Exploiting Fluorescence Spectroscopy To Identify Magnetic

Exploiting Fluorescence Spectroscopy To Identify Magnetic IonicLiquids Suitable for the Isolation of Oligonucleotides

Kalyan Santra, Kevin D. Clark, Nishith Maity, Jacob W. Petrich,* and Jared L. Anderson

Department of Chemistry, Iowa State University, Ames, Iowa 50011, United States

*S Supporting Information

ABSTRACT: Magnetic ionic liquids (MILs), which incor-porate paramagnetic ions, promise to minimize manual userintervention, decrease extraction times, and facilitate rapidrecovery of the analyte-enriched extraction solvent. If,however, fluorescence is employed in the downstream analysisof an analyte tagged with a fluorophore, the paramagnetic ionmay quench fluorescence by introducing new nonradiativeprocesses. Thus, it is necessary to employ a paramagnetic ionthat offers a compromise between possessing a high magneticmoment and not introducing new nonradiative channels.Mn(II), Fe(III), Co(II), and Ni(II) are considered incombination with phosphonium cations and anionic ligandsbased upon halides or hexafluoroacetylacetonate. Among thepossibilities examined, MILs containing Mn(II) provide the best alternative for a model system involving DNA.

■ INTRODUCTION

Nucleic acids play a myriad of roles in biological systems togovern the growth development of all living organisms.Modern DNA detection and sequencing technologies havemade possible the extremely rapid acquisition of geneticinformation and are poised to drive the expansion of nucleicacid-based diagnostics,1 personalized medicine,2 and biomarkerdiscovery.3 As a result, the isolation and purification of DNAfrom biological samples has become a significant bottleneck innucleic acid analysis. Conventional phenol−chloroformliquid−liquid extraction (LLE) or silica-based solid-phaseextraction (SPE) methods are limited by their ability to isolatea sufficient quantity of highly pure DNA that is suitable forsensitive downstream bioanalytical techniques, such aspolymerase chain reaction (PCR) amplification and fluores-cence imaging.4,5 Apart from variable DNA recoveries andpurities,6 the time-consuming and laborious sample handlingprocedures in LLE and SPE methods severely reduce samplethroughput.7

Recently, DNA extraction methods involving ionic liquids(ILs) have shown great promise to improve the speed,efficiency, and specificity of DNA isolation from biologicalsamples.8,9 ILs are a class of molten salts comprised of organic/inorganic cations and anions with melting temperatures at orbelow 100 °C. The unique ability to customize the structure ofIL cations and anions through accessible synthetic methods isan attractive feature of these solvents that has led to theirsuccessful implementation as DNA extraction solvents,10 PCRadditives,11 and nucleic acid preservation media.12 Carefuldesign of the IL structure has also generated a new class ofsolvents know as magnetic ionic liquids (MILs) that contain

paramagnetic components in their chemical structure.13−15

Unlike ferrofluids that are colloidal suspensions of magneticparticles in a carrier solvent, MILs are neat liquids that respondto applied magnetic fields.16 The convergence of the liquidnature, magnetic susceptibility, and tunable structure of MILsrepresents a distinct advantage relative to conventional ILs byproviding a solvent that can be employed in magnet-basedsample preparation workflows to minimize manual userintervention, decrease extraction times, and facilitate rapidrecovery of the analyte-enriched extraction solvent.The extraction of DNA using MIL solvents has recently

been demonstrated as a rapid, magnet-based alternative toconventional DNA sample preparation methods. Severalphosphonium and ammonium-based MILs with anioniccomponents based on paramagnetic tetrahaloferrate(III)complexes were investigated to reveal that MILs with differentchemical structures provided unique DNA solvation/extractioncapabilities.17 To capitalize on the rapid and selectiveextractions afforded by MIL solvents, a method coupling theextraction procedure with downstream PCR amplification andgel electrophoresis was developed to analyze directly the DNAenriched within the MIL.18 Although it was possible tomitigate PCR inhibition caused by the Fe(III) component ofthe MIL by designing an appropriate PCR buffer system, theFe(III)-based MILs precluded real-time fluorescence detectionof the accumulation of PCR amplification products (i.e., real-time quantitative PCR or qPCR), even under identical buffer

