Shih-Chung Wei

Diagnostic Devices for Isothermal Nucleic Acid Amplification

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Aptamer-based colorimetric detection of platelet-derived growth factor using unmodified goldnanoparticles

We developed a simple method for the detection of platelet-derived growth factors (PDGFs) based on base stacking effect coupled with an unmodified gold nanoparticle (AuNP) indicator. In the absence of a target, an aptamer probe and a capture probe stably co-exist in a solution, as it is difficult to sustain an interaction between both these probes due to the short 8bp duplex. However, when a target protein binds to the aptamer probe, the strong base stacking effect can lead to a favorable and stable interaction between the aptamer and capture probes. Hence, the capture probe dissociates from the AuNP surfaces, inducing AuNP aggregation. Compared with other AuNP-based aptasensors for PDGFs, using this base stacking effect can overcome a structured-aptamer methods limitation of requiring thiolated-aptamer-modified AuNPs. Under optimal detection conditions, this label-free colorimetric sensor could detect PDGFs down to 6nM with high selectivity in the presence of other interferring proteins. This simple detection approach provides viable methods for a structured-aptamer sensing protocol.

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.

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.

Gold nanorods as probes in two-photon fluorescence correlation spectroscopy: emerging applications and potential artifacts.

Owing to the highly efficient two‐photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two‐photon fluorescence correlation spectroscopy (TP‐FCS). This property, along with the previous successful use as a contrast agent in two‐photon fluorescence imaging, suggests a potential application in TP‐FCS as well. Although the FCS measurement becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power. Here, we investigate various photophysical processes of gold nanorods to determine the cause of such a sensitive power dependency. This understanding provides a basis for choosing appropriate FCS models to recover reasonable physical parameters. Although the correlation function amplitude G(0) is 32 times lower when the excitation power increases from 20 µW to 1.12 mW, the application of a saturation‐modified FCS model yields very good fit to each data set and the fitted concentration of 0.64 nM is comparable to the 0.7 nM given by the inductively coupled plasma mass spectrometry measurement. The FCS assay appears to be an efficient method for the quantification of gold nanorods when correctly interpreted. However, even with the saturation considered in the fitting model, the fitted rotational and translational diffusion rates are getting faster as the power increases. This indicates that other effects such as photothermal effects may raise the local temperature, and thus increasing the rotational and translational diffusion rate. Microsc. Res. Tech. 76:882–889, 2013.

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.

Characteristic investigation of scanning surface plasmon microscopy for nucleotide functionalized nanoarray.

A calculation based on surface plasmon coupling condition and Maxwell-Garnett equation was performed for predicting the coupling angle shift and thin film thickness in scanning surface plasmon microscopy (SSPM). The refractive index sensitivity and lateral resolution of an SSPM system was also investigated. The limit of detection of angle shift was 0.01°, the limit of quantification of angle shift was 0.03°, and the sensitivity was around 0.12° shift per nm ZnO film when the film thickness was less than 22.6 nm. Two partially connected Au nano-discs with a center-to-center distance of 1.1 μm could be identified as two peaks. The system was applied to image nanostructure defects and a virus-probe functionalized nanoarray. We expect the potential application in nanobiosensors with further optimization in the future.

Metallic tip enhanced fluorescence for DNA replication monitoring

We have successfully performed localized loop-mediated isothermal reactions of hepatitis B virus (HBV) and hepatitis C virus (HCV) on the apex (50~100 nm) of metallic tips coated with Bst polymerases. The SYBR green molecules binding to the new formed HBV DNA inside the optical near fields were excited by two-photon fluorescence microscopy, and directly imaged in far field. Another reporter primer is used for HCV replication detection. Preliminary results are presented in this manuscript.

Nanodots array rapidly fabricated by Dip-Pen Nanolithography with temperature and humidity control

This study demonstrates the advantage of Dip-Pen Nanolithography (DPN) as a research and design tool for metal nano-structure fabrications. We design two different gold nano-structures, which are fabricated by DPN etching method with temperature and humidity control. The plasmon resonance frequencies of both structures are measured with dark field scattering spectroscopy. Our results show that with temperature and humidity control, DPN is highly potential in developing photonic circuit, solar cell and biomedical devices due to the rapid fabrication and cost effectiveness.