Evaluation of a nanophosphor lateral-flow assay for self-testing for herpes simplex virus type 2 seropositivity.

Herpes Simplex Virus Type 2 (HSV-2) is a common human pathogen that causes life-long illness. The US prevalence of HSV-2 infection is 11.9% for individuals between 15 and 49 years of age. Individuals with HSV-2 infection are more likely to contract and spread other sexually-transmitted infections. Eighty percent of individuals with HSV-2 are unaware of their infection, in part because of the social stigma associated with in-clinic testing for sexually-transmitted infections. We conducted an initial evaluation of a prototype smartphone-based serological lateral-flow immunoassay (LFA) for HSV-2 infection that uses strontium aluminate persistent luminescent nanoparticles (nanophosphors) as reporters. When applied to a test panel of 21 human plasma/serum samples varying in anti-HSV titer, the nanophosphor HSV-2 LFA had 96.7% sensitivity and 100% specificity for detection of HSV-2 infection. The sensitivity of the nanophosphor HSV-2 LFA was higher than that of commercially-available rapid HSV-2 assays tested with the same panel. Analysis of the iPhone nanophosphor HSV-2 LFA strip images with our custom smartphone app gave greater reproducibility compared to ImageJ analysis of strip images. The smartphone-based nanophosphor LFA technology shows promise for private self-testing for sexually-transmitted infections (STI).

A low-cost smartphone-based platform for highly sensitive point-of-care testing with persistent luminescent phosphors.

Through their computational power and connectivity, smartphones are poised to rapidly expand telemedicine and transform healthcare by enabling better personal health monitoring and rapid diagnostics. Recently, a variety of platforms have been developed to enable smartphone-based point-of-care testing using imaging-based readout with the smartphone camera as the detector. Fluorescent reporters have been shown to improve the sensitivity of assays over colorimetric labels, but fluorescence readout necessitates incorporating optical hardware into the detection system, adding to the cost and complexity of the device. Here we present a simple, low-cost smartphone-based detection platform for highly sensitive luminescence imaging readout of point-of-care tests run with persistent luminescent phosphors as reporters. The extremely bright and long-lived emission of persistent phosphors allows sensitive analyte detection with a smartphone by a facile time-gated imaging strategy. Phosphors are first briefly excited with the phone's camera flash, followed by switching off the flash, and subsequent imaging of phosphor luminescence with the camera. Using this approach, we demonstrate detection of human chorionic gonadotropin using a lateral flow assay and the smartphone platform with strontium aluminate nanoparticles as reporters, giving a detection limit of ≈45 pg mL-1 (1.2 pM) in buffer. Time-gated imaging on a smartphone can be readily adapted for sensitive and potentially quantitative testing using other point-of-care formats, and is workable with a variety of persistent luminescent materials.

Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays.

In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 10(6) virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings.

An embedded microretroreflector-based microfluidic immunoassay platform.

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 µm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.

Persistent luminescence strontium aluminate nanoparticles as reporters in lateral flow assays.

Demand for highly sensitive, robust diagnostics and environmental monitoring methods has led to extensive research in improving reporter technologies. Inorganic phosphorescent materials exhibiting persistent luminescence are commonly found in electroluminescent displays and glowing paints but are not widely used as reporters in diagnostic assays. Persistent luminescence nanoparticles (PLNPs) offer advantages over conventional photoluminescent probes, including the potential for enhanced sensitivity by collecting time-resolved measurements or images with decreased background autofluorescence while eliminating the need for expensive optical hardware, superior resistance to photobleaching, amenability to quantitation, and facile bioconjugation schemes. We isolated rare-earth doped strontium aluminate PLNPs from larger-particle commercial materials by wet milling and differential sedimentation and water-stabilized the particles by silica encapsulation using a modified Stöber process. Surface treatment with aldehyde silane followed by reductive amination with heterobifunctional amine-poly(ethylene glycol)-carboxyl allowed covalent attachment of proteins to the particles using standard carbodiimide chemistry. NeutrAvidin PLNPs were used in lateral flow assays (LFAs) with biotinylated lysozyme as a model analyte in buffer and monoclonal anti-lysozyme HyHEL-5 antibodies at the test line. Preliminary experiments revealed a limit of detection below 100 pg/mL using the NeutrAvidin PLNPs, which was approximately an order of magnitude more sensitive than colloidal gold.

