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.

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.

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.

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.

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.

Solid-Phase Microextraction of DNA from Mycobacteria in Artificial Sputum Samples To Enable Visual Detection Using Isothermal Amplification.

Point-of-care (POC) technologies for the detection of pathogens in clinical samples are highly valued due to their speed, ease of use, and cost-effectiveness. Furthermore, they are ideally suited for resource-limited settings where expensive and sophisticated laboratory equipment may not be readily available. In this study, a rapid method based on solid-phase microextraction (SPME) of mycobacterial DNA with subsequent isothermal amplification and visual detection was developed. Direct coupling of the SPME desorption solution (1 M NaCl) to the isothermal reaction system was achieved to circumvent dilution steps and improve detection limits. Using this method, DNA was preconcentrated from lysed mycobacteria in just 2 min, subjected to isothermal multiple-self-matching-initiated amplification (IMSA), and the amplicons were detected visually. With a total analysis times of less than 2 h, the optimized method was capable of extracting and visually detecting mycobacterial DNA from artificial sputum samples containing clinically relevant concentrations of mycobacteria (107 colony forming units/mL), demonstrating its potential for future POC applications.

Ionic liquids: solvents and sorbents in sample preparation.

The applications of ionic liquids (ILs) and IL-derived sorbents are rapidly expanding. By careful selection of the cation and anion components, the physicochemical properties of ILs can be altered to meet the requirements of specific applications. Reports of IL solvents possessing high selectivity for specific analytes are numerous and continue to motivate the development of new IL-based sample preparation methods that are faster, more selective, and environmentally benign compared to conventional organic solvents. The advantages of ILs have also been exploited in solid/polymer formats in which ordinarily nonspecific sorbents are functionalized with IL moieties in order to impart selectivity for an analyte or analyte class. Furthermore, new ILs that incorporate a paramagnetic component into the IL structure, known as magnetic ionic liquids (MILs), have emerged as useful solvents for bioanalytical applications. In this rapidly changing field, this Review focuses on the applications of ILs and IL-based sorbents in sample preparation with a special emphasis on liquid phase extraction techniques using ILs and MILs, IL-based solid-phase extraction, ILs in mass spectrometry, and biological applications.

Selective and Efficient RNA Analysis by Solid-Phase Microextraction.

In this study, a solid-phase microextraction (SPME) method was developed for the purification of mRNA (mRNA) from complex biological samples using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for quantification. The chemical composition of the polymeric ionic liquid (PIL) and a polyacrylate (PA) SPME sorbent coating was optimized to enhance the extraction performance. Of the studied SPME sorbent coatings, the PIL containing carboxylic acid moieties in the monomer and halide-based anions extracted the highest amount of mRNA from aqueous solutions, whereas the native PA fiber showed the lowest extraction efficiency. On the basis of RT-qPCR data, electrostatic interactions and an ion-exchange mechanism between the negatively charged phosphate backbone of RNA and the PIL cation framework were the major driving forces for mRNA extraction. The optimized PIL-based SPME method purified a high quantity of mRNA from crude yeast cell lysate compared to a phenol/chloroform extraction method. The reusability and robustness of PIL-based SPME for RNA analysis represents a significant advantage over conventional silica-based solid-phase RNA extraction kits. The selectivity of the SPME method toward mRNA was enhanced by functionalizing the PA sorbent with oligo dT20 using carbodiimide-based amide linker chemistry. The oligo dT20-modified PA sorbent coating demonstrated superior extraction performance than the native PA sorbent coating with quantification cycle (Cq) values 33.74 ± 0.24 and 39, respectively. The modified PA sorbent extracted sufficient mRNA from total RNA at concentrations as low as 5 ng µL-1 in aqueous solutions without the use of organic solvents and time-consuming multiple centrifugation steps that are required in traditional RNA extraction methods.

Ion-Tagged Oligonucleotides Coupled with a Magnetic Liquid Support for the Sequence-Specific Capture of DNA.

The isolation of specific nucleic acid sequences is a major bottleneck in molecular diagnostics. Magnetic beads/particles are typically used as solid supports for the capture of DNA targets to improve sample throughput but aggregate over time resulting in lower capture efficiency and obstruction of liquid handling devices. Herein, we describe a particle-free approach to sequence-specific DNA extraction using a magnetic liquid support and ion-tagged oligonucleotide (ITO) probes. ITO conjugates were synthesized with the highest yields ever achieved for the radical thiol-ene coupling of a substrate and oligonucleotide. In addition to distinguishing nucleotide mismatches, the ITO and magnetic liquid-based approach was more sensitive than a commercial magnetic bead-based method for the capture of target DNA from a pool of interfering genomic DNA.

