Facile Preconcentration of Cell-Free DNA in Human Plasma by Ion-Specific Poly Ionic Sorbents Featuring an Anion Exchange Mechanism.

The expanding horizon of diagnostic and therapeutic applications involving nucleic acids (NA) requires novel tools for purification, including minimal sample preparation. In this work, thin-film microextraction devices featuring five poly ionic sorbents were examined as anion exchange extraction phases for the rapid purification of NAs. Each sorbent is composed of a nonionic cross-linker and a methacrylate monomer containing a core tetra-alkyl ammonium moiety with an alkyl, anionic, or cationic residue. Extraction devices were produced through the application of the prepolymer sorbent mixture onto a functionalized nitinol metal support followed by photoinduced free-radical polymerization. The miniaturized extraction devices (10 mm × 3.5 mm) were directly immersed into aqueous samples to isolate NAs via electrostatic interactions with the polycation. The ammonium methacrylate (AMA) monomer containing a propyl trimethylammonium group (AMA-C3N(CH3)3) exhibited the highest affinity for DNA, with 80 ± 10% of DNA being isolated. Recovery of DNA from the sorbents required the introduction of ions in an aqueous solution to exchange the anionic biopolymer from the polycationic moiety. An investigation of three anion species revealed that the AMA-C3N(CH3)3 sorbent showed the highest recoveries, with the perchlorate anion producing a preconcentration factor of 4.36 ± 0.86 while requiring only 250 mM NaClO4. A directly compatible quantitative polymerase chain reaction assay was developed to quantify the recovery of spiked DNA with lengths of 830, 204, and 98 base pairs in heat-treated human plasma. The AMA-C3N(CH3)3 sorbent was uninhibited by the complex human plasma matrix and enabled high preconcentration factors for the spiked DNA at a biologically relevant concentration of 10 pg/mL. While Qiagen's circulating cell-free DNA MinElute extraction kit enabled higher preconcentration of all analytes, the methodology described in this work requires fewer steps, less user intervention, and minimal equipment requirements to isolate DNA, making it more amenable for high-throughput and low resource applications.

Batch Scale Production of 3D Printed Extraction Sorbents Using a Low-Cost Modification to a Desktop Printer.

This study reports a simple modification to a commercial resin 3D printer that significantly reduces the amount of prepolymer material needed for the production of extraction sorbents. The modified printing platform is demonstrated in the printing of two imidazolium-based ionic liquid (IL) monomers. Two geometries resembling a blade-type polymeric ionic liquid (PIL) sorbent used in thin-film microextraction and a fiber-type sorbent used in solid-phase microextraction (SPME) were printed. The SPME PIL sorbents were used to extract 10 organic contaminants, including plasticizers, antimicrobial agents, UV filters, and pesticides, from water followed by high-performance liquid chromatographic (HPLC) analysis. To compare the extraction performance of the SPME sorbents, seven fibers printed with the same prepolymer composition from the same printing batch as well as different batches were evaluated. The results revealed highly reproducible extraction efficiencies for all tested sorbents with no statistical difference in their extraction performance. Method validation showed acceptable linearity (R2 > 0.92) for all analytes with limits of detection and limits of quantification ranging from 0.13 to 45 µg L-1 and 0.43 to 150 µg L-1, respectively.

Comparing π-complexation capabilities of ionic liquids containing silver(I) and copper(I) ions by headspace single drop microextraction in combination with high-performance liquid chromatography.

