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.

The clinical need for ultra-sensitive molecular analysis has motivated the development of several endpoint assay technologies capable of single molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular recognition agents for the analyte of interest. In contrast, a kinetic assay with single molecule readout could distinguish between low abundance, high affinity (specific analyte) and high abundance, low affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid phase sensor with a large area approaching that of leading bead-based endpoint assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and de-binding as well as the distribution of binding event durations are determined. We observe a limit of detection of 15 femtomolar for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.

Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 µL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies/ml, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.

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 today’s 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.