Evaluation of supercritical CO2 sterilization efficacy for sanitizing personal protective equipment from the coronavirus SARS-CoV-2.

The purpose of this research is to evaluate the supercritical carbon dioxide (scCO2) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a "worst-case" regarding challenged PPE. The inactivation of the microbes by scCO2 sterilization with NovaKill or H2O2 sterilant was investigated as a function of exposure times ranging from 5 to 90 min with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO2-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO2 sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.

Development of an integrated fingerstick blood self-collection device for radiation countermeasures.

We report the development of system for packaging critical components of the traditional collection kit to make an integrated fingerstick blood collector for self-collecting blood samples of 100 µl or more for radiation countermeasures. A miniaturized vacuum tube system (VacuStor system) has been developed to facilitate liquid reagent storage, simple operation and reduced sample contamination. Vacuum shelf life of the VacuStor tube has been analyzed by the ideal gas law and gas permeation theory, and multiple ways to extend vacuum shelf life beyond one year have been demonstrated, including low temperature storage, Parylene barrier coating and container vacuum bag sealing. Self-collection was also demonstrated by healthy donors without any previous fingerstick collection experience. The collected blood samples showed similar behavior in terms of gene expression and cytogenetic biodosimetry assays comparing to the traditionally collected samples. The integrated collector may alleviate the sample collection bottleneck for radiation countermeasures following a large-scale nuclear event, and may be useful in other applications with its self-collection and liquid reagent sample preprocessing capabilities.

Analysis of Saliva Gene Expression during Head and Neck Cancer Radiotherapy: A Pilot Study.

Saliva, a biological fluid, is a promising candidate for novel approaches to prognosis, clinical diagnosis, monitoring and management of patients with both oral and systemic diseases. However, to date, saliva has not been widely investigated as a biomarker for radiation exposure. Since white blood cells are also present in saliva, it should theoretically be possible to investigate the transcriptional biomarkers of radiation exposure classically studied in whole blood. Therefore, we collected whole blood and saliva samples from eight head and neck cancer patients before the start of radiation treatment, at mid-treatment and after treatment. We then used a panel of five genes: BAX, BBC3, CDKN1A, DDB2 and MDM2, designated for assessing radiation dose in whole blood to evaluate gene expression changes that can occur during radiotherapy. The results revealed that the expression of the five genes did not change in whole blood. However, in saliva, CDKN1A and DDB2 were significantly overexpressed at the end, compared to the start, of radiotherapy, and MDM2 was significantly underexpressed between mid-treatment and at the end of treatment. Interestingly, CDKN1A and DDB2 expressions also showed an increasing monotonic relationship with total radiation dose received during radiotherapy. To our knowledge, these results show for the first time the ability to detect gene expression changes in saliva after head and neck cancer radiotherapy, and pave the way for further promising studies validating saliva as a minimally invasive means of biofluid collection to directly measure radiation dose escalation during treatment.

A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system.

We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 µL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.

An integratable microfluidic cartridge for forensic swab samples lysis.

Fully automated rapid forensic DNA analysis requires integrating several multistep processes onto a single microfluidic platform, including substrate lysis, extraction of DNA from the released lysate solution, multiplexed PCR amplification of STR loci, separation of PCR products by capillary electrophoresis, and analysis for allelic peak calling. Over the past several years, most of the rapid DNA analysis systems developed started with the reference swab sample lysate and involved an off-chip lysis of collected substrates. As a result of advancement in technology and chemistry, addition of a microfluidic module for swab sample lysis has been achieved in a few of the rapid DNA analysis systems. However, recent reports on integrated rapid DNA analysis systems with swab-in and answer-out capability lack any quantitative and qualitative characterization of the swab-in sample lysis module, which is important for downstream forensic sample processing. Maximal collection and subsequent recovery of the biological material from the crime scene is one of the first and critical steps in forensic DNA technology. Herein we present the design, fabrication and characterization of an integratable swab lysis cartridge module and the test results obtained from different types of commonly used forensic swab samples, including buccal, saliva, and blood swab samples, demonstrating the compatibility with different downstream DNA extraction chemistries. This swab lysis cartridge module is easy to operate, compatible with both forensic and microfluidic requirements, and ready to be integrated with our existing automated rapid forensic DNA analysis system. Following the characterization of the swab lysis module, an integrated run from buccal swab sample-in to the microchip CE electropherogram-out was demonstrated on the integrated prototype instrument. Therefore, in this study, we demonstrate that this swab lysis cartridge module is: (1) functionally, comparable with routine benchtop lysis, (2) compatible with various types of swab samples and chemistries, and (3) integratable to achieve a micro total analysis system (µTAS) for rapid DNA analysis.

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile.

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.