Investigation of dual-layer membrane cloaking method by surface plasmon resonance for direct chronoamperometric immunoassay of serum sample.
ABSTRACT A "dual-layer membrane cloaking" (DLMC) method was developed to construct disposable electrochemical immunosensor for direct determination of serum sample. Mouse IgG (MIgG) molecules were firstly immobilized on a substrate. After the formation of a didodecyldimethylammonium bromide (DDAB) membrane on the MIgG modified substrate, an additional bovine serum albumin (BSA) thin layer was formed to build a BSA/DDAB dual-layer membrane (DLM). When alkaline phosphatase conjugated anti-mouse IgG antibodies (anti-MIgG-ALP) in human serum were incubated on the substrate, anti-MIgG-ALP was recognized specifically by the immobilized MIgG while all nonspecifically adsorbed proteins were selectively removed together with BSA/DDAB DLM by 5% Triton X-100 (v/v) before final measurements. The BSA/DDAB DLM was characterized and optimized by surface plasmon resonance (SPR) technique, and further employed in a disposable immunoassay based on an ITO chip. Under optimal conditions, MIgG in human serum was directly detected in the range of 2.0-18.0 ng mL(-1) without dilution or separation. A limit of detection as low as 0.922 ng mL(-1) (6.15 pM) was obtained. The proposed DLMC method can efficiently prevent the penetration of matrix proteins through single cloaking membrane and completely eliminate nonspecific adsorption. It has great potential in providing a versatile way for direct determination of serum sample with ultra-sensitivity.
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ABSTRACT: Self-assembled monolayers (SAMs) of thioglycolic acid, 3-mercaptopropionic acid, 11-mercaptoundecanoic acid, 16-mercaptohexadecanoic acid and bis(carboxydecyl) disulfide are formed on gold electrodes, respectively. The surface pKas of the ω-terminal acid groups in these monolayers are determined using electrochemical titration, to be 6.1, 5.3, 7.3, 7.9 and 7.3, respectively. The effect of chain length on the surface pKa values of ω-carboxy alkanethiol SAMs is described. The SAM has a strong influence on the heterogeneous electron transfer rate constant, ks , of the electroactive probe Fe(CN)63−. The ks values of Fe(CN)63−, determined with cyclic voltammetry at the pH values of the inflection point of the electrochemical titration curves for the four monolayers with carbon number 2, 3, 11 and 16, are calculated to be 7.4 × 10−3, 4.6 × 10−3, 4.4 × 10−3 and 2.6 × 10−3 cm s−1, respectively. The effect of time of immersion for monolayer formation on the defects present in the SAMs formed is discussed. The SAMs of bis(carboxydecyl) disulfide and 11-mercaptoundecanoic acid show the same surface pKa and ks values and thus possess the same surface properties.Physical Chemistry Chemical Physics 01/2001; 3(17):3769-3773. · 4.20 Impact Factor
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ABSTRACT: Hydrophobically substituted dextran (dextran phenoxy, DexP) and dextran phenoxy−poly(ethylene oxide) copolymers (DexP−PEO) have been used to modify the surface of polystyrene latex particles. To avoid polymer desorption in the presence of hydrophobic species such as proteins, the adsorbed layer was stabilized by chemical cross-linking and then characterized in terms of adsorbed amount, thickness, and stability. The interfacial concentration in anchoring phenoxy groups and the PEO grafting density were both varied, and their effects on nonspecific bovine serum albumin (BSA) adsorption were examined. It was found that the most important parameter in preventing BSA adsorption is the number of interactions between the adsorbed dextran and the surface, even in the presence of DexP−PEO layers with high grafting ratios of PEO chains. We also examined the ability of dextran layers to bind specifically concanavalin A (Con A) as the Con A molecule exhibits a good specific affinity for glucose-containing carbohydrates. Flocculation of DexP-modified particles by Con A was observed in the course of the experiments. All of these results are discussed in relation to the importance of polymer architecture and surface−protein interactions in protein rejection by dextran and dextran−PEO coatings.Langmuir 06/2001; 17(14). · 4.38 Impact Factor
- Analytical Chemistry 11/1979; 51(13):2282-2283. · 5.83 Impact Factor