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.

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.

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.

Arabidopsis thaliana ITS sequence-specific DNA extraction by ion-tagged oligonucleotides coupled with a magnetic ionic liquid.

This study reports a follow-up investigation on the capture of specific DNA sequences using ion-tagged oligonucleotides (ITOs) and magnetic ionic liquids (MIL). Five allylimidazolium salts bearing octyl substituents ([AOIM+]-ITOs) were used for the selective extraction of the internal transcribed spacer region (ITS) from Arabidopsis thaliana. In this work, the ability of the [AOIM+]-ITOs to enhance the extraction of longer target sequences (~ 700 bp) of plant origin was shown. Moreover, the independence of the probe binding position and the importance of complementarity to the target region for the extraction performance were demonstrated. To test the specificity of the ITOs, the same experiments were performed using the ITS region from another plant species, with a lower target capture for the probes which were specific for the A. thaliana sequence. Finally, extraction in the presence of interferences (heterogenous DNA, primary and secondary metabolites, proteins) provided interesting and insightful results. This work illustrates the feasibility and versatility of these probes when coupled to MILs for rapid, cost-effective, and environmentally sensitive sample preparation in the extraction of specific target sequences from different origins. Graphical abstract.

Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification.

Nucleic acid extraction and purification represents a major bottleneck in DNA analysis. Traditional methods for DNA purification often require reagents that may inhibit quantitative polymerase chain reaction (qPCR) if not sufficiently removed from the sample. Approaches that employ magnetic beads may exhibit lower extraction efficiencies due to sedimentation and aggregation. In this study, four hydrophobic magnetic ionic liquids (MILs) were investigated as DNA extraction solvents with the goal of improving DNA enrichment factors and compatibility with downstream bioanalytical techniques. By designing custom qPCR buffers, we directly incorporated DNA-enriched MILs including trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(hfacac)3-]), [P6,6,6,14+] tris(hexafluoroacetylaceto)colbaltate(II) ([Co(hfacac)3-]), [P6,6,6,14+] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac)3-]), or [P6,6,6,14+] tetrakis(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac)4-]) into reaction systems, thereby circumventing the need for time-consuming DNA recovery steps. Incorporating MILs into the reaction buffer did not significantly impact the amplification efficiency of the reaction (91.1%). High enrichment factors were achieved using the [P6,6,6,14+][Ni(hfacac)3-] MIL for the extraction of single-stranded and double-stranded DNA with extraction times as short as 2 min. When compared to a commercial magnetic bead-based platform, the [P6,6,6,14+][Ni(hfacac)3-] MIL was capable of producing higher enrichment factors for single-stranded DNA and similar enrichment factors for double-stranded DNA. The MIL-based method was applied for the extraction and direct qPCR amplification of mutation prone-KRAS oncogene fragment in plasma samples. Graphical abstract Magnetic ionic liquid solvents are shown to preconcentrate sufficient KRAS DNA template from an aqueous solution in as short as 2 min without using chaotropic salts or toxic organic solvents. By using custom-designed qPCR buffers, DNA can be directly amplified and quantified from four MILs examined in this study.

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.

Simple and efficient isolation of plant genomic DNA using magnetic ionic liquids.

BACKGROUND: Plant DNA isolation and purification is a time-consuming and laborious process relative to epithelial and viral DNA sample preparation due to the cell wall. The lysis of plant cells to free intracellular DNA normally requires high temperatures, chemical surfactants, and mechanical separation of plant tissue prior to a DNA purification step. Traditional DNA purification methods also do not aid themselves towards fieldwork due to the numerous chemical and bulky equipment requirements. RESULTS: In this study, intact plant tissue was coated by hydrophobic magnetic ionic liquids (MILs) and ionic liquids (ILs) and allowed to incubate under static conditions or dispersed in a suspension buffer to facilitate cell disruption and DNA extraction. The DNA-enriched MIL or IL was successfully integrated into the qPCR buffer without inhibiting the reaction. The two aforementioned advantages of ILs and MILs allow plant DNA sample preparation to occur in one minute or less without the aid of elevated temperatures or chemical surfactants that typically inhibit enzymatic amplification methods. MIL or IL-coated plant tissue could be successfully integrated into a qPCR assay without the need for custom enzymes or manual DNA isolation/purification steps that are required for conventional methods. CONCLUSIONS: The limited amount of equipment, chemicals, and time required to disrupt plant cells while simultaneously extracting DNA using MILs makes the described procedure ideal for fieldwork and lab work in low resource environments.

Magnetic ionic liquids as microRNA extraction solvents and additives for the exponential amplification reaction.