Selective π-complexation capabilities of silver(I) and copper(I) ions can be effectively facilitated in ionic liquids. To understand the effects of environmental factors that influence the π-complexation of these metal ions with analytes, techniques that employ small volumes of ionic liquid that can be readily analyzed are desired. In this study, headspace single drop microextraction coupled with HPLC is used to investigate a diverse set of environmental factors on the metal ion-mediated complexation with aromatic compounds in ionic liquid media. Silver(I) and copper(I) bis[(trifluoromethyl)sulfonyl]imide salts were both studied by dissolving them in the 1-decyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ionic liquid and employing the mixture as extraction media for aromatic compounds. Water and acetonitrile within the sample solution were observed to interfere with the complexation of silver(I) ions and aromatic compounds, while ethylene glycol and triethylene glycol did not. The temperature and extraction times were optimized to fully facilitate the π-complexation capabilities of metal ions in ionic liquid media. Partition coefficients between the sample headspace and metal ion were determined using a three-phase equilibria model. Although no discernable difference in analyte partitioning between the headspace and ionic liquid solvent was observed, analyte partition coefficients to silver(I) ion tended to be greater compared to copper(I) ion.

Characterization and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Thin Film Microextraction Enables Rapid Isolation and Recovery of DNA for Downstream Amplification Assays.

Nucleic acid analysis has been at the forefront of the COVID-19 global health crisis where millions of diagnostic tests have been used to determine disease status as well as sequencing techniques that monitor the evolving genome of SARS-CoV-2. In this study, we report the development of a sample preparation method that decreases the time required for DNA isolation while significantly increasing the sensitivity of downstream analysis. Functionalized planar supports are modified with a polymeric ionic liquid sorbent coating to form thin film microextraction (TFME) devices. The extraction devices are shown to have a high affinity for DNA while also exhibiting high reproducibility and reusability. Using quantitative polymerase chain reaction (qPCR) analysis, the TFME devices are shown to require low equilibration times while achieving higher preconcentration factors than solid-phase microextraction (SPME) by extracting larger masses of DNA. Rapid desorption kinetics enable higher DNA recoveries using desorption solutions that are less inhibitory to qPCR and loop-mediated isothermal amplification (LAMP). To demonstrate the advantageous features of the TFME platform, a customized leuco crystal violet LAMP assay is used for visual detection of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial oral fluid samples. When coupled to the TFME platform, 100% of LAMP reactions were positive for SARS-CoV-2 compared to 66.7% obtained by SPME for a clinically relevant concentration of 4.80 × 106 DNA copies/mL.

Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection.

Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.

Selective extraction of low-abundance BRAF V600E mutation from plasma, urine, and sputum using ion-tagged oligonucleotides and magnetic ionic liquids.

Sequence-specific DNA extractions have the potential to improve the detection of low-abundance mutations from complex matrices, making them ideal for circulating tumor DNA analysis during the early stages of cancer. Ion-tagged oligonucleotides (ITOs) are oligonucleotides modified with an allylimidazolium salt via thiolene click chemistry. The allylimidazolium-based tag allows the ITO-DNA duplex to be selectively captured by a hydrophobic magnetic ionic liquid (MIL). In this study, the selectivity of the ITO-MIL method was examined by extracting low abundance of the BRAF V600E mutation-a common single-nucleotide polymorphism associated with several different cancers-from diluted human plasma, artificial urine, and diluted artificial sputum. Quantitative polymerase chain reaction (qPCR) was not able to distinguish a 9% BRAF V600E standard (50 fg·µL-1 BRAF V600E, 500 fg·µL-1 wild-type BRAF) from the 100% wild-type BRAF (50 fg·µL-1) standard. However, introducing the ITO-MIL extraction prior to qPCR allowed for samples consisting of 0.1% BRAF V600E (50 fg·µL-1 V600E BRAF, 50,000 fg·µL-1 wild-type BRAF) to be distinguished from the 100% wild-type BRAF standard. Ion-tagged oligonucleotides (ITOs) are combined with magnetic ionic liquids (MILs) to extract low-abundance BRAF V600E mutation from diluted human plasma, artificial urine, and diluted artificial sputum. The ITO-MIL extraction prior to qPCR allowed for samples consisting of 0.1% BRAF V600E to be distinguished from the 100% wild-type BRAF standard.