Received: June 11, 2018Revised: July 12, 2018Published: July 13, 2018

Article

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conditions, because they strongly quenched the fluorescence ofthe tagged DNA.Since fluorescence detection is central to quantification or

imaging applications in DNA analysis, the identification ofMIL extraction solvents that are compatible with fluorescence-based DNA assays is highly desirable. As the undesirablefluorescence quenching may arise from intersystem crossingpromoted by spin−orbit coupling induced through the “heavy-atom” effect by the paramagnetic atom, excited-state electrontransfer to or from the fluorescent label to any component ofthe MIL, or from Forster energy transfer from the label, suchan identification requires assessing these quenching mecha-nisms as a function of various paramagnetic metals. Here, weexamine the roles of the paramagnetic species Fe(III), Co(II),Mn(II), and Ni(II) constituting part of a halide anion orhexafluoroacetylacetonate (hfacac) anion. These anions differstructurally from each other considerably and hence vary theinteractions between the paramagnetic ions and the fluorescenttag (cyanine5 carboxylic acid, Cy5). Quenching studies are

employed to evaluate the interactions of MILs with the Cy5 toidentify MILs that are appropriate for direct spectroscopic-based analysis of extracted DNA and possibly other biologicalmaterials such as RNA or proteins.

■ MATERIALS AND METHODS

The ILs, MILs, and fluorophores used for spectroscopicmeasurements are presented in Figure 1. (See the caption tothe figure for abbreviations.) Hydrophobic MILs containingmetal-halide anions were synthesized according to previouslyreported procedures.14,19 Briefly, the corresponding metal-halide salt was mixed with [P66614

+][Cl−] at a 2:1 mole ratio(for Co and Mn-based MILs) or 1:1 mole ratio (for Fe(III)-based MILs) in methanol for 24 h. After solvent evaporation,the MIL was dried in a vacuum oven for 48 h. Syntheses of[P66614

+][Co(hfacac)3−], [P66614

+][Ni(hfacac)3−], and

[P66614+][Mn(hfacac)3

−] were performed as reported.20 Forpurification, the hfacac-based MILs were dissolved in diethylether and washed five times with 40 mL of deionized water or

Figure 1. Structures of the IL, MILs, and fluorophores. (1) Phosphonium chloride, [P66614+][Cl−]. (2) Phosphonium cobalt(II) chloride,

[P66614+]2[CoCl4

2−]; phosphonium manganese(II) chloride, [P66614+]2[MnCl4

2−]; and phosphonium iron(III) chloride, [P66614+][FeCl4

2−]. (3)Phosphonium cobalt(II) hexafluoroacetylacetonate, [P66614

+][Co(hfacac)3−]; phosphonium nickel(II) hexafluoroacetylacetonate, [P66614

+][Ni-(hfacac)3

−]; and phosphonium manganese(II) hexafluoroacetylacetonate, [P66614+][Mn(hfacac)3

−]. (4) Coumarin 153, C153. (5) Cyanine5carboxylic acid, Cy5. Experiments using C153 are discussed in the Supporting Information.

The Journal of Physical Chemistry B Article

DOI: 10.1021/acs.jpcb.8b05580J. Phys. Chem. B 2018, 122, 7747−7756

7748

until the addition of AgNO3 to the water layer yielded noprecipitation. All MILs were dried at 50 °C in vacuo for 48 hprior to spectroscopic measurements.Cy5 and Coumarin 153 (C153) were obtained from

