Rattikan Chantiwas

Flexible fabrication and applications of polymer nanochannels and nanoslits.

Fluidic devices that employ nanoscale structures (<100 nm in one or two dimensions, slits or channels, respectively) are generating great interest due to the unique properties afforded by this size domain compared to their micro-scale counterparts. Examples of interesting nanoscale phenomena include the ability to preconcentrate ionic species at extremely high levels due to ion selective migration, unique molecular separation modalities, confined environments to allow biopolymer stretching and elongation and solid-phase bioreactions that are not constrained by mass transport artifacts. Indeed, many examples in the literature have demonstrated these unique opportunities, although predominately using glass, fused silica or silicon as the substrate material. Polymer microfluidics has established itself as an alternative to glass, fused silica, or silicon-based fluidic devices. The primary advantages arising from the use of polymers are the diverse fabrication protocols that can be used to produce the desired structures, the extensive array of physiochemical properties associated with different polymeric materials, and the simple and robust modification strategies that can be employed to alter the substrates surface chemistry. However, while the strengths of polymer microfluidics is currently being realized, the evolution of polymer-based nanofluidics has only recently been reported. In this critical review, the opportunities afforded by polymer-based nanofluidics will be discussed using both elastomeric and thermoplastic materials. In particular, various fabrication modalities will be discussed along with the nanometre size domains that they can achieve for both elastomer and thermoplastic materials. Different polymer substrates that can be used for nanofluidics will be presented along with comparisons to inorganic nanodevices and the consequences of material differences on the fabrication and operation of nanofluidic devices (257 references).

Complete plastic nanofluidic devices for DNA analysis via direct imprinting with polymer stamps

Development of all polymer-based nanofluidic devices using replication technologies, which is a prerequisite for providing devices for a larger user base, is hampered by undesired substrate deformation associated with the replication of multi-scale structures. Therefore, most nanofluidic devices have been fabricated in glass-like substrates or in a polymer resist layer coated on a substrate. This letter presents a rapid, high fidelity direct imprinting process to build polymer nanofluidic devices in a single step. Undesired substrate deformation during imprinting was significantly reduced through the use of a polymer stamp made from a UV-curable resin. The integrity of the enclosed all polymer-based nanofluidic system was verified by a fluorescein filling experiment and translocation/stretching of λ-DNA molecules through the nanochannels. It was also found that the funnel-like design of the nanochannel inlet significantly improved the entrance of DNA molecules into nanochannels compared to an abrupt nanochannel/microfluidic network interface.

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

Influence of Immobilized Biomolecules on Magnetic Bead Plug Formation and Retention in Capillary Electrophoresis

Significant changes in the formation and retention of magnetic bead plugs in a capillary during electrophoresis were studied, and it was demonstrated that these effects were due to the type of biological molecule immobilized on the surface of these beads. Three biological molecules, an antibody, an oligonucleotide, and alkaline phosphatase (AP), were attached to otherwise identical streptavidin‐coated magnetic beads through biotin‐avidin binding in order to isolate differences in bead immobilization in a magnetic field resulting from the type of biological molecule immobilized on the bead surface. AP was also attached to the magnetic beads using epoxy groups on the bead surfaces (instead of avidin‐biotin binding) to study the impact of immobilization chemistry. The formation and retention of magnetic bead plugs were studied quantitatively using light scattering detection of magnetic particles eluting from the bead plugs and qualitatively using microscopy. Both the types of biomolecule immobilized on the magnetic bead surface and the chemistry used to link the biomolecule to the magnetic bead impacted the formation and retention of the bead plugs.

Rapid simultaneous determination of four indole compounds in dietary supplements by micellar electrokinetic chromatography with a dilute and shoot step

A simple micellar electrokinetic chromatography (MEKC) method with UV detection was developed for the simultaneous determination of indole-3-carbinol, indole-3-acetonitrile, indole-3-acetic acid and 3,3′-diindolylmethane. These compounds are potentially used in cancer prevention. Investigation of solvent effects (methanol and dimethylformamide) on MEKC analysis was carried out. A dilute and shoot strategy was used for sample preparation to reduce the time required for multiple steps such as solvent evaporation. The final conditions were electrokinetic injection for 3.0 s at 423 V cm−1 and 20.0 mM borate buffer (pH 9.00) containing 20.0 mM SDS. Analysis was rapid, achieved in less than 4.5 min. Linear calibration curves for the indole compounds in the range 5–200 μg mL−1 (r2 > 0.999) were obtained. Intra- and inter-day precisions were 2.0–7.9% RSD, with LOQs in the range of 1.5–4.0 μg mL−1 and recoveries in the range of 90–110% (n = 5).