Exploring physical activity trends and lesson context of incarcerated youth in a sport-leadership program.

Incarcerated youth are one subset of the population in dire need of physical activity interventions. As inactivity within incarcerated populations has become a mounting public health concern, several sport-based physical activity and fitness programs within prisons have emerged. The purpose of this study is to explore physical activity levels and lesson context of a sport-leadership program in one juvenile detention center. Participants in this study were 27 incarcerated males (Mage = 19.3), imprisoned in one juvenile detention center. Participants participated in 20, 40-min sport-leadership lessons over the course of 3 months. Each lesson was live coded using the System for Observing Instructional Time (SOFIT) to examine physical activity levels and lesson context. Over the course of 20 lessons, participants engaged in moderate to vigorous physical activity (MVPA) for 61% of lesson time (24.5 min). Further, over 23% of lesson time was spent vigorously (9.5 min). Specific to lesson context, 18% of total lesson time (7.5 min) was spent in management whereas 7.96% (3.1 min) was consumed by knowledge acquisition. Furthermore, a vast majority of each session (70%) was spent in gameplay (28.04 min). This study is the first to adopt the SOFIT systematic observation instrument to evaluate and quantify MVPA and lesson context during a sport-leadership program within a juvenile detention center. Results from this study indicate incarcerated youth who participated in this program engaged in MVPA for more than 60% of lesson time. It was reported that overall MVPA of participants within programming slightly decreased across the 20 total lessons.

Using a Chromatographic Pseudophase Model To Elucidate the Mechanism of Olefin Separation by Silver(I) Ions in Ionic Liquids.

Silver(I) ions undergo selective olefin complexation and have been utilized in various olefin/paraffin separation techniques such as argentation chromatography and facilitated transport membranes. Ionic liquids (ILs) are solvents known for their low vapor pressure, high thermal stability, low melting points, and ability to promote a favorable solvation environment for silver(I) ion-olefin interactions. To develop highly selective separation systems, a fundamental understanding of analyte partitioning to the stationary phase and the thermodynamic driving forces behind solvation is required. In this study, a chromatographic model treating silver(I) ions as a pseudophase is constructed and employed for the first time to investigate the olefin separation mechanism in silver(I) salt/IL mixtures. Stationary phases containing varying amounts of noncoordinated silver(I) salt ([Ag+][NTf2-]) dissolved in the 1-decyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([C10MIM+][NTf2-]) IL are utilized to determine the partition coefficients of various analytes including alkanes, alkenes, alkynes, aromatics, aldehyde, esters, and ketones. As ligand coordination to silver(I) ions is known to lower its olefin complexation capability, this study also examines two different types of coordinated silver(I) ion pseudophases, namely, monocoordinated silver(I) salt ([Ag+(1-decyl-2-methylimidazole, DMIM)][NTf2-]) and dicoordinated silver(I) salt ([Ag+(1-methylimidazole, MIM)(DMIM)][NTf2-]). The extent of olefin partitioning to the coordinated silver(I) ion pseudophases over the carrier gas and IL decreased by up to two orders of magnitude. Values for enthalpy, entropy, and free energy of solvation were determined for the three silver(I) ion-containing systems. Olefin retention was observed to be enthalpically dominated, while ligand coordination to the silver(I) ion pseudophase resulted in variations for both enthalpic and entropic contributions to the free energy of solvation. The developed model can be used to study chemical changes that occur in silver(I) ions over time as well as identify optimal silver(I) salt/IL mixtures that yield high olefin selectivity.

Sequence-Specific Detection of ORF1a, BRAF, and ompW DNA Sequences with Loop Mediated Isothermal Amplification on Lateral Flow Immunoassay Strips Enabled by Molecular Beacons.

