Colorimetric Detection of Fentanyl Powder on Surfaces Using a Supramolecular Displacement Assay.

Fentanyl is a potent synthetic opioid with an alarmingly low lethal dosage of 2 mg. The equipment necessary to detect fentanyl in field settings (e.g., hand-held spectrometers) is restricted to highly trained, well-funded, and specialized personnel. Established point-of-need technologies, such as lateral flow immunochromatographic strips, are available; however, they often involve multiple contact-based steps (e.g., collection, mixing) that pose a higher risk to users handling unknown substances. Herein, we developed a colorimetric displacement assay capable of contactless detection of fentanyl in liquid or solid samples. The basis of our assay relies on the presence of fentanyl to displace a redox mediator, ferrocene carboxylic acid, inclusively bound in the cavity of a supramolecular host, CB[7]. The displacement is only possible in the presence of high affinity binding guests, like fentanyl (KA ∼ 106 M-1). The liberated redox guest can then react with indicator reagents that are free in solution, producing either: (i) a distinct blue color to indicate the presence of fentanyl or (ii) a pale blue tint in the absence of fentanyl. We demonstrate rapid and specific detection of fentanyl free base and fentanyl derivatives (e.g., acetyl fentanyl and furanyl fentanyl) against a panel of 9 other common drugs of abuse (e.g., morphine, cocaine, and heroin). Furthermore, we highlight the intended use of this assay by testing grains of fentanyl derivatives on a surface with a drop (i.e., 25 µL) of the assay reagent. We anticipate that this approach can be applied broadly to identify the presence of fentanyl at the point of need.

Generating signals at converging liquid fronts to create line-format readouts of soluble assay products in three-dimensional paper-based devices.

The correct interpretation of the result from a point-of-care device is crucial for an accurate and rapid diagnosis to guide subsequent treatment. Lateral flow tests (LFTs) use a well-established format that was designed to simplify the user experience. However, the LFT device architecture is inherently limited to detecting analytes that can be captured by molecular recognition. Microfluidic paper-based analytical devices (µPADs), like LFTs, have the potential to be used in diagnostic applications at the point of care. However, µPADs have not gained significant traction outside of academic laboratories, in part, because they have often demonstrated a lack of homogeneous shape or color in signal outputs, which consequently can lead to inaccurate interpretation of results by users. Here, we demonstrate a new class of µPADs that form colorimetric signals at the interfaces of converging liquid fronts (i.e., lines) to control where colorimetric signals are formed without relying on capture techniques. We demonstrate our approach by developing assays for three classes of analytes-an ion, an enzyme, and a small molecule-to measure using iron(III), acetylcholinesterase, and lactate, respectively. Additionally, we show these devices have the potential to support multiplexed assays by generating multiple lines in a common readout zone. These results highlight the ability of this new paper-based device architecture to aid the interpretation of assays that create soluble products by using flow to constrain those colorimetric products in a familiar, line-format output.

Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP-KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3-to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3-25 nM) exhibited the lowest limits of detection (ca. µg mL-1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual's immune response can impact the interpretation of assay results.

Clinical Correlations of Positive Herpes Simplex PCR in Cerebrospinal Fluid.

OBJECTIVES: Herpes simplex viruses (HSV) can produce encephalitis (HSE), which requires early detection, typically using polymerase chain reaction (PCR) in cerebrospinal fluid (CSF). However, other neurological conditions not directly caused by HSV may also present with a positive HSV PCR in the CSF (NCNHPCR+). We aimed to analyze the clinical features of both groups of patients (HSE vs. NCNHPCR+) and to consider the potential relevance of this finding in the latter. METHODS: A retrospective analysis of clinical presentation, workup (CSF, EEG, and MRI) and outcome of patients with an HSV+ result in CSF was conducted from Jan-2007 to Sep-2015 in our institution. Patients under 18 years and those with nonencephalitic HSV associated disorders were excluded. Group comparison between HSE and NCNHPCR+ patients was conducted using parametric and nonparametric tests accordingly. RESULTS: Sixteen HSE and 23 NCNHPCR+ patients were included. Patients with HSE presented a higher incidence of headache (87.5% vs. 43.5%; P=0.008), meningeal symptoms (50% vs. 17.4%; P=0.04), pleocytosis (75% vs. 18%; P=0.001), EEG abnormalities (46.67% vs. 22%; P=0.02) and typical MRI findings (50% vs. 0%; P<0.001), whereas 35% of patients with NCNHPCR+ had an underlying immunologic disorder (35% vs. 0%; P=0.012). CONCLUSIONS: The pathogenic role of HSV in NCNHPCR+ is uncertain. This finding must be interpreted in the appropriate clinical, EEG, and neuroimaging context. Immunocompromise and neuroinflammation states could be related to a higher presence of HSV in CSF.