A simple method using two-step hot embossing technique with shrinking for fabrication of cross microchannels on PMMA substrate and its application to electrophoretic separation of amino acids in functional drinks

This work presents a simple hot embossing method with a shrinking procedure to produce cross-shape microchannels on poly(methyl methacrylate) (PMMA) substrate for the fabrication of an electrophoresis chip. The proposed method employed a simple two-step hot embossing technique, carried out consecutively on the same piece of substrate to make the crossing channels. Studies of embossing conditions, i.e. temperature, pressure and time, were carried out to investigate their effects on the dimension of the microchannels. Applying a simple shrinking procedure reduced the size of the channels from 700±20µm wide×150±5µm deep to 250±10µm wide×30±2µm deep, i.e. 80% and 64% reduction in the depth and width, respectively. Thermal fusion was employed to bond the PMMA substrate with a PMMA cover plate to produce the microfluidic device. Replication of microchip was achieved by precise control of conditions in the fabrication process (pressure, temperature and time), resulting in lower than 7% RSD of channel dimension, width and depth (n =10 devices). The method was simple and robust without the use of expensive equipment to construct the microstructure on a thermoplastic substrate. The PMMA microchip was used for demonstration of amine functionalization on the PMMA surface, measurement of electroosmotic flow and for electrophoretic separation of amino acids in functional drink samples. The precision of migration time and peak area of the amino acids, Lys, Ile and Phe at 125μM to 500μM, were in the range 3.2-4.2% RSD (n=9 devices) and 4.5-5.3% RSD (n=9 devices), respectively.

Simple and rapid screening of the thiocyanate level in saliva for the identification of smokers and non-smokers by capillary electrophoresis with contactless conductivity detection

A simple and rapid capillary electrophoresis method with contactless conductivity detection for direct determination of thiocyanate in saliva to differentiate between smokers and non-smokers is proposed. Simple saliva preparation was performed by centrifugation and the dilute and shoot method (1:1, v/v) with a running buffer prior to hydrodynamic injection. The running buffer was 20.0 mM His/MES at pH 6.00 containing 0.1 mM CTAB as the electroosmotic flow modifier. Maleate anions were employed as the internal standard for the precision of the response factor of electrophoresis separation which was carried out at −8.0 kV (266 V cm−1) with a total analysis time of 3.5 min per sample. A linear calibration curve for thiocyanate in the concentration range 0.2–4.0 mM was obtained (r2 > 0.999). The limit of quantification was 0.12 mM which is sufficient for the quantitative determination of thiocyanate in saliva for the purpose of identification of smokers and non-smokers. Intra-day and inter-day precisions were 0.7–1.7% RSD and 2.4–6.4% RSD, respectively. Accuracy based on the percentage recovery of spiked saliva samples was in the range of 72–141% (n = 18). The measured thiocyanate levels were significantly different to classify non-smokers from smokers; a cut-off value of 1.0 mM is proposed to separate the two groups. This method is convenient and rapid, suitable for screening of a large number of subjects, such as in clinical work or health-care surveys.

Development of a sequential injection-liquid microextraction procedure with GC-FID for analysis of short-chain fatty acids in palm oil mill effluent

