Direct Measurement of Near-Wall Molecular Transport Rate in a Microchannel and Its Dependence on Diffusivity.

Solute transport in a narrow space is the most elemental process in chromatography and biological pattern formation. However, the observation of such transport has been quite difficult, and theoretical investigations have therefore preponderated. Here, using a space- and time-resolved surface plasmon resonance (SPR) method, we measured the nanoscale near-wall (next to the wall) transport rate in a narrow channel after a solution and its solvent had come into contact. By combining the SPR method with a capillary injection method, which enables two solution plugs to flow immediately after they have made contact, we were able to measure the solute concentration evolution at the channel wall. We tested three combinations of two plugs of solution-water-glucose, sodium chloride-water, and glucose-sodium chloride-and succeeded in measuring diffusion-coefficient-dependent changes in the concentration of solute flowing through a rectangular microchannel in less than 0.4 s. A numerical analysis of this system revealed the acceleration of the solute/solution boundary moving on the wall and its deceleration at the center of the channel cross section. The observed experimental transport rate agreed with the numerical result quantitatively. These results show that the solute transport followed a laminar flow with a no-slip model and that the molecules were transported in the order of their diffusivity. In the third combination, when the two solutions made contact and started flowing, the interdiffusion of the solutes resulted in temporal concentrations lower than either of the solutions before contact, which indicated that the contact between the two solutions quickly led to separation by the advection-diffusion processes. We found that such a concentration profile could actually be measured. Our techniques are simple and applicable to a wide range of molecules; the method opens the way to direct observation of the space-time near-wall solute transport process and can be used for the rapid determination of diffusivity.

Dopamine detection on activated reaction field consisting of graphene-integrated silicon photonic cavity.

Graphene is widely recognized as an outstanding and multi-functional material in various application fields such as electronics, photonics, mechanics, and life sciences. We propose a neurotransmitter sensor with ultra-small volume for detecting the photonic light-matter response. Such detection can be achieved using surface-activated monolayer graphene sheets and CMOS-compatible silicon-photonic circuits. Patterned pieces of CVD-grown graphene are integrated on the top of a silicon micro-ring resonator, which induce the adsorption of catecholamine molecules originated from the π-stacking effect. We used dopamine to demonstrate such detection and examine the sensitivity of graphene-dopamine coupling. To avoid high optical insertion loss and degradation of resonance characteristics caused by a graphene's extremely high optical absorption coefficient in the near infrared region, a ring resonator with adjusted coupling design is used to compensate for the drawbacks. Owing to the advanced nano-sensing platform and measurement system, an activated graphene-sensing surface of only ∼30 µm2/ch enables π coupling to dopamine with enough sensitivity to detect less than 10-µM solution concentration. The detection mechanism through the surface reaction is also verified by optical simulation and atomic force microscopy measurement, revealing that the flowing dopamine molecules can only occupy the outermost surface of graphene. We expect this sensor to contribute to the development of an innovative label-free and disposable bio-sensing platform with accurate, sensitive, and fast response.

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.

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.

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.

Data Processing of SPR Curve Data to Maximize the Extraction of Changes in Electrochemical SPR Measurements.

We developed a novel measuring and data-processing method for performing electrochemical surface plasmon resonance (EC-SPR) on sensor surfaces for which detecting a specific SPR angle is difficult, such as a polymer having a non-uniform thickness with coloration. SPR measurements are used in medicine and basic research as an analytical method capable of molecular detection without labeling. However, SPR is not good for detecting small molecules with small refractive index changes. The proposed EC-SPR, which combines SPR measurements with an electrochemical reaction, makes it possible to measure small molecules without increasing the number of measurement steps. A drawback of EC-SPR is that it is difficult to detect a specific SPR angle on electron mediators, and it was found that it may not be possible to capture all the features produced. The novel method we describe here is different from the conventional one in which a specific SPR angle is obtained from an SPR curve; rather, it processes the SPR curve itself and can efficiently aggregate the feature displacements in the SPR curves that are dispersed through multiple angles. As an application, we used our method to detect small concentrations of H2O2 (LOD 0.7 µM) and glutamate (LOD 5 µM).

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.

On-chip graphene oxide aptasensor for multiple protein detection.

The versatility of an on-chip graphene oxide (GO) aptasensor was successfully confirmed by the detection of three different proteins, namely, thrombin (TB), prostate specific antigen (PSA), and hemagglutinin (HA), simply by changing the aptamers but with the sensor composition remaining the same. The results indicate that both DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. Molecular selectivity and concentration dependence were investigated in relation to TB and PSA detection by using a dual, triple, and quintuple microchannel configuration. The multiple target detection of TB and PSA on a single chip was also demonstrated by using a 2×3 linear-array GO aptasensor. This work enables us to apply this sensor to the development of a multicomponent analysis system for a wide variety of targets by choosing appropriate aptamers.

Molecular design for enhanced sensitivity of a FRET aptasensor built on the graphene oxide surface.

We designed a biomolecular probe for highly sensitive protein detection by modifying an aptamer with a DNA spacer. The spacer controls the distance between a fluorescence dye and a quencher, which is crucial for FRET-based sensors. We successfully demonstrated an improvement in the sensitivity of an on-chip graphene oxide aptasensor.