A reliable aptamer array prepared by repeating inkjet-spotting toward on-site measurement.

A preparation protocol is proposed for a reliable aptamer array utilizing an ink-jet spotter. We accumulated streptavidin and biotinylated-aptamer in this order on a biotinylated-polyethylene glycol-coated gold substrate to prepare an aptamer array. The aptamer array was prepared with an alternate spotting structure where each aptamer spot was placed between reference spots formed with blocking solution thus suppressing contamination from neighboring spots during the blocking and washing processes. Four aptamer spots were prepared in a small area of 1×4.8mm(2) with five reference spots made of blocking solution. We evaluated the thrombin binding ability of the spotted aptamer array using a multi-analysis surface plasmon resonance sensor. We prepared a disposable capillary-driven flow chip designed for on-site measurement (Miura et al., 2010) with our aptamer array and detected thrombin from phosphate-buffered saline at concentrations of 50ngmL(-1) and 1µgmL(-1) (equivalent to 1.35 and 27nM, respectively). A correlation was observed between the refractive index shift and thrombin concentration. This implies that our array preparation protocol meets the requirement for the preparation of a one-time-use chip for on-site measurement.

Floating chip mounting system driven by repulsive force of permanent magnets for multiple on-site SPR immunoassay measurements.

We have developed a measurement chip installation/removal mechanism for a surface plasmon resonance (SPR) immunoassay analysis instrument designed for frequent testing, which requires a rapid and easy technique for changing chips. The key components of the mechanism are refractive index matching gel coated on the rear of the SPR chip and a float that presses the chip down. The refractive index matching gel made it possible to optically couple the chip and the prism of the SPR instrument easily via elastic deformation with no air bubbles. The float has an autonomous attitude control function that keeps the chip parallel in relation to the SPR instrument by employing the repulsive force of permanent magnets between the float and a float guide located in the SPR instrument. This function is realized by balancing the upward elastic force of the gel and the downward force of the float, which experiences a leveling force from the float guide. This system makes it possible to start an SPR measurement immediately after chip installation and to remove the chip immediately after the measurement with a simple and easy method that does not require any fine adjustment. Our sensor chip, which we installed using this mounting system, successfully performed an immunoassay measurement on a model antigen (spiked human-IgG) in a model real sample (non-homogenized milk) that included many kinds of interfering foreign substances without any sample pre-treatment. The ease of the chip installation/removal operation and simple measurement procedure are suitable for frequent on-site agricultural, environmental and medical testing.

Cooperative suction by vertical capillary array pump for controlling flow profiles of microfluidic sensor chips.

A passive pump consisting of integrated vertical capillaries has been developed for a microfluidic chip as an useful component with an excellent flow volume and flow rate. A fluidic chip built into a passive pump was used by connecting the bottoms of all the capillaries to a top surface consisting of a thin layer channel in the microfluidic chip where the thin layer channel depth was smaller than the capillary radius. As a result the vertical capillaries drew fluid cooperatively rather than independently, thus exerting the maximum suction efficiency at every instance. This meant that a flow rate was realized that exhibited little variation and without any external power or operation. A microfluidic chip built into this passive pump had the ability to achieve a quasi-steady rather than a rapidly decreasing flow rate, which is a universal flow characteristic in an ordinary capillary.

Passive fluidic chip composed of integrated vertical capillary tubes developed for on-site SPR immunoassay analysis targeting real samples.

We have successfully developed a surface plasmon resonance (SPR) measurement system for the on-site immunoassay of real samples. The system is composed of a portable SPR instrument (290 mm(W) × 160 mm(D) × 120 mm(H)) and a microfluidic immunoassay chip (16 mm(W) × 16 mm(D) × 4 mm(H)) that needs no external pump system. An integrated vertical capillary tube functions as a large volume (150 µL) passive pump and a waste reservoir that has sufficient capacity for several refill operations. An immunoassay was carried out that employed the direct injection of a buffer and a test sample in sequence into a microfluidic chip that included 9 antibody bands and 10 reference reagent bands immobilized in the flow channel. By subtracting a reliable averaged reference sensorgram from the antibody, we effectively reduced the influence of the non-specific binding, and then our chip successfully detected the specific binding of spiked IgG in non-homogeneous milk. IgG is a model antigen that is certain not to be present in non-homogeneous milk, and non-homogeneous milk is a model of real sample that includes many interfering foreign substances that induce non-specific binding. The direct injection of a real sample with no pretreatment enabled us to complete the entire immunoassay in several minutes. This ease of operation and short measuring time are acceptable for on-site agricultural, environmental and medical testing.