Short-chain fatty acids, such as acetic, propionic, butyric, iso-valeric and valeric acids, play an important role in methanogenesis activity for biogas production processes. Thus, simple and rapid procedures for monitoring the levels of short-chain fatty acids are requisite for sustaining biogas production. This work presents the development of a sequential injection-liquid microextraction (SI-LME) procedure with GC-FID analysis for determination of short-chain fatty acids. GC-FID was employed for detection of the short-chain fatty acids. Calibration curves were linear with good coefficients of determination (r2>0.999), using methacrylic acid as the internal standard. Limits of quantification (LOQ) were in the range of 0.03-0.19mM. The SI-LME procedure employed tert-butyl methyl ether (TBME) as the extracting solvent. Various SI-LME conditions were investigated and optimized to obtain the highest recovery of extraction. With these optimized conditions, an extraction recovery of the five key short-chain fatty acids of 67-90% was obtained, with less than 2% RSD (n=3). The final SI-LME procedure employed two fluidic zones of TBME with a single aqueous fluidic zone of sample sandwiched between the TBME zones, with 5 cycles of flow reversal at a flow rate of 5µL/s for the extraction process. Intra- and inter-day precision values were 0.5-4.0% RSD and 3.3-4.8% RSD, respectively. Accuracy based on percentage of sample recovery were in the range of 69-96, 102-107, and 82-101% (n=4) for acetic, propionic and butyric acids, respectively. The proposed method was applied for the measurement of short-chain fatty acids in palm oil mill effluents used in biogas production in a factory performing palm oil extraction process. The SI-LME method provides improved extraction performance with high precision, and is both simple and rapid with its economical extraction technique. The SI-LME procedure with GC-FID has strong potential for use as a quality control process for monitoring short-chain fatty acid levels in biogas production.

Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase

p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to yield 3,4-dihydroxyphenylacetate (DHPA). In this study, we investigated whether variants of the oxygenase component (C2) could catalyze hydroxylation of 4-hydroxyphenylethylamines to synthesize catecholamine derivatives. Single turnover product analysis showed that the R263D variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%). The enzyme was also found to have dual substrate charge specificity because it can also maintain reasonable hydroxylation efficiency of HPA (86%). This property is different from the R263E variant, which can hydroxylate HPA (73%) but not tyramine. The R263A variant can hydroxylate HPA (72%) and tyramine to a small extent (7%). Stopped-flow experiments indicated that tyramine and HPA prefer binding to R263D after C4a-hydroperoxy-FMN formation, while tyramine cannot bind to the wild-type or R263E enzymes. Data also indicate that the hydroxylation rate constant is the rate-limiting step. The R263D variant was used as a starting enzyme for further mutation to obtain other variants for the synthesis of additional catecholamine drugs. The R263D/Y398D double mutant enzyme showed interesting results in that it was able to catalyze the hydroxylation of octopamine to form norepinephrine. However, the enzyme still lacked stereo-selectivity in its reaction.

Chemiluminescence detection with microfluidics for innovative in situ measurement of unbound cobalt ions in dynamic equilibrium with bound ions in binding study with polyethyleneimine and its functionalized nanoparticles

This work reports a novel method for in situ measurement of binding of cobalt ions to polyethyleneimine (PEI) and polyethyleneimine-functionalized poly (methyl methacrylate) nanoparticles (PEI-NPs) using simple microfluidics with a chemiluminescence detection system. The catalytic effect of free cobalt ion in solution on the luminol-hydrogen peroxide chemiluminescence was employed for the detection of unbound cobalt in dynamic equilibrium with cobalt bound to PEI or PEI-NPs. Many binding measurements lead to incorrect estimation of free metal ions due to insufficient separation of bound and free ions. The catalytic activity of only unbound cobalt ion on the luminol reaction was demonstrated by observing that PEI and PEI-NPs alone did not give chemiluminescence. Also, both Co-PEI and Co-PEI-NPs complexes gave no chemiluminescence when cobalt ion is fully bound with excess PEI or PEI-NPs. In addition diethylenetriamine (dien) as a model ligand to completely bind the cobalt ions was also employed as further confirmation. The chemiluminescence measurement employing microfluidics was then successfully applied for the measurement of binding cobalt ion to PEI and PEI-NPs. This in situ measurement of binding does not require filtration of the two species. As there is no perturbation of equilibrium, an accurate binding measurement can therefore be successfully performed. Experimental parameters, such as concentrations of polymers and cobalt ions, and equilibration time were investigated. Analysis of the experimental data employed the binding equation derived assuming independent and equivalent binding sites of the polymer for the metal ions. Also the binding constant of cobalt ions with PEI-NPs is first reported employing chemiluminescence detection. This work provides quantitative determination of the binding constant and total binding capacity of PEI and PEI-NPs with cobalt ions using chemiluminescence detection and microfluidics as an innovative in situ measurement of the unbound cobalt ions.