Preparation Of Novel DNA-based Diagnostic Probes And Their Applications In Vitro Tumor Detection

The incidence rate of malignant tumor increases year by year due to the deterioration of living environment and the unhealthy life style of people,so it is important to improve the sensitivity and specificity of vitro tumor detection.In recent years,the detection of tumor cells and tumor biomarkers have attracted wide attention among researchers.They have designed a variety of probes for the detection of genes,bio-molecules and tumor cells of the tumor.However,current technologies are often unable to take into account the sensitivity and easy operation,or some of the detection methods demand for stringent instruments.In view of these problems,we designed two noval diagnostic probes based on DNA in this paper which can be simply operated with high sensitivity in tumor diagnosis.In order to improve the sensitivity of raman cellular imaging of tumor cells,we prepared GCTs(Au@Au core-shell SERS tags)mediated by DNA,which are used for target imaging of tumor cells.By controlling the thickness of the shell,we have firstly investigated the size-dependent SERS activity of GCNPs(Au@Au core-shell SERS nanoparticles)and found that under the same laser wavelength,the maximum Raman enhancement was achieved at a certain threshold of particle size(~76 nm).The nanogap-containing GCTs were prepared via the functionalization of hyaluronic acid(HA)on GCNPs,which can recognize CD44 receptor,a tumor-associated surface biomarker.According to our results,GCTs showed good dispersity and stability during the whole process.The internal nanogap enabled a good control of the hot spots' distribution,thus lead to a good uniformity and reproducibility of SERS signal.And they can be readily functionalized to permit simultaneous multiplexed determination of two or more biomarkers on the cell surface.For further implementation of the quantitative detection of tumors,and in order to improve the sensitivity of tumor detection by the detection of markers,we combined RCA-based origami technology with conventional magnetic bead-based ELISA method,and improve on the basis to fully take advantage of the high amplification efficiency of RCA and the strong controllability of DNA origami.DNA origami based-system for detection of tumor markers with signal amplification ability has been designed.In our work,we have prepared DNA belts which were then characterized by agarose gel electrophoresis and AFM.The pure product DNA belts occurred to be ~16nm in width,~1nm in height.DNA belts were then employed for detection of the specific detection of tumor markers in order to further reduce the detection limit of tumor markers.The two probes prepared in this paper are simple and easily to operate,the detecting instrument is easy to obtain,and the identification sensitivity and specificity of tumor cells and tumor markers are high.Experiments show that,the as-prepared GCTs not only have good biocompatibility and stability compared with the fluorescent probes,but also show good uniformity and reproducibility of SERS signal,thus they are ideal candidates for simultaneous bioimaging and SERS detection for the early diagnosis of cancers.On this basis,DNA origami based-detection system with signal amplification ability have achieved the sensitivity of 50 aM for tumor markers detection,which is comparable to the nanoparticle-based biobar-code approach and two orders of magnitude greater than immuno-PCR and immuno-RCA methods.It is of vital significance to improve specificity and sensitivity in the early detection of tumor.