Lumiprobe and Exciton, Inc., respectively. Cy5-tagged DNA(5′-Cy5-ACAGACTGATGTTGA-3′, subsequently referred asCy5-DNA) was obtained from Integrated DNA Technologies(Coralville, IA). All chemicals and organic solvents (FischerScientific, HPLC or ACS grade) were used as received. Forfluorescence quenching studies of phosphonium, metal-halideMILs, about 1 μM stock solution of Cy5 was prepared indimethyl sulfoxide (DMSO). The neat MILs are dissolved inthe stock dye solution to keep the concentration of the dyeidentical throughout the experiments. The concentrations ofthe metal, halide-based MILs were varied between 0 and 5 mMfor [P66614

+]2[CoCl42−], 0−30 mM for [P66614

+][FeCl4−], and

0−70 mM for [P66614+]2[MnCl4

2−]. Similarly, for phospho-nium metal hfacac MILs, the solutions were prepared in a ∼1μM stock solution of Cy5 in methanol. The concentration ofmetal hfacac-based MILs was kept between 0 and 30 mM for[P66614

+][Co(hfacac)3−], 0−30 mM for [P66614

+][Ni-(hfacac)3

−], and 0−150 mM for [P66614+][Mn(hfacac)3

−].For the control experiment with [P66614

+][Cl−] (phosphoniumchloride IL), the solutions were prepared in a ∼1 μM stocksolution of Cy5 in methanol and the concentrations were keptwithin 0−30 mM. For the control experiments with metalchloride salts, solutions were prepared in a ∼1 μM stocksolution of C153 in DMSO and in a ∼1 μM stock solution ofCy5 in DMSO. The concentrations were 0−5 mM for FeCl3 insolution with C153, 0−100 for MnCl2 in solution with C153,0−100 mM for FeCl3 in solution with Cy5, and 0−150 mM forMnCl2 in solution with Cy5. To study the fluorescence of Cy5and Cy5-DNA in neat MILs, the samples were prepared bydissolving the fluorophore or Cy5-DNA in a small volume(∼10 μL) of methanol and added dropwise to the MILs. Thesamples were then dried at room temperature to constant massusing a Mettler Toledo NewClassic MF MS105 microbalance(Columbus, OH) with a 0.01 mg readability. The finalconcentrations of the fluorophore or the Cy5-DNA weremaintained near 1 μM.Steady-state absorption spectra were recorded using an

Agilent 8453 UV−visible spectrometer (Agilent Technologies)with 1 nm resolution. Steady-state fluorescence spectra wereobtained on a Fluoromax-4 spectrometer (Horiba Scientific)and corrected for lamp spectral intensity and detectorresponse. A 3 mm path-length quartz cuvette was used forabsorption and fluorescence measurements. Samples contain-ing Cy5 and Cy5-DNA were excited at 600 nm; thosecontaining C153, 400 nm.Time-resolved data were collected using a home-made, time-

correlated, single-photon counting (TCSPC) instrument usinga SPC-630 TCSPC module (Becker & Hickl GmbH). Acollimated Fianium pulsed laser (Fianium Ltd., Southampton,U.K.) at a 2 MHz repetition rate was used to excite the sampleat 550 nm. The excitation beam was vertically polarized.Emission was detected at the “magic angle” (54.7°) withrespect to the excitation using a 590 nm long-pass filter. Theinstrument response function (IRF) was measured bycollecting scattered light at 550 nm (without the emissionfilter). The full width at half-maximum of the instrumentfunction was typically ∼120 ps. TCSPC data were collected in1024 channels (bins), providing a time resolution of 19.51 ps/channel, and a full-scale time window of 19.98 ns. The number

of counts in the peak channel was set to 65 535 unlessotherwise indicated. A 3 mm path-length quartz cuvette wasused for the lifetime measurements.

