Wen-Hsin Hsieh

On‐line SERS Detection of Single Bacterium Using Novel SERS Nanoprobes and A Microfluidic Dielectrophoresis Device

The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB-DEP-Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL(-1) . In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.

Using a fiber optic particle plasmon resonance biosensor to determine kinetic constants of antigen-antibody binding reaction.

In this paper, one simple and label-free biosensing method has been developed for determining the binding kinetic constants of antiovalbumin antibody (anti-OVA) and anti-mouse IgG antibody using the fiber optic particle plasmon resonance (FOPPR) biosensor. The FOPPR sensor is based on gold-nanoparticle-modified optical fiber, where the gold nanoparticle surface has been modified by a mixed self-assembled monolayer for conjugation of a molecular probe reporter (ovalbumin or mouse IgG) to dock with the corresponding analyte species such as anti-OVA or anti-mouse IgG. The binding process, occurring when an analyte reacts with a probe molecule immobilized on the optical fiber, can be monitored in real-time. In addition, by assuming a Langmuir-type adsorption isotherm to measure the initial binding rate, the quantitative determination of binding kinetic constants, the association and dissociation rate constants, yields k(a) of (7.21 ± 0.4) × 10(3) M(-1) s(-1) and k(d) of (2.97 ± 0.1) × 10(-3) s(-1) for OVA/anti-OVA and k(a) of (1.45 ± 0.2) × 10(6) M(-1) s(-1) and k(d) of (2.97 ± 0.6) × 10(-2) s(-1) for mouse IgG/anti-mouse IgG. We demonstrate that the FOPPR biosensor can study real-time biomolecular interactions.

Using ac-field-induced electro-osmosis to accelerate biomolecular binding in fiber-optic sensing chips with microstructures.

This article reports the use of ac-field-induced charges at the corners of microstructures on fiber-optic sensing chips to generate electro-osmotic vortex flows in flow cell channels that can accelerate solute binding on the fiber. The sensing chip made of a cyclic olefin copolymer COC substrate contained a flow cell channel of dimensions 15 mm x 1 mm x 1 mm. A partially unclad optical fiber was placed within the channel. Relief-like strip structures of 25-mum thickness fabricated on the channel bottom were produced with an injection-molding process. The external electric field lines penetrating through the corners of the plastic microstructures induce charges on the corner surfaces to build up electrical double layers. When a high-frequency ac field (approximately 100 kHz) is used to flip the field polarities quickly, neutralization of the induced charge cannot be accomplished. The electrical double layer is therefore sustained. When absorbed charges in the double layer are driven by the external field, electro-osmotic flows are generated. The unclad portion of the fiber was coated with biotin-functionalized gold nanoparticles. The streptavidin solution was filled in the channel from the feeding tube, and the ac field (approximately 50 V/cm) was subsequently turned on for 30 s. The ac-field-induced electro-osmotic flows can accelerate solute transport in the sensing channel to enhance the binding kinetics of streptavidin molecules with biotin probes implanted on the gold nanoparticle surface. As a result, the fiber-optic localized plasmon resonance (FO-LPR) sensing signal becomes steady as soon as the external field is turned off. In contrast, the signal cannot reach steady state until 200-300 s in a typical static sensing cell. A significant reduction in the sensing response time is demonstrated. The binding assay of streptavidin with immobilized biotin on gold nanoparticle-coated sensing fibers was validated using this mixing device. The detection limit for streptavidin of approximately 10(-11) M is close to the reported values obtained using static cells. Similarly, the sensing response time of an orchid Odontoglossum ringspot virus (ORSV) sample was reduced from 1000 to 330 s when an external field was applied to mix the fluid for 60 s, even though the detection limit was maintained.

Enhanced sensitivity in injection-molded guided-mode-resonance sensors via low-index cavity layers

We present an investigation on the use of low-index cavity layers to enhance the sensitivity of injection-molded guided-mode resonance (GMR) sensors. By adjusting the sputtering parameters, a low-index cavity layer is created at the interface between the waveguide layer and the substrate. Refractive index measurements show that a sensitivity enhancement of up to 220% is achieved with a cavity layer, in comparison to a reference GMR sensor without a cavity layer. Finite-element-method simulations were performed, and the results indicate that the cavities significantly redistribute the resonance mode profile and thus enhances the sensitivity. The present investigation demonstrates a new method for enhancing the sensitivity of injection-molded GMR sensors for high-sensitivity label-free biosensing.

