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11-30-2018 Capture, Concentration and Detection of in Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification Stephanie A. Hice Iowa State University, [email protected]

Kevin D. Clark Iowa State University

Jared L. Anderson Iowa State University, [email protected]

Byron F. Brehm-Stecher Iowa State University, [email protected] Follow this and additional works at: https://lib.dr.iastate.edu/fshn_ag_pubs Part of the Commons, Commons, Human and Clinical Nutrition Commons, and Infectious Disease Commons, and the Molecular, Genetic, and Biochemical Nutrition Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ fshn_ag_pubs/215. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the and Human Nutrition at Iowa State University Digital Repository. It has been accepted for inclusion in Food Science and Human Nutrition Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Capture, Concentration and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification

Abstract We have previously investigated the extraction and concentration of bacteria from model systems using magnetic ionic liquid (MIL) solvents, while retaining their viability. Here, we combine MIL-based sample preparation with isothermal amplification and detection of Salmonella-specific DNA using Recombinase Polymerase Amplification (RPA). After initial developmental work with Serratia marcescens in , Salmonella Typhimurium ATCC 14028 was inoculated in water, 2% milk, almond milk or liquid egg samples and extracted using one of two MILs, including: trihexyl(tetradecyl)phosphonium cobalt(II) hexafluoroacetylacetonate ([P66614+][Co(hfacac)3–]) and trihexyl(tetradecyl)phosphonium nickel(II) hexafluoroacetylacetonate ([P66614+][Ni(hfacac)3–]). Viable cells were recovered from the MIL extraction phase after the addition of modified LB broth, followed by a 20 min isothermal RPA assay. Amplification was carried out using supersaturated sodium acetate heat packs and results compared to those using a conventional laboratory thermocycler set to a single temperature. Results were visualized using either gel electrophoresis or nucleic acid lateral flow immunoassay (NALFIA). The ombc ined MIL-RPA approach enabled detection of Salmonella at levels as low as 103 CFU mL-1. MIL-based sample preparation required less than 5 min to capture and concentrate sufficient cells for detection using RPA, which (including NALFIA or gel-based analysis) required approximately 30 - 45 min. Our results suggest the utility of MILs for the rapid extraction and concentration of pathogenic in food samples, providing a means for physical enrichment that is compatible with downstream analysis using RPA.

Disciplines Food Chemistry | Food Processing | Food Science | Human and Clinical Nutrition | Immunology and Infectious Disease | Molecular, Genetic, and Biochemical Nutrition

Comments This accepted article is published as Hice, S.A.,Clark, K.D., Anderson,J.L., Brehm-Stecher,B.F., Capture, Concentration and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification. Analytical Chemistry, 2018. DOI: 10.1021/acs.analchem.8b04751. Posted with permission.

This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/fshn_ag_pubs/215 Subscriber access provided by Iowa State University | Library Article Capture, Concentration and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification Stephanie A. Hice, Kevin D. Clark, Jared L. Anderson, and Byron F. Brehm-Stecher Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04751 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 14 Analytical Chemistry

1 2 3 4 5 6 7 Capture, Concentration and Detection of Salmonella in Foods Using 8 Magnetic Ionic Liquids and Recombinase Polymerase Amplification 9 10 Stephanie A. Hice1‡, Kevin D. Clark2†‡, Jared L. Anderson2*, and Byron F. Brehm-Stecher1* 11 12 Department of Food Science and Human Nutrition1, Department of Chemistry2, Iowa State University, Ames, Iowa 50011, 13 United States 14 15 ABSTRACT: We have previously investigated the extraction and concentration of bacteria from model systems using magnetic ionic 16 liquid (MIL) solvents, while retaining their viability. Here, we combine MIL-based sample preparation with isothermal amplification 17 and detection of Salmonella-specific DNA using Recombinase Polymerase Amplification (RPA). After initial developmental work 18 with Serratia marcescens in water, Salmonella Typhimurium ATCC 14028 was inoculated in water, 2% milk, almond milk or liquid 19 egg samples and extracted using one of two MILs, including: trihexyl(tetradecyl)phosphonium cobalt(II) hexafluoroacetylacetonate + – + – 20 ([P66614 ][Co(hfacac)3 ]) and trihexyl(tetradecyl)phosphonium nickel(II) hexafluoroacetylacetonate ([P66614 ][Ni(hfacac)3 ]). Viable 21 cells were recovered from the MIL extraction phase after the addition of modified LB broth, followed by a 20 min isothermal RPA 22 assay. Amplification was carried out using supersaturated sodium acetate heat packs and results compared to those using a 23 conventional laboratory thermocycler set to a single temperature. Results were visualized using either gel electrophoresis or nucleic 24 acid lateral flow immunoassay (NALFIA). The combined MIL-RPA approach enabled detection of Salmonella at levels as low as 103 CFU mL-1. MIL-based sample preparation required less than 5 min to capture and concentrate sufficient cells for detection using 25 RPA, which (including NALFIA or gel-based analysis) required approximately 30 - 45 min. Our results suggest the utility of MILs 26 for the rapid extraction and concentration of pathogenic microorganisms in food samples, providing a means for physical enrichment 27 that is compatible with downstream analysis using RPA. 28 29 30 31 Introduction: Salmonella is a ubiquitous, gram-negative to denature and separate duplex DNA (dsDNA), RPA 32 bacterium that is widespread in the environment and is a accomplishes this at lower temperatures using an , 7 33 contaminant in various foods, food ingredients, and in industrial recombinase. While RPA maintains several similarities to food processing environments.1 Infection typically results from PCR, including exponential amplification of target sequences, 34 ingestion of tainted food products, including consumption of a major difference between the two detection methods is the 35 contaminated poultry, eggs and dairy products. The Centers for temperature profile used for amplification. RPA is an 36 Disease Control and Prevention (CDC) estimates that isothermal process, which obviates the need for a thermal 37 nontyphoidal Salmonella spp. are responsible for 1.2 million cycler, allowing use of simple, small and inexpensive heating 38 cases of illness, 19,000 hospitalizations and nearly 380 deaths devices. The optimal temperature of RPA has been reported to 39 in the United States annually, resulting in an economic burden be 37 – 42 °C, but amplification of specific products has been 40 greater than 3.4 billion USD.2 Therefore, rapid, streamlined and demonstrated at temperatures ranging from 25 – 45 °C.8 41 field-deployable methods for detection of Salmonella spp. and Suitable product amplification can be achieved in less than 20 42 other foodborne pathogens are crucial for ensuring the safety min with RPA, as time-consuming ramping between separate 43 and quality of the foodstream. denaturation, annealing and extension temperatures is not ,9 44 Current detection techniques used by the required. Together, these attributes make RPA advantageous include standard culture methods and polymerase for use in resource-poor environments. 