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.

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We report on chemiluminescence resonance energy transfer (CRET) between graphene nanosheets and chemiluminescent donors. In contrast to fluorescence resonance energy transfer, CRET occurs via nonradiative dipole-dipole transfer of energy from a chemiluminescent donor to a suitable acceptor molecule without an external excitation source. We designed a graphene-based CRET platform for homogeneous immunoassay of C-reactive protein (CRP), a key marker for human inflammation and cardiovascular diseases, using a luminol/hydrogen peroxide chemiluminescence (CL) reaction catalyzed by horseradish peroxidase. According to our results, anti-CRP antibody conjugated to graphene nanosheets enabled the capture of CRP at the concentration above 1.6 ng mL(-1). In the CRET platform, graphene played a key role as an energy acceptor, which was more efficient than graphene oxide, while luminol served as a donor to graphene, triggering the CRET phenomenon between luminol and graphene. The graphene-based CRET platform was successfully applied to the detection of CRP in human serum samples in the range observed during acute inflammatory stress.

We report a new method for the micropatterning of multiple proteins and cells with micrometer-scale precision. Microscope projection photolithography based on a new protein-friendly photoresist, poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP), was used for the fabrication of multicomponent protein/cell arrays. Microscope projection lithography allows precise registration between multiple patterns as well as facile fabrication of microscale features. Thin films of PDMP became soluble in near-neutral physiological buffer solutions upon UV exposure and exhibited excellent resistance to protein adsorption and cell adhesion. By harnessing advantages in microscope projection photolithography and properties of PDMP thin films, we could successfully fabricate protein arrays composed of multiple proteins. Furthermore, we could extend this method for the patterning of two different types of immune cells for the potential study of immune cell interactions. This technique will in general be useful for protein chip fabrication and high-throughput cell-cell communication study.

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Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

The development of real-time innocuous blood diagnosis has been a long-standing goal in healthcare; an improved, miniature, all-in-one point-of-care testing (POCT) system with low cost and simplified operation is highly desired. Here, we present a one-touch-activated blood multidiagnostic system (OBMS) involving the synergistic integration of a hollow microneedle and paper-based sensor, providing a number of unique characteristics for simplifying the design of microsystems and enhancing user performance. In this OBMS, all functions of blood collection, serum separation, and detection were sequentially automated in one single device that only required one-touch activation by finger-power without additional operations. For the first time, we successfully demonstrated the operation of this system in vivo in glucose and cholesterol diagnosis, showing a great possibility for human clinical application and commercialization. Additionally, this novel system offers a new approach for the use of microneedles and paper sensors as promising intelligent elements in future real-time healthcare monitoring devices.

A highly rapid, one-step immunoassay of high sensitivity C-reactive protein (hsCRP) using a biosensor with a vertical flow immunoassay (VFA) was developed. The VFA biosensor was primarily composed of a sample pad, conjugate pad, FTH film and nitrocellulose (NC) membrane, which were all vertically stacked upon one another. Anti-hsCRP and secondary antibodies were consecutively immobilized on the NC membrane at the position below the holes. Gold nanoparticles (AuNPs) conjugated with another anti-hsCRP antibody were encapsulated in the conjugation pad. Various assay conditions, including the size of the hole and the sample volume, were optimized. Under optimized conditions, hsCRP concentrations from 0.01 to 10 μg mL(-1) were detected within 2 min. In comparison with a lateral flow assay (LFA) system, the VFA sensor showed a gradual increase of signal in a concentration-dependent manner without a hook effect in the tested range.