Lai-Kwan Chau

Sol–gel encapsulation of lactate dehydrogenase for optical sensing of l-lactate

Sol-gel encapsulation of lactate dehydrogenase and its cofactor can be employed as a disposable sensor for L-lactate. The sensor utilized the changes in absorbance or fluorescence from the reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. Although, problems such as diminished enzymatic activity and/or leaching of enzyme from the sol-gel matrix occurred, the sol-gel process is sufficiently mild to permit retention of enzymatic activity. The apparent activity of LDH in the sensor is at least 10% of that of the dissolved enzyme. The sensor has a linear dynamic range over the normal physiological L-lactate level and has a long-term storage stability of at least 3 weeks.

Single‐Domain Antibody‐Conjugated Nanoaggregate‐Embedded Beads for Targeted Detection of Pathogenic Bacteria

The rapid screening of pathogenic bacteria remains a key issue in the diagnosis of infectious diseases, food safety, and public health assurance. In particular, the emergence of drug-resistant bacteria presents great challenges to the health care sector. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for one of the better-known hospital-acquired infections. S. aureus is a common pathogen that can colonize various areas of the human anatomy. Healthy individuals may carry MRSA asymptomatically, but patients with a compromised immune system are at a greater risk of symptomatic secondary infection. Due to the drug-resistant nature of S. aureus, preventive measures, such as routine patient screening, remain the most effective way to control the spread of this bacterium in clinical environments. In the clinical setting, routine analysis of pathogenic bacteria typically involves biochemical characterization of cultured microorganisms taken from contaminated sources. Standardized procedures are time consuming, which thus highlights the need for a rapid and targeted detection methodology. Advances in nanotechnology and biotechnology offer new possibilities for the rapid screening of harmful microorganisms. Surface-enhanced Raman scattering (SERS) has been demonstrated to achieve ultra-high sensitivity detection in many bioanalytical assays. Herein, we combine the high sensitivity of a newly developed SERS nanoprobe with the high specificity of single-domain antibody (sdAb) to achieve the targeted detection of a single bacterial pathogen, S. aureus. The ability of metallic nanostructures to localized surface plasmon resonance (LSPR) under appropriate electromagnetic field excitation is largely responsible for the SERS effect. The LSPR is strongly dependent on the size and shape of the nanostructures. It has been demonstrated that extremely high SERS enhancement can be achieved when nanostructures are closely spaced, which allows their LSPR to couple. At the optimal interparticle spacing, LSPR coupling results in the capacitive enhancement of the Raman effect for molecules located between particles in the SERS “hot sites”, which thus enables the detection of very few molecules under optimal excitation conditions. A specially designed SERS nanoprobe called a nanoaggregateembedded bead (NAEB) was fabricated with this optimization in mind. NAEBs are fabricated by controlled formation of small Au nanoparticle (NP) aggregates that are subsequently encapsulated in a protective silica shell (Scheme 1a), and fully utilize the advantage of LSPR coupling of a small NP aggregate; as a result each nanosized bead is an ultrahigh sensitivity SERS nanoprobe. Raman reporter molecules (R6G) were incorporated into the nanoaggregate during the formation process to give each NAEB a unique Raman signature. Compared with other SERS-based bioanalytical applications that utilize Ag or Au NPs, NAEBs have the added advantage of stability. Without the protective silica shell, even passivated Au or Ag NPs immersed in biological buffers are prone to parasitic signals from adsorption of the molecules in the biological fluids or loss of signals due to the desorption of the Raman reporter molecule. This problem was recognized by the groups of Liz-Marzan, Natan, Nie, and Brown, who pioneered the work on encapsulating Au NPs with a protective silica shell to improve their stability. In these earlier works, encapsulation was done without deliberate aggregation, which resulted in a relatively low [a] P.-J. Huang, Prof. L.-K. Chau Department of Chemistry and Biochemistry National Chung Cheng University 168 University Road Min-Hsiung, Chia-Yi (Taiwan) Fax: (+886)5-2721040 E-mail : chelkc@ccu.edu.tw [b] Dr. L.-L. Tay, Dr. J. Tanha, S. Ryan Institute for Microstructural Sciences and Institute for Biological Sciences National Research Council Canada Ottawa, ON K1A 0R6 (Canada) Fax: (+1) 952-6337 E-mail : lilin.tay@nrc-cnrc.gc.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901397.

