Abstract
This thesis focuses on developing a sensitive gold nanoparticle (AuNP)–based amplification and magnetic separation method for the detection of oligonucleotide sequences and mercuric ion. In the study on oligonucleotide sequences analysis, the assay relies on (i) the sandwich-type binding of two designed probe sequences that specifically recognize the target oligonucleotide sequences, (ii) magnetic bead separation, and (iii) AuNP-based ICP-MS amplification detection. To enhance the analytical signal and minimize the background signal resulting from nonspecific binding, we performed a series of experiments to evaluate the effects of various parameters (the concentration of the capture probe; the time required for hybridization; the number of washings required to eliminate nonspecific binding). Under the optimized conditions, the detection limit was 80 zmol (corresponding to 1.6 fM of the target sequence in a sample volume of 50 μL). Compared with the “gold standard” methodology (plaque assay) for the quantification of dengue virus, our method has the capability to allow early detection of dengue virus in complicated and small-volume samples, with high specificity, good analytical sensitivity, and superior time-effectiveness. Moreover, this simple, high sensitive and selective method was also applied to simultaneously quantify two different types (type 1 and 2) of dengue viruses through the utility of AuNP and AgNP probes. Based on the experimental results, our developed nanoprobing technique for quantifying and typing viruses was proven to be applicable for the simultaneously determination of two types of analyte oligonucleotides in 50 μL of samples with detection limit of 50 femtomolar. Because the involvement of a shorter hybridization and ICP-MS measurement, a relatively simple and rapid (ca. 2 h) procedure for the quantification and typing of two viruses was provided. In the study on mercuric ion analysis, we have developed a sensitive gold-nanoparticle-based (AuNP-based) graphite furnace atomic absorption spectrometry (GFAAS) amplification and magnetic separation method for the detection of mercuric ions (Hg2+). The assay relies on (i) a sandwich-type structure containing two thymine–thymine (T–T) mismatches for selectively recognizing Hg2+ ions; (ii) magnetic beads for homogeneous separation; and (iii) AuNP-based GFAAS amplification detection. The limit of detection (LOD) of this assay is 0.45 nM (0.09 □g L-1) □ one order of magnitude lower than the United States Environmental Protection Agency (US EPA) limit for Hg2+ in drinking water. Furthermore, because a shorter hybridization step and a simpler AuNP-based GFAAS amplification detection were employed, a faster analytical run time allowing us to analyze a batch of 24 samples within 0.5 h. We demonstrated the feasibility of the developed approach for the determination of Hg2+ in urine and aqueous environmental samples. In addition, to further improve the analytical sensitivity, a large DNA–gold-nanoparticle (AuNP) probe coupling with the analytical principle described above was also established for the detection of Hg2+. Our findings indicated that using large AuNP (55 nm) probe could increase the ΔTm and provide a sharper melting profile of the sandwich structure when compared with that of AuNP (20 nm). That is, using a larger AuNP probe to detect the Hg2+ can improve the analytical sensitivity of the detection system. Under the optimized conditions, the detection limit of Hg2+ was 28 pM (5.6 ng L-1) and the obtained relative standard deviations (RSD) for different Hg2+ concentrations were in the range of 4.1% to 13.2%. The acceptable recoveries obtained for both spiked tap water (88.7%) and urine samples (97.3%) indicated that this approach could be used to accurately determine Hg2+ concentrations in both samples.