Alexander P. Reddington

LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular interactions

Label-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the Interferometric Reflectance Imaging Sensor (IRIS) using an inexpensive and durable multi-color LED illumination source to monitor protein-protein and DNA-DNA interactions. We demonstrate the capability of this system to dynamically monitor antigen-antibody interactions with a noise floor of 5.2 pg/mm(2) and DNA single mismatch detection under denaturing conditions in an array format. Our experiments show that this platform has comparable sensitivity to high-end label-free biosensors at a much lower cost with the capability to be translated to field-deployable applications.

Multiplexed Method to Calibrate and Quantitate Fluorescence Signal for Allergen-Specific IgE

Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody-antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.

Silicon biochips for dual label-free and fluorescence detection: Application to protein microarray development

A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500 nm SiO2 layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100 nm SiO2 providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fluorescence detection of the molecular interaction counterparts. Such a combined chip is of high practical utility during assay development process to image arrays, check consistency and quality of the protein array, quantify the amount of immobilized probes and finally detect fluorescence of bioassays.

Interferometric silicon biochips for label and label‐free DNA and protein microarrays

Protein and DNA microarrays hold the promise to revolutionize the field of molecular diagnostics. Traditional microarray applications employ labeled detection strategies based on the use of fluorescent and chemiluminescent secondary antibodies. However, the development of high throughput, sensitive, label‐free detection techniques is attracting attention as they do not require labeled reactants and provide quantitative information on binding kinetics. In this article, we will provide an overview of the recent authors work in label and label‐free sensing platforms employing silicon/silicon oxide (Si/SiO2) substrates for interferometric and/or fluorescence detection of microarrays. The review will focus on applications of Si/SiO2 with controlled oxide layers to (i) enhance the fluorescence intensity by optical interferences, (ii) quantify with sub‐nanometer accuracy the axial locations of fluorophore‐labeled probes tethered to the surface, and (iii) detect protein–protein interactions label free. Different methods of biofunctionalization of the sensing surface will be discussed. In particular, organosilanization reactions for monodimensional coatings and polymeric coatings will be extensively reviewed. Finally, the importance of calibration of protein microarrays through the dual use of labeled and label‐free detection schemes on the same chip will be illustrated.

An Interferometric Reflectance Imaging Sensor for Point of Care Viral Diagnostics

The use of in vitro diagnostic devices is transitioning from the laboratory to the primary care setting to address early disease detection needs. Time critical viral diagnoses are often made without support due to the experimental time required in todays standard tests. Available rapid point of care (POC) viral tests are less reliable, requiring a follow-on confirmatory test before conclusions can be drawn. The development of a reliable POC viral test for the primary care setting would decrease the time for diagnosis leading to a lower chance of transmission and improve recovery. The single particle interferometric reflectance imaging sensor (SP-IRIS) has been shown to be a sensitive and specific-detection platform in serum and whole blood. This paper presents a step towards a POC viral assay through a SP-IRIS prototype with automated data acquisition and analysis and a simple, easy-to-use software interface. Decreasing operation complexity highlights the potential of SP-IRIS as a sensitive and specific POC diagnostic tool. With the integration of a microfluidic cartridge, this automated instrument will allow an untrained user to run a sample-to-answer viral assay in the POC setting.

Self-referencing substrates for optical interferometric biosensors

Optical interference is a powerful technique for monitoring surface topography or refractive index changes in a thin film layer. Reflectance spectroscopy provides label-free biosensing capability by monitoring small variations in interference signature resulting from optical path length changes from surface-adsorbed biomolecules. Spectral reflectance data can be acquired either by broad wavelength illumination and spectroscopy at a single point, thus necessitating scanning, or by varying the wavelength of illumination and imaging the reflected intensity allowing for acquisition of a spectral image of a large field of view simultaneously. In imaging modalities, intensity fluctuations of the illuminating light source couple into the detected signal, increasing the noise in measured surface profiles. This article introduces a simple technique for eliminating the effects of illumination light power fluctuations by fabricating on-substrate self-reference regions to measure and normalize for the incident intensity, simplifying the overall platform for reflection or transmission-based imaging biosensors. Experimental results demonstrate that the sensitivity performance using self-referencing is equivalent or better than an optimized system with an external reference.

Integrated imaging instrument for self-calibrated fluorescence protein microarrays

Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur

The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation. Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time. Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches. Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Multiplexed, rapid, point of care device to quantify allergen-specific IgE

Allergy is a disorder of the immune system characterized by a maladaptive immune response to harmless environmental antigens (“allergens”). Allergy affects nearly 40–50 million people in the US, with total estimated costs attributable to asthma care at