Nano Fluorescent Probe For Intracellular Simultaneous Determination Of Tumor Markers

Cancer is a leading cause of death worldwide and accounted for several millions ofdeaths every year. The survival of cancer patients is strongly associated with the tumorstage at the time of diagnosis. Identifying the cancer at cell level in early stage beforemetastasis holds great promise to increase the survival of cancer patients. Toward thisgoal, a major focus of research is to estimate the abnormalities of gene expression inliving cells. Tumor mRNA as a specific marker has been widely used to assess themigration of tumor cells locally or into the bloodstream. Changes in the levels of tumormRNA expression have been correlated with tumor burden and malignant progression.Detection of tumor mRNA markers in intact cancer cells provides new tools foridentifying cancer cells in clinical samples.Recently, a variety of techniques have been exploited to monitor tumor mRNA.Among these methods, fluorescence imaging analysis offers an appealing approach forthe detection of cancer at cell level, which may be of prognostic significance. Manyfluorescence probes have been synthesized for detection and imaging of mRNA incancer cells and most of the research focuses on detecting single mRNA, which mayyield“false positive”results and limits the development of intracellular mRNA imagingand detection. Notably, cancer is associated with multiple tumor mRNAs and somemRNA markers are expressed in normal cells. Simultaneous detection of multipletargets brings new opportunities for improving the accuracy for early cancer detectionover single marker assays. Although various types of platform for multiple targetsdetection have been developed, none have been applied for the imaging of three or moremarkers in living cells. Therefore, it is highly desirable to develop multiple tumormRNAs for intracellular imaging which could promote the progress of early cancerdetection.In this study, the recent research progress on the application of gold nanoprobes inbiotechnology and multiplex analysis of tumor markers are reviewed. Based on theabove, we carried out the following work: 1. We developed a multicolor fluorescence nanoprobe to simultaneously detectthree intracellular tumor mRNAs based on nanoflares. The nanoprobe consists of goldnanoparticles (Au NPs) functionalized with a dense shell of recognition sequences(synthetic oligonucleotides) hybridized to three short dye-terminated reporter sequencesvia gold-thiol bond formation. The recognition sequences contained 21-base recognitionelement to three specific mRNA transcripts: c-myc mRNA, TK1 mRNA and GalNAc-TmRNA, which were rarely reported to be imaged in living cells previously, though theywere important markers in cancer cells. We succeessfully applied the nanoprobe forsimultaneously detecting multiple mRNAs in cancer cell lines (MCF-7 & HepG2) andtheir normal cell lines (MCF-10A & HL-7702).2. Quantum dots (QDs) have several distinctive advantages over conventionalorganic dyes such as high quantum yields, narrow emission peaks and highphoto/chemical stability. In particular, QDs can be excited with a single excitation light,which makes QDs appealing for the design of multiplex analysis systems. In this paper,we used green, yellow and red emission of CdTe quantum dots labelled molecularbeacons assembled on the gold nanoparticles to detect different tumor mRNAs. QDsstay in the“quenched”state in the absence of targets but fluorescence was restoredwhen they meet the specific targets. This strategy combines the unique fluorescenceproperties of QDs and good specificity of molecular beacons. We anticipate that thenanoprobe will provide new opportunities for detection and imaging of multiplemarkers in living cells.