Rapid preconcentration of viable bacteria using magnetic ionic liquid for PCR amplification and culture-based diagnostics
11046991 · 2021-06-29
Assignee
Inventors
Cpc classification
C12Q1/24
CHEMISTRY; METALLURGY
G01N33/54333
PHYSICS
G01N33/48735
PHYSICS
C12Q1/04
CHEMISTRY; METALLURGY
H01F1/447
ELECTRICITY
International classification
C12Q1/04
CHEMISTRY; METALLURGY
G01N33/543
PHYSICS
C12Q1/24
CHEMISTRY; METALLURGY
Abstract
In this disclosure, transition and rare earth metal-based magnetic ionic liquids (MILs) are successfully prepared in a two-step synthesis and used to extract viable bacteria from a liquid sample. The disclosed MILs are extremely hydrophobic MILs and were insoluble in aqueous solution at 0.01% (v/v). Furthermore, these MILs were miscible in a variety of polar and non-polar organic solvents. Moreover, these MILs possess low viscosity and increased magnetic susceptibility. These MILs possess unique characteristics that can have great potential uses in various chemical applications such as extraction solvents in LLE, liquid electrochromic materials (Co-based MILs), and novel reaction media for organic synthesis.
Claims
1. A magnetic ionic liquid, comprising a paramagnetic anionic component and a cationic component, wherein the cationic component has a general formula (I)
[(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+] (I) wherein each of the R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently a C.sub.2-C.sub.20 unsubstituted alkyl, and at least two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are the same; the paramagnetic anionic component has the following general formula (II),
[M(Y).sub.x.sup.−] (II) wherein M is Co, Mn, Ni, Dy, Nd, Gd ion or a combination thereof; and Y is a chelating agent having the general formula (III), ##STR00011## each of the R.sup.10 and R.sup.11 are independently a CH.sub.3, CHF.sub.2, CH.sub.2F, or CF.sub.3 group; and x is 3 or 4.
2. A magnetic ionic liquid, comprising a paramagnetic anionic component and a cationic component, wherein the cationic component has a general formula (I)
[(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+] (I) wherein each of the R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently a C.sub.2-C.sub.20 unsubstituted alkyl, and at least two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are the same; the paramagnetic anionic component is [Co(hfacac).sub.3.sup.−], [Ni(hfacac).sub.3.sup.−], ([Mn(hfacac).sub.3.sup.−]), ([Dy(hfacac).sub.4.sup.−]), ([Gd(hfacac).sub.4.sup.−]), ([Nd(hfacac).sub.4.sup.−]), or combination thereof, wherein hfacac is ##STR00012## wherein each of the R.sup.10 and R.sup.11 are independently a substituted or unsubstituted methyl, phenyl, thiophenyl, napthyl, alkyl, or aryl group; and x is 3 or 4.
3. The magnetic ionic liquid of claim 1, wherein for the cationic component, at least one of R.sup.1-R.sup.4 group is different from the others.
4. The magnetic ionic liquid of claim 1, wherein the cationic component is [(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+], wherein each of the R.sup.1, R.sup.2, and R.sup.3 is independently a C.sub.6 alkyl, and R.sup.4 is a C.sub.14 alkyl.
5. The magnetic ionic liquid of claim 1, wherein the magnetic ionic liquid is water insoluble.
6. The magnetic ionic liquid of claim 1, wherein the magnetic ionic liquid has a viscosity of from about 150 cp to about 1,000 cp at the temperature of 23.7° C.
7. The magnetic ionic liquid of claim 1, wherein the magnetic ionic liquid has a magnetic susceptibility of from about 2.5μ.sub.B to about 10.0μ.sub.B, measured by a SQUID magnetometer.
8. The magnetic ionic liquid of claim 1, wherein the magnetic ionic liquid is soluble in hexane, heptane, toluene, and benzene at 10% (v/v) MIL to solvent ratio, in acetone, acetonitrile, chloroform, dichloromethane, dioxane, ethanol, ethyl acetate, diethyl ether, methanol, or isopropyl alcohol at 20% (v/v) MIL to solvent ratio, or in hexane, heptane, toluene, and benzene at 20% (v/v) MIL to solvent ratio.
9. The magnetic ionic liquid of claim 1, wherein the magnetic ionic liquid keeps a bacterium viable.
10. A method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium from a sample, comprising: contacting a sample with a magnetic ionic liquid of claim 1 for the period of a contacting time, wherein the sample comprises a viable bacterium; and the magnetic ionic liquid extracts the bacterium from the sample.
11. The method of claim 10, wherein the method further comprises detecting the bacteria by a culture-based or nucleic acid-based method for pathogen detection.
12. The method of claim 10, wherein the bacterium is a Gram-negative bacterium, E. coli, Gram positive bacterium, M. smegmatis, or a combination thereof.
13. The method of claim 10, wherein the sample is a heterogeneous aqueous solution comprising food, milk, juices, biological fluid, blood, environmental water or soil, or any suspended solid.
14. The method of claim 11, wherein the detecting comprises using a nucleic acid-based method.
15. The method of claim 11, wherein the detecting comprises using PCR amplification in a PCR reagent mixture for a gene or genes in the bacterium.
16. The method of claim 11, wherein the detecting comprises using reverse transcription PCR (RT-PCR) for mRNA in the bacterium.
17. The method of claim 11, wherein the detecting comprises using mass/flow cytometry.
18. The method of claim 11, wherein the detecting comprises using a culture-based method.
19. The method of claim 10, wherein the method further comprises back-extracting the bacterium from the magnetic ionic liquid to a back extraction solution.
20. The method of claim 19, wherein the back extraction solution is a nutrient broth, salt solution, or aqueous medium that recovers the bacteria from the MIL.
21. The method of claim 10, wherein the contacting time is from about 30 second to about 10 min.
22. The method of claim 10, wherein the method further comprises separating the magnetic ionic liquid from the sample by a magnetic field.
23. The method of claim 10, wherein the method further comprises separating the magnetic ionic liquid from the sample by a magnetic field of from about 0.1 tesla to about 2 tesla.
24. The method of claim 10, wherein the bacterium is viable in the MTh.
25. The magnetic ionic liquid of claim 2, wherein for the cationic component, at least one of R.sup.1-R.sup.4 group is different from the others.
26. The magnetic ionic liquid of claim 2, wherein the cationic component is [(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+], wherein each of the R.sup.1, R.sup.2, and R.sup.3 is independently a C.sub.6 alkyl, and R.sup.4 is a C.sub.14 alkyl.
27. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid is water insoluble.
28. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid has a viscosity of from about 150 cp to about 1,000 cp at the temperature of 23.7° C.
29. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid has a magnetic susceptibility of from about 2.5μ.sub.B to about 10.0μ.sub.B, measured by a SQUID magnetometer.
30. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid is soluble in hexane, heptane, toluene, and benzene at 10% (v/v) MIL to solvent ratio, in acetone, acetonitrile, chloroform, dichloromethane, dioxane, ethanol, ethyl acetate, diethyl ether, methanol, or isopropyl alcohol at 20% (v/v) MTh to solvent ratio, or in hexane, heptane, toluene, and benzene at 20% (v/v) MTh to solvent ratio.
31. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid keeps a bacterium viable.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing or photograph executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
(23) To circumvent the challenges for using the prior art MILs and produce hydrophobic MILs with low viscosity, a new class of MILs is obtained by pairing a hydrophobic cation with a weakly coordinating but also hydrophobic anion that can chelate with paramagnetic metal centers.
(24) In this disclosure, a two-step synthesis is disclosed to create low melting, room temperature transition and rare earth metal-based MILs. Transition metals, such as Co(II), Mn(II), and Ni(II) were incorporated into the MIL structure to create trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)cobaltate(II) ([P.sub.66614.sup.+][Co(hfacac).sub.3.sup.−]), [P.sub.66614.sup.+] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac).sub.3.sup.−]), [P.sub.66614.sup.+] tris(hexafluoroacetylaceto)nickelate(II) ([Ni(hfacac).sub.3.sup.−]) MILs. Three exemplary rare earth metal centers, dysprosium(III), gadolinium(III) and neodymium(III), were also used to prepare [P.sub.66614.sup.+] tetrakis(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac).sub.4.sup.−]), [P.sub.66614.sup.+] tetrakis(hexafluoroacetylaceto)gadolinate(III) ([Gd(hfacac).sub.4.sup.−]), and [P.sub.66614.sup.+] tetrakis(hexafluoroacetylaceto)neodymate(III) ([Nd(hfacac).sub.4.sup.−]) MILs.