45 chain reaction (PCR), due to the selectivity, reliability and Although RPA products can be 46 regulatory acceptance of these techniques. However, culture- visualized using gel electrophoresis, gels require specialized 47 based methods for Salmonella may require between several equipment and are time-consuming, taking upwards of 35 min 48 days to more than a week, depending on the sample.3,4 To to get results. As an alternative, disposable, colorimetric lateral 49 address these time-to-result issues, the food industry relies PCR flow devices can be used for rapid (<10 min) amplicon 50 as a means for rapid screening of food samples for detection. These paper- or nitrocellulose-based devices rely on 51 contamination. Although PCR has gained wide acceptance in capillary action and therefore have no requirement for power. 52 the food industry over the past 20 years, the thermal cyclers Nucleic acid lateral flow immunoassays (NALFIA) like those 53 needed for this approach can be expensive and most systems used in this work with RPA are typically based on a sandwich- 5 10 54 remain bench-bound or have limited portability. type assay. Like RPA itself, these NALFIA devices are Recently, recombinase portable, economical and simple to use outside of laboratory 55 polymerase amplification (RPA) has demonstrated promise as settings – characteristics that make them ideal for use in the 56 an alternative means for the rapid detection of microbial field. While nucleic acid 57 pathogens and viruses.6 Unlike PCR, which relies on high heat amplification techniques are highly specific, their successful 58 59 60 ACS Paragon Plus Environment Analytical Chemistry Page 2 of 14

use in foods may be limited by low pathogen levels.11 Without Experimental Section 1 a suitable means for capture, concentration and purification of 2 bacteria prior to downstream analysis (“extraction” in purely Magnetic Ionic Liquids: The structures of the three MIL 3 chemical terms), the detection of pathogens in contaminated solvents examined in this work are shown in Fig. 1, panel a. 4 food samples may suffer from lack of reproducibility or from Synthesis and characterization of the MILs was performed as 15 5 poor sensitivity due to inhibitory substances carried over from previously described. MIL solvents were purified by liquid- liquid extraction with acetonitrile/hexane and dried in vacuo. 6 the sample matrix. Cultural enrichment is commonly used prior to detection, allowing dilution of food matrix-associated assay MILs were kept for long-term storage in capped glass vials and 7 inhibitors, recovery of stressed or injured cells and generation MILs were stored in a dessicator for at least 24 h prior to use. 8 of a detectable threshold of cells. However, key drawbacks 9 include increased assay time, outgrowth of target organisms by Bacteria and Culture Conditions: Serratia marcescens 10 competitive microflora and loss of information on initial (originally from Carolina Biological Supply Company, 11 pathogen load.3 While filtration- and Burlington, NC, USA) was sourced from a teaching lab at Iowa 12 centrifugation-based sample preparation techniques can enable State. Salmonella enterica subspecies enterica ser. 13 physical enrichment of cells, clogging of filters or co-isolation Typhimurium ATCC 14028 and Escherichia coli ATCC 25922 14 of particles or debris that may interfere with assay performance were from the American Type Culture Collection (ATCC, 11 Manassas, VA, USA). Overnight cultures (10 mL) of S. 15 can be problematic. Magnetic techniques can also be used for marcescens were grown at 25 °C in 250 mL glass Erlenmeyer 16 capture, concentration and purification of cells from complex food matrices, including the use of magnetic microbeads or flasks containing Luria Bertani (LB) broth (Becton, Dickinson 17 nanoparticle substrates functionalized with pathogen-specific and Company [BD], Franklin Lakes, NJ, USA) supplemented 18 antibodies. Other means for functionalizing substrates for cell with 1% (wt/vol) glycerol to accelerate production of the red 19 capture include the use of cationic charge, which is non- pigment prodigiosion.21. Flasks were incubated with shaking at 20 selective, or those that depend on semi-selective cell-ligand 190 rpm in a Shel Lab Shaking Incubator (Sheldon 21 interactions, such as lectins, antimicrobial peptides or Manufacturing, Inc., Cornelius, OR, USA). S. Typhimurium 22 antibiotics.12 In these approaches, functionalized magnetic and E. coli were grown in 14 mL polystyrene round-bottom 23 beads or nanoparticles are dispersed throughout a sample slurry tubes (Corning Inc., Corning, NY, USA) containing 10 mL 24 where they encounter and bind to bacteria. A magnetic field is Tryptic Soy Broth (TSB) (BD), and incubated at 37 °C. 11 25 then applied for physical isolation of the cell-enriched sorbent. Organisms were enumerated using Tryptic Soy Agar (TSA) plates (BD). 26 Drawbacks to particulate magnetic sorbents such as microbeads may include aggregation, diffusion- or suspension-based 27 limitations or poor access to microscopic physical niches where Pasteurized Liquid Food Products: Two-percent milk (Hy- 28 bacteria may be present. These issues may result in lower Vee Reduced Milk), almond milk (Hy-Vee All Natural 29 extraction efficiencies and/or clogging of microfluidic systems. Original) and a liquid egg product (Hy-Vee 99% Real Egg) 30 These problems can be addressed, but not completely avoided, were purchased from a local grocery store (Hy-Vee, Ames, IA) 31 through the use of functionalized nanoparticles.11 for evaluation of MIL-based capture in liquid food products. All 32 foods were evaluated before the “Sell by”, “Best if used by” or 33 Magnetic ionic liquids (MILs) are paramagnetic “Use by” dates listed on their packaging. 