High Resolution Ion Mobility Enables the Structural Characterization of Atropisomers of GDC-6036, a KRAS G12C Covalent Inhibitor.

GDC-6036 is a covalent KRAS G12C inhibitor that demonstrates high potency and selectivity. Structurally, GDC-6036 consists of several motifs that make the analytical characterization of this molecule challenging, including a highly basic pyrrolidine motif bonded to a quinazoline ring via an ether bond and an atropisomeric carbon-carbon bond between functionalized pyridine and quinazoline groups. Structurally, the desired atropisomer was synthesized via an atroposelective Negishi coupling with very high yield. However, having a direct way to analyze and confirm the presence of the atropisomeric species remained challenging in routine analytical workflows. In this study, both variable temperature nuclear magnetic resonance (VT-NMR) and two different approaches of in-line ion mobility coupled to liquid chromatography mass spectrometry (LC-MS) workflows were evaluated for the characterization of GDC-6036 and its undesired atropisomer (Compound B) to support synthetic route development. Briefly, both VT-NMR and traveling wave ion mobility spectrometry (TWIMS) enabled by structures for lossless ion manipulation (SLIM) technology coupled to high resolution MS (HRMS) are able to elucidate the structures of the atropisomers in a complex mixture. Drift tube IMS (DTIMS) was also evaluated, but lacked the resolving power to demonstrate separation between the two species in a mixture, but did show slight differences in their arrival times when multiplexed and injected separately. The determined resolving power (Rp) by multiplexing the ions via DTIMS was 67.3 and 60.5 for GDC-6036 and Compound B, respectively, while the two peak resolving power (Rpp) was determined to be 0.41, indicating inadequate resolution between the two species. Alternatively, the SLIM-IM studies showed Rp of 103.8 and 99.4, with a Rpp of 2.64, indicating good separation between the atropisomers. Furthermore, the CCS/z for GDC-6036 and Compound B was determined to be 231.2 Å2/z and 235.0 Å2/z, respectively. Quantitative experiments demonstrate linearity (R2 >0.99) for both GDC-6036 and Compound B while maintaining separation via SLIM-IM. Spike recoveries of one atropisomer relative to the other yielded strong recoveries (98.7% to 102.5%) while maintaining reproducibility (<7% RSD). The study herein describes the analytical process for evaluating new technologies and strategies for implementation in routine biopharmaceutical characterization workflows.

Advances in Mutation Detection Using Loop-Mediated Isothermal Amplification.

Detection of mutations and single-nucleotide polymorphisms is highly important for diagnostic applications. Loop-mediated isothermal amplification (LAMP) is a powerful technique for the rapid and sensitive detection of nucleic acids. However, LAMP traditionally does not possess the ability to resolve single-nucleotide differences within the target sequence. Because of its speed and isothermal nature, LAMP is ideally suited for point-of-care applications in resource-limited settings. Recently, different approaches have been developed and applied to enable single-nucleotide differentiation within target sequences. This Mini-Review highlights advancements in mutation detection using LAMP. Methods involving primer design and modification to enable sequence differentiation are discussed. In addition, the development of probe-based detection methods for mutation detection are also covered.

Modification of polyacrylate sorbent coatings with carbodiimide crosslinker chemistry for sequence-selective DNA extraction using solid-phase microextraction.

Selective DNA extraction is immensely useful for the isolation and detection of low-abundance sequences. Oligonucleotide-modified substrates are often used to capture sequences of interest for downstream analysis. In this study, we explore the chemical modification of commercial-available polyacrylate solid-phase microextraction fibers for selective DNA analysis using carbodiimide crosslinker chemistry. Reproducible modification conditions are found and the fibers were subsequently applied for selective DNA analysis. Several experimental parameters such as stir-rate, desorption time, and buffer-type are optimized. The developed method was able to selectively extract the target DNA sequence (260 bp) in the presence of 100-fold excess interfering salmon testes DNA.

Magnetic Ionic Liquids as Solvents for RNA Extraction and Preservation.