(25) It was observed that all of the exemplary MILs disclosed herein have a water solubility of less than 0.01% (v/v), making them ideal for MIL-based applications in aqueous systems. Furthermore, these MILs are found to be soluble in nearly 15 organic solvents. Viscosities of the synthesized MILs ranged from 276.5 centipoise (cP) to 927.9 cP at 23.7° C., making them among the least viscous hydrophobic MILs ever reported. Thermal properties of these exemplary MILs were investigated by monitoring the onset of volatilization/decomposition using flame ionization detection with thermal stabilities ranging from 130-225° C. and suitable for a wide range of applications. In addition, incorporation of Gd(III) and Dy(III) metal centers produced MILs with magnetic moments (μ.sub.eff) of 7.7 and 9.7 Bohr magnetons (μ.sub.B), respectively, as determined by superconducting quantum interference device (SQUID) magnetometry. This new class of MILs possess high hydrophobicity, low melting points, low viscosity, and high magnetic susceptibility making them ideal solvents for many applications ranging from catalysis to microfluidic applications where the MIL can be readily controlled and manipulated within the device.
(26) The present disclosure relates to a new class of MILs comprising a transition or rare earth metal complex hydrophobic anion and a hydrophobic cation that can chelate with paramagnetic metal centers, a method to synthesize the disclosed MILs, and a method to use the disclosed MILs to extract a viable bacterium from a sample. The embodiments of the disclosed MILs, synthesis thereof, or methods thereof are not limited to any particular metal ion, chelating species, synthesis method, bacterium, or extraction method which can vary and are understood by skilled artisans based on the present disclosure. It is further to be understood that all terminology used herein is for describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.
(27) Numeric ranges recited within the specification are inclusive of the numbers within the defined range. Throughout this disclosure, various aspects of the disclosed MILs, synthesis thereof, and methods are presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed MILs and methods. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).
(28) So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosed MILs and methods pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the disclosed MILs and methods without undue experimentation. The preferred materials and methods are described herein. In describing and claiming the embodiments of the disclosed MILs and methods, the following terminology will be used in accordance with the definitions set out below.
(29) The term “about,” as used herein, refers to variations in size, distance or any other types of measurements that can be resulted from the inherent heterogeneous nature of the measured objects and imprecise nature of the measurements themselves. The term “about” also encompasses variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods, and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities.
(30) The term “independently” means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.
(31) As used herein, “substituted” refers to an organic group as defined below (i.e., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to carbon(s) or hydrogen(s) atom replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. A substituted group can be substituted with 1, 2, 3, 4, 5, or 6 substituents.
(32) Substituted ring groups include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl, and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups are as defined herein.
(33) As used herein, the term “alkyl” or “alkyl groups” refers to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or “cycloalkyl” or “alicyclic” or “carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups).
(34) Unless otherwise specified, the term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls.” As used herein, the term “substituted alkyls” refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.
(35) In some embodiments, substituted alkyls can include a heterocyclic group. As used herein, the term “heterocyclic group” includes closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur or oxygen. Heterocyclic groups may be saturated or unsaturated. Exemplary heterocyclic groups include, but are not limited to, aziridine, ethylene oxide (epoxides, oxiranes), thiirane (episulfides), dioxirane, azetidine, oxetane, thietane, dioxetane, dithietane, dithiete, azolidine, pyrrolidine, pyrroline, oxolane, dihydrofuran, and furan.
(36) Alkenyl groups or alkenes are straight chain, branched, or cyclic alkyl groups having 2 to about 30 carbon atoms, and further including at least one double bond. In some embodiments, alkenyl groups have from 2 to about 20 carbon, or typically, from 2 to 10 carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups may be substituted similarly to alkyl groups.
(37) As used herein, the terms “alkylene”, cycloalkylene“, alkynylene, and alkenylene”, alone or as part of another substituent, refer to a divalent radical derived from an alkyl, cycloalkyl, or alkenyl group, respectively, as exemplified by —CH.sub.2CH.sub.2CH.sub.2—. For alkylene, cycloalkylene, alkynylene, and alkenylene groups, no orientation of the linking group is implied.
(38) As used herein, “aryl” or “aromatic” groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups include monocyclic, bicyclic, and polycyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl, florenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodimets, aryl groups contain 6-14 carbons, in others from 6 to 12 or 6-10 carbon atoms in the ring portions of the groups. The phrase “aryl groups” includes groups containing fused rings, such as fused aromatic-aliphatic ring systems. Aryl groups may be substituted or unsubstituted.
(39) In one aspect, the present disclosure relates to a magnetic ionic liquid, the magnetic ionic liquid comprises a paramagnetic anionic component and a cationic component, wherein the cationic component has a general formula (I)
[(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+] (I)
wherein each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an unsubstituted or substituted alkyl; the paramagnetic anionic component has the following general formula (II),
[M(Y).sub.x.sup.−] (II)
wherein M is transition metal or rare earth metal ion; and Y is a chelating agent having the general formula (III),
(40) ##STR00003##
each of R.sup.10 and R.sup.11 is independently a substituted or unsubstituted methyl, phenyl, thiophenyl, napthyl, alkyl, or aryl group; and x is 3 or 4.
(41) In some embodiments, in the magnetic ionic liquid disclosed herein, M is a transition metal ion. In some other embodiments, M is Co, Mn, Ni, or combination thereof.
(42) In some embodiments, in the magnetic ionic liquid disclosed herein, M is a rare earth metal ion. In some other embodiments, M is Dy, Nd, Gd, or a combination thereof. In yet some other embodiments, M is a mixture of a transition metal ion and rare earth ion. In some other embodiments, M is Co, Mn, Ni, Dy, Nd, Gd ion, or a combination thereof.
(43) In some embodiments, for the magnetic ionic liquid disclosed herein, Rth and RH are independently a methyl, phenyl, thiophenyl, napthyl, alkyl, or aryl group substituted by one or more electron withdrawing halogens or other groups. In some other embodiments, R.sup.10 and R.sup.11 are independently a C1-C4 alkyl group substituted by one or more electron withdrawing halogens or other groups. In some other embodiments, R.sup.10 and R.sup.11 are independently a CH.sub.3, CHF.sub.2, CH.sub.2F, or CF.sub.3 group. In yet some other embodiments, R.sup.10 and R.sup.11 are independently a CF.sub.3 group.
(44) In some other embodiments, for the magnetic ionic liquid disclosed herein, the anionic component is [Co(hfacac).sub.3.sup.−], [Ni(hfacac).sub.3.sup.−], ([Mn(hfacac).sub.3.sup.−]), ([Dy(hfacac).sub.4.sup.−]), ([Gd(hfacac).sub.4.sup.−]), ([Nd(hfacac).sub.4.sup.−]), or combination thereof, wherein hfacac is
(45) ##STR00004##
(46) In some embodiments, for the magnetic ionic liquid disclosed herein, the cationic component is [(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+], wherein each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an unsubstituted or substituted alkyl. In other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an straight-chain or branched alkyl. In other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an C.sub.2-C.sub.20 unsubstituted alkyl. In other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an C.sub.2-C.sub.20 straight-chain or branched alkyl. In some other embodiments, at least one of R.sup.1-R.sup.4 group is different from the others in the cationic component. In some other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an C.sub.2-C.sub.20 unsubstituted alkyl, and at least two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are the same. In yet some other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an C.sub.2-C.sub.20 unsubstituted alkyl, and three of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are the same. In some other embodiments, each of R.sup.1, R.sup.2, and R.sup.3 is independently a C.sub.6 alkyl, and R.sup.4 is a C.sub.14 alkyl. In some other embodiments, each of R.sup.1, R.sup.2, and R.sup.3 is independently a straight-chain C.sub.6 alkyl, and R.sup.4 is a straight-chain C.sub.14 alkyl.
(47) In some embodiments, the magnetic ionic liquid disclosed herein is water insoluble, indicated by exhibiting no observable change in color or pH of either the MIL or aqueous phase, or by that the MIL droplets still responded readily to an external magnetic field after three days of suspension in the aqueous phase. In other embodiments, the magnetic ionic liquid disclosed herein has a solubility of less than about 0.01% (v/v) in water. In other embodiments, the magnetic ionic liquid disclosed herein has a solubility of less than about 0.05% (v/v), about 0.04% (v/v), about 0.03% (v/v), about 0.02% (v/v), about 0.009% (v/v), about 0.008% (v/v), about 0.007% (v/v), about 0.006% (v/v), about 0.005% (v/v), about 0.004% (v/v), about 0.003% (v/v), about 0.002% (v/v), about 0.001% (v/v), or any value therein between in water.
(48) In some embodiments, the magnetic ionic liquid disclosed herein has a viscosity of from about 150 cp to about 1,000 cp at the temperature of 23.7° C. In some other embodiments, the magnetic ionic liquid disclosed herein has a viscosity of from about 200 cp to about 950 cp, from about 250 cp to about 900 cp, from about 300 cp to about 850 cp, from about 350 cp to about 800 cp, from about 400 cp to about 750 cp, from about 450 cp to about 700 cp, from about 500 cp to about 650 cp, from about 550 cp to about 600 cp, about 900 cp, about 800 cp, about 700 cp, about 600 cp, about 500 cp, about 400 cp, about 300 cp, about 200 cp, or any value therein between at the temperature of 23.7° C.
