Omprakash Nacham

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.

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.

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.

Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual solvents in pharmaceuticals

Graphical abstract Figure. No Caption available. HighlightsTwo ionic liquids (ILs), 1‐butyl‐3‐methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]imide ([P66614][NTf2]) were examined as contemporary diluents for residual solvent analysis using static headspace gas chromatography (SHS‐GC) coupled with flame ionization detection (FID).A 25‐fold improvement in limit of detection (LD) was observed with respect to traditional HS‐GC diluents, such as N‐methylpyrrolidone (NMP).The established IL‐based method demonstrated LDs ranging from 5.8 parts‐per‐million (ppm) to 20 ppm of residual solvents in drug substances.The analytical performance was demonstrated by determining the repeatability, accuracy, and linearity of the method.Linear ranges of up to two orders of magnitude were obtained for class 3 solvents.Excellent analyte recoveries were obtained in the presence of three different active pharmaceutical ingredients. Abstract In this study, two ionic liquids (ILs), 1‐butyl‐3‐methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]imide ([P66614][NTf2]) were examined as contemporary diluents for residual solvent analysis using static headspace gas chromatography (SHS‐GC) coupled with flame ionization detection (FID). ILs are a class of non‐molecular solvents featuring negligible vapor pressure and high thermal stabilities. Owing to these favorable properties, ILs have potential to enable superior sensitivity and reduced interference, compared to conventional organic diluents, at high headspace incubation temperatures. By employing the [BMIM][NTf2] IL as a diluent, a 25‐fold improvement in limit of detection (LOD) was observed with respect to traditional HS‐GC diluents, such as N‐methylpyrrolidone (NMP). The established IL‐based method demonstrated LODs ranging from 5.8 parts‐per‐million (ppm) to 20 ppm of residual solvents in drug substances. The optimization of headspace extraction conditions was performed prior to method validation. An incubation temperature of 140 °C and a 15 min incubation time provided the best sensitivity for the analysis. Under optimized experimental conditions, the mass of residual solvents partitioned in the headspace was higher when using [BMIM][NTf2] than NMP as a diluent. The analytical performance was demonstrated by determining the repeatability, accuracy, and linearity of the method. Linear ranges of up to two orders of magnitude were obtained for class 3 solvents. Excellent analyte recoveries were obtained in the presence of three different active pharmaceutical ingredients. Owing to its robustness, high throughput, and superior sensitivity, the HS‐GC IL‐based method can be used as an alternative to existing residual solvent methods.

Synthesis and characterization of the physicochemical and magnetic properties for perfluoroalkyl ester and Fe(III) carboxylate-based hydrophobic magnetic ionic liquids

Magnetic ionic liquids (MILs) are a new class of ionic liquids (ILs) that incorporate a paramagnetic component in their chemical structure. Although imidazolium-based MILs can be synthesized using inexpensive and relatively straightforward procedures, these compounds often are water soluble which limits their usefulness in aqueous applications. In this study, two classes of hydrophobic MILs, including perfluorobutyryl ester-based and Fe(III) carboxylate-based MILs, were synthesized and characterized. Functionalization of the cation with fluorinated substituents yielded MILs that were insoluble in aqueous solution at concentrations as low as 0.1% (w/v). In contrast to conventional MILs that rely on paramagnetic anions, Fe(III) carboxylate-based MILs were prepared featuring carboxylate ligands in the cationic moiety capable of chelating a paramagnetic Fe(III) center. The hydrophobic character of the Fe(III) carboxylate-based MILs was subsequently controlled by incorporating the bis[(trifluoromethyl)sulfonyl]imide ([NTf2−]) anion, resulting in MILs that were insoluble in aqueous solutions at 0.1% (w/v). This synthetic strategy has the potential to impart dual functionality to MILs by providing the flexibility to incorporate a task specific anion without sacrificing paramagnetic properties. The molar magnetic susceptibilities (χm) and effective magnetic moments (μeff) of the studied MILs were determined using superconducting quantum interference device (SQUID) magnetometry. Consistent with the Curie–Weiss law, a linear relationship between temperature and inverse magnetic susceptibility (χm−1) was observed for the hydrophobic MILs. The μeff values of the MILs examined in this study ranged from 3.56 to 8.06 Bohr magnetons (μB).

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.