James P. Landry

Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein-Ligand Reactions

We study the kinetic effect of extrinsic fluorescent labeling agents on protein-ligand binding affinity and find that the kinetics is related to the loss or change of protein function when proteins are fluorescent-labeled.

Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format

We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in.×3 in. (2.54 cm×7.62 cm) microscope slide.

Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy.

We developed an oblique-incidence reflectivity difference (OI-RD) scanning microscope for label-free imaging of microarrays of biomolecules upon solid substrates. We demonstrate that hybridization reactions in an oligonucleotide microarray fabricated upon a glass slide can be detected by such an OI-RD microscope.

A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays

We describe a novel scanning optical microscope based on a polarization-modulated nulling ellipsometry. The new microscope employs a combination of scanning mirror and sample translation and thus enables high-throughput label-free detection of biomolecular microarrays with more than 10 000 protein or small-molecule targets. For illustration, we show the image of a 2760-spot protein microarray on a functionalized glass slide obtained with such a microscope. The new scanning microscope is also capable of determining, in parallel, the real-time binding kinetics of multiple molecular species under aqueous conditions.

Simultaneous measurement of 10,000 protein-ligand affinity constants using microarray-based kinetic constant assays.

Fluorescence-based endpoint detection of microarrays with 10,000 or more molecular targets is a most useful tool for high-throughput profiling of biomolecular interactions, including screening large molecular libraries for novel protein ligands. However, endpoint fluorescence data such as images of reacted microarrays contain little information on kinetic rate constants, and the reliability of endpoint data as measures of binding affinity depends on reaction conditions and postreaction processing. We here report a simultaneous measurement of binding curves of a protein probe with 10,000 molecular targets in a microarray with an ellipsometry-based (label-free) optical scanner. The reaction rate constants extracted from these curves (k(on), k(off), and k(a)=k(on)/k(off)) are used to characterize the probe-target interactions instead of the endpoints. This work advances the microarray technology to a new milestone, namely, from an endpoint assay to a kinetic constant assay platform. The throughput of this binding curve assay platform is comparable to those at the National Institutes of Health Molecular Library Screening Centers, making it a practical method in screening compound libraries for novel ligands and for system-wide affinity profiling of proteins, viruses, or whole cells against diverse molecular targets.

Macromolecular scaffolds for immobilizing small molecule microarrays in label-free detection of protein-ligand interactions on solid support

We explored two macromolecular scaffolds, bovine serum albumin (BSA) and polyvinyl alcohol (PVA), as chemically complementary platforms for immobilizing small molecule compounds on functionalized glass slides. We conjugated biotin molecules to BSA and amine-derivatized PVA and subsequently immobilized the conjugates on epoxy-functionalized glass slides through reaction of free amine residues on BSA and PVA with surface-bound epoxy groups. We studied binding reactions of such immobilized small molecule targets with solution-phase protein probes using an oblique-incidence reflectivity difference scanning optical microscope. The results showed that both BSA and amine-derivatized PVA were effective and efficient as carriers of small molecules with NHS residues and fluoric residues and for immobilization on epoxy-coated solid surfaces. A significant fraction of the conjugated small molecules retain their innate chemical activity.

Protein reactions with surface-bound molecular targets detected by oblique-incidence reflectivity difference microscopes.

We applied oblique-incidence reflectivity difference microscopes (a form of polarization-modulated nulling ellipsometry) to detection of biomolecular microarrays without external labeling in a study of protein reactions with surface-immobilized targets. We show that the optical reflectivity difference signals can be quantitatively related to changes in surface mass density of molecular layers as a result of the reactions. Our experimental results demonstrate the feasibility of using oblique-incidence reflectivity difference microscopes for high-throughput proteomics research such as screening unlabeled protein probes against libraries of surface-immobilized small molecules.

Fluorescent labeling agents change binding profiles of glycan-binding proteins.

Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four β1-4-linked galactosides, three β1-3-linked galactosides, one β-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin-glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of protein-ligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein-glycan interactions.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Incidence-angle dependence of optical reflectivity difference from an ultrathin film on solid surface

We studied the incidence-angle dependence of the optical reflectivity difference in response to ultrathin films on transparent and opaque substrates. We found that the classical three-layer model reproduces the experimentally obtained angular dependence for a monolayer of xenon on Nb(110) and for a monolayer of protein molecules on functionalized glass. We explore the enhancement of the optical response near the Brewster angle (or its equivalent for opaque substrates) in thin film detection.