(49) In some embodiments, the magnetic ionic liquid disclosed herein has a thermal stability indicated by an onset of decomposition starting at about 110° C. or above. In some embodiments, the magnetic ionic liquid disclosed herein has a thermal stability indicated by an onset of decomposition starting at about 120° C., about 100° C., about 95° C., about 90° C., about 85° C., about 80° C., about 75° C., about 70° C., about 65° C., about 60° C., about 55° C., about 50° C., about 45° C., or any value therein between.
(50) In some embodiments, the magnetic ionic liquid disclosed herein has a magnetic susceptibility from about 2.5μ.sub.B to about 10.0μ.sub.B, measured by a Quantum Design MPMS SQUID magnetometer. In some embodiments, the magnetic ionic liquid disclosed herein has a magnetic susceptibility from about 0.5μ.sub.B to about 3.0μ.sub.B, from about 2μ.sub.B to about 10μ.sub.B, from about 1μ.sub.B to about 5μ.sub.B, from about 1μ.sub.B to about 10.0μ.sub.B, from about 2μ.sub.B to about 10μ.sub.B, from about 3μ.sub.B to about 10.0μ.sub.B, from about 1μ.sub.B to about 5μ.sub.B, from about 5μ.sub.B to about 10.0μ.sub.B, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.2, or any value therein between as measured by a Quantum Design MPMS SQUID magnetometer.
(51) In some embodiments, the magnetic ionic liquid disclosed herein is soluble in hexane, heptane, toluene, and benzene at 10% (v/v) MIL to solvent ratio, in acetone, acetonitrile, chloroform, dichloromethane, dioxane, ethanol, ethyl acetate, diethyl ether, methanol, or isopropyl alcohol at 20% (v/v) MIL to solvent ratio, or in hexane, heptane, toluene, and benzene at 20% (v/v) MIL to solvent ratio.
(52) In some embodiments, the magnetic ionic liquid has a solubility of greater than about 10% (v/v) in an organic solvent (except DMSO). In some other embodiments, the magnetic ionic liquid has a solubility of greater than about 20% (v/v) in an organic solvent (except DMSO). In some other embodiments, the magnetic ionic liquid has a solubility of greater than about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v), about 9% (v/v), about 10% (v/v), about 11% (v/v), about 12% (v/v), about 13% (v/v), about 14% (v/v), about 15% (v/v), about 16% (v/v), about 17% (v/v), about 19% (v/v), or any value therein between in an organic solvent (except DMSO).
(53) In some embodiments, the magnetic ionic liquid disclosed herein can extract a viable bacterium from an aqueous solution comprising the viable bacterium. In some embodiments, the magnetic ionic liquid disclosed herein can preconcentrate a viable bacterium from an aqueous solution comprising the viable bacterium. As used herein, “preconcentrate” means that the bacterium has a higher concentration in the MIL than in the aqueous solution after the MIL is mixed with the aqueous solution.
(54) In some embodiments, the magnetic ionic liquid disclosed herein is not toxic to a bacterium. In some other embodiments, the magnetic ionic liquid disclosed herein is capable of keeping a bacterium viable. As used herein, “viable” means that the bacterium shows no difference in its proliferation, as determined by counting colonies or other routine methods, after being extracted by MILs, when compared to a standard plate in which the bacteria were not exposed to MIL.
(55) In some embodiments, the bacterium is a gram negative bacterium. In some other embodiments, the bacterium is a gram positive bacterium. In yet some other embodiments, the bacterium is E. coli. In some other embodiments, the bacterium is M. smegmatis.
(56) In another aspect, the present disclosure relates to a method of synthesizing a magnetic ionic liquid, the method comprises mixing ammonium hydroxide, an alcohol, a chelating agent, and metal salt in a reaction vessel for the period of a reaction time at a reaction temperature to produce a corresponding chelated metal anionic component, and mixing a cationic component and the anionic component at a second temperature to produce a magnetic ionic liquid, wherein the metal salt is a paramagnetic transition or rare earth metal salt; the anionic component has the general formula, [M(Y).sub.x.sup.−], wherein M is transition metal or rare earth metal ion; and the chelating agent comprising a negative ion, Y having the general formula (III),
(57) ##STR00005##
each of R.sup.10 and R.sup.11 is independently a substituted or unsubstituted methyl, phenyl, thiophenyl, napthyl, alkyl or aryl group; and x is 3 or 4; the cationic component is [(PR.sup.1R.sup.2R.sup.3R.sup.4).sup.+], wherein each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an unsubstituted alkyl; the vessel is capped, and the chelating agent is added slowly to the vessel containing the alcohol and ammonia hydroxide before the addition of the metal salt.
(58) In some embodiments, the alcohol is ethanol.
(59) In some embodiments, the metal salt is a metal halide salt. In some other embodiments, the metal is a metal chloride salt.
(60) In some embodiments, after the first mixing step starts, any manual or mechanical method can be used during the reaction time to maximize the mixing of the reactants. In some embodiments, vortexing is used. In some embodiments, mechanical stirring is used.
(61) In some embodiments, the reaction time is from about 1 hour to 6 hours. In some other embodiments, the reaction time is from about 30 minutes to 24 hours, about 30 minutes to 1 hours, about 30 minutes to 2 hours, about 30 minutes to 3 hours, about 30 minutes to 4 hours, about 30 minutes to 5 hours, about 30 minutes to 6 hours, about 30 minutes to 7 hours, about 30 minutes to 8 hours, about 30 minutes to 10 hours, about 30 minutes to 12 hours, about 30 minutes to 15 hours, about 30 minutes to 17 hours, about 30 minutes to 20 hours, about 1 hour, about 3 hours, about 5 hours, about 8 hours, about 10 hours, about 15 hours, about 18 hours, about 20 hours, or any value therein between.
(62) In some other embodiments, the reaction temperature or the second temperature is from about 15 to about 40° C. In other embodiments, the reaction temperature or the second temperature is a room temperature, about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 40° C., or any value therein between.
(63) In some embodiments, the method of synthesizing MILs disclosed herein, further comprises mixing an alcohol and the chelating agent in the vessel. In some other embodiments, the alcohol used for the synthesis is ethanol.
(64) In some embodiments, for the method of synthesizing MILs disclosed herein, the yield of producing the anionic component is greater than 75%.
(65) In some embodiments, for the method of synthesizing MILs disclosed herein, the produced magnetic ionic liquid is one of the MILs disclosed herein.
(66) In some embodiments, for the method of synthesizing MILs disclosed herein, the metal salt is Dy, Nd, Gd metal chloride, or a combination thereof. In some embodiments, for the method of synthesizing MILs disclosed herein, the metal salt is Dy, Nd, Gd metal chloride, or a combination thereof. In yet some other embodiments, the metal salt is a mixture of a transition metal chloride and rare earth chloride. In some other embodiments, the metal salt is Co, Mn, Ni, Dy, Nd, Gd chloride, or a combination thereof.
(67) In some embodiments, for the method of synthesizing MILs disclosed herein, each of R.sup.10 and R.sup.11 is independently a methyl, phenyl, thiophenyl, napthyl, alkyl, or aryl group substituted by one or more electron drawing halogens or other groups. In some other embodiments, each of R.sup.10 and R.sup.11 is independently a C.sub.1-C.sub.4 alkyl group substituted by one or more electron withdrawing halogens or other groups. In some other embodiments, each of R.sup.10 and R.sup.11 is independently a CH.sub.3, CHF.sub.2, CH.sub.2F, or CF.sub.3 group. In yet some other embodiments, each of R.sup.10 and R.sup.11 is independently a CF.sub.3 group.
(68) In some other embodiments, for the method of synthesizing MILs disclosed herein, the produced anionic component is [Co(hfacac).sub.3.sup.−], [Ni(hfacac).sub.3.sup.−], ([Mn(hfacac).sub.3.sup.−]), ([Dy(hfacac).sub.4.sup.−]), ([Gd(hfacac).sub.4.sup.−]), ([Nd(hfacac).sub.4.sup.−]), or combination thereof, wherein hfacac is
(69) ##STR00006##
(70) In some embodiments, for the method of synthesizing MILs disclosed herein, the cationic component is [(PR.sup.1R.sup.2R.sup.3R.sup.4)+], wherein each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently an unsubstituted or substituted alkyl. In other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently a straight-chain or branched alkyl. In other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently a C.sub.2-C.sub.20 unsubstituted alkyl. In other embodiments, each of R.sub.1, R.sup.2, R.sup.3, and R.sup.4 is independently a C.sub.2-C.sub.20 straight-chain or branched alkyl. In some other embodiments, at least one of R.sup.1-R.sup.4 group is different from the others in the cationic component. In some other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently a C.sub.2-C.sub.20 unsubstituted alkyl, and at least two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are the same. In yet some other embodiments, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is independently a C.sub.2-C.sub.20 unsubstituted alkyl, and three of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are the same. In some other embodiments, each of R.sup.1, R.sup.2, and R.sup.3 is independently a C.sub.6 alkyl, and R.sup.4 is a C.sub.14 alkyl. In some other embodiments, each of R.sup.1, R.sup.2, and R.sup.3 is independently a straight-chain C.sub.6 alkyl, and R.sup.4 is a straight-chain C.sub.14 alkyl.