■ RESULTS AND DISCUSSION

Role of Forster Resonant Energy Transfer. The rate ofnonradiative energy transfer, kET, from a fluorescent donor toa n a c c e p t o r w a s d e s c r i b e d b y F o r s t e r : 2 1− 2 4

( )kR

RET1 6

D

0=τ

,where τD is the unquenched fluorescence

lifetime of the donor, R is the distance between donor andacceptor; and R0 is referred to as the “critical distance,” definedby

Rn N

f9000 ln(10)

128( ) ( ) d0

6 D2

5 4A 0

D A4∫

ϕ κ

πν ν ν ν= ϵ

∞−

(1)

where f D(ν ) = F(ν)/∫ 0∞F(ν ) dν is the fluorescence intensity of

the unquenched donor normalized to unit area on awavenumber scale. The other parameters are: ϕD, thefluorescence quantum yield of the donor; κ2, the orientationfactor, assumed to be 2/3 for randomly oriented donors andacceptors; ϵA, the decadic molar extinction coefficient; n, therefractive index of the medium; and NA, Avogadro’s number.Quantifying the rate of nonradiative energy transfer thusreduces to evaluating R0, which for random or nearly randomlyoriented samples is determined by the overlap of the emissionspectrum of the donor and the absorption spectrum of theacceptor. Figure 2 provides an example for Cy5 and Co(II).

We have evaluated R0 for Cy5 with Mn(II), Fe(III), Co(II),and Ni(II) and hfacac and [P66614

+], with or without theparamagnetic ions. Values of R0 are compiled in Table 1.Co(II) is the only component of the MILs investigated herethat has a significant R0: for the isolated chloride salt of Co(II),R0 = 58.1 Å, larger than nearly a factor of 2 or more than anyother ligand. The ability, then, of Fe(III), for example, toquench Cy5 so efficiently17,18 must lie in mechanisms otherthan nonradiative energy transfer. Distinguishing the two,intersystem crossing and excited-state electron transfer, mostlikely lies beyond the scope of this investigation. But it is

Figure 2. Fluorescence and absorption data required for thecomputation of the Forster overlap integral indicated in eq 1. Thefluorescence spectrum of Cy5 (red) is plotted to have unit area on thewavenumber scale. The absorption of Co(II) is given in blue. Asufficient amount of Cl− was used in the preparation of the solutionsto ensure a tetrahedral arrangement of the halide about the metalcation.



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possible, however, to quantify the efficiency of quenching bythe paramagnetic ions, which is done in the following sections.Quantifying Bimolecular Quenching of Fluorescence.

Bimolecular quenching processes may occur, for example, bythe collisions of the excited-state fluorophore with othermolecules, which enhance nonradiative rates or introduce newones. It may also occur through the formation of non-fluorescent ground-state complexes, which is referred to asstatic quenching. Both collisional and static quenching can bequantified by Stern−Volmer equations.25−39 Collisionalquenching (also referred to as dynamic quenching) can bequantified by a plot of fluorescence intensity or fluorescencelifetime as a function of quencher concentration

F

FK1 Q0 0D

τ

τ= = + [ ]

(2)

where F and F0 are the integrated fluorescence intensities ofthe corrected spectra when concentrations of the quencher are[Q] and 0, respectively, and τ and τ0 are the fluorescencelifetimes of the fluorophore at the respective concentrations.KD is the Stern−Volmer quenching constant or dynamicquenching constant.In the case where the quencher forms a nonfluorescent

ground-state complex with the fluorophore, the fluorescenceintensity also decreases. The mechanism of the decrease isdifferent, however, because in the simplest example of theprocess, the ground-state complex is nonfluorescent, and theuncomplexed fluorophores have the unquenched lifetime.(Another signature of this process is that the absorptionspectrum changes with respect to that of the uncomplexedfluorophore.) This type of quenching is described by a staticquenching model, whose form is identical to that of eq 2 ifintensities are used

F

FK1 Q and 10S

0τ

τ= + [ ] =

(3)

where KS is the association constant or the static quenchingconstant. Clearly, however, it is possible for quenching to occur

by both collisions of the quencher with the excited-statefluorophore and its complexes with the ground-statefluorophore. In such cases, the Stern−Volmer plot shows anupward curvature, which is nonlinear in the quencherconcentration

F

FK K K(1 Q )(1 Q ) and 1 Q0D S

0D

τ

τ= + [ ] + [ ] = + [ ]

(4)

