Wei-Chuan Shih

Microfluidic surface-enhanced Raman scattering sensor with monolithically integrated nanoporous gold disk arrays for rapid and label-free biomolecular detection

Abstract. We present a microfluidic surface-enhanced Raman scattering (SERS) sensor for rapid and label-free biomolecular detection. Our sensor design mitigates a common limiting factor in microfluidic SERS sensors that utilize integrated nanostructures: low-efficiency transport of biomolecules to nanostructured surface which adversely impacts sensitivity. Our strategy is to increase the total usable nanostructured surface area, which provides more adsorption sites for biomolecules. Specifically, a nanoporous gold disk (NPGD) array, a highly effective SERS substrate, has been monolithically integrated inside a microfluidic chip. Individual NPGD is known to feature an order of magnitude larger surface area than its projected disk area. The increased surface area arises from nanoscale pores and ligaments three-dimensionally distributed in the NPGD, which manifest themselves as high-density SERS hot-spots. High-density NPGD arrays further guarantee large coverage of these hot-spots on the microchannel floor. The sensor performance has been demonstrated using Rhodamine 6G to quantify spatial uniformity and determine the shortest detection time. Next, the sensor is applied to detect two biomolecules, dopamine and urea, with unprecedented detection limit and speed compared to other existing microfluidic SERS sensors. The sensor holds great promise in point-of-care applications for various biomolecular detections.

METHOD OF STAMPING SURFACE-ENHANCE RAMAN SPECTROSCOPY FOR LABEL-FREE, MULTIPLEXED, MOLECULAR SENSING AND IMAGING

Abstract. We report stamping surface-enhanced Raman spectroscopy (S-SERS) for label-free, multiplexed, molecular sensing and large-area, high-resolution molecular imaging on a flexible, nonplasmonic surface without solution-phase molecule transfer. In this technique, a polydimethylsiloxane (PDMS) thin film and nanoporous gold disk SERS substrate play the roles as molecule carrier and Raman signal enhancer, respectively. After stamping the SERS substrate onto the PDMS film, SERS measurements can be directly taken from the “sandwiched” target molecules. The performance of S-SERS is evaluated by the detection of Rhodamine 6G, urea, and its mixture with acetaminophen, in a physiologically relevant concentration range, along with the corresponding SERS spectroscopic maps. S-SERS features simple sample preparation, low cost, and high reproducibility, which could lead to SERS-based sensing and imaging for point-of-care and forensics applications.

Intrinsic Raman spectroscopy for quantitative biological spectroscopy Part I: Theory and simulations

We present a novel technique, intrinsic Raman spectroscopy (IRS), to correct turbidity-induced Raman spectral distortions, resulting in the intrinsic Raman spectrum that would be observed in the absence of scattering and absorption. We develop an expression relating the observed and intrinsic Raman spectra through diffuse reflectance using the photon migration depiction of light transport. Numerical simulations are employed to validate the theoretical results and study the dependence of this expression on sample size and elastic scattering anisotropy.

Performance of line-scan Raman microscopy for high-throughput chemical imaging of cell population

We evaluate the performance of line-scan Raman microscopy (LSRM), a versatile label-free technique, for high-throughput chemical imaging of cell population. We provide detailed design and configuration of a home-built LSRM system developed in our laboratory. By exploiting parallel acquisition, the LSRM system achieves a significant throughput advantage over conventional point-scan Raman microscopy by projecting a laser line onto the sample and imaging the Raman scattered light from the entire line using a grating spectrograph and a charge-coupled device (CCD) camera. Two-dimensional chemical maps can be generated by scanning the projected line in the transverse direction. The resolution in the x and y direction has been characterized to be ~600-800 nm for 785 nm laser excitation. Our system enables rapid classification of microparticles with similar shape, size, and refractive index based on their chemical composition. An equivalent imaging throughput of 100 microparticles/s for 1 μm polystyrene beads has been achieved. We demonstrate the application of LSRM to imaging bacterial spores by identifying endogenous calcium dipicolinate. We also demonstrate that LSRM enables the study of intact microalgal cells at the colonial level and the identification of intra- and extracellular chemical constituents and metabolites, such as chlorophyll, carotenoids, lipids, and hydrocarbons. We conclude that LSRM can be an effective and practical tool for obtaining endogenous microscopic chemical and molecular information from cell population.