Selective detection of a catecholamine against electroactive interferents using an interdigitated heteroarray electrode consisting of a metal oxide electrode and a metal band electrode.

We developed an interdigitated array electrode (IDAE) consisting of a metal oxide electrode and a metal band heteroelectrode and employed it for the selective detection of catecholamines. We used an indium-tin oxide (ITO) film as the oxidation electrode of the IDAE because the ITO was able to suppress response currents from L-ascorbic acid (AA) and uric acid (UA), which are major electroactive interferents in biological fluids. However, the ITO film also suppresses the reduction of quinones including oxidized catecholamines. We developed a simple technique for fabricating our hetero IDAE, which also preserves the electrochemical properties of the ITO. When we compared hetero ITO-gold, homo ITO-ITO, and carbon-carbon IDAEs, we found that the hetero IDAE provided both high sensitivity and selectivity for DA detection. We achieved high selectivities for DA against AA and UA. The ratios of the response currents of AA and UA to DA were calculated as 6 and 5%, respectively.

Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector.

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.

Separate detection of BTX mixture gas by a microfluidic device using a function of nanosized pores of mesoporous silica adsorbent.

We achieved separate detection of the components of 10 ppm of a benzene, toluene, and o-xylene mixture gas by using mesoporous silica powder incorporated in our microfluidic device. The device consists of concentration and detection cells formed of 3 cm x 1 cm Pyrex plates. We first introduced the mixture gas into the concentration cell where it was adsorbed on an adsorbent in a channel formed in the cell. We then raised the temperature using a thin-film heater and introduced the desorbed gas into the detection cell. Here, we measured the changes in the absorption spectra of the mixture gas in the detection cell. We found that the mixture ratio of the compounds in the desorbed gas varies with time because the thermal desorption property of each compound is different from that of the adsorbent. We analyzed the thermal desorption mechanism by comparing two types of silica adsorbents with different pore structures. We found that an adsorbent that has pores with a periodic and uniform nanosized column shape provides better component separation. We concluded that the uniform pore structure might cause the adsorbate molecules to exhibit a homogeneous adsorption state thus revealing the desorption properties of the gas more clearly.

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance.

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.

Air-cooled cold trap channel integrated in a microfluidic device for monitoring airborne BTEX with an improved detection limit.

We integrated an air-cooled cold trap (CT) channel in a microfluidic device for monitoring airborne benzene, toluene, ethylbenzene, and xylene (BTEX) gases and demonstrated its effect on improving the detection limit of the microfluidic device. The device consists of concentration and detection cells formed of 3 x 1 cm Pyrex plates. We first introduced a sample gas into the concentration cell, and the gas was adsorbed onto an adsorbent in the channel. We then raised the temperature using a thin-film heater and introduced the desorbed gas into the detection cell. To prevent dilution of the gas before detection, we propose an improvement to the concentration cell structure that involves the integration of the CT channel. We examined the CT effect by comparing three types of concentration cell with different channel structures. We found that we could detect a gas concentration about 2 orders of magnitude lower than in our previous work by optimizing the channel structure and integrating a CT channel. As an example of BTEX detection,we obtained a 0.05 ppm detection limit for toluene gas with a sampling time of 30 min.

Differential measurement with a microfluidic device for the highly selective continuous measurement of histamine released from rat basophilic leukemia cells (RBL-2H3).

We fabricated a micro-fluidic device for the highly selective detection of the histamine released from rat basophilic leukemia (RBL) 2H3 cells. The device has two thin layer flow channels, each with one working electrode. One electrode was modified with Os-polyvinylpyridine based mediator containing horseradish peroxidase (Os-gel-HRP) and histamine oxidase (HAOx), the other was modified with Os-gel-HRP without any HAOx. We employed the device for differential measurement by using the HAOx modified electrode for detection and the unmodified electrode as a reference. The detection limit was greatly improved from 190 to 25 nM since the baseline noise level was suppressed. We used differential measurement to observe the histamine released from RBL-2H3 cells when stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) as an antigen. We injected 5 microM of histamine solution into our device and it remained stable for more than 8 h.