Jane Pepper

A label-free optical technique for detecting small molecule interactions

A novel approach for the label-free detection of molecular interactions is presented in which a colorimetric resonant grating is used as a surface binding platform. The grating, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or radioactive label. The detection technique is capable of detecting the addition and removal of small molecules as they interact with receptor molecules on the sensor surface or enzymes in the solution surrounding the sensor. Two assays are presented to exemplify the detection of small molecule interactions with the biosensor. First, an avidin receptor layer is used to detect 244 Da biotin binding. Second, a protease assay is performed in which a 136 Da p-nitroanilide (pNA) moeity is cleaved from an immobilized substrate. Because the sensor structure can be embedded in the plastic surfaces of microtiter plates or the glass surfaces of microarray slides, it is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. Screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.

Design, fabrication and vapor characterization of a microfabricated flexural plate resonator sensor and application to integrated sensor arrays

A chemical vapor detection and biosensor array based on microfabricated silicon resonators coated with thin film polymer sorption layers is described. The resonators within the array are micro-electromechanical (MEM) flexural plate wave (FPW) sensors that have been miniaturized to allow many independently addressable sensors to be integrated within a single silicon chip. The target analyte of an individual sensor within the chip is selected by placing a polymer coating onto the resonating membrane. Detection is performed by monitoring changes in the frequency and damping factor of the resonance as the coating interacts with the environment. This work documents vapor response characterization of an individual sensor element within an array and reports on the operation of an eight-element sensor array. Polymer coatings targeted toward detection of chemical weapon agents have been applied to the sensor and chemical vapor exposure tests using two chemical weapon simulants and four vapor phase interferents have been performed. Data describing temperature dependence, long-term/short-term drift stability, detection limits, detection linearity and vapor selectivity will be presented. The use of resonant damping information is shown to provide the ability to discriminate between vapor analytes that produce equal resonant frequency shifts. # 2001 Elsevier Science B.V. All rights reserved.

Detection of proteins and intact microorganisms using microfabricated flexural plate silicon resonator arrays

We are developing biosensor arrays that are based on microfabricated silicon flexural plate wave (FPW) resonators coated with molecular recognition chemistry. The resonators within the micro-chemical analysis array (CANARY) are micro-electromechanical (MEM) sensors that have been miniaturized to allow many independently addressable sensors to be integrated within a single silicon chip. The target analyte of an individual sensor within the chip is selectively detected by depositing molecular recognition component (or “coating”) onto the sensor surface, and monitoring changes in the frequency and phase of the resonance as the coating interacts with the analyte. The ultimate goal of this project is integration of hundreds of miniature resonators within a single chip for detection of biological species. As proof of concept demonstration, we describe here the detection of proteins and intact microorganisms using 2-element and 8-element CANARY sensor chips and address electronics. Preliminary results of sensitivity, selectivity, and surface regeneration methods of the sensor are presented. Detection of proteins and microorganisms with the CANARY sensor were confirmed by optical measurements.

A plastic colorimetric resonant optical biosensor for multiparallel detection of label-free biochemical interactions

A high sensitivity plastic biosensor based on detection of changes in optical density on the surface of a narrow bandwidth guided mode resonant filter is demonstrated. Using sub-micron microreplication of a master sensor surface structure on continuous sheets of plastic film, the sensor can be produced inexpensively over large surface areas. In this work, the sensor structure is incorporated into standard 96-well microtiter plates and used to perform a protein-protein affinity assay. A surface receptor immobilization protocol demonstrating low nonspecific binding is used to detect an antibody with 8.3 nM sensitivity. By measuring the kinetic interaction of a protein-protein binding pair simultaneously at several concentrations, the affinity binding constant can be quickly determined.

Colorimetric resonant reflection as a direct biochemical assay technique

A novel approach for the detection of molecular interactions is presented in which a colorimetric resonant diffractive grating surface is used as a surface binding platform. A guided mode resonant phenomenon is used to produce an optical structure that, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or particle label. It is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. High throughput screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.

Chemical vapor detection using microfabricated flexural plate silicon resonator arrays

This presentation will describe the design, fabrication, and testing of 2-element, and 8-element micro-Chemical Analysis Array ((mu) CANARY) sensor chips and address electronics. The detailed performance characterization of (mu) CANARY sensors for vapor and liquid phase detection will be presented. For vapor detection, NRL has applied polymer receptor coatings targeted at detection of chemical weapon agents, and has performed extensive chemical vapor exposure tests using two chemical weapon simulants and four vapor phase interferents. Data describing temperature dependence, long term/short term drift stability, detection limits, detection linearity, and vapor selectivity will be presented.