Internal and external morphology-dependent plasmonic resonance in monolithic nanoporous gold nanoparticles

We report morphology-dependent plasmonic resonance in monolithic nanoporous gold nanoparticles with a nanoscale internal porous network produced by the combination of lithographic patterning and dealloying. Timed dealloying and post-dealloying thermal annealing techniques have been employed to precisely control the morphological evolution. We found that prolonged dealloying time caused further pore coarsening to increase by ∼4 nm, whereas thermal annealing induced both pore coalescence and disk shrinkage, which eventually led to pore elimination. Both types of morphological changes caused a blueshift in the major plasmonic extinction band of up to 200 nm, in contrast to the redshift (∼50 nm) observed in semi-infinite NPG thin films. In addition, a greater blueshift was observed in a higher Au atomic content starting alloy. The tunable plasmonic properties have great potential in surface-enhanced spectroscopy and optical sensing.

Reagent- and separation-free measurements of urine creatinine concentration using stamping surface enhanced Raman scattering (S-SERS).

We report a novel reagent- and separation-free method for urine creatinine concentration measurement using stamping surface enhanced Raman scattering (S-SERS) technique with nanoporous gold disk (NPGD) plasmonic substrates, a label-free, multiplexed molecular sensing and imaging technique recently developed by us. The performance of this new technology is evaluated by the detection and quantification of creatinine spiked in three different liquids: creatinine in water, mixture of creatinine and urea in water, and creatinine in artificial urine within physiologically relevant concentration ranges. Moreover, the potential application of our method is demonstrated by creatinine concentration measurements in urine samples collected from a mouse model of nephritis. The limit of detection of creatinine was 13.2 nM (0.15 µg/dl) and 0.68 mg/dl in water and urine, respectively. Our method would provide an alternative tool for rapid, cost-effective, and reliable urine analysis for non-invasive diagnosis and monitoring of renal function.

Morphological control and plasmonic tuning of nanoporous gold disks by surface modifications

We report a surface modification protocol to control nanoporous gold (NPG) disk morphology and tune its plasmonic resonance. Enlarged pore size up to ∼20 nm within 60 s dealloying time has been achieved by adsorbing halides onto alloy surfaces in-between two dealloying steps. In addition, plasmonic resonance has been significantly red-shifted by up to ∼258 nm by the surface modification. Furthermore, with the enlarged pore size, small gold nanoparticles have been effectively loaded into the pores to enhance the performance of surface-enhanced Raman scattering (SERS) due to hot spot formation between the original nanoporous network and loaded nanoparticles.

High-speed hyperspectral Raman imaging for label-free compositional microanalysis

We present high-speed hyperspectral Raman imaging with integrated active-illumination for label-free compositional microanalysis. We show that high-quality Raman spectra can be acquired from as many as ~1,000 spots/sec semi-randomly distributed among a ~100x100 μm(2) area without mechanical scanning. We demonstrate rapid data acquisition from three types of samples: 1) uniform, strong Raman scatterers, e.g., silicon substrates; 2) non-uniform, medium-strength Raman scatterers, e.g., polymer microparticles; and, 3) non-uniform, relatively weak Raman scatterers, e.g., bacterial spores. We compare the system performance to that of point-scan with an electron-multiplied CCD camera, as implemented in some commercial systems. The results suggest that our system not only provides significant imaging speed advantage for various types of samples, but also permits substantially longer integration time per spot, leading to superior signal-to-noise ratio data. Our system enables the rapid collection of high quality Raman spectra for reliable and robust compositional microanalysis that are potentially transformative in applications such as semiconductor material and device, polymer blend and biomedicine.

Label-free, in situ SERS monitoring of individual DNA hybridization in microfluidics

We present label-free, in situ monitoring of individual DNA hybridization in microfluidics. By immobilizing molecular sentinel probes on nanoporous gold disks, we demonstrate sensitivity approaching the single-molecule limit via surface-enhanced Raman scattering which provides robust signals without photobleaching for more than an hour. We further demonstrate that a target concentration as low as 20 pM can be detected within 10 min under diffusion-limited transport.

Intrinsic Raman spectroscopy for quantitative biological spectroscopy Part II: Experimental applications

We demonstrate the effectiveness of intrinsic Raman spectroscopy (IRS) at reducing errors caused by absorption and scattering. Physical tissue models, solutions of varying absorption and scattering coefficients with known concentrations of Raman scatterers, are studied. We show significant improvement in prediction error by implementing IRS to predict concentrations of Raman scatterers using both ordinary least squares regression (OLS) and partial least squares regression (PLS). In particular, we show that IRS provides a robust calibration model that does not increase in error when applied to samples with optical properties outside the range of calibration.