Exploiting Magnetic Ionic Liquids to Develop a One-step Method for Plant DNA Isolation

1 4 E X T E C H 2 0 1 8

O_7:

Applications of Two New Ambient Ionization Technologies for

Direct Sample Analysis: PaperSpray and Flowprobe

David J. Borts , Laura E. Burns, Michaela Lerch Iowa State University, USA

3DSHUVSUD\PDVVVSHFWURPHWU\3606DQGOLTXLGPLFURMXQFWLRQVXUIDFHVDPSOLQJSUREH/0-663DUHWZRUHODWLYHO\QHZDPELHQWLRQL]DWLRQtechnologies that enable rapid and direct analysis of samples with little to no sample preparation and no chromatographic separation. PS-MS is well suited for quanti-WDWLYHDQDO\VLVRI[HQRELRWLFVLQELRORJLFDOćXLGVZKLOH/0-663IDFLOLWDWHVUDSLGTXDOLWDWLYHDQDO\VLVRIVDPSOHVXVLQJDOLTXLGPLFURMXQFWLRQZLWKDVDPSOH surface. We have developed a number of quantitative bioanalytical methods using PS-MS, including quantitation of multiple NSAID drugs in equine and porcine plasma, and quantitation of an antifungal drug in equine tears and plasma. For NSAIDs in equine plasma, PS-MS quantitative results were compared with those generated using an established, validated, LC-MS method and shown to be equivalent. Each PS-MS method was validated with respect to selec-WLYLW\OLQHDULW\DFFXUDF\SUHFLVLRQPDWUL[HIIHFWDQGFDUU\RYHU0HWKRGYDOLGDWLRQUHVXOWVDQGĆJXUHVRIPHULWZLOOEHSUHVHQWHGDVZLOOUHVXOWVIURPDQDO\VLV of authentic unknown samples. We have also developed a method using LMJ-SSP for rapid, untargeted screening for veterinary drug residues and xenobi-RWLFVLQIRRGDQGFRPSDQLRQDQLPDOWLVVXHV([DPSOHVRIDSSOLFDWLRQVRIWKLVPHWKRGLQFOXGHLGHQWLĆFDWLRQRIWKHYHWHULQDU\DQHVWKHWLFSHQWREDUELWDODQG WKHDQWLVHL]XUHFRPSRXQGSKHQ\WRLQVLPXOWDQHRXVO\LQFDQLQHOLYHULGHQWLĆFDWLRQRIWKHJURZWKDQGOHDQQHVVSURPRWLQJGUXJUDFWRSDPLQHLQSRUFLQHOLYHU DQGLGHQWLĆFDWLRQRIWKH16$,'GUXJćXQL[LQLQDSRUFLQHPXVFOHKRPRJHQDWHVPHDU&RQVLGHUDWLRQVUHJDUGLQJSDUDPHWHUVHOHFWLRQDQGRSWLPL]DWLRQZKHQ LPSOHPHQWLQJWKH/0-663DVSDUWRIDQXQWDUJHWHGVFUHHQLQJZRUNćRZZLOOEHUHYLHZHG)RU3606PHWKRGVD9HOR[DXWRPDWHGSDSHUVSUD\VRXUFH 3URVROLD,QF,QGLDQDSROLV,186$ZDVLQWHUIDFHGZLWKD4([DFWLYH)RFXVTXDGUXSROHRUELWUDSPDVVVSHFWURPHWHU7KHUPR)LVKHU6FLHQWLĆF6DQ-RVH &$86$)RUWKH/0-663ZRUNDćRZSUREH3URVROLD,QF,QGLDQDSROLV,186$ZDVLQWHUIDFHGZLWKD4([DFWLYH)RFXVPDVVVSHFWURPHWHU $FNQRZOHGJHPHQW'DYLG%RUWVè'HSDUWPHQWDO6WDUWXS)XQGV,RZD6WDWH8QLYHUVLW\

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Ashley N. Bowers 0DU¯D-RV«7UXMLOOR5RGU¯JXH]2PSUDNDVK1DFKDP-DUHG/$QGHUVRQ Iowa State University, USA

7KHH[WUDFWLRQDQGSXULĆFDWLRQRIWRWDOULERQXFOHLFDFLGWRWDO51$IURPFRPSOH[ELRORJLFDOVDPSOHPDWULFHVLVHVVHQWLDOIRUQXPHURXVGRZQVWUHDP applications in molecular biology such as quantitative reverse-transcription polymerase chain reaction (RT-qPCR), electrophoresis, protein expression, VHTXHQFLQJWUDQVIHFWLRQDQGJHQHWKHUDS\7UDGLWLRQDOO\51$H[WUDFWLRQSURWRFROVXVHRUJDQLFVROYHQWVZKLFKSRVHHQYLURQPHQWDOKD]DUGVDQGDUH often time-consuming as they can contain multiple steps. Commercial kits have been developed to address these issues. However, they are usually YHU\H[SHQVLYHDQGPD\UHTXLUHVSHFLDOL]HGHTXLSPHQWWRXVH,QWKLVVWXG\DVLPSOHHQYLURQPHQWDOO\IULHQGO\DQGUDSLGH[WUDFWLRQWHFKQLTXHKDVEHHQ developed for the effective determination of total RNA from aqueous solutions. Six ionic liquids (ILs) were tested as possible extraction solvents in an in situ ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) technique involving aqueous solutions spiked with total RNA, ionic liquid, and OLWKLXPELV>WULćXRURPHWK\OVXOIRQ\O@LPLGH/L17IDVWKHDQLRQH[FKDQJHUHDJHQW7RWDO51$LVSUHFRQFHQWUDWHGLQWRWKH,/H[WUDFWLRQSKDVHDQGLQGLUHFW DQDO\VLVRQWKHDPRXQWRIWRWDO51$H[WUDFWHGLVSHUIRUPHGE\LQMHFWLQJDQDOLTXRWRIWKHXSSHUDTXHRXVSKDVHDIWHUH[WUDFWLRQWRKLJKSHUIRUPDQFHOLTXLG chromatography with diode array detection following a new ion-pair reversed-phase method (IP-RP-HPLC).