Loop-mediated isothermal amplification (LAMP) holds great potential for point-of-care (POC) diagnostics due to its speed and sensitivity. However, differentiation between spurious amplification and amplification of the target sequence is a challenge. Herein, we develop the use of molecular beacon (MB) probes for the sequence-specific detection of LAMP on commercially available lateral flow immunoassay (LFIA) strips. The detection of three unique DNA sequences, including ORF1a from SARS-CoV-2, is demonstrated. In addition, the method is capable of detecting clinically relevant single-nucleotide polymorphisms (BRAF V600E). For all sequences tested, the LFIA method offers similar sensitivity to fluorescence detection using a qPCR instrument. We also demonstrate the coupling of the method with solid-phase microextraction to enable isolation and detection of the target sequences from human plasma, pond water, and artificial saliva. Lastly, a 3D printed device is designed and implemented to prevent contamination caused by opening the reaction containers after LAMP.

Polymeric metal-containing ionic liquid sorbent coating for the determination of amines using headspace solid-phase microextraction.

This study describes the design, synthesis, and application of polymeric ionic liquid sorbent coatings featuring nickel metal centers for the determination of volatile and semivolatile amines from water samples using headspace solid-phase microextraction. The examined polymeric ionic liquid (PIL) sorbent coatings were composed of two ionic liquid monomers (tetra(3-vinylimidazolium)nickel bis[(trifluoromethyl)sulfonyl]imide [Ni2+ (VIM)4 ] 2[NTf2 - ] and 1-vinyl-3-hexylimidazolium [HVIM+ ][NTf2 - ]), and an ionic liquid cross-linker (1,12-di(3-vinylimidazolium)dodecane  [(VIM)2 C12 2+ ] 2[NTf2 - ]). With these ionic liquid monomers and cross-linkers, three different types of coatings were prepared: PIL 1 based on the neat [Ni2+ (VIM)4 ] 2[NTf2 - ] monomer, PIL 2 consisting of the [Ni2+ (VIM)4 ] 2[NTf2 - ] monomer with addition of cross-linker, and PIL 3 composed of the [HVIM+ ][NTf2 - ] monomer and cross-linker. Analytical performance of the prepared sorbent coatings using headspace solid-phase microextraction gas chromatography-mass spectrometry was compared with the polydimethylsiloxane and polyacrylate commercial coatings. The PIL 2 sorbent coating yielded the highest enrichment factors ranging from 5500 to over 160 000 for the target analytes. The developed headspace solid-phase microextraction gas chromatography-mass spectrometry method was applied for the analysis of real samples (the concentration of amines was 200 µg/L), producing relative recovery values in the range of 90.9-120.0% (PIL 1) and 83.0-122.7% (PIL 2) from tap water, and 84.8-112.4% (PIL 1) and 79.2-119.3% (PIL 2) from lake water.

Solid-Phase Microextraction Enables Isolation of BRAF V600E Circulating Tumor DNA from Human Plasma for Detection with a Molecular Beacon Loop-Mediated Isothermal Amplification Assay.

Circulating tumor DNA (ctDNA) is a promising biomarker that can provide a wealth of information regarding the genetic makeup of cancer as well as provide a guide for monitoring treatment. Methods for rapid and accurate profiling of ctDNA are highly desirable in order to obtain the necessary information from this biomarker. However, isolation of ctDNA and its subsequent analysis remains a challenge due to the dependence on expensive and specialized equipment. In order to enable widespread implementation of ctDNA analysis, there is a need for low-cost and highly accurate methods that can be performed by nonexpert users. In this study, an assay is developed that exploits the high specificity of molecular beacon (MB) probes with the speed and simplicity of loop-mediated isothermal amplification (LAMP) for the detection of the BRAF V600E single-nucleotide polymorphism (SNP). Furthermore, solid-phase microextraction (SPME) is applied for the successful isolation of clinically relevant concentrations (73.26 fM) of ctDNA from human plasma. In addition, the individual effects of plasma salts and protein on the extraction of ctDNA with SPME are explored. The performed work expands the use of MB-LAMP for SNP detection as well as demonstrates SPME as a sample preparation tool for nucleic acid analysis in plasma.