34 molten salts comprised of organic/inorganic cations and anions that exhibit melting points at or below 100 °C. Similar to MIL-Based Extraction of Viable Bacteria: A representative 35 schematic for the MIL-based extraction of bacteria is depicted 36 conventional ionic liquids (ILs), MILs possess negligible vapor pressures at ambient temperatures and tunable physicochemical in Fig. 1, panel b. A 1 mL aliquot of diluted cell suspension, 37 properties including viscosity, solvent miscibility, and solvation artificially spiked milk, almond milk or liquid egg product was 38 capabilities.13,14,15 Owing to their tunable chemical structures added to a 2 mL or 4 mL screw cap glass vial. A small volume 39 and susceptibility to magnetic fields, MILs have been applied of MIL (e.g., 15 µL) was added and vortexed vigorously for 30 40 for the analysis of hormones in biological fluids,16 acidic s to create a cell-capturing microdroplet dispersion. With some 41 pharmaceuticals and endocrine disrupters,17 and the extraction samples (e.g. the egg product, due to viscosity and foaming), a 42 and preservation of nucleic acids.18,19 Very recently, MILs were magnet was applied externally to concentrate the cell-enriched 43 also investigated as solvents for the preconcentration of viable MIL, although this step was not necessary with some samples, as the hydrophobic, denser-than-water MIL droplets were able 44 bacteria for culture and PCR-based detection.20 By dispersing to sink to the bottom of the extraction vial. After gravity-based 45 the hydrophobic MIL in an aqueous suspension of Escherichia coli K12, viable cells were rapidly extracted and concentrated deposition or magnetic extraction, the aqueous phase was then 46 discarded and the MIL was subjected to a brief wash step using 47 for downstream analysis using qPCR. However, the ability of MIL solvents to extract industrially-relevant foodborne 1 mL of nuclease-free water (Integrated DNA Technologies, 48 pathogens, such as Salmonella Typhimurium, in complex food Coralville, IA, USA), to ensure adequate removal of residual 49 matrices was not tested. In this study, we report development of bacteria that were not captured by the MIL microdroplets. 50 a method for the preconcentration and detection of S. Recovery of viable cells from the MIL extraction phase was 51 Typhimurium that capitalizes on the rapid and cell-compatible achieved through a “back-extraction” step that involved 52 extraction capabilities of MIL solvents and the portability, vortexing the cell-enriched MIL in 1 mL or 200 µL of a nutritive 53 simplicity and rapidity of a RPA detection platform. medium comprised of tryptone (20 g/L; “2x tryptone”) and NaCl (10 g/L; “1x NaCl”) for 2 min. After back-extraction, 54 captured bacteria were detected using microbiological culture 55 or RPA. Prior to RPA, the cell-enriched back-extraction media 56 was heated at 100 °C for 10 min for cell lysis and release of 57 target nucleic acids. The MIL-RPA method was compared to a 58 59 60 ACS Paragon Plus Environment Page 3 of 14 Analytical Chemistry

commercial nucleic acid sample preparation approach using the amplification. Sample tubes were inverted vigorously 10 times, 1 PrepMan Ultra Sample Preparation Reagent (PMU; Life vortexed for 10 s, followed by centrifugation for 5 s to draw the 2 Technologies, Carlsbad, CA, USA) according to the sample to the base of the tube. This mixing process was 3 manufacturer’s instructions. repeated after 4 min of incubation, and after completion of 4 incubation. For both the TwistAmp Basic and TwistFlow 5 Plating and Enumeration: Following back-extraction, Salmonella kits, reactions were incubated using a GeneAmp aliquots of the cell-enriched modified LB media were serially 6 PCR System 9700 (Applied Biosystems, Foster City, CA, USA) diluted in 0.1% peptone water. A 10 µL aliquot of each dilution at 40 °C for 20 min. S. Typhimurium was tested for each RPA 7 was applied to the appropriate lane on square, gridded TSA kit, with E. coli serving as a negative control. 8 plates as described previously.22 The track plates were then 9 tilted to an angle of~80° for 15 min to allow the deposited liquid Gel Electrophoresis: After heating, RPA products generated 10 to travel toward the opposite end of the plate. Plates were using the TwistAmp Basic kit were mixed with 10 µL of 6X 11 incubated for 48 h at 25 °C (S. marcescens) or for 24 h at 37 °C bromophenol blue/xylene cyanol FF loading dye and loaded on 12 (S. Typhimurium). Colonies were manually counted for a 1% agarose gel stained with either SYBR Safe DNA Gel Stain 13 determination of the number of CFUs in each sample. The (Thermo Fisher Scientific) or GelRed (Biotium, Fremont, CA, 14 enrichment factor (EF) for the MIL-based method was USA). Using 1X TBE as the running buffer, samples were 15 calculated using Equation 1, where CMIL represents the electrophoresed using a Mupid-2Plus Submarine concentration of bacteria in suspension following MIL-based Electrophoresis System (Mupid Co., Ltd., Tokyo, JP), for 35 16 extraction and CS is the concentration of bacteria in the initial min at 100 V. Bands were visualized using either a Safe Imager 17 sample. 2.0 Blue Light Transilluminator (Thermo Fisher Scientific), or 18 an Azure Biosystems c300 imaging system (Azure Biosystems, 19 CMIL Dublin, CA, USA) at 302 nm with a 20 s exposure time. EF = (1) 20 CS 21 Lateral Flow Assay: Single-tube amplification products Recombinase Polymerase Amplification: RPA TwistAmp 22 generated using the TwistFlow Salmonella kit were added Basic and TwistFlow Salmonella were purchased from 23 directly to a nucleic acid lateral flow immunoassay (NALFIA) TwistDx (Cambridge, UK). RPA was carried out according to disposable cartridge (Ustar Biotechnologies (Hangzhou) Ltd., 24 the manufacturer’s instructions and results were visualized 25 Hangzhou, CN). The NALFIA relies on visual detection of a using either gel electrophoresis (TwistAmp Basic kit) or a test band facilitated by the extension of biotin and 6- 26 chromatographic lateral flow assay (TwistFlow Salmonella carboxyfluorescein (6-FAM) labeled primers during RPA. The 27 kit). Using the TwistAmp Basic kit, a 340 bp region of the amplification product is visible by eye as a result of aggregation 28 putative dienelactone hydrolase gene (DLH) was amplified of streptavidin-conjugated gold nanoparticles, which bind to the 29 using the following primers: (Forward primer) 5’-GCC GGG biotin-labeled 5’ end of the double-stranded amplicon. The 30 CAG CRA TTA TTC TGC ATG AA-3’and (Reverse primer) terminal 6-FAM group of the amplicon is also selectively 31 5’-TGG CGT ATA CGG GAA CCG TAA TAG CA-3’. An in captured by the anti-FAM antibody, which is embedded in the silico analysis of this primer set using the Primer-Blast tool 32 test line on the lateral flow strip. A control line consisting of (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi)23 33 biotin-conjugated BSA exhibits strong affinity for any indicated that within the Salmonella enterica I database (NCBI remaining streptavidin-conjugated gold nanoparticles and can 34 taxonomy ID: 59201), the primer set matched several 35 be visualized for a valid assay. Generation of a red band at the subspecies I serotypes, including the top three disease-causing test position indicates successful amplification of the double- 36 serotypes identified in the most recent Centers for Disease stranded product, sandwiched between the bound anti-FAM 37 Control and Prevention (CDC) Salmonella Surveillance antibody and the streptavidin-conjugated gold nanoparticles. A 24 38 Report. These include S. Enteritidis (225 hits within the S. positive result was recorded if both the control and test bands 39 enterica I database), S. Typhimurium (57 hits) and S. Newport were identified within 10 min, whereas detection of only the 40 (21 hits). Because the Primer-Blast software does not control band indicated a negative result. 