X. D. Zhu

Studies of alkali adsorption on Rh(111) using optical second-harmonic generation

Dramatic changes were observed in the optical second-harmonic generation from the Rh(111) crystal surface as the surface was covered with alkali atoms. For low alkali coverage, these changes are explained qualitatively in terms of optical transitions between alkali atomic-derived electronic states and, for high coverage, in terms of the plasmon resonance of the alkali layer. The results establish the surface specificity of second-harmonic generation to the topmost 1 or 2 atomic layers of alkali metals.

Selective launching of higher-order modes into an optical fiber with an optical phase shifter.

We demonstrate a simple, effective technique for launching higher-order modes into a double-mode optical fiber by using an optical phase shifter at the input end. The suppression of the lowest-order mode was measured (but not limited) to be 27 dB.

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.

Correlation between thermal desorption spectroscopy and optical second harmonic generation for monitoring surface coverages

Abstract Optical second harmonic generation was used to monitor CO coverages on Cu(100) and Ni(111) in an ultrahigh vacuum chamber. In both cases, the adsorption kinetics were found to obey the simple Langmuir model. In monitoring CO coverages remained on Cu(100) and Ni(111) during a thermal desorption process, optical second harmonic generation correlates well with thermal desorption spectroscopy.

Vibrational and electronic spectroscopy of pyridine and benzene adsorbed on the Rh(111) crystal surface

We report the vibrational and electronic spectra for pyridine and benzene adsorbed on the Rh(111) crystal surface obtained by high‐resolution electron energy loss spectroscopy (HREELS). Low‐energy electron diffraction (LEED), thermal desorption spectroscopy (TDS), and optical second harmonic generation (SHG) have also been used to provide complementary information. Pyridine adsorption on Rh(111) was studied over the 77–450 K temperature range. At 77 K, multilayers of pyridine are observed with a vibrational spectrum similar to that of liquid pyridine. Between 185 and 230 K, HREELS and TDS indicate that both physisorbed and chemisorbed pyridine species are present on the surface. The physisorbed species desorbs at 295 K, while the chemisorbed species is stable until it decomposes on the surface at 400 K. We propose that the chemisorbed species is an α‐pyridyl complex as thermal desorption spectroscopy indicates partial dehydrogenation of this pyridine surface species. Electronic energy loss spectra for bot...

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.