Hong Yan Song

Comparative Study of Random and Oriented Antibody Immobilization as Measured by Dual Polarization Interferometry and Surface Plasmon Resonance Spectroscopy

Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.

Identifying Novel Type ZBGs and Nonhydroxamate HDAC Inhibitors Through a SVM Based Virtual Screening Approach

Histone deacetylase inhibitors (HDACi) have been successfully used for the treatment of cancers and other diseases. Search for novel type ZBGs and development of non‐hydroxamate HDACi has become a focus in current research. To complement this, it is desirable to explore a virtual screening (VS) tool capable of identifying different types of potential inhibitors from large compound libraries with high yields and low false‐hit rates similar to HTS. This work explored the use of support vector machines (SVM) combined with our newly developed putative non‐inhibitor generation method as such a tool. SVM trained by 702 pre‐2008 hydroxamate HDACi and 64334 putative non‐HDACi showed good yields and low false‐hit rates in cross‐validation test and independent test using 220 diverse types of HDACi reported since 2008. The SVM hit rates in scanning 13.56 M PubChem and 168K MDDR compounds are comparable to HTS rates. Further structural analysis of SVM virtual hits suggests its potential for identification of non‐hydroxamate HDACi. From this analysis, a series of novel ZBG and cap groups were proposed for HDACi design.

Development of Localized Surface Plasmon Resonance-Based Point-of-Care System

This paper describes our point-of-care system development based on localized surface plasmon resonance (LSPR). Although LSPR has been a hot research area for a few decades, there are several bottlenecks which hampered its application for point-of-care (POC) medical diagnostics. The first is the detection sensitivity shortage when the direct LSPR wavelength shift is used for sensing, the second is the mass fabrication of durable metal nanostructures on the substrate, and the third is the microfluidic chip and the POC system which have to be combined with LSPR chips in a seamless but cost-effective way. To solve the above challenges, several novel technologies are initiated and successfully implemented in this work. To increase the sensitivity of the LSPR detection, we use plasmonic field to excite the fluorescence dyes conjugated to the analyte rather than directly detecting the LSPR wavelength shift upon analyte bonding. This method can enhance the biomarker detection sensitivity 10 to 100 times upon careful design of the metal nanostructures and the location of the fluorescence dyes in the bioassay. To mass fabricate the metal nanostructures, a 4″ nickel mold is fabricated by electroplating and employed for UV nanoimprinting lithography. Our technology achieves high yield on wafer-level mass fabrication of the designed metal nanostructures. In terms of the surface modification of the bioassay, the orientation of the capturing antibody is controlled to enhance the sensitivity of biomarker detections, and an antifouling polymer is synthesized inside the gold nanoholes. To accomplish the cost-effective point-of-care system, a plastic multichamber microfluidic chip is fabricated, which contains the metal nanostructures, microfluidic channels, and trenches for controlling the sample flow. The microfluidic chip is inserted into a point-of-care system which consists of micropumps to control the microfluidic flow, a light source for fluorescence excitation, a camera system for fluorescence detection, and software to automate the POC system and to analyze the results. We believe this highly sensitive LSPR point-of-care system has ample applications on medical diagnostics.

End-on Covalent Antibody Immobilization on Dual Polarization Interferometry Sensor Chip for Enhanced Immuno-sensing

End-on immobilization of antibody is an important technology to greatly improve the sensitivity of immuno-sensing. The end-on, or constant fragment (Fc) site-specifically oriented antibodies expose more active antigen binding sites (Fab) and tend to capture more analytes from the solution. In this study, dual polarization interferometry (DPI), which is the only available equipment that can concurrently obtain the mass, thickness, density, and refractive index in real time, was applied to investigate two end-on antibody immobilization methods, as exemplified by prostate-specific antigen (PSA) antibody. In the first method, antibody was immobilized via the saccharide chain linked to Fc portion of the antibody, by chelation to the surface-bound boronic acid. In the second method, antibody was partially reduced by tris(2-carboxyethyl) phosphine (TCEP) under mild conditions, followed by covalent conjugation to the surface via thiol-maleimide reaction. Time-resolved measurement from DPI verifies the end-on conformation of the antibody on the sensor surface. The second method shows better detection performance, with enhanced sensitivity and reproducibility than the first method, due to the optimal alignment of the antibody. Finally, these two methods were compared with the protein G-based antibody orientation control and random antibody immobilization in terms of the mass, thickness, density, refractive index, reproducibility, and stability. The detailed antibody immobilization parameters obtained in this paper are of great importance in the developing of solid–liquid phase immunosensors with enhanced detection sensitivity.

