMosaic Virus with no need of stringen isolation and purification of the virus prtc1es and RNAas well, Which benefited from the ProIninen SPecificity and selectivity of molecular beacon.W sense Probe (MB2) was also twsized for control eXPerimens. The resuh twedthat MB designed in consisten the the comPlndny sequence of a sechon of theconservative regions of TMV-RNA was suitable fOr the direet assay of TMV-RNA. Anothercontrl exPeriIned had been pefformed to testify that our resuit was re1iable or not.In chaPter 4, a novel biotinyated MB (5'-MCCT AGC TCT AAA TCG CTA TGGTCG CGC (biotindTAG GMCYL-3') was designed and Synthesized based on thesequence of 6-aCtin. Aner investigaAng the SPectri of MB, the interation of am with itScDNA in sofution was also stUdied. An oPical DNA biosensor based on strcytavidin-biotininteraCtion was proposed by bridge driobilization...