Enzyme-linked immunosorbent assay using thin-layered microfluidics with perfect capture of the target protein.

We developed a process for enzyme-linked immunosorbent assay on a glass microchip via the use of a thin-layered microfluidic channel. This channel possesses a high aspect ratio (width/depth ∼200) and has an antibody layer immobilized directly on the channel surface. A depth of several microns and an excessive width and length (mm scale) of the channel provide a large-volume capacity (102 nL) and maximum capture efficiency of the analyte for a high level of detection sensitivity (102 pg mL-1). The developed reusable immunosensor has demonstrated high-performance characteristics by requiring less than 50 µL of sample and providing analysis in less than 25 min. This new method could impact the development of point-of-care devices for biomedical applications.

From Extended Nanofluidics to an Autonomous Solar-Light-Driven Micro Fuel-Cell Device.

Autonomous micro/nano mechanical, chemical, and biomedical sensors require persistent power sources scaled to their size. Realization of autonomous micro-power sources is a challenging task, as it requires combination of wireless energy supply, conversion, storage, and delivery to the sensor. Herein, we realized a solar-light-driven power source that consists of a micro fuel cell (µFC) and a photocatalytic micro fuel generator (µFG) integrated on a single microfluidic chip. The µFG produces hydrogen by photocatalytic water splitting under solar light. The hydrogen fuel is then consumed by the µFC to generate electricity. Importantly, the by-product water returns back to the photocatalytic µFG via recirculation loop without losses. Both devices rely on novel phenomena in extended-nano-fluidic channels that ensure ultra-fast proton transport. As a proof of concept, we demonstrate that µFG/µFC source achieves remarkable energy density of ca. 17.2 mWh cm-2 at room temperature.

Extended-nano chromatography.

A novel liquid chromatographic method utilizing the extended-nano space called extended-nano chromatography, encompassing the 10-1000nm region, has emerged recently. It utilizes an extended-nano fluidic channel, which is fabricated on a glass chip, as a separation column. The advantages of extended-nano chromatography are that it uses extremely small sample volumes (attoliter to femtoliter) and demonstrate high separation efficiency. In this review, the fundamentals of extended-nano chromatography are summarized. Instrumentations to realize attoliter sample injections and sensitive detection methods are described. A fabrication method for nanochannel separation columns, including substrate-bonding and surface modification, is also introduced. A highly efficient separation was performed within several seconds, as predicted by the theory. Future perspectives, including living single cell analysis and ultrahigh performance separation, are also discussed.

On-Chip Step-Mixing in a T-Nanomixer for Liquid Chromatography in Extended-Nanochannels.

Miniaturization of liquid chromatography separation columns is a key trend in chemical and biochemical areas, particularly in genomics, proteomics, and single-cell analysis. The work at this level relies upon a novel analytical platform that can deal with sample volumes that are much smaller than a cell. An extended-nanospace is within a scale of 101-103 nm and defines the space between a single molecule and normal liquid. Our group has realized high-performance liquid chromatography (HPLC) separation in extended-nanospace with sample injections of hundreds of attoliters and a separation efficiency of hundreds of thousands of plates/m that can overcome the limitations of a conventional packed column by a magnitude of several orders. However, gradient flow is needed to improve the separation performance, and in this work we present reversed-phase chromatography with step-mixing in extended-nanospace and describe its application. Six fluorescently labeled amino acids were separated in 16 s, followed by separation of 17 labeled amino acids in only 50 s with a plate height for most of the peaks of less than 1 µm.

Femtoliter high-performance liquid chromatography using extended-nano channels.