(71) In yet another aspect, the present disclosure relates to a method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium from a sample, the method comprises contacting a sample with a magnetic ionic liquid for the period of a contacting time, wherein the sample comprises a viable bacterium; and the magnetic ionic liquid extracts the bacterium from the sample.
(72) As used herein, “extract” means that the bacterium in the sample migrates onto or into the MIL after the contacting step starts. As used herein, a “sample” can be one originated directly from any food, milk, juice, biological fluid, blood, environmental water, soil, clinical collection, person, or animal, with or without any sample preparation or treatment procedure. A “sample” can also be a mixture of water and a specimen collected from any food, clinical, environmental, human, or animal source that comprises a bacterium. A “sample” can be any specimen that comprises a viable bacterium.
(73) In some embodiments, after the contacting step, any manual or mechanical method can be used during the contact time to maximize the mixing of the MIL and the sample. In some embodiments, vortexing is used. In some embodiments, mechanical stirring is used.
(74) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the magnetic ionic liquid is one of the MILs disclosed herein.
(75) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the magnetic ionic liquid has a higher concentration of the viable bacteria than the sample, after the contacting step. In some embodiments, the ratio of the bacterium concentration in the magnetic ionic liquid to one in the sample is from about 1:1 to about 50:1. In some embodiments, the ratio of the bacterium concentration in the magnetic ionic liquid to one in the sample is from about 1:10 to about 1:1. In some embodiments, the bacterium concentration in the magnetic ionic liquid is higher than one in the sample after the contact time. In some embodiments, the bacterium concentration in the magnetic ionic liquid can be lower than in the sample after the contact time.
(76) In some embodiments, the ratio of the bacterium concentration in the magnetic ionic liquid to one in the sample is from about 1:1 to about 2:1, from about 1:1 to about 5:1, from about 1:1 to about 10:1, from about 1:1 to about 20:1, from about 1:1 to about 30:1, from about 1:1 to about 40:1, about 1:1, about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:, about 35:1, about 40:1, about 50:1, or any value therein between.
(77) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the weight ratio between the magnetic ionic liquid and the sample is between about 1:10 to 1:100. In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the weight ratio between the magnetic ionic liquid and the sample is between about 1:10 to 1:20, between about 1:10 to 1:30, between about 1:10 to 1:40, between about 1:10 to 1:50, between about 1:10 to 1:60, between about 1:70 to 1:80, between about 1:10 to 1:90, between about 10:1 to 1:10, between about 1:20 to 1:50, between about 1:20 to 1:100, between about 1:40 to 1:80, between about 1:50 to 1:10, between about 1:10 to 1:20, about 10:1, about 5:1, about 1:1, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, or any value therein between.
(78) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the bacterium has a concentration of at least 1.68×10.sup.4 CFU/mL in the sample.
(79) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the bacterium is a Gram-negative bacterium or Gram-positive bacterium. In some other embodiments, the bacterium is a Gram-negative or E. coli. In yet some other embodiments, the bacterium is a Gram-positive bacteria or M. smegmatis.
(80) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, wherein the sample is a heterogeneous aqueous solution. In some other embodiments, the sample is a heterogeneous aqueous solution comprising food, milk, juices, biological fluid, blood, environmental water or soil, or any suspended solid. In yet some other embodiments, the sample is an aqueous solution comprising food, milk, juice, biological fluid, blood, environmental water, or soil. In some other embodiments, the sample is an aqueous solution comprising or suspended with any material that host a viable bacterium.
(81) In some embodiments, for the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium, the contact time for the extracting step is from about 30 seconds to about 10 min. In some embodiments, the contact time is from about 1 minute to about 1 hour, from 1 minute to about 2 hours, from 1 minute to about 5 hours, from about 1 hour to 24 hours, about 5 minutes, about 10 minutes, about 2 minutes, about 1 hour, about 2 hours, about 5 hours, about 10 hours, or any value therein between.
(82) In some embodiments, during the contact time, a manual or mechanical method is utilized to maximize the contact between the MIL and the sample for the whole contact time or only for a part of the contact time.
(83) In some embodiments, the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium further comprises mixing the sample and magnetic ionic liquid during contact time through manual or mechanical agitation after the contacting step starts. Vortexing and hand shaking are examples of agitation to maximize the interaction between the MIL and the sample.
(84) In some embodiments, the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium further comprises separating the magnetic ionic liquid from the sample by a magnetic field. In some other embodiments, the method further comprises separating the magnetic ionic liquid from the sample by a magnetic field of from about 0.1 tesla to about 2 tesla.
(85) In some embodiments, the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium further comprises back-extracting the bacterium from the magnetic ionic liquid to a back extraction solution. In some embodiments, the back extraction solution is a nutrient broth, salt solution, or aqueous medium that recovers the bacteria from the MIL as one skilled in the art would employ.
(86) In some embodiments, the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium further comprises enriching, culturing, or multiplying the bacterium extracted from the sample by the MIL. The techniques are any one of those that would be used by one skilled in the art to increase population of a bacterium.
(87) In some embodiments, the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium further comprises detecting, identifying, quantifying, or a combination thereof the bacterium.
(88) In some other embodiments, the method further comprises detecting, identifying, quantifying, or a combination thereof the bacterium using PCR amplification in a PCR reagent mixture for a gene or genes in the bacterium. In some other embodiments, the method further comprises detecting, identifying, quantifying, or a combination thereof the bacterium using mass/flow cytometry. In yet some other embodiments, the method further comprises detecting, identifying, quantifying, or a combination thereof the bacterium using a culture-based method.
(89) In some embodiments, the method further comprises detecting, identifying, quantifying, or a combination thereof the bacterium using reverse transcription PCR (RT-PCR) for mRNA in the bacterium. The RT-PCR technique for this purpose is similar to one described in the prior art, such as in “Sheridan, G. E. C., et al. “Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coli Cells”, Applied and Environmental Microbiology 64, 4 (1998).
(90) In some embodiments, the method of extracting, detecting, identifying, quantifying, or a combination thereof a viable bacterium has a lower detection limit than the PCR amplification method alone. In some other embodiments, the method has a lower detection limit than a culture based method alone.
(91) Herein, we describe a class of magnetic ionic liquids (MILs) that have very low water solubility, tunable chemical structure, low viscosity, suitable hydrophobicity and greater magnetic susceptibility. Compared to the prior art MILs, the disclosed MILs have improved properties to be used in various applications. One of the unique properties for the disclosed MILs is their ability to isolate, extract, and/or concentrate viable bacteria, such as E. coli, from an aqueous sample.
(92) The bacteria extracted or preconcentrated from an aqueous sample using the disclosed MILs can be analyzed by conventional microbiological culture and PCR amplification. However, using the disclosed MILs for extraction or preconcentration can speed up the detection, identification, or quantification of the bacteria, because the MILs can preconcentrate the bacteria or eliminate other factors that might interfere or prevent the detection of the bacteria. By dispersing hydrophobic MILs in an aqueous sample comprising bacteria cells, such as E. coli cells, the bacteria can be rapidly extracted and isolated using an applied magnetic field. The extracted cells or derivatives thereof were recovered from the MIL extraction phase by agitation in a nutrient broth and subsequently cultured on selective agar for detection. Interestingly, the enrichment of the bacteria, such as E. coli by MILs was dependent upon the identity of the paramagnetic metal incorporated into the chemical structure of the MIL, providing a basis for the design of MILs to exhibit enhanced cell extraction performance. Under optimized conditions, the MIL comprised of a trihexyl(tetradecyl)phosphonium cation ([P.sub.6661.sup.+]) and Ni(II) hexafluoroacetylacetonate-based anion ([Ni(hfacac).sub.3.sup.−]) was capable of enriching sufficient viable cells for the detection of E. coli at concentrations as low as 1.68×10.sup.4 CFUs mL.sup.−1 in aqueous solution with an extraction/recovery procedure of less than 10 min. The MIL-based extraction method was also coupled with PCR amplification for the rapid analysis of E. coli, demonstrating the compatibility of MILs with both culture-based and nucleic acid-based methodologies for pathogen detection.
(93) Extracting or concentrating bacteria from a complex sample is one of the approaches to improve the existing bacteria testing throughput, since doing so decreases the amount of time to grow the requisite colonies needed for proper identification and quantification. Therefore, extracting or concentrating of bacteria from complex sample matrices using MILs, also called “preconcentration”, has great potential applications.