The Effect of a Rapid Assessment Zone on Emergency Department Operations and Throughput.

STUDY OBJECTIVE: We examine the effects of a front-end flow model designated the rapid assessment zone on multiple emergency department (ED) operational metrics. METHODS: This was a retrospective, before-after study of consecutive patient visits at an urban community ED. Six-month periods were compared before and after an intervention in 2017 that changed patient flow and the intake process. A lead nurse role splits patient flow immediately on patient arrival according to only age and chief complaint, allowing direct bedding without the bottlenecks of vital sign measurement, full triage assessment, or Emergency Severity Index assignment. A new patient care area (designated rapid assessment zone) preferentially expedites treatment of patients likely to remain ambulatory and serves as flexible acute care space when needed by individual cases and the ED. The outcomes measured were ED length of stay, arrival-to-provider time, the rate of leaving before treatment completion, and the rate of leaving before being seen. Data were analyzed with nonparametric testing, χ2 analysis, and multiple linear regression, controlling for patient visit characteristics, ED daily census volumes, and measurements of boarding patients. RESULTS: We analyzed 43,847 visits in the preintervention and 44,792 visits in the postintervention periods. The intervention was associated with the following changes: median ED length of stay from 203 to 171 minutes (-15.8%), median arrival-to-provider time from 28 to 13 minutes (-53.6%), leaving before treatment completion from 1.0% to 0.8% (-20%), and leaving before being seen from 3.1% to 0.5% (-84%). Regression analysis accounting for multiple confounders demonstrated that the reduced length of stay after rapid assessment zone implementation persisted across Emergency Severity Index levels 2 to 5 and all ED daily census levels. CONCLUSION: The rapid assessment zone model aims to decrease front-end bottlenecks and minimize serial intake assessments at a high-volume, urban ED. It was associated with improved patient throughput and decreased early patient departure. It may represent a useful model for similar centers.

Simultaneous cell lysis and DNA extraction from whole blood using magnetic ionic liquids.

Conventional DNA sample preparation methods involve tedious sample handling steps that require numerous inhibitors of the polymerase chain reaction (PCR) and instrumentation to implement. These disadvantages limit the applicability of conventional cell lysis and DNA extraction methods in high-throughput applications, particularly in forensics and clinical laboratories. To overcome these drawbacks, a series of nine hydrophobic magnetic ionic liquids (MILs) previously shown to preconcentrate DNA were explored as cell lysis reagents. The MILs were found to lyse white blood cells from whole blood, 2-fold diluted blood, and dry blood samples while simultaneously extracting human genomic DNA. The identity of metal ion incorporated within the MIL appears to cause hemolysis while the cationic component further reduces the cell's integrity. Over 500 pg of human genomic DNA was isolated from 50 µL of whole blood using the trioctylbenzylammonium tris(hexafluoroacetylaceto)nickelate(II) ([N8,8,8,Bz+][Ni(hfacac)3-]) MIL, and 800 pg DNA was isolated from a dry blood samples using the trihexyl(tetradecyl)phosphonium tris(phenyltrifluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(Phfacac)3-]) MIL following a 1-min vortex step. A rapid, one-step cell lysis and DNA extraction from blood is ideal for settings that seek high-throughput analysis while minimizing the potential for contamination.Graphical abstract.

Magnetic ionic liquids: interactions with bacterial cells, behavior in aqueous suspension, and broader applications.

