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)42+][Glu–]2) and tetra(1-octylimidazole)cobaltate(II) di(l-aspartate) ([Co(OIM)42+][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 KRASfragment, 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 ± 1.44 from pure water and 4.02 ± 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

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

Investigating the effect of ligand and cation on the properties of metal fluorinated acetylacetonate based magnetic ionic liquids

Magnetic ionic liquids (MILs) are a subclass of ionic liquids that possess a paramagnetic metal within their chemical structure, making them susceptible to external magnetic fields. A total of twenty-four (24) MILs were prepared and characterized to investigate the effect of the ligand, cation and anion on the physiochemical properties of acetylacetonate-based MILs. It was found that thermal stabilities as high as 260 °C could be achieved by incorporating aromatic moieties in the anion structure. Additionally, the magnetic moment could be modulated by simply changing the transition metal in the anion. Magnetic moment values of 2.8 μB, 4.5 μBand 5.6 μB were obtained by using Ni(II), Co(II), and Mn(II) as the metal centers, respectively. Furthermore, the viscosity of the MILs could be tailored from a few hundred centipoise to several thousand centipoise, increasing their potential applications in numerous interdisciplinary fields. Moreover, the MILs synthesized in this study were found to be insoluble in water at a MIL-to-solvent ratio of 0.01% (w/v), making them potentially useful in targeted separations, where very hydrophobic solvents are highly desired

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

Second language learners' perceptions of school climate and educational satisfaction and their relationships to academic achievement and the mediational effects of gender.

The purposes of this study were (1) to compare perceptions of school climate measured by 10 subscales of the NASSP School Climate Survey and student satisfaction measured by 8 subscales of the Student Satisfaction Survey of language-minority students to those of non-language-minority students and (2) to investigate whether students' perceptions of school climate, student satisfaction, gender, or language group affected students' academic achievement. The sample population included ninth-grade students at Nogales High School. Students were classified as second-language learners according to the district's Identification, Assessment, and Program Placement flow chart. The study used four independent predictors of reading and math achievement: language group, gender, school climate, and student satisfaction. A multiple regression analysis tested relationships between the predicated variable and independent predictors. The Pearson product-moment correlation determined the relationship between student satisfaction and perception of school climate. A two-way analysis of variance (ANOVA) distinguished whether satisfaction and perceptions of school climate were different for second-language learners and non-second-language learners and whether there was a difference in satisfaction and perceptions of school climate by gender among second-language learners and non-second-language learners. The findings of this study indicated a moderate correlation between student satisfaction and perceptions of school climate. Statistically significant differences were found by language group for student satisfaction and for satisfaction by gender among non-second-language learners. No statistically significant differences were found by language group for school climate, for gender among second-language learners for satisfaction or school climate, or between perceptions of school climate for boys and girls among non-second-language learners. The parameter estimates showed that gender, school climate, and language group were significant independent predictors of reading achievement, while student satisfaction was not. Language group was the only significant independent predictor of mathematics achievement

Development of isolation and detection methods for the analysis of DNA and bacteria

Nucleic acids are essential biopolymers that have served as diagnostic biomarkers for many applications. Traditional nucleic acid isolation methods typically rely on nucleic acid adsorption onto silica particles and require multiple centrifugation steps, organic solvents and significant user intervention. Additionally, popular detection methods, such as polymerase chain reaction (PCR), suffer from long analysis times and require complex thermal cycling equipment. These drawbacks limit the utility of traditional isolation/detection methods for rapid in-field testing and point-of-care applications. The work presented in this dissertation addresses challenges associated with nucleic acid isolation and detection through the development of solid-phase microextraction (SPME) and isothermal amplification methodologies. SPME is an alternative sample preparation methodology that circumvents many limitations of conventional methods. A rapid SPME method was developed and optimized to enable the isolation of DNA from mycobacterium smegmatis in artificial sputum samples. For detecting the extracted DNA, a colorimetric isothermal multiple-self-matching initiated amplification (IMSA) assay was designed using hydroxy napthol blue (HNB). A custom buffer was designed to allow direct coupling of the 1 M NaCl SPME desorption solution with the IMSA assay. The custom buffer was successfully applied in multiple studies for the detection of a variety of DNA sequences. A significant limitation of isothermal amplification methodologies is the lack of sequence-specificity in the detection method. Traditional methods rely on colorimetric indicators that indirectly detect amplification. Molecular beacons (MB) were employed along with HNB to specifically detect loop-mediated isothermal amplification (LAMP) using a transilluminator. The high specificity of the method was demonstrated by discriminating two sequences that varied by a single nucleotide. To further demonstrate the applicability of MB-LAMP, an assay was designed for the detection of BRAF V600E, a mutation present in 90% of melanomas. Two MBs were employed containing a FAM (wild-type) or HEX (mutant) fluorophore to identify each sequence. A plate reader was employed in order to simply the detection scheme. Additionally, SPME was successfully applied to isolate the mutant sequence from human plasma and enable detection with the MB-LAMP assay. The specific detection of LAMP was further simplified by employing MBs and a biotinylated primer in a lateral-flow immunoassay format. This approach eliminated the need for additional equipment in the detection step. The versatility of the method was demonstrated through the detection of three independent sequences. Additionally, the high specificity of the method was highlighted through the differentiation of wild type and mutant BRAF V600E. The developed detection scheme was also compatible with SPME and enabled detection from pond water, human plasma, and artificial saliva. A disadvantage to the previously used SPME extraction phases is the lack of specificity, as the primary interaction with the nucleic acid was anion exchange. To circumvent this, a sequence-specific SPME sorbent was developed in collaboration with Millipore Sigma. A commercially-available polyacrylate SPME fiber was modified with an amine-labeled oligonucleotide complementary to a DNA target using carbodiimide coupling chemistry. Several parameters were optimized including desorption time and the use of Exonuclease III to minimize carryover. While detection of nucleic acids from pathogens is often sufficient in many cases, certain applications such as food safety require knowledge of the viability of the detected organisms. Traditional methods rely on pre-enrichment cultures that are time consuming to perform, decrease the sample-to-answer time. Magnetic ionic-liquids have been previously shown to be capable of enriching viable bacteria from a variety of food matrices. In the study presented within this dissertation, the physiological effects of magnetic ionic liquids on a variety of bacteria were evaluated. Salmonella and E. coli O157:H7 that were captured by magnetic ionic liquids were plated on selective and non-selective agars to evaluate whether or not cellular injury occurred. Additionally, the ability for magnetic ionic liquids to capture a wide range of Gram-negative bacteria was also evaluated