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O_9:

Advances in Pre-Analytical Sample Preparation: Unclogging the Bottleneck from Sample to Answer

Byron Brehm-Stecher

Rapid Microbial Detection and Control Laboratory, Iowa State University, USA

Successful detection of pathogens in foods involves the seamless integration of three interdependent steps: 1) statistically validated sampling, 2) pre-an-alytical sample preparation and 3) detection. Weak links in any of these three steps will propagate through the system and degrade the end result. In a worst-case scenario, this could lead to false-negative results. “Upstream” methods for sampling have long been established, and the past decade has seen a revolution in development of the hardware and reagents needed for truly rapid detection of pathogens. However, even the most sophisticated detection methods cannot reach their full potential without next-level advances in pre-analytical sample preparation, which is still the key bottleneck in getting from sample to answer when detecting pathogens in foods. Further, sample preparation is not only of critical importance for detection of pathogens in foods, but also for detection of any analyte (chemical, biochemical, cellular, etc.) distributed in any medium. Where appropriate, this talk will address sample prepara-tion-related motivations, problems - and potentially, solutions - that are common to even very different disciplines such as chemistry and food science.

O_10 :

Exploiting Magnetic Ionic Liquids to Develop a One-step Method for Plant DNA Isolation

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Nowadays, there is an increasing demand for fast sustainable, and reliable plant biomolecular analyses. Plant DNA can be used for several fast screening applications including, for instance, DNA barcoding for food traceability [1]. In general, DNA isolation requires multiple and time-consuming steps to UHPRYHFHOOXODULQWHUIHUHQFHVZLWKWKHFRQVHTXHQFHWKDWVDPSOHSUHSDUDWLRQLVWKHPDLQERWWOHQHFNLQDELRDQDO\WLFDOZRUNćRZ1HZSHUVSHFWLYHVKDYH EHHQRSHQHGE\WKHXVHRI0DJQHWLF,RQLF/LTXLGV0,/VSURSRVHGLQE\$QGHUVRQèVJURXSDVQHZPDWHULDOVIRUVHOHFWLYHH[WUDFWLRQDQGSXULĆFDWLRQ of nucleic acids [2]. Hydrophobic MILs can be used in magnet-assisted Dispersive Liquid-Liquid Microextraction (maDLLME) by dispersing the MILs LQWRYHU\VPDOOPLFURGURSOHWVLQDZDWHUVROXWLRQFRQWDLQLQJWKHQXFOHLFDFLGVDQGE\UHFRYHULQJWKH0,/VZLWKWKHDSSOLFDWLRQRIDPDJQHWLFĆHOG7KLV DSSURDFKUHVXOWVLQUHGXFHGH[WUDFWLRQWLPHDQGLQFUHDVHGH[WUDFWLRQHIĆFLHQF\WRZDUGQXFOHLFDFLGV6RIDULWKDVEHHQDSSOLHGWR'1$SXULĆHGRULQ complex solutions containing proteins) and to bacterial cell lysate, but, to date, no applications on plants have been reported [3]. In the present study, three ORZYLVFRVLW\K\GURSKRELF0,/VFRQWDLQLQJ&R0QDQG1L>@ZHUHWHVWHGIRUWKHLVRODWLRQRISODQW'1$3UHOLPLQDU\WHVWVZHUHFDUULHGRXWRQSXULĆHG DNA from Arabidopsis thaliana (L.) Heynh. while subsequent experiments were performed directly on the plant material. The isolation of the DNA was SHUIRUPHGE\PD'//0(DIWHUO\VLVRIWKHSODQWFHOOVDQGWKH'1$ZDVWKHQUHFRYHUHGE\EDFNH[WUDFWLRQ7KHLVRODWHG'1$ZDVVXEMHFWHGWRTXDOLWDWLYHDQG TXDQWLWDWLYHDQDO\VHVZLWKLQWHUHVWLQJUHVXOWVLQWHUPVRI0,/VEDVHGPHWKRGH[WUDFWLRQSHUIRUPDQFH,QWKHĆHOGRISODQWPROHFXODUELRORJ\WKHDSSOLFDWLRQ of maDLLME with MILs could represent a powerful tool for fast, sustainable and potentially automatable screening methods involving DNA.>@*DOLPEHUWL

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