Kevin D. Clark

Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis.

DNA extraction represents a significant bottleneck in nucleic acid analysis. In this study, hydrophobic magnetic ionic liquids (MILs) were synthesized and employed as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique DNA extraction capabilities when applied toward a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) ([(C8)3BnN(+)][FeCl3Br(-)]) MIL, while the dicationic 1,12-di(3-hexadecylbenzimidazolium)dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) ([(C16BnIM)2C12(2+)][NTf2(-), FeCl3Br(-)]) MIL produced higher extraction efficiencies for larger DNA molecules. The MIL-based method was also employed for the extraction of DNA from a complex matrix containing albumin, revealing a competitive extraction behavior for the trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P6,6,6,14(+)][FeCl4(-)]) MIL in contrast to the [(C8)3BnN(+)][FeCl3Br(-)] MIL, which resulted in significantly less coextraction of albumin. The MIL-DNA method was employed for the extraction of plasmid DNA from bacterial cell lysate. DNA of sufficient quality and quantity for polymerase chain reaction (PCR) amplification was recovered from the MIL extraction phase, demonstrating the feasibility of MIL-based DNA sample preparation prior to downstream analysis.

Magnetic ionic liquids in analytical chemistry: A review

Magnetic ionic liquids (MILs) have recently generated a cascade of innovative applications in numerous areas of analytical chemistry. By incorporating a paramagnetic component within the cation or anion, MILs exhibit a strong response toward external magnetic fields. Careful design of the MIL structure has yielded magnetoactive compounds with unique physicochemical properties including high magnetic moments, enhanced hydrophobicity, and the ability to solvate a broad range of molecules. The structural tunability and paramagnetic properties of MILs have enabled magnet-based technologies that can easily be added to the analytical method workflow, complement needed extraction requirements, or target specific analytes. This review highlights the application of MILs in analytical chemistry and examines the important structural features of MILs that largely influence their physicochemical and magnetic properties.

Rapid and sensitive analysis of microcystins using ionic liquid-based in situ dispersive liquid–liquid microextraction

Three structurally different ionic liquids (ILs), namely 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-(6-hydroxyethyl)-3-methylimidazolium chloride ([HeOHMIM][Cl]) and 1-benzyl-3-(2-hydroxyethyl)imidazolium bromide ([BeEOHIM][Br]), were applied as extraction solvents using in situ dispersive liquid-liquid microextraction (in situ DLLME) for the preconcentration of two microcystin variants, microcystin-RR (MC-RR) and microcystin-LR (MC-LR) from aqueous samples. Extraction parameters including sample solution pH, ratio of IL to metathesis reagent, sample volume, IL quantity, and salt concentration were optimized to achieve the best extraction efficiency. The [BeEOHIM][Br] IL, which contains both an aromatic moiety and a hydroxyl group within its chemical structure, exhibited superior extraction efficiency compared to the other two ILs. The analytical performance of the [BeEOHIM][Br] IL as an extraction solvent for in situ DLLME of microcystins was investigated using HPLC-UV and HPLC-MS. The limits of detection (LODs) for MC-RR and MC-LR were 0.7μgL(-1) using UV detection with a linear range from 1 to 50μgL(-1). The separation method was successfully adapted for ESI-MS/SIM detection, wherein the LODs for MC-RR and MC-LR were greatly improved to 0.005 and 0.003μgL(-1), respectively. The accuracy of the method was demonstrated by examining the relative recovery using tap water and river water and produced recoveries ranging from 45.0 to 109.7% and from 46.3 to 103.2%, respectively.

Magnetic ionic liquids as PCR-compatible solvents for DNA extraction from biological samples

A polymerase chain reaction (PCR) buffer was systematically designed to relieve the inhibition caused by hydrophobic magnetic ionic liquids (MILs). We describe a simple, rapid method for MIL-based plasmid DNA extraction from crude bacterial cell lysate in which DNA-enriched MIL is transferred directly to a PCR tube for analysis.

Preservation of DNA in nuclease-rich samples using magnetic ionic liquids

Nucleic acids are important diagnostic molecules for a variety of applications, but are exceedingly sensitive to enzymatic degradation by nucleases. Very recently, hydrophobic magnetic ionic liquids (MILs) have shown considerable promise in the area of DNA extraction. Here, we show that MILs can also serve as DNA preservation media in nuclease-rich environments. DNA samples treated with deoxyribonuclease I (DNase I) were found to retain their molecular weight for up to 72 h at room temperature within the benzyltrioctylammonium bromotrichloroferrate(III) ([N888Bn+][FeCl3Br−]) and trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P66614+][FeCl4−]) MILs, whereas DNA in aqueous samples suffered complete enzymatic degradation under similar conditions. Using a single drop extraction (SDE) technique, DNase I was found to partition between aqueous solution and MIL with a smaller amount of the enzyme extracted by the [N888Bn+][FeCl3Br−] MIL relative to the [P66614+][FeCl4−] MIL. Plasmid DNA (pDNA) exhibited structural stability for up to 1 week in the [N888Bn+][FeCl3Br−] and [P66614+][FeCl4−] MILs, even when treated with 20 U of DNase I. pDNA stored within the MIL solvent under these conditions was successfully amplified by polymerase chain reaction (PCR), whereas pDNA in aqueous solutions of DNase I yielded no detectable amplicon. Furthermore, pDNA stored within the trihexyl(tetradecyl)phosphonium tetrachloromanganate(II) ([P66614+]2[MnCl42−]) MIL was capable of conveying antibiotic resistance to competent E. coli following 24 h incubation with DNase I at room temperature, demonstrating that the biological activity of pDNA was preserved.