Photoconversion of Spiropyran to Merocyanine in a Monolayer Observed Using Nanosecond Pump-Probe Brewster Angle Reflectometry

A new apparatus for nanosecond-time-resolved Brewster angle reflectometry is described that can be used to measure transient angle-resolved reflectivity changes in thin films and monolayers in a single pulsed laser shot. In order to achieve this, a cylindrical lens is placed in the probe beam path replacing the goniometer that is usually used for angular scanning in other systems. Using two synchronized nanosecond pulsed lasers in pump-probe configuration it is possible to measure the kinetics of photoinduced conformational changes by altering the delay between pump and probe pulses. The system was used to observe nanosecond time-resolved photodynamics in a spiropyran monolayer at the air-water interface. After UV excitation the spiropyran converted to its merocyanine form in two stages. The first stage occurred with a timescale close to the instrument time resolution (tens of nanoseconds) whereas the second stage occurred over a few hundred nanoseconds.

QUANTIFICATION OF TRANSIENT ABSORPTION IN PHOTO-REACTIVE MONOLAYERS USING REFLECTOMETRY IN THE VICINITY OF BREWSTER ANGLE

A method is presented that enables the determination of transient absorption in Langmuir films made from a monolayer of spiropyran on water. This is achievable even though the optical pathlength of such a monolayer is < 10-9 m. The approach is to monitor reflectivity changes close to the Brewster angle, where the background of reflected light is minimized. This is the key to the sensitivity of the method. Relatively speaking the small changes in reflectivity due to changes in both real and imaginary parts of the refractive index are easier to observe with the intrinsically low backgrounds at the Brewster angle. Notably using Fresnel equations we can show that the real and imaginary parts of the refractive index can be independently assessed for ultrathin films and monolayers using the approach presented.

Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor α and Specificity Protein 1 with Composite DNA Elements

Estrogen receptor α (ERα) and Specificity protein 1 (Sp1) are transcription factors (TF) that are involved in regulating progesterone receptor (PR) gene expression through cooperative interactions with DNA. The natural composite DNA +571 ERE/Sp1 site in promoter A of the progesterone receptor contains a half-site of estrogen response elements (½ERE) upstream of two Sp1 binding sites (the proximal Sp1 (Sp1/P) and distal Sp1 (Sp1/D)) with a 4 bp spacer. Here, we have developed a protocol for studying the cooperative interaction of Sp1 and ERα with the composite DNA of +571 ERE/Sp1 site using Biacore T200, a high sensitivity surface plasmon resonance spectroscopy. With this protocol, we have concluded that Sp1 binding enhances the overall ERα binding to the composite DNA. We have also determined the optimal spacer distance between the ½ERE and Sp1/D for the best cooperative protein binding. This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor-DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators.

Optical detection of photo-activated protein adsorption using Brewster angle microscopy

Bovine serum albumin (BSA) was space selectively immobilized onto a quartz substrate using photo-reactions initiated by pulsed laser irradiation of benzophenone derivatives tethered by silane coupling to the quartz. The excitation of the benzophenone with 266 nm light yields the pi-pi* triplet state with a quantum yield of 1. This triplet state then reacts with BSA in buffer solution in contact with the modified quartz. The triplet state may form a ketyl radical by reaction with the solution or it may abstract a proton directly from the BSA becoming covalently tethered to the surface. The entire deposition process was monitored in real time, in-situ using a Brewster angle microscope (BAM).