Preparation And Application Of New Nanoprobes In DNA Detection

Detection of DNA has become increasingly important in a variety of areas including medical diagnostics, food safety and anti-bioterrorism. Conventional polymerase chain reaction (PCR) is extremely sensitive. However, because of the high cost of equipment, complicated operation and contamination, it is not suitable for wide-spread real-time detection. Much effort has been devoted to the development of more cost-effective, more convenient, more sensitive,(toward the detection of several DNA molecules) real-time detection method.Nanomaterials provide almost unlimited combinations of various compositions, sizes, dimensions and shapes of materials, which can be tailored to couple different biomolecules in order to develop nanoprobes with desired properties. Recombination of different methods based on various nanoprobes will provide more amplification strategies, thus new chance for DNA detection.Despite tremendous progress, there remain several challenges for nanoprobe-based DNA detection technologies:(1) the use of nanomaterials always introduces heterogeneous interfaces in bioassays that are usually in homogenous solution, which might result in several problems (e.g. slow binding kinetics and low recognition efficiency);(2) many ultrasensitive assays require multiple steps that significantly increase the operation complexity;(3) elimination of non-specific binding is critically important to avoid interferences, particularly for detection in complex biological matrixes;(4) the ultrahigh amplification offered by nanomaterials often lacks generality, therefore bioassay reactions should be individually optimized. Therefore, design of new nanoprobes and new detection strategies is critical.Based on these conditions, we started our research in the development of new nanoprobes in DNA detection:(1) We have prepared metal-organic hybrid particles (MOHPs) with sub-stoichiometric metal contents. MOHPs are constructed by an organic molecule (E-4,4’-di(N-(2-aminophenyl)amino)stilbene, ED APS) and metal ions (Cu(Ⅱ) or Fe(Ⅲ)). Organic molecule particles (OMPs) of EDAPS were first prepared by precipitation in the preparation of MOHPs. Then metal ions were added. The introduction of coordination interaction resulted in a heat-assisted morphology transformation to sphere-shaped MOHPs. The size of MOHPs could be tuned by the tuning of precipitation speed, while the metal contents in MOHPs is tunable through both the initial precipitation speed and the initial concentration of metal ions. The final yielded MOHPs contained sub-stoichiometric metal contents, which is between that of OMPs and coordination polymer particles (CPPs). MOHPs provide new opportunity toward the control of particle properties through the tuning of particle composition. The characterization of MOHPs and MOHP preparation process also provides important insight into the inter-relationship between the seemingly disparate classes of particles (OMPs and CPPs).(2) DNA probe based on MOHPs (DNA-modified MOHPs, DNA-MOHPs) have been prepared. Every DNA-MOHP contained many EDAPS molecules and relatively less metal ions. Aqueous suspension of DNA-MOHP is stabilized by the increase of surface charge from metal ions. In a typical assay, hybridization with target DNA could bind DNA-MOHPs to the surface of DNA-modified glass slide. Then, blue fluorescence could be observed through the dissolving of DNA-MOHPs. Compared with organic fluorescent dyes, better sensitivity was achieved. While compared with the harsh synthetic condition of quantum dots, the preparation of MOHP was much easier. Additionally, the detection system based on DNA-MOHPs could differentiate complementary DNA strand with DNA strands containing single-base mismatches.(3) Aminated polystyrene nanospheres (APSNSs) were synthesized and used for the preparation of positively charged DNA-modified APSNSs (DNA-APSNSs). Based on hybridization-driven agglutination of DNA nanoprobes (DNA-APSNSs), we developed an ultrafast kinetic DNA hybridization assay system. In this system, the onset time of a visually identifiable, turbidity-definitive, and kinetic threshold state (state of threshold turbidity) was employed in the characterization of DNA hybridization kinetics, and a fast DNA assay within the timeframe of minutes was achieved. By the onset time of threshold turbidity, DNA quantification could be performed. Additionally, DNA strands with single-base mismatches could be differentiated with complementary target DNA. The judicious selection of the size and surface groups of APSNSs ensured the ultrafast DNA hybridization assay. The positive surface charge fundamentally improved the hybridization kinetics caused by the screening and reducing of the electrostatic repulsion between negatively charged DNA strands. More importantly, the kinetic visual protocol complements conventionally used thermodynamic strategies and provides an entry point for the circumvention of assay issues associated with ill-defined thermodynamic endpoints.