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We present nanohole arrays in a gold film integrated with a six-channel microfluidic chip for parallel measurements of molecular binding kinetics. Surface plasmon resonance effects in the nanohole arrays enable real-time, label-free measurements of molecular binding events in each channel, while adjacent negative reference channels can record measurement artifacts such as bulk solution index changes, temperature variations, or changing light absorption in the liquid. With the use of this platform, streptavidin--biotin specific binding kinetics are measured at various concentrations with negative controls. A high-density microarray of 252 biosensing pixels is also demonstrated with a packing density of 106 sensing elements/[cm.sup.2], which can potentially be coupled with a massively parallel array of microfluidic channels for protein microarray applications.

This work aims for ultrasensitive detection of target proteins in complex biological matrixes based on aptamer microarrays. Two extensively studied aptamers (HTQ and HTDQ) that bind distinct epitopes of thrombin are chosen for the microarray study. Although HTQ and HTDQ have nanomolar to subnanomolar affinities, it is found that either aptamer when applied directly has difficulty in detecting a few nanomoles per liter thrombin in the presence of a 10- or 100-fold (w/w) excess of serum total protein (STP). By investigating dodecyl (12-carbon) and oligodeoxythymidine (oligo(dT)) spacers, we observe both spacers enhance the microarray signal response, but oligo(dT) is strikingly better than dodecyl. Moreover, we discover that a microarray spot coprinted with the two distinct aptamers (HTQ and HTDQ) functions like a bivalent molecular construct and exhibits an avidity effect. With the synergy of oligo(dT) spacers and the avidity effect, detection of picomolar-range thrombin in the presence of either 10% unlabeled serum or a 10 000-fold excess of labeled serum total protein is achieved. It corresponds to a 100-1000-fold sensitivity enhancement as compared to using an individual aptamer without a spacer.

The melanocortin-1 receptor (MC1R) is a key regulator of pigmentation in mammals and is tightly linked to an increased risk of skin cancers, including melanoma, in humans. Physiologically activated by [alpha]-melanocyte stimulating hormone [alpha]MSH), MC1R function can be antagonized by a secreted factor, agouti signal protein (ASP), which is responsible for the lighter phenotypes in mammals (including humans), and is also associated with increased risk of skin cancer. It is therefore of great interest to characterize the molecular effects elicited by those MC1R ligands. In this study, we determined the gene expression profiles of murine melan-a melanocytes treated with ASP or [alpha]MSH over a 4-day time course using genome-wide oligonucleotide microarrays. As expected, there were significant reductions in expression of numerous melanogenic proteins elicited by ASP, which correlates with its inhibition of pigmentation. ASP also unexpectedly modulated the expression of genes involved in various other cellular pathways, including glutathione synthesis and redox metabolism. Many genes up-regulated by ASP are involved in morphogenesis (especially in nervous system development), cell adhesion, and extracellular matrix-receptor interactions. Concomitantly, ASP enhanced the migratory potential and the invasiveness of melanocytic cells in vitro. These results demonstrate the role of ASP in the dedifferentiation of melanocytes, identify pigment-related genes targeted by ASP and by [alpha]MSH, and provide insights into the pleiotropic molecular effects of MC1R signaling that may function during development and may affect skin cancer risk. pigmentation | skin cancer

In the assembly of microarrays and microarray-based chemical assays and enzymatic bioassays, most approaches use pins for contact spotting. Acoustic dispensing is a technology capable of nanoliter transfers by using acoustic energy to eject liquid sample from an open source well. Although typically used for well plate transfers, when applied to microarraying, it avoids the drawbacks of undesired physical contact with the sample; difficulty in assembling multicomponent reactions on a chip by readdressing, a rigid mode of printing that lacks patterning capabilities; and time-consuming wash steps. We demonstrated the utility of acoustic dispensing by delivering human cathepsin L in a drop-on-drop fashion into individual 50-nanoliter, prespotted reaction volumes to activate enzyme reactions at targeted positions on a microarray. We generated variable-sized spots ranging from 200 to 750 [micro]m (and higher) and handled the transfer of fluorescent bead suspensions with increasing source well concentrations of 0.1 to 10 x [l0.sup.8] beads/mL in a linear fashion. There are no tips that can clog, and liquid dispensing CVs are generally below 5%. This platform expands the toolbox for generating analytical arrays and meets needs associated with spatially addressed assembly of multicomponent microarrays on the nanoliter scale.