Previously, we demonstrated capture and concentration of Salmonella enterica subspecies enterica ser. Typhimurium using magnetic ionic liquids (MILs), followed by rapid isothermal detection of captured cells via recombinase polymerase amplification (RPA). Here, we report work intended to explore the broader potential of MILs as novel pre-analytical capture reagents in food safety and related applications. Specifically, we evaluated the capacity of the ([P66614+][Ni(hfacac)3-]) ("Ni(II)") MIL to bind a wider range of human pathogens using a panel of Salmonella and Escherichia coli O157:H7 isolates, including a "deep rough" strain of S. Minnesota. We extended this exploration further to include other members of the family Enterobacteriaceae of food safety and clinical or agricultural significance. Both the Ni(II) MIL and the ([P66614+][Dy(hfacac)4-]) ("Dy(III)") MIL were evaluated for their effects on cell viability and structure-function relationships behind observed antimicrobial activities of the Dy(III) MIL were determined. Next, we used flow imaging microscopy (FIM) of Ni(II) MIL dispersions made in model liquid media to examine the impact of increasing ionic complexity on MIL droplet properties as a first step towards understanding the impact of suspension medium properties on MIL dispersion behavior. Finally, we used FIM to examine interactions between the Ni(II) MIL and Serratia marcescens, providing insights into how the MIL may act to capture and concentrate Gram-negative bacteria in aqueous samples, including food suspensions. Together, our results provide further characterization of bacteria-MIL interactions and support the broader utility of the Ni(II) MIL as a cell-friendly capture reagent for sample preparation prior to cultural or molecular analyses. Graphical abstract.

Fluorescence quenching of the SYBR Green I-dsDNA complex by in situ generated magnetic ionic liquids.

Magnetic ionic liquids (MILs) with metal-containing cations are promising extraction solvents that provide fast and high efficiency extraction of DNA. Hydrophobic MILs can be generated in situ in a methodology called in situ dispersive liquid-liquid microextraction. To consolidate the sample preparation workflow, it is desirable to directly use the DNA-enriched MIL microdroplet in the subsequent analytical detection technique. Fluorescence-based techniques employed for DNA detection often utilize SYBR Green I, a DNA binding dye that exhibits optimal fluorescence when bound to double-stranded DNA. However, the MIL may hinder the fluorescence signal of the SYBR Green I-dsDNA complex due to quenching. In this study, MILs with metal-containing cations were selected and their fluorescence quenching effects evaluated using FÓ§rster Resonance Energy Transfer and quantified using Stern-Volmer models. The MILs were based on N-substituted imidazole ligands (with butyl- and benzyl- groups as substituents) coordinated to Ni2+ or Co2+ metal centers as cations, and paired with chloride anions. The effects of NiCl2 and CoCl2 salts and of the 1-butyl-3-methylimidazolium chloride ionic liquid on the fluorophore complex were also studied to understand the components of the MIL structure that are responsible for quenching. The metal within the MIL chemical structure was found to be the main component contributing to fluorescence quenching. FÓ§rster critical distances between 11.9 and 18.8 Å were obtained for the MILs, indicating that quenching is likely not due to non-radiative energy transfer but rather to spin-orbit coupling or excited-state electron transfer. The MILs were able to be directly used in qPCR and fluorescence emission measurements using a microplate reader for detection, demonstrating their applicability in fluorescence-based detection methods. Graphical abstract.

Are We Acute-Care or Recovery-Oriented? Exploring Ideals and Practices Expressed Within the Substance Use Treatment and Correctional Systems.

AIM: The aim of this study was to explore whether treatment and probation professionals describe ideals and practices more aligned with the recovery-oriented systems of care (ROSC) model or the acute-care model. Methods: Semi-structured individual interviews were used to gather qualitative data on the ideals and practices of nine probation professionals and nine treatment professionals. Results: Directed content analysis revealed that all treatment professionals interviewed and eight out of nine probation professionals described more ideals and practices in line with the ROSC model than those in line with the acute-care model. Of all the meaning units coded for model alignment, 81.7% aligned with ROSC and 18.3% with acute care. Of the meaning units coded as ROSC, 51.4% were from treatment professionals and 48.6% from probation professionals. Of the meaning units coded as acute care, 30.2% came from treatment professionals and 69.8% from probation professionals. In building a ROSC, it seems the concern is less about buy in for recovery-oriented characteristics and more about shedding characteristics of the acute-care model. Although professionals have many ideals and practices in line with the ROSC model, some acute-care characteristics linger and could continue to exist without intervention.

