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We have developed a microsystem for the detection of organophosphorus (OP) compounds using acetylcholine esterase (AchE) and choline oxidase (ChOx) bienzyme bioelectrocatalysis. Because AchE is irreversibly inhibited by OP pesticides, the change in AchE activity with OP treatment can be traced to determine OP concentration. Polymer-associated ChOx immobilization on the working electrode surface and magnetic microparticle (MP)-assisted AchE deposition methods were employed to create an AchE–ChOx bienzyme-modified biosensing system. ChOx was immobilized on the micropatterned electrodes using poly(L-lysine), glutaraldehyde, and amine-rich interfacial surface. AchE was immobilized on the MP surface via Schiff's base formation, and the enzyme-modified MPs were deposited on the working electrode using a magnet under the microfluidic channel. The bioelectrocatalytic reaction between AchE–ChOx bienzyme cascade and the ferrocenyl electron shuttle was successfully used to detect OP with the developed microchip. This provides a self-contained and relatively easy method for OP detection. It requires minimal time and a small sample size, and has potential analytic applications in pesticides and chemical warfare agents.

A Love wave-based biosensor with a 440 MHz operating frequency was developed for simultaneous detection of two different concentrations of anti-dinitrophenyl-keyhole limpet hemocyanin (Anti-DNP-KLH) rabbit immunoglobulin G (IgG) in a single sensor. The sensor was composed of surface acoustic wave (SAW) reflective delay lines built from interdigital transducer (IDT) and several reflectors, a poly(methyl methacrylate) (PMMA) waveguide layer, and two sensitive films. To extract optimal device parameters, coupling of mode (COM) modeling was carried. According to the device parameters determined, the Love wave biosensor was fabricated and then wirelessly characterized by a network analyzer. Binding of anti-DNP IgG to DNP induced a change in the time positions of the original reflection peaks mainly due to the mass loading effect. The measured time positions were matched well with the predicted values from COM modeling. The sensitivities evaluated from the first and second sensitive films were 167.9 and 44.8 deg·µg-1·ml-1, respectively.

A Love wave-based wireless immunosensor with a 440 MHz operating frequency was developed on a 41� YX LiNbO3 piezoelectric substrate. The developed sensor was composed of a shear horizontal surface acoustic wave (SH SAW) reflective delay line, a poly(methyl methacrylate) (PMMA) waveguide layer, and a 2,4-dinitrophenyl (DNP) receptor layer that specifically responds to an anti-DNP immunoglobulin G (IgG). Fabricated devices were wirelessly tested using a network analyzer. Four reflection peaks with large signal/noise (S/N) ratios and sharp reflection peaks were observed in the time domain, and are attributed to three shorted grating reflectors and DNPbound gold film. With 10 mW radio frequency (RF) power from the network analyzer, a readout distance of � 1 m was observed. The binding of IgG to DNP receptor molecules induced a change in the Love wave velocity owing to a mass loading effect, resulting in phase shifts of the original reflection peaks. The phase shifts increased linearly with increasing IgG concentration. Fast response time and long-term stability of the developed sensor in a liquid environment were observed. The phase shifts depending on different PMMA thicknesses were also investigated to identify the optimal PMMA waveguide thickness. # 2009 The Japan Society of Applied Physics

This paper presents the development of a hand-operated lab-on-a-chip (LOC) with latch mechanism for portable immunosensing applications. To control the transportation of multiple liquids without any external equipment, a capillary attraction force is used for filling liquid. Hydraulic pressure which is caused by the elastic deformation of the cover caps of the reservoir is used for exchanging liquids in the reaction channel. To prevent unwanted backward flow, the latches of the LOC, which consist of reversed mushroom-shaped elastic cover caps and rigid locking components, are used to retain the internal pressure in the reservoir. The electrochemical immunoassay measuring antibody concentrations are performed within the fabricated LOC to highlight its portability to be used as a diagnostic system. The immunosening mechanism applied to this LOC is the electrochemical signaling from the antigen?antibody interaction with the bioelectrocatalyzed enzymatic signal amplification. The proposed signaling strategy is based on the backfilling method, which does not require any complicated processes such as antibody labeling steps or any labeled secondary antibodies. As a modeling test of the immunoassay, the anti-dinitrophenyl (DNP) antibody with an enzymatic catalysis was selected as the target analyte.

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.