Microretroreflector-sedimentation immunoassays for pathogen detection.

Point-of-care detection of pathogens is medically valuable but poses challenging trade-offs between instrument complexity and clinical and analytical sensitivity. Here we introduce a diagnostic platform utilizing lithographically fabricated micron-scale forms of cubic retroreflectors, arguably one of the most optically detectable human artifacts, as reporter labels for use in sensitive immunoassays. We demonstrate the applicability of this novel optical label in a simple assay format in which retroreflector cubes are first mixed with the sample. The cubes are then allowed to settle onto an immuno-capture surface, followed by inversion for gravity-driven removal of nonspecifically bound cubes. Cubes bridged to the capture surface by the analyte are detected using inexpensive, low-numerical aperture optics. For model bacterial and viral pathogens, sensitivity in 10% human serum was found to be 10(4) bacterial cells/mL and 10(4) virus particles/mL, consistent with clinical utility.

Retroreflective imaging system for optical labeling and detection of microorganisms.

A retroreflective imaging system for imaging microscopic targets over macroscopic sampling areas is introduced. Detection of microorganism-bound retroreflector (RR) targets across millimeter-scale samples is implemented according to retroreflection directionality, collimation, and contrast design characteristics. Retroreflection directionality is considered for corner-cube (CC) and spherical geometries. Spherical-RRs improve directionality and reliability. Retroreflection collimation is considered for spherical-RRs. Retroreflective images for micro-CC-RRs and micro-spherical-RRs with varying refractive indices show optimal results for high refractive index BaTiO3 micro-spherical-RRs. A differential imaging technique improves retroreflection contrast by 35 dB. High refractive index micro-spherical-RRs and differential imaging, together, can detect microscopic RR targets across macroscopic areas.

Monolithic NPG nanoparticles with large surface area, tunable plasmonics, and high-density internal hot-spots.

Plasmonic metal nanostructures have shown great potential in sensing, photovoltaics, imaging and biomedicine, principally due to the enhancement of local electric field by light-excited surface plasmons, i.e., collective oscillation of conduction band electrons. Thin films of nanoporous gold have received a great deal of interest due to the unique 3-dimensional bicontinuous nanostructures with high specific surface area. However, in the form of semi-infinite thin films, nanoporous gold exhibits weak plasmonic extinction and little tunability in the plasmon resonance, because the pore size is much smaller than the wavelength of light. Here we show that by making nanoporous gold in the form of disks of sub-wavelength diameter and sub-100 nm thickness, these limitations can be overcome. Nanoporous gold disks not only possess large specific surface area but also high-density, internal plasmonic "hot-spots" with impressive electric field enhancement, which greatly promotes plasmon-matter interactions as evidenced by spectral shifts in the surface plasmon resonance. In addition, the plasmonic resonance of nanoporous gold disks can be easily tuned from 900 to 1850 nm by changing the disk diameter from 300 to 700 nm. Furthermore, nanoporous gold disks can be fabricated as either bound on a surface or as non-aggregating colloidal suspension with high stability.

Label-free, in situ SERS monitoring of individual DNA hybridization in microfluidics.

We present label-free, in situ monitoring of individual DNA hybridization in microfluidics. By immobilizing molecular sentinel probes on nanoporous gold disks, we demonstrate sensitivity approaching the single-molecule limit via surface-enhanced Raman scattering which provides robust signals without photobleaching for more than an hour. We further demonstrate that a target concentration as low as 20 pM can be detected within 10 min under diffusion-limited transport.