Equation 4 can be transformed into the following linear form

F FK

/

/(1 Q )0

0S

τ τ= + [ ]

(5)

Alternatively, the first part of eq 4 can be modified so that itdoes not use the lifetime data

F

FK1 Q0app= + [ ]

(6)

where Kapp = (KD + KS) + KDKS[Q]. The apparent quenchingconstant Kapp is calculated using eq 6 and plotted against [Q]to obtain the KD and KS from the slope and the intercept.If the association of the fluorophore with the quencher is

weak, then an apparent static quenching is observed along withthe dynamic quenching. Then, we have

F

FK V V(1 Q ) exp( Q ) exp( Q )0D

0τ

τ= + [ ] [ ] = [ ]

(7)

where V is the volume of the sphere of action within which thequencher can quench the excited fluorophore. If, however, theinteraction of the quencher with the fluorophore is very strong,V can be incorporated into eq 5

F FK V

/

/(1 Q ) exp( Q )0

0S

τ τ= + [ ] [ ]

(8)

Fluorescence Quenching of Cy5 by PhosphoniumMetal-Halide MILs. The steady-state and the time-resolvedStern−Volmer plots for fluorescence quenching of Cy5 inDMSO are presented in Figures 3 and 4 and in Figure S1 forthe [P66614

+]2[CoCl42−], [P66614

+]2[MnCl42−], and [P66614

+]-[FeCl4

−] MILs, respectively. The quenching parametersobtained from eqs 2 to 8 are summarized in Table 2. Amongthese three MILs, [P66614

+]2[CoCl42−] is the most efficient

quencher of Cy5 fluorescence. A plot (Figure 3d) of F0/Fagainst its concentration is highly nonlinear with an upwardcurvature, whereas a similar plot using τ0/τ plot (Figure 3e) islinear with a nonzero slope. From this complex quenching ofCy5 by [P66614

+]2[CoCl42−], the dynamic quenching constant

can be extracted: KD = 82 ± 3 M−1. The plot (Figure 3f) of(F0/F)/(τ0/τ) using eq 8 yields KS = 1200 ± 100 M−1 and V =520 ± 30 M−1, indicating a very large contribution fromground-state complex formation with the quencher. Also, asexpected, the absorption spectrum at 600 nm (Figure 3a) issignificantly altered in the presence of the quencher. Amongthe three MILs, the least efficient quencher of Cy5 in DMSO is[P66614

+]2[MnCl42−]. The plot of τ0/τ against its concentration

(Figure 4e) is linear, providing a dynamic quenching constant,KD = 0.2 ± 0.1 M−1, which is negligible compared to thatobtained for [P66614

+]2[CoCl42−]. The plot obtained from eq 5

provides the static quenching constant, KS = 1 ± 1 M−1,indicating a negligible interaction of [P66614

+]2[MnCl42−] with

Cy5. Finally, the time-resolved Stern−Volmer plot (FigureS1e) for [P66614

+][FeCl4−] yields KD = 6.0 ± 0.2 M−1, whereas

Table 1. Forster Critical Distances

quencher R0 (Å)

Mn(II)a 16.1

Fe(III)a 26.9

Co(II)a 58.1

Ni(II)a 33.6

hfacac 15.9

[P66614+][Cl−] 10.2

[P66614+][hfacac−] 17.0


−] 26.7

[P66614+][Co(hfacac)3

−] 22.2

[P66614+][Ni(hfacac)3

−] 33.8

[P66614+]2[CoCl4

2−] in DMSOb 57.3

[P66614+]2[MnCl4

2−] 14.2

[P66614+][FeCl4

−] 20.7aA sufficient amount of Cl− (i.e., ∼6 M) was used in the preparationof the solutions to ensure a tetrahedral arrangement of the halideabout the metal cation. bMethanol was the solvent for themeasurement of Forster critical distances in all cases except for[P66614

+]2[CoCl42−], where DMSO was required. In methanol, the

solution took on an uncharacteristic pink color, and R0 = 33.5 Å. Notethat R0 for [P66614

+]2[CoCl42−] in DMSO is essentially equal to that

for Co(II) in methanol.



