High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles.

We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2-3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103 particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(-). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

Microfluidic affinity selection of active SARS-CoV-2 virus particles.

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus's S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip's surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.

Analytical Technologies for Liquid Biopsy of Subcellular Materials.

Liquid biopsy markers, which can be secured from a simple blood draw or other biological samples, are used to manage a variety of diseases and even monitor for bacterial or viral infections. Although there are several different types of liquid biopsy markers, the subcellular ones, including cell-free DNA, microRNA, extracellular vesicles, and viral particles, are evolving in terms of their utility. A challenge with liquid biopsy markers is that they must be enriched from the biological sample prior to analysis because they are a vast minority in a mixed population, and potential interferences may be present in the sample matrix that can inhibit profiling the molecular cargo from the subcellular marker. In this article, we discuss existing and developing analytical enrichment platforms used to isolate subcellular liquid biopsy markers, and discuss their figures of merit such as recovery, throughput, and purity.

Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules.

We report a simple method for tailoring the size of in-plane nanopores fabricated in thermoplastics for single-molecule sensing. The in-plane pores were fabricated via nanoimprint lithography (NIL) from resin stamps, which were generated from Si masters. We could reduce the size of the in-plane nanopores from 30 to ∼10 nm during the thermal fusion bonding (TFB) step, which places a cover plate over the imprinted polymer substrate under a controlled pressure and temperature to form the relevant nanofluidic devices. Increased pressures during TFB caused the cross-sectional area of the in-plane pore to be reduced. The in-plane nanopores prepared with different TFB pressures were utilized to detect single-λ-DNA molecules via resistive pulse sensing, which showed a higher current amplitude in devices bonded at higher pressures. Using this method, we also show the ability to tune the pore size to detect single-stranded (ss) RNA molecules and single ribonucleotide adenosine monophosphate (rAMP). However, due to the small size of the pores required for detection of the ssRNA and rAMPs, the surface charge arising from carboxylate groups generated during O2 plasma oxidation of the surfaces of the nanopores to make them wettable had to be reduced to allow translocation of coions. This was accomplished using EDC/NHS coupling chemistry and ethanolamine. This simple modification chemistry increased the event frequency from ∼1 s-1 to >136 s-1 for an ssRNA concentration of 100 nM.

Mercury levels in hair samples of dentists: A comparative study in Sri Lanka.

AIM: Elemental mercury is widely used in dentistry as dental amalgam. Hair samples constitute preferred biomarkers to determine the index of mercury exposure. In the absence of any published studies, the aim of the present study was to ascertain the level of mercury in a selected sample of dentists and controls in Sri Lanka. METHODS: Hair samples (.7 g) from dentists working in and around the Colombo district in Sri Lanka (n = 50) and controls (n = 50) were digested with sulfuric acid-potassium permanganate solution, and reduced to metallic mercury with stannous chloride. Digested samples were analyzed for mercury using an atomic absorption spectrophotometer. RESULTS: The average mercury contents of the test and control samples (n = 50 each) were 5.36 ± 2.64 ppb and 3.1 ± 1.99 ppb, respectively. Based on the estimated ratio of mercury in hair to blood in humans as 250:1, the average content of mercury in the blood of the two groups could be .02 ppb and .01 ppb, respectively. The number of years in dentistry and number of amalgam restorations performed within 1 week were not correlated with the hair concentration of mercury. CONCLUSIONS: As the values of both groups were well below 10 ng/mL (10 ppb), within the concentrations considered as normal, mercury use was not considered an occupational hazard for dentists.

Microfluidic-based solid phase extraction of cell free DNA.

Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 → 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (µSPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the µSPE of cfDNA. The µSPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The µSPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire µSPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The µSPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100-700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the µSPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. µSPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.