Yaw-Kuen Li

Silicon nanowire field-effect-transistor based biosensors: From sensitive to ultra-sensitive

Silicon nanowire field effect transistors (SiNW-FETs) have shown great promise as biosensors in highly sensitive, selective, real-time and label-free measurements. While applications of SiNW-FETs for detection of biological species have been described in several publications, less attention has been devoted to summarize the conjugating methods involved in linking organic bio-receptors with the inorganic transducer and the strategies of improving the sensitivity of devices. This article attempts to focus on summarizing the various organic immobilization approaches and discussing various sensitivity improving strategies, that include (I) reducing non-specific binding, (II) alignment of the probes, (III) enhancing signals by charge reporter, (IV) novel architecture structures, and (V) sensing in the sub-threshold regime.

CMOS chip as luminescent sensor for biochemical reactions

We describe a novel biochemical sensing method and its potential new biosensing applications. A light-sensitive complementary metal oxide semiconductor (CMOS) chip prepared through a standard 0.5-/spl mu/m CMOS process was developed for measuring biochemical reactions. A light producing enzymatic reaction catalyzed by horseradish peroxidase (HRP) was designed as a platform reaction to determine the concentration of hydrogen peroxide (H/sub 2/O/sub 2/) by the CMOS chip with a standard semiconductor parameter analyzer (HP4145). The kinetics of enzymatic reaction were determined and compared with a standard and sophisticated fluorometer (Hitachi F-4500) in a biochemical laboratory. Similar results were obtained by both instruments. Using glucose oxidase as an example, we further demonstrated that the HRP platform can be used to determine other H/sub 2/O/sub 2/ producing reactions with the CMOS system. The result points to an important application of the CMOS chip in biological measurements and in diagnosis of various health factors.

Water-soluble germanium nanoparticles cause necrotic cell death and the damage can be attenuated by blocking the transduction of necrotic signaling pathway

Water-soluble germanium nanoparticles (wsGeNPs) with allyamine-conjugated surfaces were fabricated and emit blue fluorescence under ultraviolet light. The wsGeNP was physically and chemically stable at various experimental conditions. Cytotoxicity of the fabricated wsGeNP was examined. MTT assay demonstrated that wsGeNP possessed high toxicity to cells and clonogenic survival assay further indicated that this effect was not resulted from retarding cell growth. Flow cytometric analysis indicated that wsGeNP did not alter the cell cycle profile but the sub-G1 fraction was absent from treated cells. Results from DNA fragmentation and propidium iodide exclusion assays also suggested that apoptotic cell death did not occur in cells treated with wsGeNP. Addition of a necrosis inhibitor, necrostatin-1, attenuated cell damage and indicated that wsGeNP caused necrotic cell death. Cell signaling leads to necrotic death was investigated. Intracellular calcium and reactive oxygen species (ROS) levels were increased upon wsGeNP treatment. These effects can be abrogated by BAPTA-AM and N-acetyl cysteine respectively, resulting in a reduction in cell damage. In addition, wsGeNP caused a decrease in mitochondrial membrane potential (MMP) which could be recovered by cyclosporine A. The cellular signaling events revealed that wsGeNP increase the cellular calcium level which enhances the production of ROS and leads to a reduction of MMP, consequentially results in necrotic cell death.

Diversity of sugar acceptor of glycosyltransferase 1 from Bacillus cereus and its application for glucoside synthesis.

Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors—fluorescein methyl ester, 17-β-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine—were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are β-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.

Preparation of High-Performance Water-Soluble Quantum Dots for Biorecognition through Fluorescence Resonance Energy Transfer

An improved method for the synthesis of high-performance and water-soluble quantum dots (QDs) involving the encapsulation of mercaptosuccinic acid coated QDs (MSA-QDs) with poly(diallyldimethylammonium chloride) (PDDA) followed by their direct photoactivation with fluorescent radiation near 295u2005K to yield PDDA-coated QDs (PDDA-QDs) has been demonstrated. The quantum yield (QY) of the PDDA-QDs was significantly improved from 0.6 (QY of MSA-QDs) to 48%. By using this synthetic strategy, highly photoluminescent PDDA-QDs of varied size were readily prepared. The surface properties of PDDA-QDs and MSA-QDs were extensively characterized. The highly luminescent and positively charged PDDA-QDs serve as a useful and convenient tool for protein adsorption. With a Δ(5)-3-ketosteroid isomerase adsorbed PDDA-QD complex, the biorecognition of steroids was demonstrated through the application of fluorescent resonance energy transfer.

Three important amino acids control the regioselectivity of flavonoid glucosidation in glycosyltransferase-1 from Bacillus cereus

Glycosyltransferase-1 from Bacillus cereus (BcGT1) catalyzes a reaction that transfers a glucosyl moiety to flavonoids, such as quercetin, kaempferol, and myricetin. The enzymatic glucosidation shows a broad substrate specificity when the reaction is catalyzed by wild-type BcGT1. Preliminary assays demonstrated that the F240A mutant significantly improves the regioselectivity of enzymatic glucosidation toward quercetin. To unveil and further to control the catalytic function of BcGT1, mutation of F240 to other amino acids, such as C, E, G, R, Y, W, and K, was performed. Among these mutants, F240A, F240G, F240R, and F240K greatly altered the regioselectivity. The quercetin-3-O-glucoside, instead of quercetin-7-O-glucoside as for the wild-type enzyme, was obtained as the major product. Among these mutants, F240R showed nearly 100xa0% product specificity but only retained 25xa0% catalytic efficiency of wild-type enzyme. From an inspection of the protein structure, we found two other amino acids, F132 and F138, together with F240, are likely to form a hydrophobic binding region, which is sufficiently spacious to accommodate substrates with varied aromatic moieties. Through the replacement of a phenylalanine by a tyrosine residue in the substrate-binding region, the mutants may be able to fix the orientation of flavonoids, presumably through the formation of a hydrogen bond between substrates and mutants. Multiple mutants—F240R_F132Y, F240R_F138Y, and F240R_F132Y_F138Y—were thus constructed for further investigation. The multiple points of mutants not only maintained the high product specificity but also significantly improved the catalytic efficiency, relative to F240R. The same product specificity was obtained when kaempferol and myricetin were used as a substrate.