A Zika Reference Panel for Molecular-Based Diagnostic Devices as a US Food and Drug Administration Response Tool to a Public Health Emergency.

In 2015, Zika virus (ZIKV) appeared as an emerging pathogen, generating a global and urgent need for accurate diagnostic devices. During this public health crisis, several nucleic acid testing (NAT)-based Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Authorization. The FDA's Center for Devices and Radiological Health, in collaboration with the FDA's Center for Biologics Evaluation and Research, responded to this Zika emergency by developing and producing a reference panel (RP) for Zika RNA (Zika FDA-RP) suitable for performance assessment of ZIKV NAT-based in vitro diagnostic devices.

Detecting Biothreat Agents: From Current Diagnostics to Developing Sensor Technologies.

Although a fundamental understanding of the pathogenicity of most biothreat agents has been elucidated and available treatments have increased substantially over the past decades, they still represent a significant public health threat in this age of (bio)terrorism, indiscriminate warfare, pollution, climate change, unchecked population growth, and globalization. The key step to almost all prevention, protection, prophylaxis, post-exposure treatment, and mitigation of any bioagent is early detection. Here, we review available methods for detecting bioagents including pathogenic bacteria and viruses along with their toxins.

Evaluation of optical detection platforms for multiplexed detection of proteins and the need for point-of-care biosensors for clinical use.

This review investigates optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks encompassing multiple, rather than single, proteins. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots) or with the use of unique identifiers/labels (such as spectral separation-different colored dyes, or unique beads-size or color).

In vitro evaluation of fluorescence glucose biosensor response.

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods.

Detecting kallikrein proteolytic activity with peptide-quantum dot nanosensors.

Contamination and adulterants in both naturally derived and synthetic drugs pose a serious threat to the worldwide medical community. Developing rapid and sensitive sensors/devices to detect these hazards is thus a continuing need. We describe a hydrophilic semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) nanosensor for monitoring the activity of kallikrein, a key proteolytic enzyme functioning at the initiation of the blood clotting cascade.

Current perspectives on the US FDA regulatory framework for intelligent drug-delivery systems.

The US FDA is the US agency responsible for regulating intelligent drug-delivery systems (IDDS). IDDS can be classified as a device, drug, biologic or combination product. In this perspective, the current regulatory framework for IDDS and future perspectives on how the field is expected to evolve from a regulatory standpoint is discussed.

Optimizing two-color semiconductor nanocrystal immunoassays in single well microtiter plate formats.

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent.

FRET-based quantum dot immunoassay for rapid and sensitive detection of Aspergillus amstelodami.

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor.

Cellular uptake and fate of PEGylated gold nanoparticles is dependent on both cell-penetration peptides and particle size.

Numerous studies have examined how the cellular delivery of gold nanoparticles (AuNPs) is influenced by different physical and chemical characteristics; however, the complex relationship between AuNP size, uptake efficiency and intracellular localization remains only partially understood. Here we examine the cellular uptake of a series of AuNPs ranging in diameter from 2.4 to 89 nm that are synthesized and made soluble with poly(ethylene glycol)-functionalized dithiolane ligands terminating in either carboxyl or methoxy groups and covalently conjugated to cell penetrating peptides.

Monitoring botulinum neurotoxin a activity with peptide-functionalized quantum dot resonance energy transfer sensors.

Botulinum neurotoxins (BoNTs) are extremely potent bacterial toxins that contaminate food supplies along with having a high potential for exploitation as bioterrorism agents. There is a continuing need to rapidly and sensitively detect exposure to these toxins and to verify their active state, as the latter directly affects diagnosis and helps provide effective treatments. We investigate the use of semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA).

Molecular electronics based nanosensors on a viral scaffold.

Assembling and interconnecting the building blocks of nanoscale devices and being able to electronically address or measure responses at the molecular level remains an important challenge for nanotechnology. Here we show the usefulness of bottom-up self-assembly for building electronic nanosensors from multiple components that have been designed to interact in a controlled manner. Cowpea mosaic virus was used as a scaffold to control the positions of gold nanoparticles.

Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays.

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical, environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently labeled protein, AlexaFluor647-chicken IgG (Alexa647-chick IgG).

Biomarkers to improve the benefit/risk balance for approved therapeutics: a US FDA perspective on personalized medicine.

