Graphene-oxide-based immunosensing through fluorescence quenching by peroxidase-catalyzed polymerization.

A graphene oxide (GO)-based immunosensor is developed for the detection of interleukin-5 (IL-5), a key cytokine associated with asthma pathology and eosinophilia. The immunosensing platform utilizes the innate fluorescence of GO, not demanding biomolecules labeled with fluorescent dyes. The GO-based immunoassay exhibits high specificity for IL-5 among other cytokines and is not affected by nonspecific proteins in human serum.

Biomimetic Nanopillar-Based Biosensor for Label-Free Detection of Influenza A Virus.

Since the first emergence of influenza viruses, they have caused the flu seasonally worldwide. Precise detection of influenza viruses is required to prevent the spreading of the disease. Herein, we developed an optical biosensor using peptide-immobilized nanopillar structures for the label-free detection of influenza viruses. The spin-on-glass nanopillar structures were fabricated by nanoimprint lithography. A sialic acid-mimic peptide, which can specifically bind to hemagglutinin on the surface of the influenza virus, was immobilized onto the nanopillars via polymerized dopamine. The constructed nanopillar sensor enabled us to detect influenza A viruses in the range of 103-105 plaque-forming units through simple measurements of reflectance. Our findings suggest that biomimetic modification of nanopillar structures can be an alternative method for the immunodiagnosis of influenza viruses.

Hierarchically Porous, Laser-Pyrolyzed Carbon Electrode from Black Photoresist for On-Chip Microsupercapacitors.

We report a laser-pyrolyzed carbon (LPC) electrode prepared from a black photoresist for an on-chip microsupercapacitor (MSC). An interdigitated LPC electrode was fabricated by direct laser writing using a high-power carbon dioxide (CO2) laser to simultaneously carbonize and pattern a spin-coated black SU-8 film. Due to the high absorption of carbon blacks in black SU-8, the laser-irradiated SU-8 surface was directly exfoliated and carbonized by a fast photo-thermal reaction. Facile laser pyrolysis of black SU-8 provides a hierarchically macroporous, graphitic carbon structure with fewer defects (ID/IG = 0.19). The experimental conditions of CO2 direct laser writing were optimized to fabricate high-quality LPCs for MSC electrodes with low sheet resistance and good porosity. A typical MSC based on an LPC electrode showed a large areal capacitance of 1.26 mF cm-2 at a scan rate of 5 mV/s, outperforming most MSCs based on thermally pyrolyzed carbon. In addition, the results revealed that the high-resolution electrode pattern in the same footprint as that of the LPC-MSCs significantly affected the rate performance of the MSCs. Consequently, the proposed laser pyrolysis technique using black SU-8 provided simple and facile fabrication of porous, graphitic carbon electrodes for high-performance on-chip MSCs without high-temperature thermal pyrolysis.

Enhanced biomolecular detection based on localized surface plasmon resonance (LSPR) using enzyme-precipitation reaction.

An enzyme-catalyzed precipitation reaction was employed as a means to increase the change in the LSPR signal after intermolecular bindings between antigens and antibodies occurred on gold nanodot surfaces. The gold nanodot array with an diameter of 175 nm and a thickness of 20 nm was fabricated on a glass wafer using thermal nanoimprint lithography. The human interleukin (hIL) 5 antibody was immobilized on the gold nanodot, followed by binding of hIL 5 to the anti-hIL 5. Subsequently, a biotinylated anti-hIL 5 and a alkaline phosphatase conjugated with streptavidin were simultaneously introduced. A mixture of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and nitro blue tetrazolium (NBT) was then used for precipitation, which resulted from the biocatalytic reaction of the alkaline phosphatase on gold nanodot. The LSPR spectra were obtained after each binding process. Using this analysis, the enzyme-catalyzed precipitation reaction on gold nanodots was found to be effective in amplifying the change in the peak wavelength of LSPR after molecular bindings.

Signal amplification by enzymatic reaction in an immunosensor based on localized surface plasmon resonance (LSPR).

An enzymatic reaction was employed as a means to enhance the sensitivity of an immunosensor based on localized surface plasmon resonance (LSPR). The reaction occurs after intermolecular binding between an antigen and an antibody on gold nano-island (NI) surfaces. For LSPR sensing, the gold NI surface was fabricated on glass substrates using vacuum evaporation and heat treatment. The interferon-γ (IFN-γ) capture antibody was immobilized on the gold NIs, followed by binding of IFN-γ to the antibody. Subsequently, a biotinylated antibody and a horseradish peroxidase (HRP) conjugated with avidin were simultaneously introduced. A solution of 4-chloro-1-naphthol (4-CN) was then used for precipitation; precipitation was the result of the enzymatic reaction catalyzed the HRP on gold NIs. The LSPR spectra were obtained after each binding process. Using this method, the enzyme-catalyzed precipitation reaction on the gold NI surface was found to effectively amplify the change in the signal of the LSPR immunosensor after intermolecular binding.

A Lipid-Nanopillar-Array-Based Immunosorbent Assay.

