George G. Daaboul

High-throughput detection and sizing of individual low-index nanoparticles and viruses for pathogen identification.

Rapid, chip-scale, and cost-effective single particle detection of biological agents is of great importance to human health and national security. We report real-time, high-throughput detection and sizing of individual, low-index polystyrene nanoparticles and H1N1 virus. Our widefield, common path interferometer detects nanoparticles and viruses over a very large sensing area, orders of magnitude larger than competing techniques. We demonstrate nanoparticle detection and sizing down to 70 nm in diameter. We clearly size discriminate nanoparticles with diameters of 70, 100, 150, and 200 nm. We also demonstrate detection and size characterization of hundreds of individual H1N1 viruses in a single experiment.

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.

Single nanoparticle detectors for biological applications

Nanoparticle research has become increasingly important in the context of bioscience and biotechnology. Practical use of nanoparticles in biology has significantly advanced our understanding about biological processes in the nanoscale as well as led to many novel diagnostic and therapeutic applications. Besides, synthetic and natural nanoparticles are of concern for their potential adverse effect on human health. Development of novel detection and characterization tools for nanoparticles will impact a broad range of disciplines in biological research from nanomedicine to nanotoxicology. In this article, we discuss the recent progress and future directions in the area of single nanoparticle detectors with an emphasis on their biological applications. A brief critical overview of electrical and mechanical detection techniques is given and a more in-depth discussion of label-free optical detection techniques is presented.

Single nanoparticle detection for multiplexed protein diagnostics with attomolar sensitivity in serum and unprocessed whole blood.

Although biomarkers exist for a range of disease diagnostics, a single low-cost platform exhibiting the required sensitivity, a large dynamic-range and multiplexing capability, and zero sample preparation remains in high demand for a variety of clinical applications. The Interferometric Reflectance Imaging Sensor (IRIS) was utilized to digitally detect and size single gold nanoparticles to identify protein biomarkers in unprocessed serum and blood samples. IRIS is a simple, inexpensive, multiplexed, high-throughput, and label-free optical biosensor that was originally used to quantify biomass captured on a surface with moderate sensitivity. Here we demonstrate detection of β-lactoglobulin, a cows milk whey protein spiked in serum (>10 orders of magnitude) and whole blood (>5 orders of magnitude), at attomolar sensitivity. The clinical utility of IRIS was demonstrated by detecting allergen-specific IgE from microliters of characterized human serum and unprocessed whole blood samples by using secondary antibodies against human IgE labeled with 40 nm gold nanoparticles. To the best of our knowledge, this level of sensitivity over a large dynamic range has not been previously demonstrated. IRIS offers four main advantages compared to existing technologies: it (i) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, (ii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, (iii) detects protein targets with conjugated very small nanoparticle tags (~40 nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and (iv) utilizes components that make the instrument inexpensive, robust, and portable. These features make IRIS an ideal candidate for clinical and diagnostic applications.

Label-free multiplexed virus detection using spectral reflectance imaging

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.

Real-Time Capture and Visualization of Individual Viruses in Complex Media.

Label-free imaging of individual viruses and nanoparticles directly in complex solutions is important for virology research and biosensing applications. A successful visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. Current approaches typically require fluorescent labeling or the use of an electron microscope, which are expensive and time-consuming to use. We have developed an imaging technique for real-time, sensitive, and label-free visualization of viruses and nanoparticles directly in complex solutions such as serum. By combining the advantages of a single-particle reflectance imaging sensor, with microfluidics, we perform real-time digital detection of individual 100 nm vesicular stomatitis viruses as they bind to an antibody microarray. Using this approach, we have shown capture and visualization of a recombinant vesicular stomatitis virus Ebola model (rVSV-ZEBOV) at 100 PFU/mL in undiluted fetal bovine serum in less than 30 min.

Digital detection of biomarkers assisted by nanoparticles: application to diagnostics

Single-molecule detection and counting is the new frontier in biomarker analysis. Here, we report on recent techniques for the digital detection of biomolecules for clinical application. First we highlight methods based on the immunocapture of proteins onto microparticles, followed by isolation of individual particles in microenvironments so that a sufficient signal is acquired for each binding event to make a binary decision, thus dramatically enhancing the signal:noise ratio. Various approaches are categorized based on the method used for particle confinement in an isolated microenvironment. We go on to describe methods for the detection of individual biological nanoparticles as well as the digital detection of proteins by artificial nanoparticle labels. The discussion of the methods emphasizes the practical considerations and their clinical applicability.

DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications.

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.

Label-Free Optical Biosensors for Virus Detection and Characterization

Label-free optical biosensors have shown promise in rapid and sensitive detection of viruses. Recently, we have demonstrated a technique termed interferometric reflectance imaging sensor (IRIS) capable of detecting viral antigens and single viruses in a high-throughput, quantitative, and label-free fashion. In this paper, we briefly review the current label-free optical methods that have demonstrated virus detection through ensemble measurements of viral antigen and emerging techniques that are capable of detecting individual viruses captured by the sensor. We then present the principle of single nanoparticle detection using IRIS and show proof-of-principle results on specific detection and shape analysis of individual viruses captured on the IRIS platform.