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.

Fiber-optic particle plasmon resonance sensor for detection of interleukin-1β in synovial fluids

A facile and label-free biosensing method has been developed for determining an osteoarthritis concerned cytokine, interleukin-1β (IL-1β), in synovial fluids. The biosensing technique, fiber-optic particle plasmon resonance (FOPPR), is based on gold nanoparticles-modified optical fiber where the gold nanoparticle surface has been modified by a mixed self-assembled monolayer for further conjugation of anti-IL-1β antibody and minimization of nonspecific adsorption. Upon binding of IL-1β to anti-IL-1β on the gold nanoparticle surface, the absorbance of the gold nanoparticle layer on the optical fiber changes and the signal change is enhanced through multiple total internal reflections along the optical fiber. Results show that the detection of IL-1β in synovial fluid by this sensor agrees quantitatively with the clinically accepted enzyme-linked immunosorbent assay (ELISA) method but a much shorter analysis time is required (<10 min). The sensor response versus log concentration of IL-1β was linear (r=0.9947) over the concentration range of 0.050-10 ng/mL and a limit of detection (LOD) of 21 pg/mL (1.2 pM) was achieved. Such a LOD for IL-1β (17 kDa) represents a major advancement in the field of real-time monitoring of low molecular weight proteins in complex biological fluids.

Preparation of colloidal gold multilayers with 3-(mercaptopropyl)-trimethoxysilane as a linker molecule

Abstract A novel strategy to prepare colloidal Au multilayers using 3-(mercaptopropyl)-trimethoxysilane (MPTMS) as a linker molecule was demonstrated. The nanostructure consists of alternate layers of ultrathin thiol-functionalized silica films and Au colloids. In which, a surface sol–gel process was used to prepare the thiol-functionalized silica films. During the self-assembly of Au colloids, a reiterative immersion–rinse approach was used to ensure maximum coverage of isolated Au colloids. The surface coverage of the first colloidal Au monolayer is ca. 45% of a close-packed monolayer. The method yields uniform layer-by-layer growth colloidal Au multilayers through self-assembly at the Au(111) planes, as judged by the ultraviolet–visible (UV–vis) spectroscopic data and the X-ray diffraction (XRD) patterns.

Direct detection of orchid viruses using nanorod-based fiber optic particle plasmon resonance immunosensor

A fiber optic particle plasmon resonance (FOPPR) immunosensor is developed for label-free detection of orchid viruses that use gold nanorods (AuNRs) as the sensing material. The AuNRs are employed to create a near-infrared sensing window to solve the color interference problem of sample matrix for direct sensing of target analyte. This work cannot be achieved using gold nanospheres (AuNSs) because the signal of sample color absorption largely overlaps the signal of molecular recognition events in the visible spectrum, making the signal interpretation much more difficult. The AuNRs are immobilized on the unclad fiber core surface, and functionalized by antibodies which can specifically recognize the corresponding Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) for rapid viral infection diagnosis. The refractive index resolution of the AuNR-FOPPR sensor is estimated to be 8×10(-6) RIU. The limits of detection (LODs) for CymMV and ORSV in leaf saps are 48 and 42 pg/mL, respectively, which are better than the LODs of 1200 pg/mL for both viruses obtained by enzyme-linked immunosorbent assay (ELISA). Exploiting the AuNR-FOPPR sensing strategy not only solves the color interference problem encountered by using AuNSs, but provides faster analysis, better reproducibility, and lower detection limit than ELISA. The sensor can distinguish between healthy and infected orchids in 10 min, and can further provide the quantitative analysis of infection level. It is potentially applicable to the quality control of orchid cultivation industry, but not limited to this, especially for creating special spectral sensing window for particular samples.