Visual Detection of Single-Nucleotide Polymorphisms Using Molecular Beacon Loop-Mediated Isothermal Amplification with Centrifuge-Free DNA Extraction.

Loop-mediated isothermal amplification (LAMP) is a powerful nucleic acid amplification technique due to its rapid and sensitive nature. These characteristics, in addition to low-cost and robustness, make LAMP an attractive alternative to polymerase chain reaction for point-of-care applications. However, sequence-specific detection remains a formidable challenge, particularly when single-nucleotide resolution is required. In this study, a LAMP method is developed for facile visual detection of single-nucleotide polymorphisms (SNPs) using molecular beacons (MBs) by exploiting the dual-fluorescence of fluorescein (6-FAM) and hydoxynaphthol blue (HNB). The method is coupled with solid-phase microextraction (SPME) to facilitate rapid extraction and detection of the target sequence. This work expands the use of MBs in LAMP for the visual detection of SNPs and facilitates the development of future LAMP assays for a wide-range of targets.

Tunable Silver-Containing Stationary Phases for Multidimensional Gas Chromatography.

To achieve high separation power of complex samples using multidimensional gas chromatography (MDGC), the selectivity of the employed stationary phases is crucial. The nonpolar × polar column combination remains the most popular column set used in MDGC. However, resolution of mixtures containing light analytes possessing very similar properties remains a formidable challenge. The development of stationary phases that offer unique separation mechanisms have the potential to significantly improve MDGC separations, particularly in resolving coeluting peaks in complex samples. For the first time, a stationary phase containing silver(I) ions was successfully designed and employed as a second-dimension column using comprehensive two-dimensional gas chromatography (GC × GC) for the separation of mixtures containing alkynes, dienes, terpenes, esters, aldehydes, and ketones. Compared with a widely used nonpolar and polar column set, the silver-based column exhibited superior performance by providing better chromatographic resolution of coeluting compounds. A mixture of unsaturated fatty acids was successfully separated using a GC × GC method in which the elution order in the second dimension was highly dependent on the number of double bonds within the analytes.

Maximizing Ion-Tagged Oligonucleotide Loading on Magnetic Ionic Liquid Supports for the Sequence-Specific Extraction of Nucleic Acids.

Targeted nucleic acid analysis requires the highly selective extraction of desired DNA fragments in order to minimize interferences from samples with abundant heterogeneous sequences. We previously reported a method based on functionalized oligonucleotide probes known as ion-tagged oligonucleotides (ITOs) that hybridize with complementary DNA targets for subsequent capture using a hydrophobic magnetic ionic liquid (MIL) support. Although the ITO-MIL approach enriched specific DNA sequences in quantities comparable to a commercial magnetic bead-based method, the modest affinity of the ITO for the hydrophobic MIL limited the yield of DNA targets, particularly when stringent wash conditions were applied to remove untargeted DNA. Here, we report the synthesis and characterization of a series of ITOs in which functional groups were installed within the cation and anion components of the tag moiety in order to facilitate loading of the ITO to the MIL support phase. In addition to hydrophobic interactions, we demonstrate that π-π stacking and fluorophilic interactions can be exploited for loading oligonucleotide probes onto MILs. Using a disubstituted ion-tagged oligonucleotide (DTO) possessing two linear C8 groups, nearly quantitative loading of the probe onto the MIL support was achieved. The enhanced stability of the DTO within the MIL solvent permitted successive wash steps without the loss of the DNA target compared to a monosubstituted ITO with a single C8 group that was susceptible to increased loss of analyte. Furthermore, the successful capture of a 120 bp KRAS fragment from human plasma samples followed by real-time quantitative polymerase chain reaction (qPCR) amplification is demonstrated.