41 accommodate degenerate bases (the DLH primer set contains an “R”, which indicates either an “A” or a “G” in this position), 42 Results and Discussion it is expected that additional Salmonella serovars will be 43 detected with this primer set. An invA target, using primers Improved MIL Extraction Conditions for Gram-Negative 44 described by Liu et al. was also investigated for use in gel Bacteria: To begin our investigation of the MIL capture 45 electrophoresis-based experiments.25 For both assays, primers process for bacteria other than the previously-reported E. coli 46 were diluted with nuclease free water from a 100 µM stock of K-12, we selected the non-pathogenic Serratia marcescens. S. 47 mixed primers to a working concentration of 10 µM. Because marcescens, a gram-negative bacterium in the same family as 48 the DLH primer set resulted in higher amplicon production, it Salmonella, produces the reddish-orange pigment prodigiosin, 49 was used in subsequent experiments. allowing its unambiguous visual detection when concentrated 50 For the TwistFlow Salmonella kit and facilitating its use as a model gram-negative bacterium in 51 (also targeting the invA gene), a master mix containing primer development of pre-analytical sample preparation methods.26 52 in rehydration buffer and nuclease-free water was prepared. Since the chemical structure of the MIL has profound Sample DNA was obtained from the MIL back-extraction, or 18,27 53 implications on its extraction behavior, three MILs were using the PMU approach as per manufacturer’s instructions. For studied for bacterial extraction (Fig. 1a). By vigorously 54 each kit, master mix, plus 1 µL of sample DNA was added to dispersing a small volume of MIL (e.g., 15 µL) in an aqueous 55 the lyophilized RPA reagents contained in a PCR tube, where suspension of S. marcescens (1×103 CFU mL−1), cells were 56 the entire volume was mixed using a pipette. Following this, 2.5 extracted into the resulting MIL microdroplets and cell- 57 µL of 280 mM magnesium acetate was added to initiate enriched microdroplets were harvested based on either MIL 58 59 60 ACS Paragon Plus Environment Analytical Chemistry Page 4 of 14

density (~1.3 g mL-1 for the Ni(II) MIL) or through 1 manipulation with an external magnetic field (Fig. 1b). 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Figure 1. (a) Structures of MILs evaluated in this study; (b) Figure 2. Recovery of S. marcescens extracted with Co(II) MIL 16 Schematic for the extraction and preconcentration of Salmonella as a function of back-extraction medium composition. 17 Typhimurium from aqueous samples, followed by downstream Percentage of initial cell load recovered from the Co(II) MIL 18 analysis using RPA amplification and microbiological culture extractant using aqueous back-extraction media of different ionic 19 detection methods. Panel b adapted from Clark et al., 2017.20 composition is shown. A suspension of S. marcescens was prepared 20 and captured with the Co(II) MIL as described in the text, then back-extracted into water (control), LB medium or 7 variations of 21 After the cell-enriched MIL was rinsed with deionized the basic LB medium recipe. Back-extraction media used: 1X YE 22 -1 -1 water, bacteria were recovered from the extraction phase using (5 g L yeast extract); 2x YE (10 g L yeast extract); 1x T (10 g 23 -1 -1 -1 Luria-Bertani nutrient broth (LB, per L: 10 g tryptone, 5 g yeast L tryptone); 2x T (20 g L tryptone); 1x T, 1x YE (10 g L 24 tryptone, 5 g L-1 yeast extract); 2x T, 2x YE (20 g L-1 tryptone, 10 extract, 10 g NaCl), plated, and incubated at 25 °C for 48 h prior 25 g L-1 yeast extract); 2x T, 1x NaCl (20 g L-1 tryptone, 10 g L-1 to enumeration. Of the three MILs studied, the Ni(II) and Co(II) NaCl). Average cell recoveries for three separate experiments are 26 MILs yielded comparable colony forming units (CFUs), 27 shown. Inset: Representative cell growth on track plates obtained whereas no growth was detected after extraction using the following the extraction of S. marcescens using Ni(II), Co(II) and 28 Dy(III) MIL (Fig. 2, inset). Despite possessing identical cation Dy(III) MILs. + 29 moieties ([P66614 ]) and ligands (hfacac), the extraction of S. 30 marcescens by MILs strongly depended on the identity of the 31 metal component. This phenomenon has also been observed Next, we investigated our MIL-based method for the 32 when using similar MIL solvents for the preconcentration of 28 preconcentration of S. Typhimurium, using the 2x T, 1x NaCl 33 nucleic acids from aqueous solution. back-extraction medium. As with S. marcescens, similar To maximize the recovery of viable bacteria 34 recoveries of viable Salmonella were observed for the Ni(II) from the MIL extraction phase, several LB-based back- and Co(II) MILs, resulting in enrichment factors of 35 extraction media varying in ionic strength and nutrient 36 approximately 12, which is comparable to previous enrichment composition were investigated (Fig. 2). For these experiments, factors for the MIL-based extraction of E. coli.20 The work 37 3 −1 suspensions of S. marcescens (1×10 CFU mL , in 1 mL 0.1% reported here represents the first use of MILs as solvents for the 38 peptone) were vortexed for 30 s with 15 µL of the Co(II) MIL, preconcentration of viable pathogenic bacteria from aqueous 39 then resuspended for a 2 min back-extraction into 1 mL of either suspensions. Because Ni(II) and Co(II) results were similar, 40 water (control), LB medium or 7 variations of the basic LB subsequent experiments toward coupling MIL-based bacterial 41 medium recipe. Back-extraction using deionized water resulted extraction with molecular detection using RPA were performed in the lowest quantity of bacteria recovered from the MIL while + - 42 using the [P66614 ] [Ni(hfacac)3 ] MIL. 43 the greatest quantity of cells was obtained with a nutrient-rich tryptone medium supplemented with NaCl (2x T, 1x NaCl, Fig. 44 Combining MIL-based Extraction with Salmonella- 2). Back-extracted samples were diluted 100-fold prior to 45 