Interferometric detection and enumeration of viral particles using Si-based microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.This work was supported in part by a research contract with the ASELSAN Research Center, Ankara, Turkey, and in part by the European Union's Horizon 2020 FET Open program under Grant 766466-INDEX. (ASELSAN Research Center, Ankara, Turkey; 766466-INDEX - European Union's Horizon 2020 FET Open program)First author draf

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.National Cancer Institute (U.S.) (P30-CA14051

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 identifying the bacterial contaminations and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards development of 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 E. coli detection utilizing interferometric reflectance imaging enhancement allowing for visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate a limit of detection of 2.2 CFU/ml from a buffer solution with no sample preparation. To the best of our knowledge, this high 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 performance in tap water also 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.First author draf

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.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

Inertio-elastic focusing of bioparticles in microchannels at high throughput

Controlled manipulation of particles from very large volumes of fluid at high throughput is critical for many biomedical, environmental and industrial applications. One promising approach is to use microfluidic technologies that rely on fluid inertia or elasticity to drive lateral migration of particles to stable equilibrium positions in a microchannel. Here, we report on a hydrodynamic approach that enables deterministic focusing of beads, mammalian cells and anisotropic hydrogel particles in a microchannel at extremely high flow rates. We show that on addition of micromolar concentrations of hyaluronic acid, the resulting fluid viscoelasticity can be used to control the focal position of particles at Reynolds numbers up to Re≈10,000 with corresponding flow rates and particle velocities up to 50 ml min[superscript −1] and 130 m s[superscript −1]. This study explores a previously unattained regime of inertio-elastic fluid flow and demonstrates bioparticle focusing at flow rates that are the highest yet achieved.National Institute for Biomedical Imaging and Bioengineering (U.S.) (P41 BioMicroElectroMechanical Systems Resource Center)National Institute for Biomedical Imaging and Bioengineering (U.S.) (P41 EB002503)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-0001

Microfluidic Blood Cell Sorting: Now and Beyond

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106971/1/smll201302907.pd

An automated Raman-based platform for the sorting of live cells by functional properties

Stable-isotope probing is widely used to study the function of microbial taxa in their natural environment, but sorting of isotopically labelled microbial cells from complex samples for subsequent genomic analysis or cultivation is still in its early infancy. Here, we introduce an optofluidic platform for automated sorting of stable-isotope-probing-labelled microbial cells, combining microfluidics, optical tweezing and Raman microspectroscopy, which yields live cells suitable for subsequent single-cell genomics, mini-metagenomics or cultivation. We describe the design and optimization of this Raman-activated cell-sorting approach, illustrate its operation with four model bacteria (two intestinal, one soil and one marine) and demonstrate its high sorting accuracy (98.3 ± 1.7%), throughput (200-500 cells h-1; 3.3-8.3 cells min-1) and compatibility with cultivation. Application of this sorting approach for the metagenomic characterization of bacteria involved in mucin degradation in the mouse colon revealed a diverse consortium of bacteria, including several members of the underexplored family Muribaculaceae, highlighting both the complexity of this niche and the potential of Raman-activated cell sorting for identifying key players in targeted processes.</p