Da-Shin Wang

Surface plasmon effects on two photon luminescence of gold nanorods

Gold nanorods emit strong photoluminescence under two photon excitation; the efficient two photon lumininescence (TPL) arises from the local field enhancement assisted by surface plasmons. The surface plasmon effects on the TPL efficiency and spectrum are investigated by measuring the TPL of gold nanorods with various aspect ratios. A large TPL efficiency is found when incident light wavelength coincides with the longitudinal surface plasmon mode of the gold nanorods. However, the emission spectra of nanorods with various aspect ratios look similar and exhibit modest surface plasmon features, which implies a major non-radiative decay of excited surface plasmons.

Density-dependent optical response of gold nanoparticle monolayers on silicon substrates

We experimentally and theoretically study the density-dependent optical properties of 20 nm gold nanoparticle monolayers on silicon at 530 nm. Both results indicate a density-dependent resonance behavior. The density-dependent resonance suggests that the optical properties of the gold nanoparticle monolayers can be drastically modulated by changing the volume ratio V(r). It also implies, in this composite system, that the total surface plasmon resonance is maximized at a certain V(r).

AN ELLIPSOMETRIC STUDY ON THE DENSITY AND FUNCTIONALITY OF ANTIBODY LAYERS IMMOBILIZED BY A RANDOMLY COVALENT METHOD AND A PROTEIN A-ORIENTED METHOD

Ellipsometry is used to quantitatively evaluate the antigen-sensing capability of immuno-surfaces. The ellipsometric measurement is a rapid, label-free, and in situ detection; however, quantification of analytes by ellipsometry is not unambiguous. The proper use of the effective thickness as a measure for the amount of analytes is addressed in this article. Taking the effective thickness as the quantification reference, we find that employing protein A to immobilize antibodies makes the antigen capture two to three times higher than the covalent immobilization technique using N-succinimidyl 4-maleimidobutyrate (GMBS). Besides, the results also suggest a uniform orientation of antibodies achieved by protein A. Increasing the density of surface antibodies would improve the sensitivity as well; however, the effect is more crucial for the protein A immuno-surface and appears to be less important for the GMBS immuno-surface. Therefore, the optimal immobilization strategy should include an orientation layer, such as protein A, and maximize the amount of oriented antibodies on the surface to further improve the efficiency for antigen detection.

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.

The studies of Schottky-diode based co-plane detector for surface plasmon resonance sensing

The Surface Plasmon Resonance (SPR) is a label-free, highly sensitive and real time sensing technique and has been extensively applied to biosensing and assay for decades. In a conventional SPR biosensor, a prism is used to create the total reflection in which the evanescent wave can excite the surface plasmon mode at the metal-dielectric interface at certain angle, at which condition the reflectivity of incident TM-polarized vanished as measured by a far-field photodetector. This is the optical detection of surface plasmon resonance. In this research, zinc oxide (ZnO) was used as the dielectric thin-film material above the gold surface on the glass substrate to form a co-plane Schottky diode; this structure is designed to be an alternative way to detect SPR. The strength of plasmonic field is possible to be monitored by measuring the photocurrent under the reverse bias. According to our experimental results, the measured photocurrents with TM-polarized illumination (representing the SPR case), TE-polarized illumination (non-SPR case) and no illumination conditions under DC -1.5V bias are -76.158mA (2.5μA), -76.085mA (3.6μA) and -76.089mA (3.4μA), respectively. Based on the results, we have demonstrated this Schottky diode based co-plane device has the potential to be used as the SPR detector and provides a possible solution for the need of a low-cost, miniaturized, electronically integrated, and portable SPR biosensor in the near future.

Development and measurement of two-photon-base fluorescence correlation spectroscopy

We presented the fluorescence correlation spectroscopy (FCS) based on two-photon microscopy system to access physical parameters that give rise to fluctuations in fluorescence signal. As given excitation volume, the diffusion coefficient of molecules due to Brownian Movement into or out of excitation volume is determined. Further, depended on autocorrelation analysis based on diffusion coefficients of molecules, the fraction of bound species from measured samples containing the dye labeled anti-rabbit IgG and rabbit IgG can be separated. The mean standard deviation of diffusion coefficient measurement in our two-photon based system is about 0.064. The changes of diffusion coefficient with 15 nM concentration solution of fluorescein are 246.173 um^2/s to 35.1626 um^2/s with different concentration solution of glycerol ranging from 2.5% to 40%. Moreover, the maximal binding fraction of kinetics mode and end mode between Anti-Rabbit IgG and Rabbit IgG is 60% and 73%, respectively.