Targeted RPA: In an effort to identify improved approaches plating to ensure that countable dilutions within the statistically for food analysis, we investigated the feasibility of coupling our 46 valid range of 25 – 250 CFU were obtained. Our results show 47 MIL-based method for capture and concentration of S. that S. marcescens cells were physically enriched by the MIL to Typhimurium from aqueous media with the speed and 48 levels between 5 and 6 times higher than their initial simplicity of RPA analysis. Briefly, after preconcentration and 49 concentration. Apart from the higher ionic strength of the best recovery of S. Typhimurium from an aqueous sample using the 50 back-extraction media, which has previously been shown to Ni(II) MIL, the back-extraction suspension was heated for 10 20 51 assist in the recovery of gram-negative bacteria, it is min at 100 °C to lyse the bacteria and release their nucleic acids 52 conceivable that the hydrophobic MIL solvent imposes stress for downstream RPA analysis. Initially, we compared two 53 on the cell in a process that is attenuated by transferring the primer sets for the amplification of nucleic acids from S. bacteria to a supportive nutrient media.29 Because the 2x T, 1x 54 Typhimurium: primers described by Liu et al.25 targeting the NaCl back-extraction medium provided the highest recoveries 55 invA gene, which codes for a involved in invasion of for S. marcescens, it was selected for use in subsequent host intestinal epithelia by Salmonella, and primers identified 56 experiments. 57 via comparative genomic analysis and amplifying a 340 bp 58 59 60 ACS Paragon Plus Environment Page 5 of 14 Analytical Chemistry

region of a putative dienelactone hydrolase gene (DLH, this amplification times. Temperatures at various points along the time- 1 study). Although both primer sets successfully amplified DNA temperature curve are 25.2 °C (initial temperature, point a), 45.4 2 from the bacteria recovered from the MIL phase, gel °C (point b), 43.7 °C (point c) and 42.1 °C (point d). Righthand 3 electrophoresis results demonstrated that the DLH-targeted panel: Comparison of DLH-RPA reactions driven with a thermal 4 primers yielded bands with higher fluorescence intensities (Fig. cycler (TC) or with sodium acetate heat packs (SA), using bacterial S1). As a control, E. coli ATCC 25922 (105 CFU mL-1) was DNA obtained using the PrepMan Ultra Sample Preparation 5 Reagent (PMU) or via MIL-based extraction (MIL). 6 extracted using the Ni(II) MIL, lysed and examined via DLH- based RPA. No amplicon was detected, indicating good 7 selectivity of the DLH primers for Salmonella (Fig. S1). 8 Comparison of Methods for Amplicon Detection, Further 9 Evaluating Use of a Power-free Heat Source for Salmonella Improvement of MIL Approach: All elements of a detection 10 -Targeted RPA: A major limitation of many nucleic acid assay (cell capture, release of nucleic acids, amplification of 11 amplification methodologies is their reliance on electricity to target DNA and product detection) may impact the quality of power a heat source such as a thermal cycler in PCR or a heat 12 the final result. With this in mind, we evaluated different block for isothermal methods. In an effort to circumvent this 13 methods for nucleic acid release in conjunction with further limitation, we examined supersaturated sodium acetate heat 14 improvement of the MIL-based workflow (larger sample vial packs - a small, portable consumer-grade novelty product used 15 size, smaller back-extraction volume) and two approaches for in handwarmers and earmuffs (Cristalheat, www.xUmp.com) - 16 amplicon detection (gel electrophoresis, nucleic acid lateral as a power-free means for driving RPA. In initial work, we flow immunoassay [NALFIA]). For direct comparison of 17 measured the internal temperature of a template-free RPA methods for release of nucleic acids prior to RPA, we compared 18 reaction tube sandwiched between two activated heat packs 19 using an OPTOCON FOTEMP1-4 fiber optic temperature heating of the cell-enriched MIL to 100C (with modifications, 20 monitoring system (Optocon AG, Dresden, Germany). Data as described below) with use the commercial reagent PMU. For 21 were collected using the FOTEMP Assistant software, exported both approaches, aqueous samples were inoculated with S. Typhimurium at concentrations ranging from 103 to 106 CFU 22 to Microsoft Excel and plotted in Prism graphing software mL-1, followed by DLH-targeted RPA. 23 (Prism 7 for Mac OS X, v. 7.0d, GraphPad Software, La Jolla, CA) (Fig. 3). The temperature of the RPA mixture increased The PMU method was used according to the 24 manufacturer’s instructions. Briefly, cells from a liquid 25 rapidly after heat pack activation, reached equilibrium between 42 °C and 44 °C (Fig. 3a) and remained within optimal RPA suspension were lysed in 200 µL of PMU reagent, followed by 26 temperature range for up to 40 min. We then tested the heating (15 min, 100C) and centrifugation to separate cellular 27 performance of heat pack-driven RPA using the DLH primer debris from the DNA-containing supernatant. The PMU method 4 -1 28 set and MIL-extracted S. Typhimurium. Intense amplicon bands facilitated consistent detection down to 10 CFU mL , while 29 were seen for sodium acetate-driven DLH-RPA with DNA detection at lower levels was inconsistent (Fig. 4 gel image, lane 30 isolated using either the PrepMan Ultra Sample Preparation 1). PMU was not evaluated for lysis of MIL-captured cells, as 31 Reagent (PMU-SA) or with MIL-extracted cells (MIL-SA) this would have introduced additional assay elements, namely 32 (Fig. 3b). Although a band was seen with the sodium acetate- use of a chemical lysis reagent and a centrifugation step. Based on observations of liquid behavior during vortexing, we 33 heated no-template control (NTC-SA), it is expected that use of lateral flow-based detection would enable differentiation of hypothesized that slight modifications to our established MIL- 34 RPA approach might result in process improvements. By 35 legitimate amplicons from spurious NTC bands sometimes seen 30,31 decreasing the sample vial size from 4 mL to 2 mL and 36 on agarose gels with RPA These results highlight the utility of sodium acetate heat packs as a viable, power-free means for decreasing the back-extraction volume from 1 mL to 200 µL, 3 -1 37 amplifying nucleic acids from microbial pathogens using RPA. we found we could lower our detection limit to 10 CFU mL , 38 presumably due to enhanced contact between the MIL and back 39 extraction solution when a smaller sample vial was used and the 40 increased concentration of bacteria in the smaller back- 41 extraction volume (Fig. 4 gel image, lane 3). These slight 42 modifications enabled us to improve the sensitivity of our streamlined “capture, concentrate, heat and amplify” MIL-RPA 43 process, without the addition of further assay elements. 44 Without these process 45 modifications, the limit of detection for our MIL-RPA approach 46 was 104 CFU mL-1 for gel electrophoresis-based visualization of 47 Salmonella DLH amplicons. 48 49 Figure 3. Evaluation of sodium acetate heat pack for power- 50 free incubation of RPA reactions. In order to assess the utility of 51 sodium acetate heat packs (inset) for portable and power-free 52 incubation of RPA reactions, we measured the internal temperature 53 of a template-free RPA reaction tube sandwiched between two 54 activated heat packs using a fiber optic temperature monitoring 55 system as described in the text. Lefthand panel: Sodium acetate heat packs were able to provide near-optimal RPA reaction 56 temperatures (reported range, 37 °C – 42 °C) over typical 57 58 59 60 ACS Paragon Plus Environment Analytical Chemistry Page 6 of 14