Single-step approach to β-cyclodextrin-bonded silica as monolithic stationary phases for CEC

A novel single-step sol-gel approach for the preparation of beta-CD-bonded silica monolithic electrochromatographic columns is established. The porous silica networks were fabricated inside fused-silica capillaries using sol-gel processing of tetramethoxysilane and an organfunctional silicon alkoxide that contains beta-CD. Scanning electron micrographs and nitrogen adsorption-desorption data showed that these functional monolithic columns have double pores structures with micrometer-size co-continuous through-pores and silica skeletons with open mesopores. The beta-CD monolithic columns have successfully been applied to the separation of several neutral and negatively charged isomers by CEC. The column performance was evaluated by using positional isomers of naphthalenedisulfonic acid as model compounds. A plate height of less than 10 mum for the first eluted isomer of naphthalenedisulfonic acid was obtained at an optimal flow rate (0.47 mm/s) of the mobile phase. Moreover, the columns have been proved to be stable for more than 100 runs during 3 months period and show reasonable column reproducibility.

Electroosmotic flow controllable coating on a capillary surface by a sol–gel process for capillary electrophoresis

A simple coating procedure employing a sol-gel process to modify the inner surface of a bare fused-silica capillary with a positively charged quaternary ammonium group is established. Scanning electron microscopic studies reveal that a smooth coating with 1 to approximately 2 microm thickness can be obtained at optimized coating conditions. With 40 mM citrate as a running electrolyte, the plot of electroosmotic flow (EOF) versus pH shows a unique three-stage EOF pattern from negative to zero and then to positive over a pH range of 2.5 to 7.0. At pH above 5.5, the direction of the EOF is from the anode to the cathode, as is the case in a bare fused-silica capillary, and the electroosmotic mobility increases as the pH increases. However, the direction of the EOF is reversed at pH below 4.0. Over the pH range of 4.0 to 5.5, zero electroosmotic mobility is obtained. Such a three-stage EOF pattern has been used to separate six aromatic acids under suppressed EOF and to separate nitrate and nitrite with the anions migrating in the same direction as the EOF. The positively charged quaternary ammonium group on the coating was also utilized to minimize the adsorption problem during the separation of five basic drugs under suppressed EOF and during the separation of four basic proteins with the cations migrate in the opposite direction as the EOF. Also, the stability and reproducibility of this column are good.

A Novel Design of Grooved Fibers for Fiber-Optic Localized Plasmon Resonance Biosensors

Bio-molecular recognition is detected by the unique optical properties of self-assembled gold nanoparticles on the unclad portions of an optical fiber whose surfaces have been modified with a receptor. To enhance the performance of the sensing platform, the sensing element is integrated with a microfluidic chip to reduce sample and reagent volume, to shorten response time and analysis time, as well as to increase sensitivity. The main purpose of the present study is to design grooves on the optical fiber for the FO-LPR microfluidic chip and investigate the effect of the groove geometry on the biochemical binding kinetics through simulations. The optical fiber is designed and termed as U-type or D-type based on the shape of the grooves. The numerical results indicate that the design of the D-type fiber exhibits efficient performance on biochemical binding. The grooves designed on the optical fiber also induce chaotic advection to enhance the mixing in the microchannel. The mixing patterns indicate that D-type grooves enhance the mixing more effectively than U-type grooves. D-type fiber with six grooves is the optimum design according to the numerical results. The experimental results show that the D-type fiber could sustain larger elongation than the U-type fiber. Furthermore, this study successfully demonstrates the feasibility of fabricating the grooved optical fibers by the femtosecond laser, and making a transmission-based FO-LPR probe for chemical sensing. The sensor resolution of the sensor implementing the D-type fiber modified by gold nanoparticles was 4.1 × 10−7 RIU, which is much more sensitive than that of U-type optical fiber (1.8 × 10−3 RIU).

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.