The uniformity of aminosilane layers typically used for the modification of hydroxyl bearing surfaces such as silicon dioxide is critical for a wide variety of applications, including biosensors. However, in spite of many studies that have been undertaken on surface silanization, there remains a paucity of easy-to-implement deposition methods reproducibly yielding smooth aminosilane monolayers. In this study, solution- and vapor-phase deposition methods for three aminoalkoxysilanes differing in the number of reactive groups (3-aminopropyl triethoxysilane (APTES), 3-aminopropyl methyl diethoxysilane (APMDES) and 3-aminopropyl dimethyl ethoxysilane (APDMES)) were assessed with the aim of identifying methods that yield highly uniform and reproducible silane layers that are resistant to minor procedural variations. Silane film quality was characterized based on measured thickness, hydrophilicity and surface roughness. Additionally, hydrolytic stability of the films was assessed via these thickness and contact angle values following desorption in water. We found that two simple solution-phase methods, an aqueous deposition of APTES and a toluene based deposition of APDMES, yielded high quality silane layers that exhibit comparable characteristics to those deposited via vapor-phase methods.

Rapid, sensitive, and selective detection of viruses is critical for applications in medical diagnostics, biosecurity, and environmental safety. In this article, we report the application of a point-defect-coupled W1 photonic crystal (PhC) waveguide biosensor to label-free optical detection of viruses. Fabricated on a silicon-on-insulator (SOI) substrate using electron-beam (e-beam) lithography and reactive-ion-etching, the PhC sensing platform allows optical detection based on resonant mode shifts in response to ambient refractive index changes produced by infiltration of target biomaterial within the holes of the PhC structure. Finite difference time domain (FDTD) calculations were performed to assist with design of the sensor, and to serve as a theoretical benchmark against which experimental results could be compared. Using Human Papillomavirus virus-like particles (VLPs) spiked in 10% fetal bovine serum as a model system, we observed a limit of detection of 1.4 nM in simple (buffer only) or complex (10% serum) sample matrices. The use of anti-VLP antibodies specific for intact VLPs with the PhC sensors provided highly selective VLP detection.

The near-ubiquity of the involvement of RNA in crucial biological processes is accepted. It is important, therefore, to study and understand the biophysical principles that regulate the function of RNA and its interactions with other molecules (e.g., proteins and antibiotics). Methods enabling the high-throughput determination of RNA–protein binding kinetics and thermodynamics would greatly accelerate understanding of these interactions. To that end, we describe the development of a real-time biomolecular interaction analysis platform based on arrayed imaging reflectometry (AIR) for multiplex analysis of RNA–protein interactions. We demonstrate the use of aqueous AIR by measuring the binding kinetics between muscleblind-like 1 (MBNL1), a splicing regulator protein that plays a pivotal role in the Myotonic Dystrophies and Huntington's Disease, and several of its RNA targets simultaneously on a microarrayed chip. Using this approach, we observe that the kinetics of MBNL1 binding isolated CUG and repeat CUG RNA sequences (as models for “normal” and “pathogenic” RNA, respectively) are different even though their steady state binding constants are similar. The ability to compare binding kinetics between RNA sequences rapidly and easily may provide insight into the molecular basis of MBNL1-RNA binding, and more generally suggests that AIR can be a powerful tool to enable the label-free, real-time analysis of biomolecular interactions in a high throughput format.

One of the critical steps in the development of an analytical technique is to confirm that its experimental response correlates with predictions derived from the theoretical framework on which it is based. This validates the technique quantitatively, and, in the case of a biosensor, facilitates a correlation of the sensor’s output signal to the concentration of the analyte being tested. Herein we report studies demonstrating that the quantitative response of Arrayed Imaging Reflectometry (AIR), a highly sensitive label-free biosensing method, is a predictable function of probe and analyte properties. We first incorporated a standard one-site Langmuir binding model describing probe-analyte interactions at the surface into the theoretical model for thickness-dependent reflectance in AIR. This established a hypothetical correlation between analyte concentration and the AIR response. Spectroscopic ellipsometry, surface plasmon resonance (SPR) and AIR were then used to validate this model for two biomedically important proteins, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). While our studies demonstrated that the 1:1 one-site Langmuir model accurately described the observed response of macro spot AIR arrays, either a two-site Langmuir model or a Sips isotherm better described the behavior of AIR microarrays. These studies confirmed the quantitative performance of AIR across a range of probe-analyte affinities. Furthermore, the methodology developed here can be extended to other label-free biosensing platforms, thus facilitating a more accurate and quantitative interpretation of the sensor response.

Uniform spot morphology is of critical importance in the fabrication and successful use of protein arrays, and solution additives are often needed in order to ensure good spot quality. While hydroxyl-bearing molecules such as glycerol have found wide usage, in our experience these reduce the efficiency of probe immobilization (particularly in the context of aldehyde-terminated surfaces). Here, we report a series of non-nucleophilic molecules that can be used as additives in order to improve spot homogeneity in protein arrays. Arrayed Imaging Reflectometry, a label-free optical biosensing technique, has been used along with spectroscopic ellipsometry to test the spot homogeneity, antibody immobilization efficiency, and activity of anti-human IgG arrays prepared with these non-nucleophilic additives on glutaraldehyde surfaces. It has been determined that 0.1% v/v 12-crown-4 has the optimum performance in MPBS buffer.