centrifuge that is incompatible with pathogen analysis in the 1 field or in resource limited settings. 2 3 MIL-RPA for detection of Salmonella in Liquid Food 4 Samples: In order to examine the application of the combined 5 MIL-RPA method in a practical setting, it was applied for the 6 detection of S. Typhimurium in food samples including milk 7 (2% milk fat) and almond milk. S. Typhimurium cells were inoculated into 1 mL samples at 105 CFU mL-1 and extracted 8 using the Ni(II) MIL under improved conditions. The combined 9 approach enabled detection of S. Typhimurium at 105 CFU mL-1 10 (Fig. 5a). The PMU method did not consistently detect S. 11 Typhimurium in spiked 2% milk samples. To explore whether 12 dilution of samples (and of potentially interfering substances 13 such as and ) might lead to improvements in both 14 PMU and MIL samples, samples were diluted with 0.1% 15 peptone water (PW) in some experiments. Interestingly, and for 16 Figure 4. MIL-RPA with Ni(II) MIL. The combined MIL and unknown reasons, PW appeared to reduce the efficacy of both 17 RPA approach was visualized using gel electrophoresis (35 min) or PMU extraction of cell DNA and MIL-based cell capture, with the diluted MIL sample not yielding a detectable band (Fig 5a, 18 lateral flow (10 min). MIL-based extraction was compared with concurrent extraction using PrepMan Ultra Sample Preparation lane 6). These results suggest examination of alternate diluents 19 Reagent (PMU). For gel electrophoresis (lefthand panel), PMU (i.e. molecular-grade water) in future experiments to explore 20 LOD was consistently identified as 104 CFU mL-1, however, bands whether such inhibition may be avoided and if a more 21 were inconsistently present at lower levels (lane 1), or were less appropriate dilution medium may represent a viable approach 22 defined than those for the Ni(II) MIL at the same level (lane 3). for reducing sample complexity, if needed. Because 23 Lanes 4, 5 represent no template controls (NTC). With lateral flow Salmonella spp. have been especially problematic in eggs, with 4 - 24 (right-hand panel) detection was achieved as low as 10 CFU mL large outbreaks occurring in 2010 (almost a half a billion eggs 1 for both PMU (strip 1) and the MIL (strip 2). 25 recalled)32 and 2018 (almost 207 million eggs recalled)33, the 26 MIL-based preconcentration method was applied to liquid egg 27 samples spiked with concentrations of S. Typhimurium ranging Because conventional gel electrophoresis is inherently from 103 to 105 CFU mL-1. The foamy nature of the liquid egg 28 laboratory-bound and time consuming, we sought to evaluate sample initially caused challenges in recovering a sufficient 29 the use of a rapid, portable alternative for amplicon detection. volume of MIL for downstream detection bacteria. However, 30 Due to its simplicity and portability, NALFIA is often used in ~1 min exposure of the sample to a 0.66 T rod magnet to the 31 resource-limited environments, or in non-laboratory settings,10 base of the 2 mL glass vial facilitated collection of the cell- 32 as it does not require electricity or laboratory equipment. Using enriched MIL solvent, enabling detection limits as low as 104 33 the Ni(II) MIL for preconcentration and extraction of aqueous CFU mL-1 (Fig. 5b). The commercial PMU method was 34 samples of S. Typhimurium at concentrations ranging from 103 concurrently compared to the MIL-based approach and 6 -1 35 to 10 CFU mL , RPA was carried out using the TwistFlow exhibited an identical detection limit using RPA and gel 36 Salmonella kit from TwistDx. Test results were determined electrophoresis. However, the PMU method resulted in less using a 5 min NALFIA step. Initially, the combined MIL-RPA- 37 intense bands than those from the MIL-based extraction method NALFIA approach facilitated detection at levels as low as 105 (Fig. 5b). The Ni(II) MIL was also applied for preconcentration 38 -1 CFU mL . Since the detection limits using NALFIA were 2 log of S. Typhimurium in liquid egg samples coupled to RPA and 39 higher than with gel electrophoresis, we investigated whether NALFIA readout. The combined MIL-RPA-NALFIA approach 40 metal ions released from the hydrophobic MIL phase (e.g., facilitated detection at levels as low as 104 CFU mL-1 in 41 Ni2+)28 during back-extraction had any influence on the inoculated liquid egg samples. Once again, this method was 42 outcome of the NALFIA step. For these experiments, PMU concurrently compared with the use of PMU, which maintained 43 samples were spiked with levels of NiCl2 ranging from 0.2 mM similar detection levels (Fig. 5c) but required a centrifugation 44 to 2 mM. Visible control and test bands were observed for all step that is not compatible with field sampling or on-site 2+ 45 NiCl2 samples, suggesting that Ni potentially released from analysis. 46 the MIL did not inhibit the NALFIA. It is important to note that the NALFIA targets the invA gene, using primers and 47 conditions developed by the manufacturer. Our choice of the 48 dienelactone hydrolase gene target for the gel electrophoresis 49 experiments may also have contributed to differences in 50 observed detection limits due to differences in amplification 51 efficiency between the two primer sets. In order to improve 52 detection limits, the back-extraction volume was decreased to 53 200 µL, resulting in detection limits for S. Typhimurium as low 54 as 103 CFU mL-1 with the Ni(II) MIL (Fig. 4). The PMU method 4 55 provided detection of Salmonella at levels as low as 10 CFU -1 56 mL (Fig. 4), but once again requires the use of a benchtop 57 58 59 60 ACS Paragon Plus Environment Page 7 of 14 Analytical Chemistry

