Search

Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in industrial and research fields, such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many of these require the use of high-throughput platforms. Although the microtiter plate remains the standard for high-throughput enzymatic bioassays, microfluidic arrays and droplet microfluidics represent emerging methods. Each has seen significant advances and offers distinct advantages; however, drawbacks in key performance metrics, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity, remain. Herein, we compare recent high-throughput platforms using the aforementioned metrics as criteria and provide insights into remaining challenges and future research trends. Advances in microtiter plates, microfluidics arrays, and droplet microfluidics show unique advantages for high-throughput enzymatic bioassays regarding key performance metrics, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity. Advances in microtiter plates focus on their integration with accessory systems to improve their overall performance, such as sensitivity and reaction rates. Advances in microfluidic arrays aim to reduce reagent consumption, increase assay throughput, and enable multi-step bioassays and reaction recovery. Advances in droplet microfluidics enable binary detection with integrated sorting, kinetic screening under continuous flow, real-time measurement via in situ droplet trapping, and multiplexing and combinatorial screening. [ABSTRACT FROM AUTHOR]

Bacterial infections, especially polymicrobial infections, remain a threat to global health and require advances in diagnostic technologies for timely and accurate identification of all causative species. Digital melt – microfluidic chip-based digital PCR combined with high resolution melt (HRM) – is an emerging method for identification and quantification of polymicrobial bacterial infections. Despite advances in recent years, existing digital melt instrumentation often delivers nonuniform temperatures across digital chips, resulting in nonuniform digital melt curves for individual bacterial species. This nonuniformity can lead to inaccurate species identification and reduce the capacity for differentiating bacterial species with similar digital melt curves. We introduce herein a new temperature calibration method for digital melt by incorporating an unamplified, synthetic DNA fragment with a known melting temperature as a calibrator. When added at a tuned concentration to an established digital melt assay amplifying the commonly targeted 16S V1 – V6 region, this calibrator produced visible low temperature calibrator melt curves across-chip along with the target bacterial melt curves. This enables alignment of the bacterial melt curves and correction of heating-induced nonuniformities. Using this calibration method, we were able to improve the uniformity of digital melt curves from three causative species of bacteria. Additionally, we assessed calibration's effects on identification accuracy by performing machine learning identification of three polymicrobial mixtures comprised of two bacteria with similar digital melt curves in different ratios. Calibration greatly improved mixture composition prediction. To the best of our knowledge, this work represents the first DNA calibrator-supplemented assay and calibration method for nanoarray digital melt. Our results suggest that this calibration method can be flexibly used to improve identification accuracy and reduce melt curve variabilities across a variety of pathogens and assays. Therefore, this calibration method has the potential to elevate the diagnostic capabilities of digital melt toward polymicrobial bacterial infections and other infectious diseases. [Display omitted] • Digital melt is an emerging method for identifying tough polymicrobial infections. • Cross-platform temperature variations reduce digital melt identification accuracy. • DNA temperature calibrator enables alignment and improves uniformity of melt curves. • Calibrated digital melt shows enhanced polymicrobial sample identification accuracy. [ABSTRACT FROM AUTHOR]

Abstract: Niobium-doped titania (TNO) films of various Nb content were deposited on glass and silicon substrates by reactive co-sputtering of Ti and Nb metal targets. Nb content in the TNO films was varied from 0 to ∼13at.% (atomic percent), corresponding to Ti1−x Nb x O2 with x =0–0.52, by modulating the Nb target power from 0 to 150W (Watts). The influence of ion bombardment on the TNO films was investigated by applying an RF substrate bias from 0 to 25W. The as-deposited TNO films were all amorphous and insulating, but after annealing at 600°C for 1h in hydrogen, they became crystalline and conductive. The annealed films crystallized into either pure anatase or mixed anatase and rutile structures. The as-deposited and the annealed films were transparent, with an average transmittance above 70%. Anatase TNO film (Ti1−0.39Nb0.39O2) with Nb 9.7at.% exhibited a dramatically reduced resistivity of 9.2×10−4 Ωcm, a carrier density of 6.6×1021 cm−3 and a carrier mobility around 1.0cm2 V−1 s−1. In contrast, the mixed-phase Ti1−0.39Nb0.39O2 showed a higher resistivity of 1.2×10−1 Ωcm. This work demonstrates that the anatase phase, oxygen vacancies, and Nb dopants are all important factors in achieving high conductivities in TNO films. [Copyright &y& Elsevier]

In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but their lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR – a single-step CRISPR-Cas-assisted assay that incoporates sample preparation with minimal manual operation via facile magnetic-based nucleic acid concentration and transport. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/μL SARS-CoV-2 RNA from a sample volume of 100 μL in < 30 min. When evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (C q values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (C q < 25) in 20 min. • POC-CRISPR is a point-of-care amenable CRISPR-based sensing assay • The 1-step assay couples magnetofluidic sample preparation and CRISPR-aided RT-RPA • The assay is automated in a low-cost plastic cartridge and a palm-sized device • POC-CRISPR is evaluated with unprocessed clinical swab eluates (n = 27) • POC-CRISPR detects SARS-CoV-2 positive samples with high viral loads in 20 min [ABSTRACT FROM AUTHOR]

The rise of highly transmissible SARS‐CoV‐2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS‐CoV‐2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS‐CoV‐2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment. [ABSTRACT FROM AUTHOR]