A high-performance liquid chromatography system with 35 fL sample volume was developed using extended-nano (10-1000 nm) fluidic channels. For many years, miniaturization and enhancement of separation performance have been important issues in separation science. Recently, we have reported an ultimate miniaturization of chromatography using extended-nano channels with extremely high separation efficiency of 7 × 106 plates per m. However, the real theoretical plate number was limited to 103 due to the short nanochannel length. In this paper, the theoretical plate number was dramatically increased by developing a new high-pressure system with a very long nanochannel. A separation experiment of two fluorescent dyes demonstrated that the theoretical plate number could be improved to 1.4 × 104, which is much higher than that with conventional HPLC. The theoretical plate number is also comparable to those of capillary monolithic columns. The extremely small sample volume of extended-nano chromatography could support innovative analytical techniques capable of analyzing a single living cell in the near future.

Reversed-phase Chromatography in an Extended Nanospace: Separating Amino Acids in Short and Long Nanochannels.

Micro- and nanofluidics has attracted much attention, particularly concerning single-cell analysis when small amounts of liquids are examined. In present work we successfully fabricated extended-nano channels that were more narrow and shorter (2 mm) as well as wider and longer (10 mm), and accomplished a reversed-phase HPLC separation of labeled amino acids on these channels after octadecylsilylation (ODS). The separation performance characteristics were compared for both types of nano spaces. At an equal amount of pressure, the longer extended-nano channels showed permeability that was one-order higher (K = 47 × 10(-14) m(2)) and separation impedance (E = 13) that was one-order lower than that of the shorter version. Also, the separation plate number for the longer channel was 4000 with a plate height of 2.5 µm. Both channels have advantages for use in single-cell analysis. The longer channel can be applied for the separation of macromolecules (proteomics), while the short version is more applicable to small molecules (amino acids).

Reversed-phase chromatography in extended-nano space for the separation of amino acids.

In this work we used reversed-phase chromatography in extended-nano channels to separate amino acids. A hydrophobic surface modification of extended-nano channels was established. A sample mixture of fluorescein and sulforhodamine B (0.5 and 0.05mM respectively) was used for the demonstration of a reversed-phase separation mode. A small amount of sample band (30fL) was injected into the separation channel, and two compounds were successfully separated. The maximum theoretical plate number of sulforhodamine B was 300,000plates/m. Two sets of 3 amino acids (3.75mM each) were separated using 0.01M citrate buffer (pH 5.5) with 0.01M sodium perchlorate and 12 and 25% of acetonitrile as a mobile phase. A successful separation (320,000plates/m with plate height of 3.2µm for serine) was accomplished.

Desktop near-field thermal-lens microscope for thermo-optical detection in microfluidics.

A new compact near-field desktop-sized diode laser thermal-lens microscope for analysis in microfluidics was proposed. A novel beam-alignment and detection systems provided high signal stability and, along with reduced number of optical elements rendered the instrument portable. The detection of nonfluorescent model species (Fe(II)-bathophenanthroline chelate) in water showed good linearity in the range of 5 × 10(-9) to 1 × 10(-4) M, and the limit of detection was 3.5 × 10(-9) M, which corresponded to 3.5 × 10(-7) absorbance units and provided a 20-fold enhancement in sensitivity compared with existing schematic.

Comparison of performance parameters of photothermal procedures in homogeneous and heterogeneous systems.

The main types of analytical procedures used in thermal-lens spectroscopy and microscopy, which are based on photometric reactions in (i) aqueous solutions, (ii) organo-aqueous mixtures, (iii) polymer-containing (nonionic surfactants or polyethylene glycol) aqueous solutions, (iv) water-organic extraction systems, and (v) two-phase aqueous extraction systems, were compared from the viewpoint of both reproducibility and sensitivity. This comparison was made by examples of the determination of cobalt and iron for batch conditions, flow determination, and detection in HPLC, flow-injection analysis (FIA), and µFIA. It was revealed that for all five types, the real analytical efficiency (a decrease in the limit of detection (LOD) as compared to spectrophotometry) is primarily determined by the reaction conditions, provided that excitation of the thermal lens is the same. Aqueous solutions provide more efficient optimization of reaction conditions than do those in organo-aqueous solutions and solvent-extraction water-organic mixtures. The best results are achieved when shifting to polymer-containing aqueous solutions and two-phase aqueous extraction systems, which decreases in the LODs by a factor of 20 - 100%.

