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The therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile. For the development of glycoproteins with therapeutic applications, selection of cell fines producing a glycoprotein with adequate glycoform is crucial. Here, we demonstrate an array-based analysis of secreted glycoproteins for rapid and efficient selection of a single cell producing a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of glycoproteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. On the basis of statistical analysis of the interrogation, single cells which are predicted to produce a desired glycoprotein are selected, retrieved, and expanded. We applied the approach to human recombinant erythropoietin (rhEPO)-producing CHO cells and verified the selection of a single CHO cell that produces rhEPO with a high sialylation degree. Human erythropoietin (hEPO) bearing highly sialylated oligosaccharide was shown to display a much longer plasma half-life, resulting in high therapeutic efficacy. This method may find widespread use in the clonal selection for the production of other glycoproteins with specific glycosylation as well as analysis of the heterogeneity in cell populations in a high-throughput manner. 10.1021/ac100992n

A label-free, activity-based assay of matrix metalloproteinase (MMP) and its inhibition was demonstrated on peptide-conjugated gold nanoparticles (AuNPs) with nonbiofouling poly(oligo(ethylene glycol) methacrylate) (pOEGMA) films using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Following surface-initiated atom-transfer radical polymerization of OEGMA on a Si/Si[O.sub.2] substrate, the MMP activity was determined by analyzing the cleaved peptide fragments using TOF-SIMS on the peptide-conjugated AuNPs. The use of nonbiofouling pOEGMA films in conjunction with AuNPs synergistically enhanced the sensitivity of assays for MMP activity and its inhibition in human serum. The detection sensitivity of MMP-7 in serum was as low as 20 ng [mL.sup.-1] (1 pmol [mL.sup.-1]), and the half-maximal inhibitory concentration ([IC.sub.50]) of minocycline, which is a MMP-7 inhibitor, was estimated to be 450 nM. It is anticipated that the developed system will be broadly useful for conducting activity-based assays of serum proteases, as well as for screening of their inhibitors, with high sensitivity in a high-throughput manner.

Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.

We demonstrate a novel magnetophoretic immunoassay of allergen-specific immunoglobulin E (IgE) based on the magnetophoretic deflection velocity of a microbead that is proportional to the associated magnetic nanoparticles under enhanced magnetic field gradient in a microchannel. In this detection scheme, two types of house dust mites, Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), were used as the model allergens. Polystyrene microbeads were conjugated with each of the mite extracts followed by incubation with serum samples. The resulting mixture was then reacted with magnetic nanoparticle-conjugated anti-human IgE for detection of allergen-specific IgE by using sandwich immuno-reactions. A ferromagnetic microstructure combined with a permanent magnet was employed to increase the magnetic field gradient (~[10.sup.4] T/m) in a microfluidic device. The magnetophoretic velocities of microbeads were measured in a microchannel under applied magnetic field, and the averaged velocity was well correlated with the concentration of allergen-specific IgE in serum. From the analysis of pooled sera obtained from 44 patients, the detection limits of the allergen-specific human IgEs for D. farinae and D. pteronyssinus were determined to be 565 (0.045 IU/mL) and 268 fM (0.021 IU/mL), respectively. These values are 1 order of magnitude lower than those by a conventional CAP system. For evaluation of reproducibility and accuracy, unknown sera were subjected to a blind test by using the developed assay system, and they were compared with the CAP system. As a result, coefficient of variance was less than 10%, and the developed method enabled a fast assay with a tiny amount of serum (~10 [micro]L).

We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOFS-IMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface-immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface-immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips.