An immunoassay, which is an indispensable analytical method both in biological research and in medical fields was successfully integrated into a “single-step” by developing a microdevice composed of a graphene oxide (GO)-containing hydrogel and a poly (dimethylsiloxane) (PDMS) microchannel array with a polyethylene glycol (PEG) coating containing a fluorescently-labelled antibody. Here we used 2-hydroxyethylmethacrylate (HEMA) as a monomer that is easily, and homogeneously, mixed with GO to synthesize the hydrogel. The fluorescence quenching and size separation functions were then optimized by controlling the ratios of HEMA and GO. Free fluorescently-labelled antibody was successfully separated from the immunoreaction mixture by the hydrogel network structure, and the fluorescence was subsequently quenched by GO. In comparison to the previously reported immunoassay system using GO, the present system achieved a very high fluorescence resonance energy transfer (FRET) efficiency (∼90%), due to the use of direct adsorption of the fluorescently-labelled antibody to the GO surface; in contrast, the former reported method relied on indirect adsorption of the fluorescently-labelled antibody via immunocomplex formation at the GO surface. Finally, the single-step immunoassay microdevice was made by combining the developed hydrogel and the PDMS microchannel with a coating containing the fluorescently-labelled antibody, and successfully applied for the single-step analysis of IgM levels in diluted human serum by simple introduction of the sample via capillary action.

The caspase-3 inhibitor assay, which is important for drug development was successfully integrated into a “single step” by solving the immobilization problem of enzymes with low activity. Here we used the soluble polyethylene glycol (PEG) coatings for enzyme immobilization to the concave-shaped PDMS surface, and substrate immobilization to the convex-shaped PDMS surface. Then these PDMS structures were combined to form a capillary-structure, which we call a combinable poly (dimethylsiloxane) (PDMS) capillary (CPC) sensor, enabling simultaneous immobilization of two different reactive reagents such as enzymes and fluorescent substrates. The present sensor was disposable, and the fluorescence response was simply obtained by introducing the sample solution by capillary action. The caspase-3 activity was able to be maintained at approximately 90% under −80 °C storage conditions even after 5 months. Importantly, the total reaction time was reduced from an hour to 3–5 min, and simultaneous acquisition of multiple data sets required to determine IC50 value was also successfully achieved using the CPC sensor array.

A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen–enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10–1000 ng mL−1 linear range, is reported.

The conventional neuraminidase inhibitor assay requiring complicated step-by-step operations was successfully integrated into a “single step” operation using a combinable poly(dimethylsiloxane) (PDMS) capillary (CPC) sensor. In the conventional neuraminidase inhibitor assay, complicated step-by-step operations including initial reaction of an inhibitor and an enzyme, and subsequent reaction with a fluorescent substrate are necessary. Furthermore, optimal pH conditions for the enzymatic reaction differ from those of fluorescence detection. Therefore, preparation of different pH buffer solutions is also essential. To simplify the neuraminidase inhibitor assay into a single-step operation, we applied our recently developed CPC sensor array. Because the CPC sensor allows independent immobilization of different reagents, such as enzymes and fluorescent substrates, simple introduction of an inhibitor solution by capillary action leads to fluorescence emissions. Here, we investigated the optimal pH that enables us to perform the neuraminidase inhibitor assay via a single step operation using the CPC sensor. When the reaction profile was investigated under the optimal pH, the total reaction time was shortened from 1 h to 15 min. Moreover, the neuraminidase inhibitor assay was validated using a typical inhibitor, N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. The inhibition constant, Ki, which was calculated on the basis of the IC50 obtained from the inhibition curve, was in good agreement with the published values. Furthermore, we demonstrated a comparison of fluorescence responses for buffer solutions versus the solution of a well-known influenza drug, Relenza. We observed successful inhibition by Relenza. In addition to the CPC-based approach, the consumption of enzyme, substrate, and inhibitor was reduced to 1% of the amount used in conventional neuraminidase inhibitor assays.