(94) All publications, patent applications, issued patents, and other documents referred to in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains and are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as incorporated by reference. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
EXAMPLES
(95) Embodiments of the disclosed MILs and methods are further defined in the following non-limiting Examples. These Examples, while indicating certain embodiments of the disclosed MILs and methods, are given by way of illustration only and should not be considered as limiting in any way. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the disclosed MILs and methods to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the disclosed MILs and methods, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
(96) Materials and Methods
(97) Reagents and Materials
(98) The reagents, ammonium hydroxide (28-30% solution in water) and 1,1,1,5,5,5-hexafluroacatelyacetone (99%), were purchased from Acros Organics (Morris Plains, N.J., USA). Gadolinium(III) chloride hexahydrate (99.9%) and manganese(II) chloride tetrahydrate (98.0-101.0%) were purchased from Alfa Aesar (Ward Hill, Mass., USA). Acetonitrile (99.9%), hexane (98.5%), methanol (99.9%), cobalt(II) chloride hexahydrate (98%), dysprosium(III) chloride hexahydrate (99.9%), nickel(II) chloride (98%), and neodymium(III) chloride hexahydrate were purchased from Sigma Aldrich (St. Louis, Mo., USA). Anhydrous diethyl ether (99.0%) was purchased from Avantor Performance Materials Inc. (Center Valley, Pa., USA). Ethanol (100%) was purchased from Decon Labs (King of Prussia, Pa., USA). Deuterated DMSO was obtained from Cambridge Isotope Laboratories (Andover, Mass., USA). All solvents and reagents were used as received without any additional drying or purification. Deionized water (18.2 MS2 cm) was obtained from a Milli-Q water purification system (Millipore, Bedford, Mass., USA). Trihexyl(tetradecyl)phosphonium chloride (>93%) was purchased from Strem Chemical (Newburyport, Mass., USA). Trihexyl(tetradecyl)phosphonium chloride was further purified by dissolving 20 g of the IL in 50 mL of acetonitrile. The acetonitrile layer was washed three times with 5 mL aliquots of hexane. Acetonitrile was subsequently evaporated off under reduced pressure followed by drying of the IL at 50° C. in a vacuum oven.
(99) Nickel(II) chloride and hexafluoroacetylacetone were purchased from Acros Organics (NJ, USA) with gadolinium(III) chloride hexahydrate and manganese(II) chloride tetrahydrate were obtained from Alfa Aesar (Haverhill, Mass., USA). Cobalt(II) chloride hexahydrate, dysprosium(III) chloride hexahydrate, and neodymium(III) hexahydrate were purchased from Sigma Aldrich (St. Louis, Mo., USA). NEB-5a Competent E. coli cells (K12 strain) with Super Optimal Broth with Catabolite Repression (SOC) outgrowth medium and Phusion High Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, Mass., USA). dNTPs were obtained from Thermo Scientific (Wilmington, Del., USA). Modified pET-32 plasmid was obtained from EMD Millipore (Billerica, Mass., USA) and primers for PCR amplification were purchased from IDT (Coralville, Iowa, USA). Miller's luria broth (LB) and agar for microbiological cultures were purchased from Fisher Scientific (Fair Lawn, N.J., USA). Carbenicillin, agarose, tris(hydroxymethyl)aminomethane and a 1 kb plus DNA ladder were purchased from P212121 (Ypsilanti, Mich., USA). SYBR Safe DNA gel stain was obtained from Life Technologies (Carlsbad, Calif., USA). All stock solutions and dilutions were made using deionized water (18.2 MS2 cm) from a Milli-Q water purification system (Millipore, Bedford, Mass., USA).
(100) Instrumentation
(101) Proton NMR spectra (.sup.1H) were recorded using a Bruker 500 MHz nuclear magnetic resonance spectrometer. Solvent peaks were used as reference values for the reporting of chemical shifts. Elemental analyses were obtained using a Perkin Elmer 2100 Series II CHN/S Analyzer (Waltham, Mass., USA). Mass spectra were obtained using an Agilent 6230 TOF LC/MS (Santa Clara, Calif., USA). Viscosity measurements were obtained using a Wells/Brookfield DV1 cone and plate viscometer using a CPA-51Z cone spindle. Each MIL was dried in a vacuum oven for 48 hours at 50° C. ensuring any water or residual solvents were completely removed from the MILs. Sample volumes of 0.5 mL were used for all MILs at a temperature (23.7° C.).
(102) All cell cultures were grown in an I24R incubator shaker (New Brunswick Scientific, Enfield, Conn., USA) with a Horizon Plasmafuge-6 (Fisher Scientific) subsequently used for cell harvesting. Vortex agitation was applied to cell suspensions and samples with a Barnstead/Thermolyne Type 16700 Mixer (Dubuque, Iowa, USA). A NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, Del., USA) was used to measure the optical density of aqueous cell suspensions. A Mettler Toledo NewClassic MF MS105 (readability=0.01 mg) microbalance was used to measure the mass of MILs. PCR experiments were conducted using a Techne FTgene2D thermal cycler (Burlington, N.J., USA) and gel electrophoresis performed on a BRL H4 Horizontal Gel Electrophoresis system (Life Technologies) using a dual output power supply (Neo/Sci, Rochester, N.Y., USA). A Safe Imager 2.0 transilluminator (Invitrogen) was used for the visualization of all agarose gels. A 0.9 T cylindrical magnet purchased from K&J magnetics (Pipersville, Pa., USA) was used to manipulate the MIL extraction solvent during extraction procedures. Colonies were cultured on LB agar plates using a Barnstead/Thermolyne Type 142300 Incubator.
(103) Transformation and Cell Cultures
(104) Competent E. coli cells were transformed with modified pET-32 plasmid DNA (pDNA) containing the 5′-methylthioadenosine phosphorylase (MTAP) gene. Briefly, 20 μL of competent cells were thawed on ice for 10 min and spiked with 94.5 ng of pDNA. After the sample tube was gently flicked and placed on ice for 30 min, the mixture was subjected to heat shock at 42° C. for 30 s and once again chilled on ice for 5 min. The cell suspension was mixed with 950 μL of room temperature SOC outgrowth medium and the mixture incubated at 37° C. for 60 min. The transformed cells were then cultured in 5 mL of LB media containing 100 μg mL.sup.−1 carbenicillin for 24 h. A 500 μL aliquot of the culture was mixed with 500 μL of sterile 50% glycerol (v/v) and stored at −80° C. for later use.
(105) For each cell extraction experiment, 2 mL of sterile LB media with 100 μg mL.sup.−1 carbenicillin was inoculated with 0.5 μL of E. coli glycerol stock and incubated overnight at 37° C. and 225 rpm. The overnight culture was centrifuged at 2820 g for 8 min and the supernatant discarded. The cells were then vortexed for 15 s in 2 mL of deionized water and centrifuged once again at 2820 g for 8 min. Following a total of three washes, the cells were re-suspended in 1 mL of deionized water. The concentration of cells within the suspension was initially determined by measuring the optical density of the sample at 600 nm (OD600), where an OD600 equal to 1.000 represents 8×10.sup.8 E. coli cells mL.sup.−1. The bacterial suspension was diluted to an OD600 value of 0.021 (1.68×107 cells mL.sup.−1), after which 10-fold serial dilutions were performed to yield the desired sample concentration. Since the sample concentrations were below the detection limit of the spectrophotometer, the final concentration of each sample (in CFUs mL.sup.−1) was obtained using a plate counting method. Briefly, 15 μL of diluted cell suspension were mixed with 985 μL of LB media and vortexed for 3 min. A 1004 aliquot of the suspension was spread onto a selective LB agar plate with 100 μg mL.sup.−1 carbenicillin and incubated overnight at 37° C. to obtain visible colonies.
(106) MIL-Based Whole Cell Extraction
(107) A general schematic for the MIL-based cell extraction procedure is depicted in
(108) Selective Culture and Colony Enumeration
(109) To detect viable bacteria recovered from the MIL extraction solvent, a culture-based method selective for E. coli transformants was employed. Following MIL-based extraction, a 100 μL aliquot of the LB back-extraction solution was plated on LB agar containing 100 μg mL.sup.−1 carbenicillin and incubated overnight at 37° C. Visible colonies were counted, tabulated, and compared to the colonies obtained for the sample prior to extraction. The enrichment factor (E.sub.F) from the MIL-based extraction was calculated by Equation 1, where C.sub.MIL is the concentration of CFUs within the MIL extraction phase and C.sub.S represents the concentration of CFUs in the bacterial suspension prior to extraction.
E.sub.F=C.sub.MIL/C.sub.S Equation 1
(110) The volume of MIL used for the calculation of C.sub.MIL was determined from the mass of the MIL transferred into the sample solution prior to extraction and the density of the MIL.
(111) PCR and Gel Electrophoresis
(112) Following the recovery of E. coli cells from the MIL extraction phase, an aliquot of the LB back-extraction solution was analyzed by PCR. Briefly, a 1 μL aliquot of cell-enriched LB back-extraction solution was mixed with 35.5 μL of deionized water, 10 μL of 5× Phusion HF buffer, 0.2 mM dNTP mix, 1 U of Phusion DNA polymerase, and 0.2 μM of each primer resulting in a total reaction volume of 50 μL. Primers for amplification of the 879 bp MTAP gene from pDNA were 5′ TGC TGT TCC AGG GAC CT 3′ and 5′ GAA TTC GGA TCC GGA CGC 3′. The thermal protocol used for amplification of the MTAP gene was as follows: 5 min initial denaturation at 95° C. followed by 30 cycles of 30 s denaturation at 95° C., 45 s annealing at 54° C., and elongation for 45 s at 72° C.