Forced-convective vitrification with liquid cryogens

Cell cryopreservation by vitrification generally requires using vitrification solutions with high concentrations of cryoprotectants (CPAs), which are toxic and induce osmotic stresses associated with the addition and removal of CPAs. To increase the cooling rate and reduce the CPA concentration required for vitrification, this study proposed an innovative approach, named forced-convective vitrification with liquid cryogens, in which liquid oxygen at a temperature below its boiling point (LOX(bbp)) was used as the cryogen to reduce the generation of insulating bubbles of gaseous oxygen and the sample was subjected to a constant velocity to remove insulation bubbles from the sample. Results show that changing the cryogen from liquid nitrogen at its boiling temperature (LN(abp)) to LOX(bbp), increasing the sample velocity and reducing the test solution volume increased the cooling rate and thereby decreased the CPA concentration required for vitrification. Using the same velocity (1.2 m/s), the cooling rate achieved with LOX(bbp) was 2.3-fold greater than that achieved with LN(abp). With LOX(bbp), the increase in the sample velocity from 0.2 to 1.2 m/s enhanced the cooling rate by 1.9 times. With LOX(bbp), a velocity of 1.2m/s and a test solution volume of 1.73 μl, the CPA concentration required for vitrification decreased to 25%. These results indicate that the new approach described here can reduce the CPA concentration required for vitrification, and thus decreases the toxicity and osmotic stresses associated with adding and removing the CPA.

Low-Cost Label-Free Bio-Detection System Using Double-Sided Grating Waveguide Couplers

Low-cost label-free bio-sensing systems have long been desired to enable rapid, sensitive, quantitative, and high-throughput biosensing for bio-medical and chemical applications. Here we present an optical bio-detection system consists of injection-molded biosensors based on double-sided grating waveguide couplers and an optical intensity-based detection platform for low-cost, real-time, and label-free biosensing. The biosensors were fabricated combining injection-molding and sputtering techniques, providing unique advantages of low-cost and reduced production time. A simple and cost-effective optical intensity-based detection system employing a low-cost light emitting diode and a simple photodetector is also developed to perform label-free biosensing. We demonstrate that a high refractive index resolution of 6.43 × 10−5 RIU is achieved with this compact bio-sensing system, showing great promises for low-cost, real-time, label-free detection in bio-medical and chemical applications.Copyright

μ-TAS for label-free biosensing with double-sided grating waveguide

In this study, we demonstrated the feasibility of a μ-TAS (micro total analysis system) for label-free biosensing with double-sided grating waveguide by measuring the refractive index of sucrose solution and immunoassay interaction. The μ-TAS consisted of a durable double-sided grating waveguide biochip and a reusable integrated microfluidic module. The grating chip and the waveguide were fabricated respectively by injection molding and sputtering technology, and therefore, featured the characteristics of low cost and high stability. In addition, the unique coupling characteristics of laser light into the double-sided grating waveguide structure reduced the need of high-precision alignment optical components. Moreover, the integrated microfluidic module was reusable and could be combined with the durable waveguide biochip to form a portable μ-TAS for biosening. Using the μ-TAS presented in this study, the refractive index resolution of sucrose solution and limitation of detection for DNP/anti-DNP interaction were measured to be 4.73×10-6 RIU and 2.67×10-7g/ml, respectively.

On-chip SERS analysis for single mimic pathogen detection using Raman-labeled nanoaggregate-embedded beads with a dielectrophoretic chip

The integration of Raman-labeled nanoaggregate-embedded beads (NAEBs) for high performance SERS analysis of single mimic pathogen on a self-designed dielectrophoretic chip is demonstrated. The Raman tags called NAEBs are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). In this work, NAEBs consisting of a Raman dye tetramethyl-rhodamine-5-isothiosyanate (TRITC) are chemically functionalized with streptavidin to detect biotin-functionalized polystyrene (PS) microspheres which mimic as pathogens. The sample solution of completely mixed streptavidin-functionalized NAEBs and biotin-functionalized PS microspheres is pumped into the microfluidic channel of a dielectrophoretic chip. By giving an AC voltage on the embedded electrodes, a single mimic pathogen can be caught via the non-contact dielectrophoretic force and suspended at the central cross of four aluminum electrodes for subsequent Raman spectroscopic detection. The SERS signal of TRITC is used as a spectral signature of specific mimic pathogen recognition, otherwise only the background Raman signal of a PS microsphere is observed. A pathogen-specific biosensor based on the dielectrophoresis-Raman spectroscopy system is developed, and the proof-ofconcept is confirmed by the specific molecular interaction model of streptavidin with biotin. Therefore, the on-chip multiplex SERS analysis of pathogens can be anticipated by employing different dye-tagged NAEBs simultaneously in a sample solution. We believe this bioassay has the ability to screen and detect multiple pathogens with minimal sample processing and handling even a small number of pathogens is present.