Search

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.

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.

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.