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Poor specificity has been a lingering problem in many microRNA profiling methods, particularly surface hybridization-based methods such as microarrays. Here, we carefully investigated surface hybridization and dissociation processes of a number of sequentially similar microRNAs against nucleic acid capture probes. Single-base mismatched microRNAs were similarly hybridized to a complementary DNA capture probe and thereby poorly discriminated during conventional stringent hybridization. Interestingly, however, mismatched microRNAs showed significantly faster dissociation from the probe than the perfectly matched microRNA. Systematic analysis of various washing conditions clearly demonstrated that extremely high specificity can be obtained by releasing non-specific microRNAs from assay surfaces during a stringent and controlled dissociation step. For instance, compared with stringent hybridization, surface dissociation control provided up to 6-fold better specificity for Let-7a detection than for other Let-7 family microRNAs. In addition, a synthetically introduced single-base mismatch on miR206 was almost completely discriminated by optimized surface dissociation of captured microRNAs, while this mismatch was barely distinguished from target miR206 during stringent hybridization. Furthermore, a single dissociation condition was successfully used to simultaneously measure four different microRNAs with extremely high specificity using melting temperature-equalized capture probes. The present study on selective dissociation of surface bound microRNAs can be easily applied to various hybridization based detection methods for improved specificity.

We developed a colorimetric method to specifically detect single-strand DNA breaks using gold nanoparticles. In our assay, broken DNA cannot stabilize gold nanoparticles to prevent salt-induced aggregation as good as intact DNA can, and this effect can be easily observed with the naked eye as a red-to-purple color change.

We developed a new detection method for L-Dopa decarboxylase (DDC) activity using gold nanoparticles (AuNPs). When L-3,4-dihydroxyphenylalanine (L-Dopa) is decarboxylated to dopamine (DA) by DDC, DA induces the aggregation of AuNPs, and the color of the AuNPs changes from red to blue.

In this paper, we demonstrate a simple technique for sequentially introducing multiple sample liquids into microchannels driven by centrifugal force combined with a hydrophobic barrier pressure and apply the technique for performing solid-phase based on-chip DNA purification. Three microchannels with varying widths, all equipped with independent sample reservoirs at the inlets, were fabricated on a hydrophobic elastomer, poly(dimethylsiloxane) (PDMS). First, glass beads were packed inside the reaction chamber, and a whole cell containing the DNA extract was introduced into the widest channel by applying centrifugal force for physical adsorption of the DNA onto the glass beads. Next, washing and elution solutions were sequentially introduced into the intermediate and narrowest microchannels, respectively, by gradually increasing the amount of centrifugal force. Through a precise manipulation of the centrifugal force, the DNA adsorbed onto the glass beads was successfully washed and eluted in a continuous manner without the need to introduce each solution manually. A stepwise injection of liquids was successfully demonstrated using multiple ink solutions, the results of which corresponded well with the theoretical analyses. As a practical application, the D1S80 locus of human genomic DNA, which is widely used for forensic purposes, was successfully purified using the microdevice introduced in this study, as demonstrated through successful target amplification. This will pave the way for the construction of a control-free valve system for realizing on-chip DNA purification, which is one of the most labor-intensive and hard-to-miniaturize components, on a greatly simplified and miniaturized platform employing hydrophobic PDMS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

Antibody immobilization on a solid support is an essential process for the development of most immune-based assay systems. The choice of the immobilization method greatly affects antibody–antigen interactions on the assay surface. For the past several years, numerous strategies have been reported to control antibody immobilization, mainly by directing the orientation, stability, and density of bound antibodies on different assay platforms. Here we discuss recent developments in antibody immobilization methods with a particular focus on the strengths and limitations of reported approaches, and thereby provide a useful guideline for the selection of suitable antibody coupling procedures.

A simple, rapid, and signal-on fluorescent assay was developed for activity analysis of DNA methyltransferase and for screening of its inhibitors based on a methylation-resistant endonuclease and SYBR Green I.

An operationally simple colorimetric way for measuring hyaluronidase activity was developed using cysteamine-bound gold nanoparticles. The addition of gold nanoparticles into hyaluronidase-containing solutions resulted in color changes, which could easily be observed with the naked eye or a UV/Vis spectrophotometer.