Aptamers are short single-stranded DNA or RNA oligo-nucleotides that can bind to a wide range of target molecules with high affinity and specificity. As nucleic acids, aptamers can undergo denaturation, but the process is reversible. As a result of this stability and the possibility of automated selection of aptamers, these oligonucleotides are highly promising capture molecules in microarray formats. In this study, his-tagged proteins and an aptamer directed against the his-tag were chosen as a model system. Different factors affect the activity of aptamers immobilized on a solid support like a microarray surface. The orientation of the immobilized aptamer plays an important role in correct aptamer folding and, thus, in effective binding of the corresponding target. Other important parameters identified in this work are the microarrays' surface charge as well as the length of the spacer between aptamer and solid support. These parameters were investigated systematically, resulting in the development of an aptamer-based microarray for detection of his-tagged proteins. The general applicability of the developed immobilization strategy was demonstrated by utilization of three different aptamers.

The microarray format is being used extensively for combinatorial screening of cellular interactions with proteins, small molecules, or biomaterials. The utility of microarray-based cell cultivation approaches may be enhanced further by incorporating biosensing elements alongside the cell-adhesive ligands to enable local detection of secreted cellular products. The concept of combining cells and sensing elements in the same microarray is demonstrated in the present paper with hepatocytes serving as a model cellular system. Robotic microarraying was employed to print arrays of 300-[micro]m-diameter collagen (I) spots alongside the antibody (Ab) spots specific to liver proteins: albumin and [alpha]l-antitrypsin ([alpha]1-AT). Protein microarrays were printed onto poly(ethylene glycol) hydrogel-coated glass slides, thus eliminating nonspecific adsorption of cells or proteins. When incubated with printed microarrays, hepatocytes became localized on collagen (I) domains but did not attach on Ab spots or elsewhere on hydrogel-coated glass substrates. Liverspecific proteins secreted by hepatocytes were captured on Ab domains in the immediate vicinity of the cells, detected with a sandwich immunofluorescent assay and quantified using a microarray scanner. Importantly, hepatic albumin and [alpha]1-AT production detected in the microarray was comparable to enzyme-linked immunosorbent assay measurements of these proteins. In the future, the juxtaposition of sensing Ab regions with cell arrays will be particularly useful for the detection of local appearance or loss of phenotype of cells interacting with the printed components of the cellular microenvironment.

Capillary neoformation is important in repair of glomerular injury of various origins. VEGF was shown to be crucial for glomerular capillary repair in glomerulonephritis (GN). We reasoned that other angiogenic factors are likewise involved in glomerular capillary remodeling and found angiopoietin 1 and -2 (ANG1 and ANG2) mRNA to be upregulated in cDNA microarrays of microdissected glomeruli of anti-Thy 1.1 GN of the rat. We then studied glomerular in situ gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 in the course of antianti-Thy 1.1 GN, which was induced by injection of OX-7 antibody. Animals were perfusion fixed at days 6 and 12 after GN induction and compared with nonnephritic controls receiving PBS. Capillary damage and repair were quantitatively analyzed using stereological techniques. Gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 was analyzed using real-time quantitative PCR from microdissected glomeruli, nonradioactive in situ hybridization, double immunofluorescence, and Western blot analysis. Glomerular capillarization assessed as length density was significantly lower at day 6 of anti-Thy 1.1 GN than in controls; it was back to normal values at day 12. ANG1 and ANG2 gene expression was markedly upregulated at day 6 of the disease compared with controls. Protein expression of ANG1 and ANG2 was confined to podocytes and that of Tie-2 to endothelial cells. At day 12 of anti-Thy 1.1 GN when capillary restoration was nearly completed, ANG1 and ANG2 gene expression returned to basal levels, whereas Tie-2 expression was still high. With the use of a combined molecular and in situ approach, the spatial and temporal gene and protein expression of the angiopoietins and their receptor was analyzed in anti-Thy 1.1 GN. The results indicate that glomerular expression of ANG1 and ANG2 and Tie-2 is differentially regulated and may contribute to healing and endothelial cell stabilization in experimental GN. vascular endothelial growth factor; microarrays; glomerular injury; repair