characteristics useful for the analysis of aqueous foods or food 1 suspensions. Unlike other recently reported MILs, which have 2 reported room temperature densities that are on par with that of 3 water34, the Ni(II) and Co(II) MILs used here had densities of 4 ~1.3 g mL-1, which lie between the densities of glycerol (1.26 g -1 -1 5 mL ) and corn syrup (~1.4 g mL ). Because the MILs used in 6 this study are both hydrophobic and denser than water, they are well-suited for analysis of aqueous solutions such as liquid 7 foods or food suspensions, and can be collected through either 8 simple density-based sedimentation or with application of an 9 external magnet. For automated and high-throughput 10 applications in the food industry, it is possible that use of a 11 strong electromagnet for post-extraction collection of MILs 12 could result in fast and uniform capture of cell-enriched MILs. 13 It may also be possible to minimize the costs and environmental 14 impacts of high-throughput use of MILs in food testing by 15 developing methods capable of recycling MILs for multiple 16 rounds of cell capture. The approach described here bridges the disciplines of food science, materials science and 17 chemistry, providing new tools for rapid and efficient extraction 18 of viable cells in support of pathogen detection efforts. Future 19 work will focus on extending MIL-RPA to additional foodborne 20 pathogens, including the gram-positive pathogen 21 monocytogenes and assessment of any negative impacts that 22 MIL-based capture may have on bacterial physiology, such as 23 injury or antimicrobial activity, with the development of 24 approaches for mitigation of these impacts, should they occur. 25 Figure 5. RPA-based detection of Salmonella Typhimurium in AUTHOR INFORMATION 26 liquid food products. The combined MIL and RPA approach was 27 applied to almond milk, milk (2% milk fat), 50:50 dilution of milk Corresponding Authors 28 (2% milk fat: 0.1% peptone water) and liquid egg product. Results *Byron F. Brehm-Stecher, Department of Food Science and 29 were visualized using gel electrophoresis (35 min) or lateral flow Human Nutrition, 536 Farm House Lane, Ames, IA 50011, USA 30 (10 min). MIL-based extraction was compared with concurrent Email: [email protected] extraction using PrepMan Ultra Sample Preparation Reagent 31 (PMU). For Salmonella-spiked almond milk (Panel a, lanes 1, 4), 32 *Jared Anderson, Department of Chemistry, Iowa State milk (2% milk fat) (Panel a, lanes 2, 5) and 50:50 milk dilution University, 1605 Gilman Hall, Ames, IA 50011, USA 33 samples (Panel a, lanes 3, 6) using gel electrophoresis and either Email: [email protected] 34 extraction method (PMU or MIL), detection was achieved at 105 35 CFU mL-1. For liquid egg samples spiked with S. Typhimurium Present Address 4 -1 36 (Panel b), PMU and MIL LOD was 10 CFU mL (Panel b, lanes †Kevin D. Clark: 5217 Beckman Institute, 405 North Mathews 3, 4). Using lateral flow (Panel c), for Salmonella-spiked liquid egg Avenue, Urbana, IL 61801 37 samples, LOD was identified 104 CFU mL-1 for both PMU (Panel 38 c, 3, 4) and MIL-extracted samples (Panel c, 5, 6). Strips 1 and 2 Author Contributions 39 are the no template control (NTC) and internal control, ‡ Stephanie A. Hice and Kevin D. Clark contributed equally to 40 respectively. this work. 41 42 ACKNOWLEDGMENT 43 Conclusions: We report further improvement of our previously We acknowledge Dr. Hyun Joong Kim of Kyung Hee University 44 described method for capture of gram-negative bacteria and the Department of Food Science and Biotechnology for development 45 first use of this approach for capture and concentration of a of the Salmonella-targeted PCR primer set used here for RPA. 46 human pathogen from aqueous suspensions, including at-risk BFBS acknowledges financial support from the Midwest Dairy foods. Post-capture growth on non-selective media suggested a Association (MDA), a NASA Iowa Space Grant Consortium 47 lack of cytotoxicity with this approach. We coupled capture and Fellowship (Award No. NNX16AL88H) to SAH, and Iowa 48 concentration of S. Typhimurium to RPA, a rapid isothermal Agriculture and Home Economics Experiment Station Project No. 49 method for DNA amplification, and found that we could drive IOW03902, sponsored by Hatch Act and State of Iowa funds. JLA 50 RPA reactions using inexpensive and regenerable sodium acknowledges funding from the Chemical Measurement and 51 acetate heat packs, eliminating the need for an external power Imaging Program at the National Science Foundation (CHE- 52 source. Salmonella RPA amplicons could be detected in <10 1709372). min using a simple chromatographic readout. Our approach is 53 REFERENCES 54 simple, streamlined and amenable to analyses in the field or in (1) Besser, J. M. Food Microbiol 2018, 71, 55-59. 55 other resource-limited environments. Apart from their magnetic (2) Hoffmann, S.; Maculloch, B.; Batz, M. 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(3) Brehm-Stecher, B.; Young, C.; Jaykus, L. A.; Tortorello, M. L. J (21) Haddix, P. L.; Jones, S.; Patel, P.; Burnham, S.; Knights, K.; 1 Food Prot 2009, 72, 1774-1789. Powell, J. N.; LaForm, A. J. Bacteriol. 2008, 190, 7453-7463. 2 (4) Agriculture, U. S. D. o. In National poultry improvement plan and (22) Jett, B. D.; Hatter, K. L.; Huycke, M. M.; Gilmore, M. S. auxiliary provisions, APHIS 91-55-088; National Poultry Improvement Biotechniques 1997, 23, 648-650. 3 Plan, Conyers, GA, 2009, pp 116-124. (23) Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; 4 (5) Brehm-Stecher, B. F.; Johnson, E. A. Rapid Methods for Detection Madden, T. L. BMC Bioinformatics 2012, 13, 134. 5 of Listeria; CRC Press: New York, 2007. (24) CDC, National Salmonella Surveillance Annual Report, 2016; 6 (6) Murinda, S. E.; Ibekwe, A. M.; Zulkaffly, S.; Cruz, A.; Park, S.; US Department of Health and Human Services, CDC: Atlanta, GA, 7 Razak, N.; Md Paudzai, F.; Ab Samad, L.; Baquir, K.; Muthaiyah, K. 2018. Foodborne Pathog. Dis. 2014, 11, 529-536. (25) Liu, H. B.; Zang, Y. X.; Du, X. J.; Li, P.; Wang, S. J Dairy Sci 8 (7) Li, J.; Macdonald, J. Biosens. Bioelectron. 2015, 64, 196-211. 2017, 100, 7016-7025. 9 (8) TwistDx. In BioTechniques, 2016, p 328. (26) Rossmanith, P.; Frühwirth, K.; Süss, B.; Schopf, E.; Wagner, M. 10 (9) Kersting, S.; Rausch, V.; Bier, F. F.; von Nickisch-Rosenegk, M. Lett Appl Microbiol 2010, 50, 230-233. 11 Microchimica Acta 2014, 181, 1715-1723. (27) Trujillo-Rodríguez, M. J.; Nacham, O.; Clark, K. D.; Pino, V.; (10) Posthuma-Trumpie, G. A.; Korf, J.; van Amerongen, A. Anal. Anderson, J. L.; Ayala, J. H.; Afonso, A. M. Anal. Chim. Acta 2016, 12 Bioanal. Chem. 2009, 393, 569-582. 934, 106-113. 13 (11) Soo, H. S., Brehm-Stecher, B.F, Jaykus, L.A., Sofos, J., Ed.; (28) Emaus, M. N.; Clark, K. D.; Hinners, P.; Anderson, J. L. Anal. 14 Woodhead Publishing: Cambridge, UK, 2013, pp 173-192. Bioanal. Chem. 2018, 410, 4135-4144. 15 (12) Dao, T. N. T.; Lee, E. Y.; Koo, B.; Jin, C. E.; Lee, T. Y.; Shin, Y. (29) Bhaganna, P.; Volkers, R. J.; Bell, A. N.; Kluge, K.; Timson, D. Anal Biochem 2018, 544, 87-92. J.; McGrath, J. W.; Ruijssenaars, H. J.; Hallsworth, J. E. Microb 16 (13) Clark, K. D.; Nacham, O.; Purslow, J. A.; Pierson, S. A.; Biotechnol 2010, 3, 701-716. 