Development of isolation and detection methods for the analysis of DNA and bacteria

Nucleic acids are essential biopolymers that have served as diagnostic biomarkers for many applications. Traditional nucleic acid isolation methods typically rely on nucleic acid adsorption onto silica particles and require multiple centrifugation steps, organic solvents and significant user intervention. Additionally, popular detection methods, such as polymerase chain reaction (PCR), suffer from long analysis times and require complex thermal cycling equipment. These drawbacks limit the utility of traditional isolation/detection methods for rapid in-field testing and point-of-care applications. The work presented in this dissertation addresses challenges associated with nucleic acid isolation and detection through the development of solid-phase microextraction (SPME) and isothermal amplification methodologies.SPME is an alternative sample preparation methodology that circumvents many limitations of conventional methods. A rapid SPME method was developed and optimized to enable the isolation of DNA from mycobacterium smegmatis in artificial sputum samples. For detecting the extracted DNA, a colorimetric isothermal multiple-self-matching initiated amplification (IMSA) assay was designed using hydroxy napthol blue (HNB). A custom buffer was designed to allow direct coupling of the 1 M NaCl SPME desorption solution with the IMSA assay. The custom buffer was successfully applied in multiple studies for the detection of a variety of DNA sequences. A significant limitation of isothermal amplification methodologies is the lack of sequence-specificity in the detection method. Traditional methods rely on colorimetric indicators that indirectly detect amplification. Molecular beacons (MB) were employed along with HNB to specifically detect loop-mediated isothermal amplification (LAMP) using a transilluminator. The high specificity of the method was demonstrated by discriminating two sequences that varied by a single nucleotide. To further demonstrate the applicability of MB-LAMP, an assay was designed for the detection of BRAF V600E, a mutation present in 90% of melanomas. Two MBs were employed containing a FAM (wild-type) or HEX (mutant) fluorophore to identify each sequence. A plate reader was employed in order to simply the detection scheme. Additionally, SPME was successfully applied to isolate the mutant sequence from human plasma and enable detection with the MB-LAMP assay. The specific detection of LAMP was further simplified by employing MBs and a biotinylated primer in a lateral-flow immunoassay format. This approach eliminated the need for additional equipment in the detection step. The versatility of the method was demonstrated through the detection of three independent sequences. Additionally, the high specificity of the method was highlighted through the differentiation of wild type and mutant BRAF V600E. The developed detection scheme was also compatible with SPME and enabled detection from pond water, human plasma, and artificial saliva. A disadvantage to the previously used SPME extraction phases is the lack of specificity, as the primary interaction with the nucleic acid was anion exchange. To circumvent this, a sequence-specific SPME sorbent was developed in collaboration with Millipore Sigma. A commercially-available polyacrylate SPME fiber was modified with an amine-labeled oligonucleotide complementary to a DNA target using carbodiimide coupling chemistry. Several parameters were optimized including desorption time and the use of Exonuclease III to minimize carryover. While detection of nucleic acids from pathogens is often sufficient in many cases, certain applications such as food safety require knowledge of the viability of the detected organisms. Traditional methods rely on pre-enrichment cultures that are time consuming to perform, decrease the sample-to-answer time. Magnetic ionic-liquids have been previously shown to be capable of enriching viable bacteria from a variety of food matrices. In the study presented within this dissertation, the physiological effects of magnetic ionic liquids on a variety of bacteria were evaluated. Salmonella and E. coli O157:H7 that were captured by magnetic ionic liquids were plated on selective and non-selective agars to evaluate whether or not cellular injury occurred. Additionally, the ability for magnetic ionic liquids to capture a wide range of Gram-negative bacteria was also evaluated.</p

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.This article is published as Varona, Marcelino, and Jared L. Anderson. "Advances in Mutation Detection Using Loop-Mediated Isothermal Amplification." ACS Omega (2021). DOI: 10.1021/acsomega.0c06093. Posted with permission.</p

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)42+][Glu–]2) and tetra(1-octylimidazole)cobaltate(II) di(l-aspartate) ([Co(OIM)42+][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.This document is the Accepted Manuscript version of a Published Work that will appear in final form in ACS Omega, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see DOI: 10.1021/acsomega.0c01098. Posted with permission.</p