Development and Applications of Capillary Electrophoresis and Functionalized Gold Nanoparticles
Date Issued
2010
Date
2010
Author(s)
Chen, Chuan-Kuo
Abstract
Three different analytical approaches are demonstrated in this thesis. First, naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and -amine derivatives were separated and detected by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF) detection using poly(ethylene oxide) (PEO) containing cetyltrimethylammonium bromide (CTAB). In the presence of CTAB and acetonitrile (ACN), adsorption of PEO on the capillary wall was suppressed, leading to generation of a fast and reproducible electroosmotic flow (EOF). In order to optimize separation resolution and speed, 100 mM Tris–borate solution (pH 7.0) containing 20 mM CTAB and 25% ACN was used to fill the capillary and to prepare 1.2% PEO that entered the capillary via EOF. The analysis of 14 NDA-amino acid and -amine derivatives by this approach was rapid (< 4 min), efficient [(0.9–6.4) × 105 theoretical plates], and sensitive [the LODs (S/N = 3) range from 9.5 to 50.5 nM]. The RSD values (n = 5) of the migration times and peak heights of the analytes for the intraday analysis were less than 1.5 and 1.2%, respectively. The practicality of this approach was validated by quantitative determination of 10 amino acids and amines in a beer samples within 4 min. Secondly, a novel, label-free, colorimetric assay – using fibrinogen (Fib) and gold nanoparticles (Au NPs) –was developed for the highly selective and sensitive detection of thrombin. Addition of fibrinogen to a solution of Au NPs (average diameter: 56 nm) led to ready conjugation, forming Fib–Au NPs through electrostatic and hydrophobic interactions. Introduction of thrombin (a serine protease) into the Fib–Au NPs solutions in the presence of excess fibrinogen induced the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. After centrifugation, the absorbance at 532nm of the supernatants decreased upon increasing the concentration of thrombin. This Fib–Au NP probe provided high sensitivity [limit of detection (LOD): 0.04 pM] for thrombin, with remarkable selectivity over other proteins and proteases. The range of linearity for the absorbance against the thrombin concentration was 0.1–10 pM (R2 = 0.96). This approach provided an LOD for thrombin that is lower than those obtainable using other nanomaterial- and aptamer-based detection methods. The utility of this Fib–Au NP probe was validated through separate analyses of thrombin and Factor Xa at picomolar levels in plasma samples—without the need for sample pretreatment. This technique appears to have practical potential in the diagnosis of diseases associated with coagulation abnormalities and cancers (e.g., pulmonary metastasis). Last, detection of DNA hybridization was demonstrated using a Fib-Au NPs-based assay. Two thrombin binding aptamers (TBAs)－TBA15 (15 bases long) and TBA27 (27 bases long)－that are specific towards thrombin were used to form a TBA15-TBA27 assembly in the presence of a complementary DNA (cDNA). The TBA15-TBA27 assembly relative to TBA15 and TBA27 provided a greater inhibition activity for thrombin, showing bivalent binding capacity. The activity of thrombin decreased upon increasing the concentration of cDNA. This new sensing strategy provides high sensitivity [limit of detection (LOD): 25 pM] and remarkable specificity for cDNA. To test the practicality, another probes [TBA15’ (P-TBA15’) and TBA27’ (P-TBA27’)] were used for the detection of the single-nucleotide polymorphism (SNP) responsible for hepatocellular carcinoma. Unlike conventional approaches, this method requires neither postsynthetic modification of the probe oligonucleotides nor precise temperature control for SNP typing.
Subjects
capillary electrophoresis (CE)
gold nanoparticles (Au NPs)
biosensor
thrombin
aptamer
single nucleotide polymorphisms (SNPs)
SDGs
Type
thesis
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