Tsung-Liang Chuang

Amplified surface plasmon resonance immunosensor for interferon-Gamma based on a streptavidin-incorporated aptamer

Interferon-gamma (IFN-γ) is associated with susceptibility to tuberculosis, which is a major public health problem worldwide. Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, this assay is still labor-intensive and time-consuming. We therefore designed a DNA aptamer hairpin structure for the detection of IFN-γ with high sensitivity and selectivity. A streptavidin DNA aptamer was incorporated into the IFN-γ binding aptamer probe for the amplified detection of the target molecules. Initially, the probe remained in the inactive configuration. The addition of IFN-γ induced the rearrangement of the aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for the streptavidin molecular recognition. Under optimized conditions, the detection limit was determined to be 33 pM, with a dynamic range from 0.3 to 333 nM, both of which were superior to those of corresponding optical sensors. Because combined aptamers are composed of nucleic acids, this optical aptasensor provided the advantages of high sensitivity, simplicity, reusability, and no further labeling or sample pre-treatment.

A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification

Abstract In this study, we report a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method for the on-site detection of the hepatitis B virus (HBV). For LAMP detection, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.0011 refractive index (RI) units after LAMP reaction. The PC-based prisms linearity and thermal responses were compared to those of a traditional glass prism to show that a PC-based prism can be used for SPR measurement. Finally, the HBV template mixed in the 10μl LAMP solution could be detected by SPRLAMP system in 17min even at the detection-limited concentration of 2fg/ml. We also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (ΔRU) at varying amplification times to establish an optimal amplification time endpoint of 25min (R 2 =0.98). In conclusion, the LAMP reaction could be detected with the SPRLAMP sensing cartridge based on direct sensing of the bulk refractive index.

Aptamer-based colorimetric detection of proteins using a branched DNA cascade amplification strategy and unmodified gold nanoparticles

A branched DNA amplification strategy was employed to design a colorimetric aptameric biosensor using unmodified gold nanoparticles (AuNPs). First, a programmed DNA dendritic nanostructure was formed using two double-stranded substrate DNAs and two single-stranded auxiliary DNAs as assembly components via a target-assisted cascade amplification reaction, and it was then captured by DNA sensing probe-stabilized AuNPs. The release of sensing probes from AuNPs led to the formation of unstable AuNPs, promoting salt-induced aggregation. By integrating the signal amplification capacity of the branched DNA cascade reaction and unmodified AuNPs as a sensing indicator, this amplified colorimetric sensing strategy allows protein detection with high sensitivity (at the femtomole level) and selectivity. The limit of detection of this approach for VEGF was lower than those of other aptamer-based detection methods. Moreover, this assay provides modification-free and enzyme-free protein detection without sophisticated instrumentation and might be generally applicable to the detection of other protein targets in the future.

Semiconductor circular ring lasers fabricated with the cryo-etching technique

GaAs-AlGaAs multiple quantum well semiconductor circular ring lasers with a /spl sigma/ shape were fabricated by using the deep UV laser-assisted cryo-etching technique. Most of the fabricated lasers had external quantum efficiencies of more than 18% which mere higher than similar devices previously reported. The modal spacing observed from the resonance spectrum near threshold was always several times that corresponding to the circular oscillation of the ring cavity. The observed modal spacing was quite consistent with the theoretical result based on a coupled-cavity model.

Nonlinear switching in an all-semiconductor-optical-amplifier loop device

An all-semiconductor-optical-amplifier loop device with a multimode interference (MMI) coupler was fabricated with the deep UV cryo-etching technique. Efficient power-dependent switching was observed. With continuous-wave signals, nonlinear switching occurred due to the combined effect of the nonlinear coupling in the MMI coupler and the lateral wave field redistribution caused by the loop structure. Simulation results showed good agreement in trend with the experimental data.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

Abstract. A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR® Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004  ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

All-optical switching with pulsed signals in an all-semiconductor-optical-amplifier loop device

Abstract Picosecond pulsed-signal operation for efficient self- and cross-switching in an all-semiconductor-optical-amplifier loop device was implemented. Although the configuration of the device is similar to a conventional nonlinear optical loop mirror, the operation principles are quite different. For cw signals, the device operation relies on the nonlinear coupling effect in a multi-mode interference waveguide amplifier and the lateral field redistribution and amplification by the active loop. However, for pulsed signals the crucial factor of asymmetric gain distribution in the loop needs to be added for the device operation. The pump–probe experiments provided efficient cross-switching results and indicated that GHz-range operation is feasible by redesigning the device configuration for pump signal injection.