Cholera, an infectious disease with global impact, is caused by pathogenic strains of the bacterium Vibrio cholerae. High-throughput functional proteomics technologies now offer the opportunity to investigate all aspects of the proteome, which has led to an increased demand for comprehensive protein expression clone resources. Genome-scale reagents for cholera would encourage comprehensive analyses of immune responses and systems-wide functional studies that could lead to improved vaccine and therapeutic strategies. Here, we report the production of the FLEXGene clone set for V. cholerae 01 biovar eltor str. N16961: a complete-genome collection of ORF clones. This collection includes 3,761 sequence-verified clones from 3,887 targeted ORFs (97%). The ORFs were captured in a recombinational cloning vector to facilitate high-throughput transfer of ORF inserts into suitable expression vectors. To demonstrate its application, [approximately equal to] 15% of the collection was transferred into the relevant expression vector and used to produce a protein microarray by transcribing, translating, and capturing the proteins in situ on the array surface with 92% success. In a second application, a method to screen for protein triggers of Toll-like receptors (TLRs) was developed. We tested in vitro-synthesized proteins for their ability to stimulate TLR5 in A549 cells. This approach appropriately identified FlaC, and previously uncharacterized TLR5 agonist activities. These data suggest that the genome-scale, fully sequenced ORF collection reported here will be useful for high-throughput functional proteomic assays, immune response studies, structure biology, and other applications. cholera | functional proteomics | immunity | ORF clones | protein microarray

In plants, multistep component systems play important roles in signal transduction in response to environmental stimuli and plant growth regulators. Arabidopsis contains six nonethylene receptor histidine kinases, and, among them, AHK1/ATHK1, AHK2, AHK3, and CRE1 were shown to be stress-responsive, suggesting their roles in the regulation of plant response to abiotic stress. Gain- and loss-of-function studies in Arabidopsis indicated that AHK1 is a positive regulator of drought and salt stress responses and abscisic acid (ABA) signaling. Microarray analysis of the ahkl mutant revealed a down-regulation of many stress--and/or ABA-inducible genes, including AREB1, ANAC, and DREB2A transcription factors and their downstream genes. These data suggest that AHK1 functions upstream of AREB1, ANAC, and DREB2A and positively controls stress responses through both ABA-dependent and ABAindependent signaling pathways. In addition, AHK1 plays important roles in plant growth because the ahkl ahk2 ahk3 triple mutant showed further reduced growth. Unlike AHK1, loss-offunction analysis of ahk2, ahk3, and crel implied that the stressresponsive AHK2, AHK3, and CRE1 act as negative regulators in ABA signaling. AHK2 and AHK3 also negatively control osmotic stress responses in Arabidopsis because ahk2, ahk3, and ahk2 ahk3 mutants were strongly tolerant to drought and salt stress due to up-regulation of many stress- and/or ABA-inducible genes. Last, cytokinin clearly mediates stress responses because it was required for CRE1 to function as a negative regulator of osmotic stress. cross-talk | microarray | osmotic stress | plant growth

Activation of macrophages and subsequent 'killing' effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-[gamma] and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the unknown pathways of LM killing independent of reactive oxygen intermediates and reactive nitrogen intermediates. One differentially expressed gene, PKC[delta], had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKC[delta] during listeriosis, we infected PKC[delta]-deficient mice with LM. PKC[delta]-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKC[delta]-deficient macrophages demonstrated that, despite elevated levels of proinflammatory cytokines and NO production, increased escape of LM from the phagosome into the cytoplasm and uncontrolled bacterial growth occurred. Taken together these data identified PKC[delta] as a critical factor for confinement of LM within macrophage phagosomes. microarray | phagosomal escape | bacterial killing

We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.

Protein chips are a powerful emerging technology with extensive biomedical applications. However, the development of optimal, economical surface materials capable of maintaining the activity of embedded proteins is a challenge. Here, we introduce a new optimized, low-cost, sol-gel biomaterial for use in protein chips with femtogram-level sensitivity. A novel protein chip material with significantly improved physical properties and sensitivity was produced using unique screening and selection methods. Using this platform, the sensitive, specific detection of the interactions between an HIV antigen and its antibody and between a cyclin-kinase protein pair was observed. This study is the first to demonstrate the detection of protein-protein interactions on sol-gel microarrays and describes an important improvement in the physical properties of sol-gel-derived protein chip materials for biomedical research.

The fabrication of arrays of G protein coupled receptors (GPCRs) and assays for screening of ligands on these arrays are described. Highly practical approach is described for fabricating membrane protein arrays.