Physical modeling of interference enhanced imaging and characterization of single nanoparticles.

Interferometric imaging schemes have gained significant interest due to their superior sensitivity over imaging techniques that are solely based on scattered signal. In this study, we outline the theoretical foundations of imaging and characterization of single nanoparticles in an interferometric microscopy scheme, examine key parameters that influence the signal, and benchmark the model against experimental findings.

Development of a High-Throughput Functional Screen Using Nanowell-Assisted Cell Patterning.

Living-cell-based screens can facilitate lead discovery of functional therapeutics of interest. A versatile and scalable method is reported that uses dense arrays of nanowells for imparting defined patterns on monolayers of cells. It is shown that this approach can coordinate a multi-component biological assay by designing and implementing a high-throughput, functional nanoliter-scale neutralization assay to identify neutralizing antibodies against HIV.

Interferometric Reflectance Imaging Sensor (IRIS)--A Platform Technology for Multiplexed Diagnostics and Digital Detection.

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses.

Highly sensitive and label-free digital detection of whole cell E. coli with Interferometric Reflectance Imaging.

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in bacterial contaminations identification and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards developing new biosensors. Biosensors offering label-free, whole bacteria detection are highly desirable over those relying on label-based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label-free Escherichia coli (E. coli) detection utilizing interferometric reflectance imaging enhancement allowing visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate an extrapolated limit of detection of 2.2 CFU/ml from experimental data in buffer solution with no sample preparation. To the best of our knowledge, this level of sensitivity for whole E. coli detection is unprecedented in label-free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non-target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor's performance in tap water proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label-free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples.

Label-Free and High-Throughput Detection of Biomolecular Interactions Using a Flatbed Scanner Biosensor.

Fluorescence based microarray detection systems provide sensitive measurements; however, variation of probe immobilization and poor repeatability negatively affect the final readout, and thus quantification capability of these systems. Here, we demonstrate a label-free and high-throughput optical biosensor that can be utilized for calibration of fluorescence microarrays. The sensor employs a commercial flatbed scanner, and we demonstrate transformation of this low cost (∼100 USD) system into an Interferometric Reflectance Imaging Sensor through hardware and software modifications. Using this sensor, we report detection of DNA hybridization and DNA directed antibody immobilization on label-free microarrays with a noise floor of ∼30 pg/mm2, and a scan speed of 5 s (50 s for 10 frames averaged) for a 2 mm × 2 mm area. This novel system may be used as a standalone label-free sensor especially in low-resource settings, as well as for quality control and calibration of microarrays in existing fluorescence-based DNA and protein detection platforms.

Digital Detection of Exosomes by Interferometric Imaging.

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30-100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.