(113) After thermal cycling, the PCR products were mixed with 10 μL of bromophenol blue tracking dye solution (30% glycerol v/v) and loaded on a 1% agarose gel stained with SYBR Safe DNA gel stain. The PCR amplicon was subjected to electrophoresis at approximately 4 V cm.sup.−1 and subsequently visualized using a transilluminator.
Example 1
(114) Preparation of Exemplary Transition Metal Based MILs
(115) Transition or rare earth metal based MILs are usually synthesized according to the steps in Scheme 1.
(116) ##STR00007## ##STR00008##
(117) The exemplary MILs synthesized in this Examples section comprise heavily alkylated phosphonium-based cations, i.e., [P.sub.66614.sup.+], exhibit high hydrophobicity with relatively low melting points due largely to its asymmetry. The [P.sub.66614.sup.+][Cl.sup.−] IL is commercially available. As shown in Scheme 1, reaction of ammonium hexafluoroacetylacetonate ([NH.sub.4.sup.+][hfacac.sup.−]) with various transition and rare earth metal centers yields the hydrophobic ammonium-based salt intermediates 1a-6a that do not dissolve in water, even at very high ratios of water to salt.
(118) Pairing of intermediate 1a to an imidazolium-based cation was carried out for preliminary viscosity and hydrophobicity testing. A metathesis reaction was performed between 1-(6-hydroxyhexyl)-3-methylimidazolium chloride [MC.sub.6OHIM.sup.+][Cl.sup.−] and intermediate 1a to form [MC.sub.6OHIM.sup.+] [Co(hfacac).sub.3.sup.−]. The resulting MIL was soluble in water as determined by an obvious color change in the aqueous solution two hours after addition of the MIL. Furthermore, the neat MIL could not be transferred with a pipette at room temperature due to its high viscosity. Pairing the cation of the hydrophobic ammonium-based Aliquat 336 with intermediate 1a also resulted in a very viscous MIL that could not be drawn into a pipette. Since the broad applicability of hydrophobic MILs is very much dependent upon the ease with which they can be transferred using traditional liquid handling methods, the [P.sub.66614.sup.+] cation was selected for preparation of the hfacac-based MILs.
(119) Previously, transition metal hexafluoroacetylacetonate MILs were created in two different synthetic pathways, both involving a three-step synthesis (H. Mehdi, K. Binnemans, K. Van Hecke, L. Van Meervelt and P. Nockemann, Chem. Commun., 2010, 46, 234-236 and P. Zhang, Y. Gong, Y. Lv, Y. Guo, Y. Wang, C. Wang and H. Li, Chem. Commun., 2012, 48, 2334-2336). In this disclosure, the creation of metal salts 1a-6a (Scheme 1) was achieved in a one-pot synthesis by reacting ammonium hydroxide, hexafluoroacetylacetone, and the metal chloride salt. Reaction yields greater than 81% were achieved after 5 hours of total reaction time. It is important to highlight in this synthesis method the need to add hexafluoroacetylacetone slowly, such as to add via a syringe, to the capped reaction vessel containing ethanol and ammonium hydroxide. The acid-base reaction between hexafluoroacetylacetone and ammonium hydroxide causes a vapor to form inside the reaction vessel. Loss of this vapor resulted in low product yields <20%, presumably due to the vaporization of both hexafluoroacetylacetone and ammonium hydroxide (which have boiling points <75° C.).
(120) Exemplary transition metal based MILs 1-3 were synthesized by dissolving 10 mmol of ammonium hydroxide in 30 mL of ethanol. The reaction vessel was then sealed with a rubber septum and 10 mmol of hexafluoroacetylacetone was added dropwise to the reaction via syringe. A white vapor was allowed to settle before adding 3.3 mmol of cobalt(II) chloride hexahydrate. The reaction was allowed to stir at room temperature for 5 hours. The solvent was removed under reduced pressure and the crude product was redissolved in 20 mL of diethyl ether and washed several times with 5 mL aliquots of deionized water until the aqueous fraction yielded no precipitate during a AgNO.sub.3 test. Diethyl ether was evaporated and the anion was allowed to dry at 50° C. overnight under reduced pressure. 1 mmol of the anion was added to 1 mmol of purified phosphonium chloride and dissolved in 30 mL of methanol. This reaction was allowed to stir overnight at room temperature. The solvent was evaporated and 20 mL of diethyl ether was added to dissolve the crude product. The ether layer was washed several times with 5 mL aliquots of deionized water until the aqueous fraction yielded no precipitate during an AgNO.sub.3 test. Ether was evaporated off and MIL 1 was dried at 50° C. overnight under reduced pressure. For MILs 2 and 3, the same procedure was followed using manganese(II) chloride tetrahydrate and nickel(II) chloride, respectively.
(121) The characterization of the intermediates and MILs are the following:
(122) 1a: Red solid. Yield 82%. TOF LC/MS: m/z (−) 680.4.
(123) 2a: Yellow solid. Yield 79%. TOF LC/MS: m/z 676.4.
(124) 3a: Green solid. Yield 81%. TOF LC/MS: m/z (−) 679.4.
(125) MIL 1: Dark red viscous liquid. Yield 92%. Elem. anal. calcd (%) C.sub.47H.sub.71CoF.sub.18O.sub.6P: C, 48.50; H, 6.15; N, 0. Found: C, 49.09; H, 6.31; N, 0.03. TOF LC/MS: m/z (+) 483.4; (−) 680.4.
(126) MIL 2: Light orange viscous liquid. Yield 91%. Elem. anal. calcd (%) C.sub.47H.sub.71MnF.sub.18O.sub.6P: C, 48.67; H, 6.17; N, 0. Found: C, 48.89; H, 6.22; N, 0.37. TOF LC/MS: m/z (+) 483.4; (−) 676.4.
(127) MIL 3: Dark green viscous liquid. Yield 90%. Elem. anal. calcd (%) C.sub.47H.sub.71NiF.sub.18O.sub.6P: C, 48.51; H, 6.15; N, 0. Found: C, 48.72; H, 6.22; N, 0.27. TOF LC/MS: m/z (+) 483.4; (−) 679.4.
(128) The synthesis of the chelated metal salt was followed by a metathesis reaction between intermediates 1a-3a and ([P.sub.66614.sup.+][Cl.sup.−]), thereby producing transition metal-based MILs 1-3 (Scheme 1) in a total of two steps.
Example 2
(129) Preparation of Exemplary Rare Earth Based MILs
(130) MILs 4-6 were synthesized by dissolving 10 mmol of ammonium hydroxide in 30 mL of ethanol. The reaction vessel was then sealed with a rubber septum and 10 mmol of hexafluoroacetylacetone was added dropwise to the reaction via syringe. A white vapor was allowed to settle before adding 2.5 mmol of dysprosium(III) chloride hexahydrate. The reaction was allowed to stir at room temperature for 5 hours. The solvent was removed under reduced pressure and the crude product was redissolved in 20 mL of diethyl ether and washed several times with 5 mL aliquots of deionized water until the aqueous fraction yielded no precipitate during an AgNO.sub.3 test. Diethyl ether was evaporated and the anion was allowed to dry at 50° C. overnight under reduced pressure. 1.2 mmol of the anion salt was added to 1 mmol of purified phosphonium chloride and dissolved in 30 mL of methanol. This reaction was allowed to stir overnight at room temperature. The solvent was evaporated and 10 mL of hexane was added to the crude product to precipitate out any unreacted anion salt and filtered off. Once more, the solvent was evaporated and 20 mL of diethyl ether was added to dissolve the crude product. The ether layer was washed several times with 5 mL aliquots of deionized water until the aqueous fraction yielded no precipitate during an AgNO.sub.3 test. Ether was evaporated off and MIL 4 was dried at 50° C. overnight under reduced pressure. For MILs 5 and 6, the same procedure was followed using gadolinium(III) chloride hexahydrate and neodymium(III) chloride hexahydrate, respectively.
(131) The characterization of the intermediates and MILs are the following:
(132) 4a: White solid. Yield 83%. TOF LC/MS: m/z (−) 992.7.
(133) 5a: White solid. Yield 82%. TOF LC/MS: m/z (−) 986.7.
(134) 6a: Pink solid. Yield 81%. TOF LC/MS: m/z (−) 972.6.
(135) MIL 1: Light gold viscous liquid. Yield 93%. Elem. anal. calcd (%) C.sub.52H.sub.72DyF.sub.24O.sub.8P.2H.sub.2O: C, 41.35; H, 5.07; N, 0. Found: C, 41.39; H, 4.74; N, 0.25 TOF LC/MS: m/z (+) 483.4; (−) 992.7.