Since infectious diseases, particularly viral infections, have threatened human health and caused huge economical losses globally, a rapid, sensitive, and selective virus detection platform is highly demanded. Enzyme-linked immunosorbent assay (ELISA) with flat solid substrates has been dominantly used in detecting whole viruses for its straightforwardness and simplicity in assay protocols, but it often suffers from limited sensitivity, poor quantification range, and a time-consuming assay procedure. Here, a lipid-nanopillar-array-based immunosorbent assay (LNAIA) is developed with a nanopillar-supported lipid bilayer substrate with fluorophore-modified antibodies for rapid, sensitive, and quantitative detection of viruses. 3D nanopillar array structures and fluid antibodies with fluorophores facilitate faster and efficient target binding and rapid fluorophore localization for quick, reliable analysis on binding events with a conventional fluorescence microscopy setup. LNAIA enables quantification of H1N1 virus that targets down to 150 virus particles with 5-orders-of-magnitude dynamic range within 25 min, which cannot be achieved with conventional ELISA platforms.

System for Fabrication of Large-Area Roll Molds by Step-and-Repeat Liquid Transfer Imprint Lithography.

The effective production of nanopatterned films generally requires a nanopatterned roll mold with a large area. We report on a novel system to fabricate large-area roll molds by recombination of smaller patterned areas in a step-and-repeat imprint lithography process. The process is accomplished in a method similar to liquid transfer imprint lithography (LTIL). The stamp roll with a smaller area takes up the liquid resist by splitting from a donor substrate or a donor roll. The resist is then transferred from a stamp roll to an acceptor roll and stitched together in a longitudinal and, if necessary, in a circumferential direction. During transfer, the nanostructured resist is UV-exposed and crosslinked directly on the acceptor roll. The acceptor roll with the stitched and recombined stamp patterns is ready to be used as a large-area roll mold for roll-based imprinting. A system for this purpose was designed, and its operation was demonstrated taking the example of an acceptor roll of 1 m length and 250 mm diameter, which was covered by 56 patterned areas. Such a system represents an elegant and efficient tool to recombine small patterned areas directly on a large roll mold and opens the way for large-area roll-based processing.

Flexible nanopillar-based immunoelectrochemical biosensor for noninvasive detection of Amyloid beta.

The noninvasive early detection of biomarkers for Alzheimer's disease (AD) is essential for the development of specific treatment strategies. This paper proposes an advanced method for fabricating highly ordered and flexible nanopillar-based electrochemical biosensors by the combination of soft/photolithography and metal evaporation. The nanopillar array (NPA) exhibits high surface area containing 1500 nm height and 500 nm diameter with 3:1 ratio. In regard with physical properties of polyurethane (PU) substrate, the developed NPA is sustainable and durable to external pressure such as bending and twisting. To manipulate the NPA surface to biocompatible, the gold was uniformly deposited on the PU substrate. The thiol chemistry which is stably modified on the gold surface as a form of self-assembled monolayer was employed for fabricating the NPA as a biocompatible chip by covalently immobilize the antibodies. The proposed nanopillar-based immunoelectrochemical biosensor exhibited good and stable electrochemical performance in ß-amyloid (Aß) detection. Moreover, we successfully confirmed the performance of the as-developed sensor using the artificial injection of Aß in human tear, with sensitivity of 0.14 ng/mL and high reproducibility (as a standard deviation below 10%). Our findings show that the developed nanopillar-based sensor exhibits reliable electrochemical characteristics and prove its potential for application as a biosensor platform for testing at the point of care.

An electrophoretic DNA extraction device using a nanofilter for molecular diagnosis of pathogens.

Rapid and efficient nucleic acid (NA) extraction and concentration are required for point-of-care analysis in order to prevent an epidemic/pandemic disease outbreak. Typical silica-based NA extraction methods have limitations such as being time-consuming, requiring human intervention, and resulting in a low recovery yield. In this study, we have developed a pathogenic DNA extraction device based on electrokinetic separation incorporated with a silicon nitride (SiNx) nanofilter, which expedites the DNA extraction procedure with advantages of being convenient, efficient, and inexpensive. This DNA extraction device consists of a computer numerical control (CNC) milled-Teflon gadget with a cis-chamber as a cell lysate reservoir and a trans-chamber as a elution solution reservoir, with the SiNx nanofilter being inserted between the two chambers. The SiNx nanofilter was fabricated using a photolithographic method in conjunction with nanoimprinting. Approximately 7.2 million nanopores of 220 nm diameter were located at the center of the nanofilter. When a DC electric field is applied through the nanopores, DNA is transferred from the cis-chamber to the trans-chamber to isolate the DNA from the cell debris. To demonstrate the DNA extraction performance, we measured the absorbances at 260 and 280 nm and performed a real-time polymerase chain reaction (real-time PCR) using the recovered DNA to verify its feasibility for downstream genetic analysis. Moreover, the DNA extraction device was successfully operated using a 1.5 V alkaline battery, which verifies the portability of the device for point-of-care testing. Such an advanced DNA extraction system can be utilized in various fields including clinical analysis, pathogen detection, forensic analysis, and on-site detection.

