Using a microarray-based assay, we studied how the substitution of amino acids in the immediate vicinity of the receptor-binding domain on a peptide affects its binding to a protein. Replicates of 802 linear peptides consisting of the variants of WTHPQFAT and LQWHPQAGK, GKFPIPLGKQSG, and NGQFQVWIPGAQK, different by one amino acid, were synthesized on a glass slide with a maskless photolithography. Using a microarray-compatible label-free optical sensor, we measured the binding curves of streptavidin with the synthesized peptides and extracted the streptavidin-peptide affinity constants. We found that (a) the substitution of one residue in the HPQ motif reduces the affinity constant K _a from 10⁸ M^-1 by at least 3-4 orders of magnitude, with an exception of HPM; (b) substitution of the immediate flanking residue on the Gln side also causes the affinity to decrease by up to 3-4 orders of magnitude, depending on the substituting residue and the second-neighboring flanking residue; (c) substitution of the flanking residues on the His side has no significant effect on the affinity, possibly due to the strong binding of streptavidin to HPQF and HPQAG motifs. We also found that some of amino acid residues located close to the C-terminus (and the solid surface) improve the yield of peptide synthesis on a glass surface and can be exploited in the fabrication of peptide microarrays.

One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD) technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR). The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.

Optical biosensors have a significant impact on various aspects of our lives. In many applications of optical biosensors, fluidic chambers play a crucial role in facilitating controlled fluid delivery. It is essential to achieve complete liquid replacement in order to obtain accurate and reliable results. However, the configurations of fluidic chambers vary across different optical biosensors, resulting in diverse fluidic volumes and flow rates, and there are no standardized guidelines for liquid replacement. In this paper, we utilize COMSOL Multiphysics, a finite element analysis software, to investigate the optimal fluid volume required for two types of fluidic chambers in the context of the oblique-incidence reflectivity difference (OI-RD) biosensor. We found that the depth of the fluidic chamber is the most crucial factor influencing the required liquid volume, with the volume being a quadratic function of the depth. Additionally, the required fluid volume is also influenced by the positions on the substrate surface bearing samples, while the flow rate has no impact on the fluid volume.

Total internal reflection (TIR) is useful for interrogating physical and chemical processes that occur at the interface between two transparent media. Yet prism-coupled TIR imaging microscopes suffer from limited sensing areas due to the fact that the interface (the object plane) is not perpendicular to the optical axis of the microscope. In this paper, we show that an electrically tunable lens can be used to rapidly and reproducibly correct the focal length of an oblique-incidence scanning microscope (OI-RD) in a prism-coupled TIR geometry. We demonstrate the performance of such a correction by acquiring an image of a protein microarray over a scan area of 4 cm² with an effective resolution of less than 20 microns. The electronic focal length tuning eliminates the mechanical movement of the illumination lens in the scanning microscope and in turn the noise and background drift associated with the motion.

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.

The oblique-incidence reflectivity difference (OI-RD) microscope is a label-free detection system for microarrays that has many successful applications in high throughput drug screening. The increase and optimization of the detection speed of the OI-RD microscope will enable it to be a potential ultra-high throughput screening tool. This work presents a series of optimization methods that can significantly reduce the time to scan an OI-RD image. The wait time for the lock-in amplifier was decreased by the proper selection of the time constant and development of a new electronic amplifier. In addition, the time for the software to acquire data and for translation stage movement was also minimized. As a result, the detection speed of the OI-RD microscope is 10 times faster than before, making the OI-RD microscope suitable for ultra-high throughput screening applications.

Label-free optical biosensors have become powerful tools in the study of biomolecular interactions without the need for labels. High throughput and low detection limit are desirable for rapid and accurate biomolecule detection. The oblique-incidence reflectivity difference (OI-RD) technique is capable of detecting thousands of biomolecular interactions in a high-throughput mode, specifically for biomolecules larger than 1000 Da. In order to enhance the detection capability of OI-RD for small molecules (typically < 500 Da), we have developed a three-dimensional biochip that utilized carboxymethyl chitosan (CMCS) functionalized slides. By investigating various factors such as sonication time, protein immobilization time, CMCS molecular weight, and glutaraldehyde (GA) functionalization time, we have achieved a detection limit of 6.8 pM for avidin (68 kDa). Furthermore, accurate detection of D-biotin with a molecular weight of 244 Da has also been achieved. This paper presents an effective solution for achieving both high throughput and low detection limits using the OI-RD technique in the field of biomolecular interaction detection.

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.