This article highlights a current US FDA perspective concerning the use of biomarker-based diagnostics for personalized medicine. Specifically, current biomarkers that have application for improving the benefit/risk profile of already approved drugs are discussed. The success of biomarkers for use in personalized medicine depends on many factors, including proper evaluation of the usefulness of the biomarker for assessing the event of interest, and the safety and effectiveness of the diagnostic device used to measure the biomarker, which includes appropriate analytical and clinical validation.

Multi-wavelength Spatial LED illumination based detector for in vitro detection of Botulinum Neurotoxin A Activity.

A portable and rapid detection system for the activity analysis of Botulinum Neurotoxins (BoNT) is needed for food safety and bio-security applications. To improve BoNT activity detection, a previously designed portable charge-coupled device (CCD) based detector was modified and equipped with a higher intensity more versatile multi-wavelength spatial light-emitting diode (LED) illumination, a faster CCD detector and the capability to simultaneously detect 30 samples. A FITC/DABCYL Förster Resonance Energy Transfer (FRET)-labeled peptide substrate (SNAP-25), with BoNT-A target cleavage site sequence was used to measure BoNT-A light chain (LcA) activity through the FITC fluorescence increase that occurs upon peptide substrate cleavage.

Detection of HIV-1 specific monoclonal antibodies using enhancement of dye-labeled antigenic peptides.

A simple bifunctional colorimetric/fluorescent sensing assay is demonstrated for the detection of HIV-1 specific antibodies. This assay makes use of a short peptide sequence coupled to an environmentally sensitive dye that absorbs and emits in the visible portion of the spectrum. The core peptide sequence is derived from the highly antigenic six-residue epitope of the HIV-1 p17 protein and is situated adjacent to a terminal cysteine residue which enables site-specific fluorescent labeling with Cy3 cyanine dye.

Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector.

A previously developed fluorescence sensing platform, combining spatial illumination using electroluminescence (EL) semiconductor strips with charge coupled device (CCD)-based detection (EL-CCD), was adapted to a new 96-well chip for colorimetric immunological assays, enhancing the capabilities of the EL-CCD platform. The modified system was demonstrated using a colorimetric-based enzyme linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin B (SEB). Limits of detection (LODs) of 3.

Fluoroimmunoassays using the NRL array biosensor.

Array-based biosensor technology offers the user the ability to detect and quantify multiple targets in multiple samples simultaneously (Analytical Sciences 23:5-10, 2007). The NRL Array Biosensor has been developed with the aim of creating a system for sensitive, rapid, on-site screening for multiple targets of interest. This system is fluorescence-based, using evanescent illumination of a waveguide, and has demonstrated the use of both sandwich and competitive immunoassays for the detection of both high and low molecular weight targets, respectively.

A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity.

Current biodetection illumination technologies (laser, LED, tungsten lamp, etc.) are based on spot illumination with additional optics required when spatial excitation is required. Herein we describe a new approach of spatial illumination based on electroluminescence (EL) semiconductor strips available in several wavelengths, greatly simplifying the biosensor design by eliminating the need for additional optics.

Toward single molecule detection of staphylococcal enterotoxin B: mobile sandwich immunoassay on gliding microtubules.

An immunoassay based on gliding microtubules (MTs) is described for the detection of staphylococcal enterotoxin B. Detection is performed in a sandwich immunoassay format. Gliding microtubules carry the antigen-specific "capture" antibody, and bound analyte is detected using a fluorescent viral scaffold as the tracer.

Long term storage of virus templated fluorescent materials for sensing applications.

Wild type, mutant, and chemically modified Cowpea mosaic viruses (CPMV) were studied for long term preservation in the presence and absence of cryoprotectants. Viral complexes were reconstituted and tested via fluorescence spectroscopy and a UV/vis-based RNase assay for structural integrity. When viruses lyophilized in the absence of cryoprotectant were rehydrated and RNase treated, UV absorption increased, indicating that the capsids were damaged.

On the quenching of semiconductor quantum dot photoluminescence by proximal gold nanoparticles.

Luminescent quantum dots (QDs) were proven to be very effective fluorescence resonance energy transfer donors with an array of organic dye acceptors, and several fluorescence resonance energy transfer based biosensing assemblies utilizing QDs have been demonstrated in the past few years. Conversely, gold nanoparticles (Au-NPs) are known for their capacity to induce strong fluorescence quenching of conventional dye donors. Using a rigid variable-length polypeptide as a bifunctional biological linker, we monitor the photoluminescence quenching of CdSe-ZnS QDs by Au-NP acceptors arrayed around the QD surface, where the center-to-center separation distance was varied over a broad range of values (approximately 50-200 Angstrom).

The array biosensor: portable, automated systems.

With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously.