A Comparison of EIS and QCM NanoMIP-Based Sensors for Morphine

In this work we have compared two different sensing platforms for the detection of morphine as an example of a low molecular weight target analyte. For this, molecularly imprinted polymer nanoparticles (NanoMIP), synthesized with an affinity towards morphine, were attached to an electrochemical impedance spectroscopy (EIS) and a quartz crystal microbalance (QCM) sensor. Assay design, sensors fabrication, analyte sensitivity and specificity were performed using similar methods. The results showed that the EIS sensor achieved a limit of detection (LOD) of 0.11 ng·mL−1, which is three orders of magnitude lower than the 0.19 µg·mL−1 achieved using the QCM sensor. Both the EIS and the QCM sensors were found to be able to specifically detect morphine in a direct assay format. However, the QCM method required conjugation of gold nanoparticles (AuNPs) to the small analyte (morphine) to amplify the signal and achieve a LOD in the µg·mL−1 range. Conversely, the EIS sensor method was labor-intensive and required extensive data handling and processing, resulting in longer analysis times (~30–40 min). In addition, whereas the QCM enables visualization of the binding events between the target molecule and the sensor in real-time, the EIS method does not allow such a feature and measurements are taken post-binding. The work also highlighted the advantages of using QCM as an automated, rapid and multiplex sensor compared to the much simpler EIS platform used in this work, though, the QCM method will require sample preparation, especially when a sensitive (ng·mL−1) detection of a small analyte is needed.

Microbiological Antibiotic Assay Validation of Gentamicin Sulfate Using Two-Dose Parallel Line Model (PLM)

Till nowadays microbiological assay is still widely used with several antibiotics that are composed of a mixture of related active compounds. However, obtaining a reasonably valid determination of the potency is dependent on the validity and the suitability of the assay design. The present work aimed to validate an assay design of an aminoglycoside antibiotic (Gentamicin Sulfate) using a two-dose Parallel Line Model agar diffusion assay in a large 8×8 rectangular plate. All preparatory procedures were done following United States Pharmacopeia and the Inhibition Zones were measured using a digital caliper to the nearest 0.01 mm. Analysis of variance of compendial requirements of regression and parallelism were found to be satisfactorily meeting the acceptance criteria. Specificity was achieved for the product under investigation with no detectable IZ that could be found for all components except the antibiotic. The validation method showed acceptable linearity of r2≥0.98. Accuracy and precision parameters showed RSD (%)<2. All relative error value estimates were below 4%. The proposed validation design for 32×32 cm antibiotic plates yielded valid results and can be projected for the routine Quality Control analysis of the antibiotic material, especially which is incorporated into a finished medicinal dosage form. Doi: 10.28991/HIJ-2021-02-04-04 Full Text: PDF

Agile design and development of a high throughput cobas® SARS-CoV-2 RT-PCR diagnostic test

Diagnostic testing is essential for management of the COVID-19 pandemic. An agile assay design methodology, optimized for the cobas® 6800/8800 system, was used to develop a dual-target, qualitative SARS-CoV-2 RT-PCR test using commercially available reagents and existing sample processing and thermocycling profiles. The limit of detection was 0.004 to 0.007 TCID50/mL for USA-WA1/2020. Assay sensitivity was confirmed for SARS-CoV-2 variants Alpha, Beta, Gamma, Delta and Kappa. The coefficients of variation of the cycle threshold number (Ct) were between 1.1 and 2.2%. There was no difference in Ct using nasopharyngeal compared to oropharyngeal swabs in universal transport medium (UTM). A small increase in Ct was observed with specimens collected in cobas® PCR medium compared to UTM. In silico analysis indicated that the dual-target test is capable of detecting all >1,800,000 SARS-CoV-2 sequences in the GISAID database. Our agile assay design approach facilitated rapid development and deployment of this SARS-CoV-2 RT-PCR test.

Rapid development of analytical methods for evaluating pandemic vaccines: a COVID-19 perspective

Vaccines are key in charting a path out of the COVID-19 pandemic. However, development of new vaccines is highly dependent on availability of analytical methods for their design and evaluation. This paper highlights the challenges presented in having to rapidly develop vaccine analytical tools during an ongoing pandemic, including the need to address progressive virus mutation and adaptation which can render initial assays unreliable or redundant. It also discusses the potential of new computational modeling techniques to model and analyze key viral proteins and their attributes to assist vaccine production and assay design. It then reviews the current range of analytical tools available for COVID-19 vaccine application, ranging from in vitro assays for immunogen characterization to assays to measure vaccine responses in vivo. Finally, it provides a future perspective for COVID-19 vaccine analytical tools and attempts to predict how the field might evolve over the next 5–10 years.

Long-Range But Not Short-Range Attraction of Male Aedes aegypti (Diptera: Culicidae) Mosquitoes to Humans

Female Aedes aegypti (Linnaeus) mosquitoes integrate multiple sensory cues to locate human hosts for blood meals. Although male Ae. aegypti swarm around and land on humans in nature to mate, direct evidence of attraction to humans is limited. Male mosquito attraction to human host cues is often undetectable in confined laboratory assays, leading to a misconception that male mosquitoes are not attracted to humans. We used semifield experiments to demonstrate robust attraction of male Ae. aegypti to humans. Human-baited traps captured up to 25% of released males within 15 min, whereas control traps without humans as bait failed to capture males. Rapid attraction to humans was further demonstrated through videography. Males swarmed around and landed on human subjects, with no activity recorded in paired unbaited controls. Finally, we confirm the lack of discernible male attraction to humans in small laboratory cages. Our experiments demonstrate that both male and female Ae. aegypti show attraction to humans, but with clear sex-specific behavioral differences at short-range. Male mosquito attraction to humans is likely to be important for mating success in wild populations and its basis should be further explored. Our results highlight the importance of arena size and assay design for mosquito behavioral research. A better understanding of host cues that attract males could help us to improve mosquito surveillance and control.

Short Assay Design for Micronucleus Detection in Human Lymphocytes

There has been a constant need to develop new and faster cytogenetic assays to measure the instability induced by genotoxic agents in the field of cytogenetic research, an example of which is the micronucleus assay. Micronuclei are fragments or complete chromosomes that remain in the cytoplasm during mitosis. With their high sensitivity and specificity detection, their presence can indicate environmental and occupational genotoxic effects. However, the prolonged periods of cell incubation this assay necessitates are costly and extensive. Hence, it is essential to develop an improved assay that can achieve standardization by being reproducible in practice. The standard protocol for the detection of micronuclei in lymphocytes uses a total assay time of 72 hours. Theoretically, it is possible to reduce the incubation period, and consequently, the total assay time, considering a lymphocyte, completes its mitosis in 24 hours. This study, after careful review of literature, proposes an experimental design to reduce the incubation period and demonstrates its usefulness in practice through the design of a collaborative trial.

Sensitive assay design for detection of anti-drug antibodies to biotherapeutics that lack an immunoglobulin Fc domain

Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. However, the evolving structural complexity in contemporary biotherapeutics creates a need for on-going innovation in assay designs for reliable detection of anti-drug antibodies, especially for biotherapeutics that may not be well-suited for testing by a bridging assay. We, therefore, initiated systematic optimization of the direct binding assay to adapt it for routine use in regulatory-compliant assays of serum anti-drug antibodies. Accordingly, we first prepared a SULFO-TAG labeled conjugate of recombinant Protein-A/G to create a sensitive electrochemiluminescent secondary detection reagent with broad reactivity to antibodies across many species. Secondly, we evaluated candidate blocker-diluents to identify ones producing the highest signal-to-noise response ratios. Lastly, we introduced use of the ratio of signal responses in biotherapeutic-coated and uncoated wells as a data transformation strategy to identify biological outliers. This alternative data normalization approach improved normality, reduced skewness, and facilitated application of a parametric screening cut point. We believe the optimized direct binding assay design employing SULFO-TAG labeled Protein-A/G represents a useful analytical design for detecting serum ADA to biotherapeutics that lack an immunoglobulin Fc domain.

The Impact of Assay Design on Medicinal Chemistry: Case Studies

A diverse range of biochemical and cellular assays are used by medicinal chemists to guide compound optimization. The data collected from these assays influence decisions taken on structure-activity relationship (SAR) campaigns. Therefore, it is paramount that medicinal chemists have a solid understanding of the strengths and limitations of each assay being used to characterize synthesized analogs. For the successful execution of a medicinal chemistry campaign, it is our contention that an early partnership among assay biologists, informaticians, and medicinal chemists must exist. Their combined skill sets are necessary to not only design and develop robust assays but also implement an effective screening cascade in which multiple orthogonal and counter assays are selected to validate the activity and target(s) of the synthesized compounds. We review multiple cases of drug and chemical probe discovery from collaborative National Center for Advancing Translational Sciences/National Institutes of Health projects and published scientific literature in which the evaluation of compounds in secondary or orthogonal assays led to the discovery of unexpected activities, forcing a reconsideration of the original assay design that was used to discover the biological activity of the compound. Using these retrospective case studies, the goal of this Perspective is to hedge toward the development of physiologically relevant assays that are able to capture the true bioactivity of compounds being developed in a medicinal chemistry campaign. Graphical Abstract