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1–2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

In this study, a highly sensitive capillary-based enzyme-linked immunosorbent assay (ELISA) has been developed for the analysis of picomolar levels of thrombin-cleaved osteopontin (trOPN), a potential biomarker for ischemic stroke, in human plasma. Using a square capillary coated with 8.5 μg/ml anti-human trOPN capture antibody for ELISA, the linear range obtained was 2 to 16 pM trOPN antigen. This concentration range was in the detection window of trOPN antigen in plasma samples. Compared with the conventional microplate-based ELISA, the current capillary technique significantly reduced the amounts of reagent from milliliter to microliter, reduced the analysis time from 8 to 3 h, and had a better sensitivity and detection limit performance from approximately 50 pM down to 2 pM of trOPN antigen. These results indicate that this capillary-based immunoassay is a potential tool for biomarker detection and may be useful in clinical trials and medical diagnostic applications.

We describe a new method for fabricating a capillary-type sensor, called a combinable poly(dimethyl siloxane) (PDMS) capillary (CPC) sensor. The method for preparing the CPC simplifies enzyme inhibitor assays into a simple, single step assay. The sample inhibitor solution is introduced by capillary action. This triggers the spontaneous dissolution of physically adsorbed fluorescent substrates, and the substrate mixes with the inhibitor. This is followed by competitive reaction with insoluble enzyme to give a fluorescence response. CPC is composed of a convex-shaped PDMS stick containing reagents immobilized in an insoluble coating, and a concave-shaped PDMS stick containing reagents immobilized in a soluble coating. Since the concave-shaped PDMS has a deeper channel than the convex structure, combining these PDMS sticks is like closing the zipper of a “freezer bag”. This allows easy fabrication of “thin and long” capillary structures containing different reagents inside the same capillary, without the need for precise alignment. This method allows the immobilization of two reactive reagents, such as enzyme and substrate required for a single step assay, which are typically very difficult to immobilize using commercially available conventional capillaries. Furthermore, by simply arraying various CPCs, the CPC sensor allows multiple assays. Here, we carried out a single-step enzyme inhibitor assay using the CPC. In addition, two independent CPCs were arrayed to demonstrate multiple assaying of a protease inhibitor.

To enhance sensitivity and facilitate easy sample introduction into a combinable poly(dimethylsiloxane) (PDMS) capillary (CPC) sensor array, PDMS was modified in bulk and on its surface to prepare “black” PDMS coated with a silver layer and self-assembled monolayer (SAM). India ink, a traditional Japanese black ink, was added to the PDMS pre-polymer for bulk modification. The surface was modified by a silver mirror reaction followed by SAM formation using cysteine. These modifications enhanced the fluorescence signals by reflecting them from the surface and reducing background interference. A decrease in the water contact angle led to enhanced sensitivity and easy sample introduction. Furthermore, a CPC sensor array for multiplex detection of serum sample components was prepared that could quantify the analytes glucose, potassium, and alkaline phosphatase (ALP). When serum samples were introduced by capillary action, the CPC sensor array showed fluorescence responses for each analyte and successfully identified the components with elevated concentrations in the serum samples.

Here we present an open-type capillary-assembled microchip (CAs-CHIP), demonstrating its ease of fabrication, and use for rapid and simultaneous sensing of different serum components; viz., glucose, cholesterol, and alkaline phosphatase (ALP) and pH. Multiple square glass capillary sensors (outer diameter, 300 μm square) were embedded into a black polydimethylsiloxane (PDMS) microchannel array (300 μm width and depth) generating an open-type CAs-CHIP into which samples were introduced by capillary action. The open-type CAs-CHIP was then embedded into a PDMS reservoir chip where PDMS oil was dropped onto both ends of the capillary to avoid evaporation of the nanoliter sample inside the capillaries. We used fluorescein-based probes to achieve simultaneous multi-component sensing by using a single fluorescence filter and obtained accurate measurements with acceptable day-to-day precision (5.3–8.8%). Discrimination between control and analyte-spiked serum samples was simultaneously accomplished for 4 analytes within 10 min.

