Nidhi Nath

Creating “Smart” Surfaces Using Stimuli Responsive Polymers

Surfaces modified with stimuli-responsive polymers (SRPs) dynamically alter their physico-chemical properties in response to changes in their environmental conditions. The triggered control of interfacial properties provided by immobilized SRPs at the solid–water interface has application in the design of biomaterials, regenerable biosensors, and microfluidic bioanalytical devices. In this article, we briefly summarize recent research in this area, followed by two recent examples of research from our laboratory on stimuli-responsive surfaces. First, we present a new assay to quantify the phase transition behavior of SRPs at the solid–water interface. This assay, which is based on the distance-dependent colorimetric properties of gold nanoparticles, provides a technically simple and convenient method to determine the effect of different variables on the lower critical solution temperature (LCST) behavior of SRPs at the solid–water interface. Second, we show that stimuli-responsive surfaces can be created by the immobilization of an elastin-like polypeptide (ELP), a thermally responsive biopolymer, on a glass surface. We exploit the phase transition of the ELP at a surface to reversibly address an ELP fusion protein to a surface. This method, which we term thermodynamically reversible addressing of proteins (TRAP), enables the reversible, spatio-temporal modulation of protein binding at the solid-liquid interface, and will enable the realization of new bioanalytical applications.

Label Free Colorimetric Biosensing Using Nanoparticles

In this review article, we discuss a class of biosensors that exploit the change in the colorimetric properties of noble metal nanoparticles in response to biomolecular binding at their surface. Several sensor fabrication techniques as well as sensor configurations are discussed with an emphasis on their strengths and limitations. We conclude by presenting the future prospects and challenges for the successful transition of this technology from the laboratory to a commercial product.

Identification and Characterization of Small Molecule Inhibitors of a Plant Homeodomain Finger

A number of histone-binding domains are implicated in cancer through improper binding of chromatin. In a clinically reported case of acute myeloid leukemia (AML), a genetic fusion protein between nucleoporin 98 and the third plant homeodomain (PHD) finger of JARID1A drives an oncogenic transcriptional program that is dependent on histone binding by the PHD finger. By exploiting the requirement for chromatin binding in oncogenesis, therapeutics targeting histone readers may represent a new paradigm in drug development. In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity. Molecular modeling and site-directed mutagenesis of JARID1A PHD3 provided insights into the biochemical basis of competitive inhibition.

Protein-protein interaction studies on protein arrays: Effect of detection strategies on signal-to-background ratios

Protein arrays hold great promise for proteome-scale analysis of protein-protein interaction networks, but the technical challenges have hindered their adoption by proteomics researchers. The crucial issue of design and fabrication of protein arrays have been addressed in several studies, but the detection strategies used for identifying protein-protein interactions have received little attention. In this study, we evaluated six different detection strategies to identify four different protein-protein interaction pairs. We discuss each detection approach in terms of signal-to-background (S/B) ratio, ease of use, and adaptability to high-throughput format. Protein arrays for this study were made by expressing both the bait proteins (proteins captured at the surface) and prey proteins (probes) in cell-free rabbit reticulocyte lysate (RRL) systems. Bait proteins were expressed as HaloTag fusions that allow covalent capture on a HaloTag ligand-coated glass without any prior protein purification step. Prey proteins were expressed and modified with either tags (protein or peptides) or labels (fluorescent or radiometric) for detection. This simple method for creating protein arrays in combination with our analyses of several detection strategies should increase the usefulness of protein array technologies.

Improving protein array performance: focus on washing and storage conditions.

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.

Immobilized gold nanoparticle sensor for label-free optical detection of biomolecular interactions

We present a new label free optical technique to study biomolecular interactions in real time on a surface. This method monitors changes in the absorbance spectrum of a monolayer of gold on glass as a function of biomolecular binding. Gold nanoparticles with a diameter of 13 nm were chemisorbed onto an amine-terminated glass surface. The absorbance spectrum of the monolayer exhibited both a red shift as well as an increase in the absorbance at peak wavelength as a function of bulk solution refractive index. The increase in absorbance at peak wavelength as a function of bulk solution refractive index. The increase in absorbance at 550 nm was employed to study the kinetics of fibrinogen adsorption on the immobilized monolayer. The result obtained with the absorbance sensor were compared with those obtained using conventional SPR. This sensor is attractive because of its simplicity: gold nanoparticles are easily prepared with high reproducibility, they can be easily immobilized on glass, and their absorbance spectrum can be easily measured using widely available UV-vis spectrophotometers. Furthermore, this technique should be easily amenable to the design of chips in an array format for application in hgih-throughput immunoassays and proteomics.

On-bead antibody-small molecule conjugation using high-capacity magnetic beads

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.

A colorimetric gold nanoparticle biosensor: effect of particle size on sensitivity

We have previously developed a label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of gold nanoparticles on glass as a function of biomolecular binding at the surface of the immobilized nanoparticles. Here, we report upon optimization of this sensor by examining the effect of particle size on the analytical sensitivity of the sensor. Increasing the diameter of the gold nanoparticles from 13 nm to 47 nm is shown to increase the sensitivity of the sensor by a factor of three for the model streptavidin-biotin receptor-ligand pair.

Noble Metal Nanoparticle Biosensors

Receptor-ligand binding assays are central to medical diagnostics, proteomics, drug discovery, environmental monitoring and food processing. A typical binding assay uses a “capture molecule” (often loosely termed as the “receptor”) which is typically an antibody, DNA, peptide, or protein, that binds to a target analyte (ligand) of interest in a sample with high affinity and specificity. Binding of the analyte to the “receptor” is coupled to a transduction step to enable detection of the binding event and quantification of the analyte concentration in the sample. Based on their mode of detection, most binding assays can be divided into two categories. The first category includes assays that require a label or tracer-a radioisotope, chromophore or fluorophore-to transduce the binding event into a quantifiable signal. The second category of analyte binding assays is label-fi-ee assays that do not require the addition of extrinsic reagents or labels. Instead, a change in a physical parameter upon analyte binding such as the mass, thickness, or refractive index, is directly transduced into a measurable signal. These two categories are broad, and some overlap exists between them. For example, recent protein engineering approaches have yielded direct fluorescence biosensors, in which binding is coupled to a change in the fluorescence of the receptor through allostery.