Development of a micro-potentiometric sensor for the microchip analysis of alkali ions.

This paper reports on the development of a micro-potentiometric sensor based on external microelectrodes introduced into a microchip. We miniaturized reference and ion-selective electrodes (ISEs) and embedded them into a plastic (PDMS) microchip; the miniaturization of ISE was attained by using a monolithic capillary-based membrane. This sensor was applied to the detection of alkali ions (Na+, K+ and NH4+) in a microflow on the microg/L level.

Thermooptical detection in microchips: from macro- to micro-scale with enhanced analytical parameters.

In this paper, we compared the methods of photothermal spectroscopy used in different spatial scales, namely thermal-lens spectrometry (TLS) and thermal-lens microscopy (TLM) to enhance the performance parameters in analytical procedures. All of the experimental results were confirmed by theoretical calculation. It was proven that the design for both TLM and TLS, despite a different scale for the effect, is governed by the same signal-generating and probing conditions (probe beam diameter at the sample should be equal to the diameter of the blooming thermal lens), and almost does not depend on the nature of the solvent. Theoretical and experimental instrumental error curves for thermal lensing were coincident. TLM obeys the same law of instrumental error as TLS and shows better repeatability for the same levels of thermal-lens signals or absorbances. TLS is more advantageous for studying low concentrations in bulk, while TLM shows much lower absolute LODs due to better repeatability for low amounts. The behavior of the thermal-lens signal with different flow rates was studied and optimum conditions, with the minimum contribution to total error, were found. These conditions are reproducible, are in agreement with the existing theory of the thermal response in thermal lensing, and do not significantly affect the design of the optimum scheme for setups. TLM showed low LODs in solvent extraction (down to 10(-8) M) and electrokinetic separation (10(-7) M), which were shown to be governed by discussed instrumental regularities, instead of by microchemistry.

Pesticide analysis by MEKC on a microchip with hydrodynamic injection from organic extract.

An integrated microchip for monitoring carbamate pesticides in environmental water using continuous flow chemical processes is under development, i. e., the integration of hydrolysis, azo-derivatization, liquid-liquid extraction, electrophoretic separation, and quantification. The separation of the derivatives of four carbamate pesticides (carbaryl, carbofuran, propoxur, and bendiocarb) extracted in the continuous flow of a 1-butanol phase was studied in a silica microchip using micellar EKC. A baseline separation of four pesticide derivatives was achieved on a silica chip using hydrodynamic injection with electroosmotic gating. Detection using a thermal lens microscope showed good linearity in the concentration range of 10(-6 )-10(-5 )M with an LOD of 5 x 10(-7) M, which is superior to that of conventional CE with UV absorption detection at a level of 10(-4) M.

Application of a micro multiphase laminar flow on a microchip for extraction and determination of derivatized carbamate pesticides.

Determination of carbamate pesticides such as carbaryl, carbofuran, propoxur and bendiocarb was demonstrated on a microchip with newly designed microchannels developed for efficient solvent extraction. The pesticides were hydrolyzed to corresponding naphthols, coupled with p-nitrobenzenediazonium fluoroborate reagent, and then extracted into 1-butanol as colored azo derivatives and detected with a thermal lens microscope. Optimum flow rates for the aqueous and organic phases were evaluated in the continuous-flow chemical process established in the microchip. The calibration lines showed good linearity in the range of concentrations of 0.03 - 3 ppm (10(-7) - 10(-5) M) and a mass detection limit down to a nanogram level was achieved that is at least two orders of magnitude lower than the LODs for conventional spectrophotometric methods. Azo derivatives of the pesticides were successfully separated and identified by micellar electrokinetic chromatography (MEKC) using a sample prepared on a bulk scale.