This report describes the fabrication and characterization of a simple and disposable capillary isoelectric focusing (cIEF) device containing a reagent-release capillary (RRC) array and poly(dimethylsiloxane) (PDMS) platform, which allows rapid (within 10 min) screening of cIEF conditions by introducing a sample solution into plural RRCs by capillary action followed by electric field application. To prepare the RRC, covalent immobilization of poly(dimethylacrylamide) (PDMA) was conducted to suppress electro-osmotic flow (EOF), followed by physical adsorption of the mixture of carrier ampholyte (CA), surfactant, labeling reagent (LR), and other additives to the PDMA surface to construct a two-layer structure inside a square glass capillary. When the sample solution containing proteins was introduced into the RRC, physically adsorbed CA, surfactant, and LR can be dissolved and released into the sample solution. Then, complexation of LR with proteins, mixing with CA and surfactant, and exposure of the PDMA surface spontaneously occurs for the IEF experiments. Here, three different RRCs that immobilize different CAs were prepared, and simultaneous cIEF experiments involving hemoglobin AFSC mixtures for choosing the best CA demonstrated the proof of concept.

A single-step enzyme-linked immunosorbent assay (ELISA) capillary immunosensor that exploits the combination of surface-bonded glucose oxidase (GOD), antibody and physically-adsorbed poly(ethyleneglycol) (PEG) membrane containing peroxidase (POD)-labeled antibody in a single capillary, is presented. The present simple detection technique involves a proximity-based two-enzyme system where the product of the first enzyme reaction is used as a substrate into the second enzyme reaction. Since the degree of antigen–antibody reaction controls the second enzymatic reaction process, amount of final fluorescent product varies with antigen concentration that leads to the making ELISA procedure single step. Sample solution that contains antigen, glucose, ascorbic acid and a fluorescent substrate (amplex red) is simply introduced into the capillary immunosensor by capillary action. In the presence of an antigen in the nanoliter sample volume, the released POD-labeled antibody forms a sandwich immunocomplex on the surface bearing the antibody. This leads to a proximity of the POD and GOD. We confirmed that the rate of conversion of a non-fluorescent probe, amplex red, to a red fluorescent dye, resorufin, is dependent on the two-enzyme proximity in a certain experimental condition. Preliminary results yielded an approximate detection limit of around subnanogram per milliliter concentration for human IgG. The relative standard deviation of the fluorescence response was ca. 4.1% for the capillary pieces prepared by cutting a long immunosensor capillary. This approach could be an enabling technology applicable to microfluidic device integration for multiple analyte sensing. Realization of such an endeavor could be very promising for drug screening and clinical diagnostic applications.

This review accounts for the current development in microfluidic immunosensing chips. The basic knowledge of immunoassay in relation to its microfluidic material substrate, fluid handling and detection mode are briefly discussed. Here, we mainly focused on the surface modification, antibody immobilization, detection, signal enhancement and multiple analyte sensing. Some of the clinically important currently implemented on the microfluidic immunoassay chips are C-reactive protein (CRP), prostate specific antigen (PSA), ferritin, vascular endothelial growth factor (VEGF), myoglobin (Myo), cardiac troponin T (cTnT), cardiac troponin I (cTnI), and creatine kinase-cardiac muscle isoform (CK-MB). The emerging microfludic immunosensor technology may be a promising prospect that can propel the improvement of clinical and medical diagnosis.

Single-drop analysis of two different real sample solutions (2 µL) while simultaneously monitoring the activity of two sets of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip (CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane (PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5–8.2 (% RSD, n = 5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested the usefulness of the CAs-CHIP in the field of drug discovery.