Detection of KRAS mutations of colorectal cancer with peptide-nucleic-acid-mediated real-time PCR clamping

ABSTRACT Colorectal cancer (CRC) is the third most common cancer in the world and its disease-specific mortality is estimated to be approximately 33% in the developed world. KRAS mutations have been shown to predict response to anti-EGFR (epidermal growth factor receptor) targeted monoclonal antibody therapy. Therefore, KRAS mutation testing of metastatic CRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Currently, the most common strategy for KRAS mutation detection consists of conventional polymerase chain reaction (PCR) and direct sequencing. However, it is a time-consuming and complicated procedure, not suitable for routine clinical test. The objective of this study is to develop and evaluate a highly sensitive and rapid method using peptide nucleic acid (PNA) oligomers mediated real-time PCR clamping for detection of KRAS mutations. The PNA-mediated PCR clamping assay can real-time detect a mutation in a sample containing 1% of the mutant allele in a mixture of wild-type genomic DNA, which also enables the accurate and rapid detection of all KRAS codon 12 and 13 mutations in a single reaction. The total assay time is short as it requires only 1.5 hours after the samples preparation. Thus, the present method offers a potential alternative to be applied in clinical samples of CRC for detection of DNA carrying KRAS mutations.

Detection of tip-enhanced fluorescence from loop-mediated isothermal amplification of hepatitis B virus by two-photon microscopy

Tip-enhanced fluorescence of localized DNA replication by loop-mediated isothermal amplification (LAMP) is a potential way to observe real-time biological reaction confined in nanometer scale. We successfully coated Bst polymerase on the apex (∼100 nm) of an atomic force microscope (AFM) tip and performed localized LAMP reaction of hepatitis B virus (HBV). By using this tip-based reaction, the replicated HBV DNA can be directly imaged to be 400∼500 nm spots by using two-photon excitation fluorescence microscopy.

Low-fouling characteristics of ultrathin zwitterionic cysteine SAMs

Surface fouling remains an exigent issue for many biological implants. Unwanted solutes adsorb to reduce device efficiency and hasten degradation while increasing the risks of microbial colonization and adverse inflammatory response. To address unwanted fouling in modern implants in vivo, surface modification with antifouling polymers has become indispensable. Recently, zwitterionic self-assembled monolayers, which contain two or more charged functional groups but are electrostatically neutral and form highly hydrated surfaces, have been the focus of many antifouling coatings. Reports using various compositions of zwitterionic polymer brushes have demonstrated ultralow fouling in the ng/cm2 range. These coatings, however, are thick and can hinder the target application of biological devices. Here, we report an ultrathin (8.52 Å) antifouling self-assembled monolayer composed of cysteine that is amenable to facile fabrication. The antifouling characteristics of the zwitterionic surfaces were evaluated against bovine serum albumin, fibrinogen, and human blood in real time using quartz crystal microbalance and surface plasmon resonance imaging. Compared to untreated gold surfaces, the ultrathin cysteine coating reduced the adsorption of bovine serum albumin by 95% (43 ng/cm2 adsorbed) after 3 h and 90% reduction after 24 h. Similarly, the cysteine self-assembled monolayer reduced the adsorption of fibrinogen as well as human blood by >90%. The surfaces were further characterized using scanning electron microscopy: protein-enhanced adsorption and cellular adsorption in human blood was found on untreated surfaces but not on the cysteine SAM-protected surfaces. These findings suggest that surfaces can be functionalized with an ultrathin layer of cysteine to resist the adsorption of key proteins, with performance comparable to zwitterionic polymer brushes. As such, cysteine surface coatings are a promising methodology to improve the long-term utility of biological devices.