(136) MIL 5: Light yellow viscous liquid. Yield 91%. Elem. anal. calcd (%) C.sub.52H.sub.72GdF.sub.24O.sub.8P.2H.sub.2O: C, 41.49; H, 5.09; N, 0. Found: C, 41.85; H, 4.67; N, 0.31 TOF LC/MS: m/z (+) 483.4; (−) 986.7.
(137) MIL 6: Light pink viscous liquid. Yield 90%. Elem. anal. calcd (%) C.sub.52H.sub.72NdF.sub.24O.sub.8P.2H.sub.2O: C, 41.85; H, 5.13; N, O. Found: C, 41.82; H, 4.53; N, 0.28 TOF LC/MS:m/z (+) 483.4; (−) 972.6.
(138) The chelation of hexafluoroacetylacetone to neodymium was also previously reported and although the crystal structure was isolated, the synthesis was limited by the solubility of the cation in the aqueous phase (H. Mehdi, K. Binnemans, K. Van Hecke, L. Van Meervelt and P. Nockemann, Chem. Commun., 2010, 46, 234-236). Furthermore, the reported procedure required reaction of the rare earth oxide NdO.sub.3 with bis(trifluoromethane)sulfonamide (HNTf.sub.2), an expensive reagent when compared to hexafluoroacetylacetone. To circumvent the limitations of this reaction, a synthesis similar to the transition metal analogues (Scheme 1) was followed to produce intermediates 4a-6a after five hours in yields greater than 80%. A subsequent metathesis reaction between 4a-6a and [P.sub.66614.sup.+] [Cl.sup.−] generated MILs 4-6 (Scheme 1). This synthetic strategy allows for the incorporation of rare earth metal centers possessing higher magnetic susceptibility, such as gadolinium and dysprosium, in a two-step synthesis.
Example 3
(139) Evaluation of Exemplary MILs
(140) Water Solubility
(141) The water solubility of these exemplary MILs was tested by pipetting a 1 μL droplet of MIL into 10 mL of deionized water to create a 0.01% (v/v) solution. After pipetting the MIL into the aqueous sample, the MIL was observed to form a wide droplet that rests on top of the solution. Vortexing the MIL droplet caused dispersion of the MIL into fine microdroplets that were suspended within the aqueous solution, ultimately settling at the bottom of the vessel. The aqueous solution exhibited no observable change in color or pH, and the MIL droplets still responded readily to an external magnetic field after three days of suspension in the aqueous phase. These are all highly attractive features required in the design of low viscosity, hydrophobic MILs that possess high magnetic susceptibility. To demonstrate the hydrophobicity of these MILs compared to other available hydrophobic MILs, 50 μL of [P.sub.66614.sup.+][FeCl.sub.4.sup.−], [P.sub.66614.sup.+][Co(hfacac).sub.3.sup.−], and [P.sub.66614+][Nd(hfacac).sub.4.sup.−] were pipetted into separate scintillation vials containing 12 mL of deionized water. Each MIL was vortexed and heated to 85° C. for 10 minutes.
(142) Solvent Miscibility
(143) Owing to their unique solvation capabilities and high thermal stability, ILs have been successfully employed in organic synthesis either as reaction media or catalysts. An evaluation of the hydrophobic MIL solubility in a wide range of organic solvents may provide a fundamental understanding into their solvent properties. This could be instrumental for designing MIL-based reaction media, where the MIL can be selectively separated from the reaction products using an external magnetic field. The solubility of the MILs prepared in this study were tested in 15 different organic solvents possessing a wide range of polarities. Table 1 shows the solubility of each exemplary MIL in the different organic solvents. The transition-metal based MILs (MILs 1-3) were fully miscible in all of the organic solvents except DMSO. A trend can be observed that the transition metal-based MILs show full miscibility in solvents with Reichardt's polarity index values ranging from 0.117 (ethyl ether)-0.762 (methanol) at a 20% (v/v) MIL to solvent ratio, with the exception of DMSO. However, as the polarity value of the solvent drops below 0.117, the transition-metal based MILs show decreased solubilities at a 10% (v/v) MIL to solvent ratio. All of the transition metal-based MILs exhibited some solubility in DMSO, however, it was observed that fine droplets of insoluble MIL remain at a 10% (v/v) MIL to solvent ratio. The rare earth-based MILs were miscible in all of the tested solvents at a 20% (v/v) MIL to solvent ratio. Furthermore, the rare earth-based MILs exhibit higher solubility in non-polar solvents such as benzene, toluene, heptane, and hexane when compared to the transition metal-based MILs. The solubility of these MILs in many different organic solvents adds to their versatility and use in numerous applications.
(144) TABLE-US-00001 TABLE 1 Physicochemical and Magnetic Properties of Transition and Rare Earth Metal-based MILs MW Viscosity μ.sub.eff MIL Abbreviation (g/mol) (cP).sup.a Solubility (μ.sub.B) 1 [P.sub.66614.sup.+][Co(hfacac).sub.3.sup.−] 1164.0 575.8 S.sup.b,c,e 4.3 2 [P.sub.66614.sup.+][Mn(hfacac).sub.3.sup.−] 1160.0 401.8 S.sup.b,c,e 5.8 3 [P.sub.66614.sup.+][Ni(hfacac).sub.3.sup.−] 1163.7 927.9 S.sup.b,c,e 2.8 4 [P.sub.66614.sup.+][Dy(hfacac).sub.4.sup.−] 1474.6 291.5 S.sup.c,d,e 9.7 5 [P.sub.66614.sup.+][Gd(hfacac).sub.4.sup.−] 1469.3 276.5 S.sup.c,d,e 7.7 6 [P.sub.66614.sup.+][Nd(hfacac).sub.4.sup.−] 1456.3 299.4 S.sup.c,d,e 2.8 .sup.aViscosity measurements were performed at 23.7° C. .sup.bSoluble in hexane, heptane, toluene, and benzene at 10% (v/v) MIL to solvent ratio. .sup.cSoluble in acetone, acetonitrile, chloroform, dichloromethane, dioxane, ethanol, ethyl acetate, diethyl ether, methanol, isopropyl alcohol at 20% (v/v) MIL to solvent ratio. .sup.dSoluble in hexane, heptane, toluene, and benzene at 20% (v/v) MIL to solvent ratio. .sup.eInsoluble in water at 0.01% (v/v) MIL to water ratio. μ.sub.eff = effective magnetic moment in Bohr magnetons (μ.sub.B) determined from magnetic susceptibility data (from Quantum design SQUID magnetometer).
(145) Viscosity
(146) Many previously synthesized hydrophobic MILs such as [P.sub.66614.sup.+] tetrachloromanganate(II) ([P.sub.66614.sup.+].sub.2[MnCl.sub.4.sup.2−]), [P.sub.66614.sup.+] tetrachloroferrate(III) ([P.sub.66614.sup.+][FeCl.sub.4.sup.−]), and [P.sub.66614] hexachlorogadolinate(III) ([P.sub.66614.sup.+].sub.3[GdCl.sub.6.sup.3−]) possess high viscosities ranging from 650-83450 cP at 25° C., which can be problematic when using them for a number of applications. In this disclosure, a strategy was implemented to lower the viscosity by creating a singly charged metal anion paired with a cation that has previously been shown to produce MILs with low viscosity. The [P.sub.66614.sup.+][FeCl.sub.4.sup.−] MIL possesses a viscosity of 650 cP at 25° C., which is much lower compared to the [P.sub.66614.sup.+]2[MnCl.sub.4.sup.2−] (75230 cP at 25° C.) and [P.sub.66614].sub.3[GdCl.sub.6.sup.3−] (18390 cP at 25° C.) MILs. The use of a β-diketonate ligand allows for an overall singly charged anion as well as high spin states for some of the metal centers. Viscosities of the exemplary MILs disclosed herein are given in Table 1. The transition metal-based MILs all exhibit higher viscosities than the rare earth MILs, with the highest viscosity of 927 cP measured for the nickel-based MIL. An increasing trend of viscosities for the transition-metal MILs can be observed with a decrease in atomic radii from manganese to nickel (401.8 cP-927.9 cP). The rare earth metal MILs possess exceedingly low viscosities at 23.7° C. (<300 cP) when compared to other [P.sub.66614] based MILs such as [P.sub.66614.sup.+][FeCl.sub.4.sup.−] and [P.sub.66614.sup.+].sub.2[MnCl.sub.4.sup.2−].
(147) Thermal Stability
(148) The thermal stability of all exemplary MILs was tested by monitoring the thermal volatilization/decomposition of the MIL when the MIL was used as a stationary phase in gas chromatography. To achieve this, an approximate 0.25-0.28 μm film of IL/MIL was immobilized on the inner wall of a fused silica capillary. The IL/MIL coated capillary was then heated slowly in a GC oven and an ultra-sensitive flame ionization detector (FID) was used to detect any volatilization/decomposition of the IL/MIL. These tests were run using a temperature program starting at 40° C. and increased at 1° C./min to 350° C. on an Agilent 6850 gas chromatograph with a flame ionization detector (Santa Clara, Calif., USA). The [P.sub.66614.sup.+] [Cl.sup.−] IL was also coated under the same conditions and was used as a reference.