17 Anderson, J. L. Anal. Chim. Acta 2016, 934, 9-21. (30) Bentahir, M.; Ambroise, J.; Delcorps, C.; Pilo, P.; Gala, J. L. Appl 18 (14) Santos, E.; Albo, J.; Irabien, A. RSC Advances 2014, 4, 40008- Environ Microbiol 2018, 84. 19 40018. (31) Rohrman, B. A.; Richards-Kortum, R. R. Lab Chip 2012, 12, 20 (15) Pierson, S. A.; Nacham, O.; Clark, K. D.; Nan, H.; Mudryk, Y.; 3082-3088. Anderson, J. L. New J. Chem. 2017, 41, 5498-5505. (32) Allard, M. W.; Luo, Y.; Strain, E.; Pettengill, J.; Timme, R.; Wang, 21 (16) Merib, J.; Spudeit, D. A.; Corazza, G.; Carasek, E.; Anderson, J. C.; Li, C.; Keys, C. E.; Zheng, J.; Stones, R.; Wilson, M. R.; Musser, 22 L. Anal. Bioanal. Chem. 2018, 1-11. S. M.; Brown, E. W. PLoS One 2013, 8, e55254. 23 (17) Chatzimitakos, T.; Binellas, C.; Maidatsi, K.; Stalikas, C. Anal. (33) CDC. https://www.cdc.gov/salmonella/braenderup-04- 24 Chim. Acta 2016, 910, 53-59. 18/index.html, 2018. (18) Clark, K. D.; Nacham, O.; Yu, H.; Li, T.; Yamsek, M. M.; (34) Santos, E.; Albo, J.; Rosatella, A.; Afonso, C. A. M.; Irabien, Á. 25 Ronning, D. R.; Anderson, J. L. Anal. Chem. 2015, 87, 1552-1559. Journal of Chemical Technology & Biotechnology 2014, 89, 866-871. 26 (19) Clark, K. D.; Sorensen, M.; Nacham, O.; Anderson, J. L. RSC 27 Adv. 2016, 6, 39846-39851. 28 (20) Clark, K. D.; Purslow, J. A.; Pierson, S. A.; Nacham, O.; 29 Anderson, J. L. Anal. Bioanal. Chem. 2017, 409, 4983-4991. 30 Insert Table of Contents artwork here 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 9 of 14 Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Magnetic ionic liquid (MIL)-based extraction and concentration of Salmonella from liquid foods was combined 26 with rapid detection of this pathogen using Recombinase Polymerase Amplification (RPA). Our results 27 demonstrate the utility of MILs for pre-analytical sample preparation of foods in support of rapid molecular 28 diagnostics for the food industry. 29 84x47mm (300 x 300 DPI) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Analytical Chemistry Page 10 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 1. (a) Structures of MILs evaluated in this study; (b) Schematic for the extraction and 27 preconcentration of Salmonella Typhimurium from aqueous samples, followed by downstream analysis using 20 28 RPA amplification and microbiological culture detection methods. Panel b adapted from Clark et al., 2017. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 11 of 14 Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Figure 2. Recovery of S. marcescens extracted with Co(II) MIL as a function of back-extraction 28 medium composition. Percentage of initial cell load recovered from the Co(II) MIL extractant using 29 aqueous back-extraction media of different ionic composition is shown. A suspension of S. marcescens was 30 prepared and captured with the Co(II) MIL as described in the text, then back-extracted into water 31 (control), LB medium or 7 variations of the basic LB medium recipe. Back-extraction media used: 1X YE (5 -1 -1 -1 -1 32 g L yeast extract); 2x YE (10 g L yeast extract); 1x T (10 g L tryptone); 2x T (20 g L tryptone); 1x -1 -1 -1 -1 33 T, 1x YE (10 g L tryptone, 5 g L yeast extract); 2x T, 2x YE (20 g L tryptone, 10 g L yeast extract); 34 2x T, 1x NaCl (20 g L-1 tryptone, 10 g L-1 NaCl). Average cell recoveries for three separate experiments 35 are shown. Inset: Representative cell growth on track plates obtained following the extraction of S. 36 marcescens using Ni(II), Co(II) and Dy(III) MILs. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Analytical Chemistry Page 12 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Figure 3. Evaluation of sodium acetate heat pack for power-free incubation of RPA reactions. In 23 order to assess the utility of sodium acetate heat packs (inset) for portable and power-free incubation of 24 RPA reactions, we measured the internal temperature of a template-free RPA reaction tube sandwiched 25 between two activated heat packs using a fiber optic temperature monitoring system as described in the 26 text. Lefthand panel: Sodium acetate heat packs were able to provide near-optimal RPA reaction temperatures (reported range, 37 °C – 42 °C) over typical amplification times. Temperatures at various 27 points along the time-temperature curve are 25.2 °C (initial temperature, point a), 45.4 °C (point b), 43.7 28 °C (point c) and 42.1 °C (point d). Righthand panel: Comparison of DLH-RPA reactions driven with a 29 thermal cycler (TC) or with sodium acetate heat packs (SA), using bacterial DNA obtained using the 30 PrepMan Ultra Sample Preparation Reagent (PMU) or via MIL-based extraction (MIL). 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 13 of 14 Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Figure 4. MIL-RPA with Ni(II) MIL. The combined MIL and RPA approach was visualized using gel 31 electrophoresis (35 min) or lateral flow (10 min). MIL-based extraction was compared with concurrent 32 extraction using PrepMan Ultra Sample Preparation Reagent (PMU). For gel electrophoresis (lefthand 33 panel), PMU LOD was consistently identified as 104 CFU mL-1, however, bands were inconsistently present 34 at lower levels (lane 1), or were less defined than those for the Ni(II) MIL at the same level (lane 3). Lanes 35 4, 5 represent no template controls (NTC). With lateral flow (right-hand panel) detection was achieved as 36 low as 104 CFU mL-1 for both PMU (strip 1) and the MIL (strip 2). 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Analytical Chemistry Page 14 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 5. RPA-based detection of Salmonella Typhimurium in liquid food products. The combined 46 MIL and RPA approach was applied to almond milk, milk (2% milk fat), 50:50 dilution of milk (2% milk fat: 47 0.1% peptone water) and liquid egg product. Results were visualized using gel electrophoresis (35 min) or lateral flow (10 min). MIL-based extraction was compared with concurrent extraction using PrepMan Ultra 48 Sample Preparation Reagent (PMU). For Salmonella-spiked almond milk (Panel a, lanes 1, 4), milk (2% 49 milk fat) (Panel a, lanes 2, 5) and 50:50 milk dilution samples (Panel a, lanes 3, 6) using gel 50 electrophoresis and either extraction method (PMU or MIL), detection was achieved at 105 CFU mL-1. For 51 liquid egg samples spiked with S. Typhimurium (Panel b), PMU and MIL LOD was 104 CFU mL-1 (Panel b, 52 lanes 3, 4). Using lateral flow (Panel c), for Salmonella-spiked liquid egg samples, LOD was identified 104 53 CFU mL-1 for both PMU (Panel c, 3, 4) and MIL-extracted samples (Panel c, 5, 6). Strips 1 and 2 are the 54 no template control (NTC) and internal control, respectively. 55 56 57 58 59 60 ACS Paragon Plus Environment