Direct electrophoretic microRNA preparation from clinical samples using nanofilter membrane.

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 108/cm2. The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

SPR imaging-based monitoring of caspase-3 activation.

The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.

A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.

In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct.

Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface.

Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(l-lysine) for sequence-specific DNA-protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein-protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.

Cuvette-based microfluidic device integrated with nanostructures for measuring dual Localized Surface Plasmon Resonance (LSPR) signals.

A spectrophotometer that uses a localized surface plasmon resonance phenomenon is a powerful measurement tool in the biotechnology and bioanalysis fields. We propose a novel cuvette design type that can be used for universal spectrophotometers. The novel cuvette design needs a few µl reagent for measuring, and also two chips for measurement can be loaded and measured at the same time. A new cuvette can easily be used several times because of sample chips to be loaded and unloaded since they are mechanically mounted by screws. Therefore, it can offer advantages to users in terms of cost and time. We verify its possibility for use in the biotechnology and bioanalysis fields by a signal enhancement and dual signal detection.

Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.

The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers.

Electrical signaling of enzyme-linked immunosorbent assays with an ion-sensitive field-effect transistor.

Optical laboratory-based immunoassays, such as enzyme-linked immunosorbent assay (ELISA) give a high sensitivity and specificity of various fatal diseases. However, these assays are no longer efficient in on-spot diagnostics of wide-spreading and contagious infections. At this point in time, portable and handhold devices play a pivotal role in infectious diseases with quick diagnostics at or near the site of the disease propagation. In this paper, we demonstrated a novel electrical immunoassay of ELISA that was not based on optical signaling but on electrical signaling. This was done by combining an ion-sensitive field-effect transistor (ISFET) with ELISA. By harnessing the catalytic reaction of alkaline phosphatase that precipitated silver particles, we effectively overcame the chronic Debye screening length issue of the ISFET. Ultimately, small signal ranging from 1 pg/mL to 10 ng/mL was immensely amplified with the ALP label, regardless of buffer conditions. The sensor platform herein surpassed a sensing capability of conventional ELISA that is considered to have a LOD on the order of ~1 ng/mL. The results were compared with those of horseradish peroxidase label, which is generally used for optical analyses in ELISA. Our newly developed ISFET-based portable sensor holds a large potential for point-of-care tools in a variety of diseases, without being limited by the need for expensive equipment such as spectrophotometers.

Sequential patterning of two fluorescent streptavidins assisted by photoactivatable biotin on an aminodextran-coated surface.

Sequential patterning of two fluorescent streptavidins (SAvs) was carried out using photopatterning of photoactivatable biotin (photobiotin) on an aminodextran surface, which was crucial for the minimization of non-specific binding. Photobiotin was bound by photoreaction to the amine groups of aminodextran. Water contact angle at each step during the preparation of the aminodextran surface was measured to investigate the hydrophilicity of the surfaces. The specific and nonspecific binding of a fluorescent SAv was investigated for the aminodextran surface and the amine-silane surface. The aminodextran surface almost entirely prevented nonspecific binding of a fluorescent SAv and was successfully used for sequential patterning of two fluorescent SAvs. The addition of ethanolamine (40 mM) in the photobiotin solution diminished blurring of pattern shape. To decrease pattern size, the UV light was focused on the aminodextran surface in an inverted microscope system. Under optimized conditions, two fluorescent SAvs array of approximately 25 µm size was obtained using a shadow mask of 100 µm hole size in the inverted microscope system.

Electrical immunosensor based on a submicron-gap interdigitated electrode and gold enhancement.

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1µm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations).

Highly sensitive biosensing using arrays of plasmonic Au nanodisks realized by nanoimprint lithography.

We describe the fabrication of elliptical Au nanodisk arrays as a localized surface plasmon resonance (LSPR) sensing substrate for clinical immunoassay via thermal nanoimprint lithography (NIL) and enhancement in the sensitivity of the detection of the prostate-specific antigen (PSA) using the precipitation of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine/nitro blue tetrazolium (BCIP/NBT), catalyzed by alkaline phosphatase. Au nanodisks were fabricated on glass through an unconventional tilted evaporation, which could preserve the thickness of imprinted resists and create an undercut beneficial to the subsequent lift-off process without any damage to pattern dimension and the glass while removing the residual polymers. To investigate the optically anisotropic property of the LSPR sensors, a probe light with linear polarization parallel to and perpendicular to the long axis of the elliptical nanodisk array was utilized, and their sensitivity to the bulk refractive index (RI) was measured as 327 and 167 nm/RIU, respectively. To our knowledge, this is the first application of enzyme-substrate reaction to sandwich immunoassay-based LSPR biosensors that previously suffered from a low sensitivity due to the short penetration depth of the plasmon field, especially when large-sized antibodies were used as bioreceptors. As a result, a large change in local refractive index because of the precipitation on the Au nanodisks amplified the wavelength shift of the LSPR peak in the vis-NIR spectrum, resulting in femtomolar detection limits, which was ∼10(5)-fold lower than the label-free detection without the enzyme precipitation. This method can be extended easily to the other clinical diagnostics with a high sensitivity.