(149)
(150) Magnetic Susceptibility
(151) Exemplary MILs possess paramagnetic behavior that provides them distinct advantages over traditional ILs by allowing them to be easily removed or separated from an immiscible phase through the application of an external magnetic field. A handheld 1/16″×1″ neodymium-based rod magnet with a surface field of 6597 Gauss is sufficiently strong to collect small droplets of MIL dispersed in aqueous media. Octahedral complexes of Co(II), Mn(II), and Ni(II) all exhibit paramagnetism at room temperature. Likewise, the rare earth metals Dy(III), Gd(III), and Nd(III) with eight coordinating species have also shown paramagnetism at ambient temperatures. Exposure to a magnetic field results in spin alignment of unpaired electrons in the 3d orbital for the transition metal MILs and the 4f orbital for rare earth MILs. Removal of the magnetic field results in random spin orientation due to thermal motion, which creates a loss of magnetization. Most paramagnetic materials exhibit an inverse relationship between magnetic susceptibility and temperature as defined by the Curie-Weiss law.
(152) The μ.sub.eff values for each exemplary MIL were determined using a Quantum Design MPMS SQUID magnetometer following procedures similar to those previously reported. (Y. Mudryk, P. Manfrinetti, V. Smetana, J. Liu, M. L. Fornasini, A. Provino, V. K. Pecharsky, G. J. Miller and K. A. Gschneidner, J. Alloys Compd, 2013, 557, 252-260).
(153)
(154)
(155)
(156) MILs exhibiting higher magnetic susceptibility were achieved by chelating rare earth gadolinium(III) and dysprosium(III) ions possessing high magnetic moments into the anion structure.
Example 4
(157) Extraction of Viable E. coli Cells Using Hydrophobic MILs
(158) The chemical structures of seven exemplary hydrophobic MILs that were used for the extraction of E. coli are shown in Table 2. To determine if MILs can extract viable E. coli cells, aqueous solutions of 1.68×10.sup.5 CFUs mL.sup.−1 were extracted with each of the seven MILs using an approach that is similar to one depicted in
(159) TABLE-US-00002 TABLE 2 Chemical Structures of the Seven Exemplary Magnetic Ionic Liquids Investigated for the Extraction of E. coli from Aqueous Samples Viable Cytotoxicity No. Cation Anion Bacteria to E. coli 1
(160) This example shows that a MIL-based method may provide a rapid approach for extracting and preconcentrating viable bacteria in which the extraction phase can be easily manipulated using a magnetic field.
Example 5
(161) Cytotoxicity of MILs
(162) Since culture-based methods are contingent upon the isolation of viable bacteria, the detection of E. coli following MIL-based cell extraction is influenced by the cytotoxicity of MILs. To study the effect of MILs on the growth of E. coli, a 1 mL aliquot of LB media was inoculated with 8.0×10.sup.2±0.5×10.sup.2 CFUs mL.sup.−1 and spiked with approximately 10 μL of a MIL. The mixture was agitated by vortex to simulate the recovery procedure after which a 100 μL aliquot of the suspension was plated on selective agar. When compared to a standard that had not been exposed to a MIL, the Ni(II), Co(II), Mn(II), Dy(III), and Nd(III)-based MILs had little to no influence on the growth of E. coli. However, the [P.sub.66614.sup.+][Gd(hfacac).sub.4.sup.−] and [P.sub.66614.sup.+][FeCl.sub.4.sup.−] MILs hindered the proliferation of cells (as indicated by fewer observable colonies), suggesting that these MILs exhibit toxicity toward E. coli K12 cells. Although the mechanism for the decrease in cell viability is unknown, these findings are consistent with E. coli toxicity that was previously observed for Fe(III)-based MILs (Clark K D, Sorensen M, Nacham O, Anderson J L. Preservation of DNA in nuclease-rich samples using magnetic ionic liquids, RSC Adv. 2016; 6:39846-51).
Example 6
(163) Effect of MIL Volume on the Extraction and Preconcentration of E. coli from Aqueous Solution
(164) The effect of MIL volume on the extraction of E. coli was studied by varying the volume of [P.sub.66614.sup.+][Ni(hfacac).sub.3.sup.−] added to an aqueous cell suspension at 1.68×10.sup.4 CFUs mL.sup.−1. Since E.sub.F values are highly dependent upon the volume of extraction phase, the exact volumes of MIL dispensed into the aqueous cell suspension were calculated from the mass of MIL added to the sample and MIL density. The calculated volumes were 2.89±0.24 μL, 5.42±0.15 μL, 9.72±0.38 μL, and 12.82±0.10 μL, corresponding to 5 μL, 10 μL, 15 μL, and 20 μL of MIL initially withdrawn into the pipette, respectively. Since the studied MILs exhibit remarkable hydrophobic character (as low as 0.01% (v/v)), their solubility in aqueous solution was negligible especially when considering the short duration of the extraction protocol. As shown in
Example 7
(165) Optimization of MIL-Based Cell Extraction
(166) The effect of extraction time on the enrichment of E. coli was investigated using the [P.sub.66614.sup.+][Ni(hfacac).sub.3.sup.−] MIL from 15 s to 300 s. As shown in
Example 8
(167) Rapid Detection of E. coli with PCR Amplification
(168) Nucleic acid-based methods (e.g., PCR) have become increasingly popular for the detection of pathogens in food, environmental, and clinical samples to increase sample throughput and achieve lower detection limits. To demonstrate the compatibility of the MIL-based cell enrichment method with PCR amplification, aqueous samples were inoculated with E. coli transformants possessing the 879 bp MTAP gene at concentrations ranging from 4×10.sup.8 to 3×10.sup.7 CFUs mL.sup.−1 and extracted using the [P.sub.66614.sup.+] [Co(hfacac).sub.3.sup.−] MIL under optimized conditions. After extraction, 0.5 μL of the cell-enriched MIL phase was directly transferred into the PCR reagent mixture and heated at 95° C. for 5 min to induce cell lysis and the release of nucleic acids into solution. As shown in
(169) To improve detection limits, cells were recovered from the MIL phase using nutrient broth and a 1 μL aliquot of LB media was analyzed by PCR amplification. The six hexafluoroacetylacetonate-based MILs were applied for the extraction of E. coli at concentrations as shown in
(170) Since the [Dy(hfacac).sub.4.sup.−] and [Nd(hfacac).sub.4.sup.−]-based MILs did not extract sufficient viable cells for culture-based detection, successful PCR amplification may have resulted from the extraction of cell-free DNA in the aqueous sample. To investigate this, 2 mL of an aqueous E. coli suspension (1.68×10.sup.5 CFUs mL.sup.−1) were passed through a sterile 0.22 μm syringe filter and the filtrate (lacking E. coli) was extracted using the Dy(III) and Nd(III)-based MILs. No amplicon was detected following PCR, indicating that cell-free DNA was not extracted by the [P.sub.66614.sup.+][Dy(hfacac).sub.4.sup.−] and [P.sub.66614.sup.+][Nd(hfacac).sub.4.sup.−] MILs in sufficient quantity for detection by PCR. Similarly, no amplicon was detected after extracting the cell filtrate using the [P.sub.66614.sup.+][Ni(hfacac).sub.3.sup.−] MIL. These findings suggest that the PCR assay is more sensitive than the culture-based method following enrichment with the [Dy(hfacac).sub.4.sup.−] and [Nd(hfacac).sub.4.sup.−]-based MILs, which is consistent with previous reports comparing the sensitivity of PCR and microbiological cultures for E. coli detection (Chapman P, Ellin M, Ashton R, Shafique W. Comparison of culture, PCR and immunoassays for detecting Escherichia coli 0157 following enrichment culture and immunomagnetic separation performed on naturally contaminated raw meat products, Int. J. Food Microbiol. 2001; 68:11-20 and Heininger A, Binder M. Schmidt S, Unertl K, Botzenhart K, Doring G, PCR and blood culture for detection of Escherichia coli bacteremia in rats, J. Clin. Microbiol. 1999; 37:2479-82). It should be noted that PCR assays are unable to distinguish between live and dead cells and, therefore, are complementary to microbiological culture for the determination of viable cells in a sample. The results demonstrate that MIL-based enrichment of bacteria is applicable for nucleic acid-based detection methods to provide increased sample throughput while simultaneously supporting culture-based assays for applications that require the identification of living microorganisms in a sample.
(171) The invention is being thus described. It will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present disclosure and all such modifications are intended to be included within the scope of the following claims.
(172) The above specification provides a description of various magnetic ionic liquids, methods of synthesizing and using the magnetic ionic liquids to extract a viable bacterium. Since many embodiments can be made without departing from the spirit and scope of the present disclosure, the invention resides in the claims.