The detection of microRNAs (miRNAs) from highly complex matrices has become an area of immense interest as their characterization in biological samples has been utilized for disease diagnosis and body fluid identification. However, conventional northern blotting miRNA detection lacks the sensitivity required to detect circulating miRNAs. Additionally, polymerase chain reaction-based methods for miRNA detection require modified oligonucleotides that are difficult to design. Exponential amplification reaction (EXPAR) is an isothermal amplification method used for miRNA detection that is simple to design but suffers from non-specific amplification that masks low concentration miRNAs. Previous studies have shown that magnetic ionic liquids (MILs) are a promising alternative to traditional nucleic acid extraction methods capable of preconcentrating DNA from complex matrices. In this study, three hydrophobic magnetic ionic liquids (MILs) were investigated as EXPAR additives and miRNA extraction solvents. The addition of MIL to the EXPAR buffer decreased the background signal from non-specific amplification and increased the reaction rate. Reactions containing MIL could detect miRNA at concentration levels down to 10 aM. In comparison, reactions that did not contain MIL could not discriminate 10 fM lethal-7a (let-7a) standards from the no trigger control (NTC). All three MILs extracted miRNA from 2-fold diluted plasma, artificial urine, and artificial saliva with only a 1 min dispersion step. By integrating the miRNA-enriched MIL into the EXPAR buffer, the extraction and detection of femtomolar concentrations of miRNA required only 10 min. In contrast, conventional spin column kits require at least 20 min to isolate miRNA, indicating that a dispersive MIL-based extraction is ideal for high throughput analysis of miRNA.

Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids.

Multiplex amplification of DNA can be highly valuable in circulating tumor DNA (ctDNA) analysis due to the sheer number of potential mutations. However, commercial ctDNA extraction methods struggle to preconcentrate low concentrations of DNA and require multiple sample handling steps. Recently, magnetic ionic liquids (MILs) have been used to extract DNA and were integrated with a quantitative polymerase chain reaction (qPCR). However, in previous studies, DNA could not be preconcentrated from plasma and only one fragment could be amplified per reaction. In this study, MILs were utilized as DNA extraction solvents and directly integrated into a multiplex-qPCR buffer to simultaneously amplify wild-type KRAS, G12S KRAS, and wild-type BRAF, three clinically-relevant genes whose mutation status can affect the success of anti-EGFR therapy. DNA was desorbed from the MIL solvent during a multiplex-PCR without having a significant effect on the amplification efficiency, and allelic discrimination of single-nucleotide polymorphisms could still be achieved. Enrichment factors over 35 for all three sequences were achieved from Tris buffer using the [N8,8,8,Bz+][Ni(hfacac)3-]) and [P6,6,6,14+][Ni(Phtfacac)3-] MILs. DNA could still be preconcentrated from 2-fold diluted human plasma using the [N8,8,8,Bz+][Ni(hfacac)3-] MIL. Extractions from undiluted plasma were reproducible with the [P6,6,6,14+][Ni(Phtfacac)3-] MIL although DNA was not preconcentrated with enrichment factors around 0.6 for all three fragments. Compared to commercial DNA extraction methods (i.e., silica-based spin columns and magnetic beads), the MIL-based extraction achieved higher enrichment factors in Tris buffer and plasma. The ability of the MIL-based dispersive liquid-liquid microextraction (DLLME) direct-multiplex-qPCR method to simultaneously achieve high enrichment factors of multiple DNA fragments from human plasma is highly promising in the field of ctDNA detection.

Selective hybridization and capture of KRAS DNA from plasma and blood using ion-tagged oligonucleotide probes coupled to magnetic ionic liquids.

Detection of circulating tumor DNA (ctDNA) presents several challenges due to single-nucleotide polymorphisms and large amounts of background DNA. Previously, we reported a sequence-specific DNA extraction procedure utilizing functionalized oligonucleotides called ion-tagged oligonucleotides (ITOs) and disubstituted ion-tagged oligonucleotides (DTOs). ITOs and DTOs are capable of hybridizing to complementary DNA for subsequent capture by a magnetic ionic liquid (MIL) through hydrophobic interactions, π-π stacking, and fluorophilic interactions. However, the performance of the ITOs and DTOs in complex sample matrices has not yet been evaluated. In this study, we compare the amount of KRAS DNA extracted using ITO and DTOs from saline, 2-fold diluted plasma, 10-fold diluted plasma, and 10-fold diluted blood. We demonstrate that ITO/DTO-MIL extraction is capable of selectively preconcentrating DNA from diluted plasma and blood without additional sample preparation steps. In comparison, streptavidin-coated magnetic beads were unable to selectively extract DNA from 10-fold diluted plasma and 10-fold diluted blood without additional sample clean-up steps. Significantly more DNA could be extracted from 2-fold diluted plasma and 10-fold diluted blood matrices using the DTO probes compared to the ITO probes, likely due to stronger interactions between the probe and MIL. The ability of the DTO-MIL method to selectively preconcentrate small concentrations of DNA from complex biological matrices suggests that this method could be beneficial for ctDNA analysis.

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.

Development of an innovative and sustainable one-step method for rapid plant DNA isolation for targeted PCR using magnetic ionic liquids.

BACKGROUND: Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids. RESULTS: In the present study, a MIL-based magnet-assisted dispersive liquid-liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues. CONCLUSIONS: The developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods.

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.