Ribonucleic acid (RNA) is particularly sensitive to enzymatic degradation by endonucleases prior to sample analysis. In-field preservation has been a challenge for RNA sample preparation. Very recently, hydrophobic magnetic ionic liquids (MIL) have shown significant promise in the area of RNA extraction. In this study, MILs were synthesized and employed as solvents for the extraction and preservation of RNA in aqueous solution. RNA samples obtained from yeast cells were extracted and preserved by the trihexyl(tetradecyl) phosphonium tris(hexafluoroacetylaceto)cobaltate(II) ([P66614 +][Co(hfacac)3 -]) and trihexyl(tetradecyl) phosphonium tris(phenyltrifluoroacetylaceto)cobaltate(II) ([P66614 +][Co(Phtfacac)3 -]) MIL with a dispersion of the supporting media, polypropylene glycol, at room temperature for up to a 7 and 15 day period, respectively. High-quality RNA treated with ribonuclease A (RNase A) was recovered from the tetra(1-octylimidazole)cobaltate(II) di(l-glutamate) ([Co(OIM)4 2+][Glu-]2) and tetra(1-octylimidazole)cobaltate(II) di(l-aspartate) ([Co(OIM)4 2+][Asp-]2) MILs after a 24 h period at room temperature. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and agarose gel electrophoresis were used to determine the effect of RNA preservation. Furthermore, the preservation mechanism was investigated by exploring the partitioning of RNase A into the MIL using high-performance liquid chromatography.

Sequence-specific preconcentration of a mutation prone KRAS fragment from plasma using ion-tagged oligonucleotides coupled to qPCR compatible magnetic ionic liquid solvents.

Circulating tumor DNA (ctDNA) is a source of mutant DNA found in plasma and holds great promise in guiding cancer diagnostics, prognostics, and treatment. However, ctDNA fragments are challenging to detect in plasma due to their low abundance compared to wild-type DNA. In this study, a series of ion-tagged oligonucleotides (ITO) were synthesized using thiol-ene click chemistry and designed to selectively anneal target DNA. The ITO-DNA duplex was subsequently captured using a hydrophobic magnetic ionic liquid (MIL) as a liquid support. Extracted target DNA was quantified by adding the DNA-enriched MIL to the quantitative polymerase chain reaction (qPCR) buffer to streamline the extraction procedure. Clinically relevant concentrations of the mutation prone KRAS fragment, which has been linked to colorectal, lung, and bladder cancer, were preconcentrated using the ITO-MIL strategy allowing for enrichment factors as high as 19.49 ±â€¯1.44 from pure water and 4.02 ±â€¯0.50 from 10-fold diluted plasma after a 1 min extraction. Preconcentration could only be achieved when adding the ITO probe to the sample validating the selectivity of the ITO in the capture process. In addition, the amplification efficiency of qPCR was not affected when performing extractions from a diluted-plasma matrix demonstrating that the ITO-MIL approach coupled to direct-qPCR can be used to quantitate DNA from complex matrices. In comparison, commercially available steptavidin-coated magnetic beads were observed to lose selectivity when performing extractions from a 10-fold diluted plasma matrix. The selectivity of the ITO-MIL method, coupled with the ability to rapidly preconcentrate clinically relevant concentrations of target DNA from 10-fold diluted plasma, suggests that this method has the potential to be applied towards the extraction of ctDNA fragments from clinical samples.

Magnetic ionic liquid-enhanced isothermal nucleic acid amplification and its application to rapid visual DNA analysis.

Isothermal nucleic acid amplification (INAA) techniques such as loop-mediated isothermal amplification (LAMP) and isothermal multiple-self-matching-initiated amplification (IMSA) constitute simple and rapid approaches for the detection of pathogens. However, due to the employment of multiple primers, the detection of LAMP and IMSA products is easily influenced by high background signals from primer dimer-based nonspecific nucleic acid amplification (NSA) products. Moreover, time-consuming sample preparation steps are often required for the isolation of sufficiently pure nucleic acid prior to INAA. To address these drawbacks, hydrophobic magnetic ionic liquids (MILs) were used to rapidly preconcentrate DNA from complex biological samples followed by direct amplification by LAMP and IMSA. Careful control of the components within the isothermal buffer permitted direct addition of DNA-enriched MIL to the INAA reaction mixture, thereby circumventing tedious purification procedures that are ordinarily required prior to downstream DNA amplification. When added directly to INAA reactions, MIL solvents released metal ions that ultimately inhibited the primer dimer-mediated NSA, resulting in a flat or decreased baseline signal in no-template control samples and short threshold time for positive reactions. Using a MIL-based single droplet DNA extraction method, MIL-enhanced INAA reaction system, and a handheld 3D printed device for visual detection of the amplified product in customized tubes, we demonstrate the potential of the MIL-based approach for the onsite analysis of DNA from pathogens.

Coupling oligonucleotides possessing a poly-cytosine tag with magnetic ionic liquids for sequence-specific DNA analysis.

Oligonucleotide probes were designed with a poly-cytosine region that facilitates stable anchoring to a magnetic ionic liquid support. By tethering a recognition sequence to the poly-C tag, the resulting diblock oligonucleotides distinguished single-nucleotide variants and captured DNA targets from interfering genomic DNA and cell lysate for qPCR amplification.