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.

Extraction and Purification of DNA from Complex Biological Sample Matrices Using Solid-Phase Microextraction Coupled with Real-Time PCR

The determination of extremely small quantities of DNA from complex biological sample matrices represents a significant bottleneck in nucleic acid analysis. In this study, polymeric ionic liquid (PIL)-based solid-phase microextraction (SPME) was applied for the extraction and purification of DNA from crude bacterial cell lysate with subsequent quantification by real-time PCR (qPCR) analysis. Using an on-fiber ultraviolet initiated polymerization technique, eight different PIL sorbent coatings were generated and their DNA extraction performance evaluated using qPCR. The PIL sorbent coating featuring halide anions and carboxylic acid groups in the cationic portion exhibited superior DNA extraction capabilities when compared to the other studied PILs and a commercial polyacrylate SPME fiber. Electrostatic interactions as well as an ion-exchange mechanism were identified as the driving forces in DNA extraction by the PIL sorbents. The selectivity of the PIL sorbent coating for DNA was demonstrated in the presence of PCR inhibitors at high concentration, where a quantifiable amount of template DNA was extracted from aqueous samples containing CaCl2 and FeCl3. Furthermore, the PIL-based SPME method was successfully applied for the extraction of DNA from crude bacterial cell lysate spiked with 1 pg mL(-1) template DNA without requiring the use of organic solvents or centrifugation steps. Following PIL-based SPME of DNA from a dilute cell lysate, the qPCR amplification efficiency was determined to be 100.3%, demonstrating the feasibility of the developed method to extract high purity DNA from complex sample matrices.

Analysis of bacterial plasmid DNA by solid-phase microextraction

The extraction and preconcentration of DNA is a critical step in the analysis of microorganisms. In this study, a polymeric ionic liquid (PIL) sorbent coating was applied for the preconcentration of plasmid DNA (pDNA) from bacterial cells using solid-phase microextraction (SPME). PIL-based SPME devices were prepared by ultraviolet photoinitiated polymerization of a dicationic ionic liquid (IL)-based cross-linker and IL monomer on a nitinol support. pDNA was extracted from buffered aqueous solution using the PIL-based sorbent coating followed by the amplification of a target gene by polymerase chain reaction (PCR). Extraction conditions for the method were optimized based on the relative intensities of PCR amplicon bands visualized on an agarose gel. Compared to a commercial polyacrylate sorbent coating, the PIL sorbent coating extracted greater quantities of pDNA. With an extraction time of 5 min, the PIL-based SPME technique was capable of preconcentrating a sufficient amount of template pDNA from a 20 ng mL−1 solution to allow detection of the amplicon on an agarose gel. Sequence analysis demonstrated that the sequence of the pDNA was unaltered following PIL-based SPME. The developed method was successfully employed for the analysis of pDNA from two different E. coli transformants in a dilute aqueous solution.

Synthesis and characterization of low viscosity hexafluoroacetylacetonate-based hydrophobic magnetic ionic liquids

Magnetic ionic liquids (MILs) are distinguished from traditional ionic liquids (ILs) by the incorporation of a paramagnetic component within their chemical structure. Hydrophobic MILs are novel solvents that can be used in many applications, including liquid–liquid extraction (LLE) and catalysis. Low viscosity and low water solubility are essential features that determine their feasibility in LLE. In this study, extremely hydrophobic MILs were synthesized by using transition and rare earth metal hexafluoroacetylacetonate chelated anions paired with the trihexyl(tetradecyl)phosphonium ([P66614+]) cation. Hydrophobic MILs exhibiting water solubilities less than 0.01% (v/v) were synthesized in a rapid two-step procedure. Furthermore, the viscosities of the MILs are among some of the lowest ever reported for hydrophobic MILs (276.5–927.9 centipoise (cP) at 23.7 °C) dramatically improving the ease of handling these liquids. For the first time, the magnetic properties of MILs possessing hexafluoroacetylacetonate chelated metal anions synthesized in this study are reported using a superconducting quantum interference device (SQUID) magnetometer. Effective magnetic moments (μeff) as high as 9.7 and 7.7 Bohr magnetons (μB) were achieved by incorporating high spin dysprosium and gadolinium ions, respectively, into the anion component of the MIL. The low viscosity, high hydrophobicity, and large magnetic susceptibility of these MILs make them highly attractive and promising solvents for separations and purification, liquid electrochromic materials, catalytic studies, as well as microfluidic applications.

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.