7750

the linear plot (Figure S1f) using eq 5 yields KS = 30 ± 2 M−1.Therefore, formation of the ground-state complex is theprimary contributor to the overall quenching, which can alsobe noted from the considerable change of absorption spectranear 600 nm. The overall quenching efficiency, however, ismuch less than that of [P66614

+]2[CoCl42−] and higher than

that of [P66614+]2[MnCl4

2−]. The extent of quenching of Cy5 inneat [P66614

+][FeCl4−] is demonstrated in Figure S2 and

exhibits a 1000-fold decrease of the fluorescence intensitycompared to that for Cy5 in methanol at the sameconcentration (∼1 μM). The fluorescence lifetime of Cy5measured in neat [P66614

+][FeCl4−] is ∼150 ps compared with

810 ps in methanol. On the other hand, the fluorescenceintensity of Cy5 in neat [P66614

+]2[MnCl42−] increases by a

factor of 2 with respect to methanol and its fluorescencelifetime is 2.04 ns (Figure S3). Not only does Mn(II) not

quench the fluorescence of Cy5 but the phosphonium-Mn(II)chloride MIL increases the fluorescence intensity and lifetimeof Cy5. These increases are most likely attributable to the highviscosity of the solvent, which reduces the flexibility40 of Cy5.It is likely that a significant nonradiative pathway of Cy5 istorsion about its double bonds (Figure 1) similar to thesituation for the well-studied molecule, stilbene.22,41,42 For neat[P66614

+][FeCl4−], the viscosity20 is 650 cP at 25 °C, whereas it

is 75 230 cP at 25 °C for [P66614+]2[MnCl4

2−].To elucidate the origin of the quenching mechanism of the

phosphonium metal-halide MILs, we constructed Stern−Volmer plots of Cy5 dye using the nonmagnetic ionic liquid,[P66614

+][Cl−], as a control (Figure 5). The Stern−Volmerplot, within experimental error, is flat, demonstrating that[P66614

+][Cl−] does not quench Cy5: KD = −1 ± 1 M−1 (Table3). This confirms that the metal ions in the three phosphonium

Figure 3. Fluorescence quenching of Cy5 in DMSO as a function of [P66614+]2[CoCl4

2−] concentration. (a) Absorption spectra. (b) Fluorescenceemission spectra, λex = 600 nm. Intensities are corrected for the absorption at the wavelength of excitation. (c) Time-resolved fluorescence decay.The IRF is the instrument response function for the apparatus. (d) Stern−Volmer plot for the integrated fluorescence intensity ratio (F0/F) as afunction of the quencher concentration. (e) Stern−Volmer plot of (τ0/τ) as a function of the quencher concentration. (f) Plot of (F0/F)/(τ0/τ) asa function of the quencher concentration.



7751

MILs that we have considered are responsible for thequenching of the Cy5 fluorescence. This observation is further

confirmed by performing quenching studies of Cy5 and C153dye with chloride salts of manganese and iron (Table S1 and

Figure 4. Fluorescence quenching of Cy5 in DMSO as a function of [P66614+]2[MnCl4

2−] concentration. (a) Absorption spectra. (b) Fluorescenceemission spectra at λex = 600 nm. Intensities are corrected for the absorption at the wavelength of excitation. (c) Time-resolved fluorescence decay.(d) Stern−Volmer plot for the integrated fluorescence intensity ratio (F0/F) as a function of the quencher concentration. (e) Stern−Volmer plot of(τ0/τ) as a function of the quencher concentration. (f) Plot of (F0/F)/(τ0/τ) as a function of the quencher concentration.

Table 2. Parameters for the Quenching of Fluorescence of Cy5 by Metal-Halide-Based MILs

anion plot typea KD (M−1) KS (M−1) V (M−1)

[CoCl42−]b τ0/τ vs [Q] 82 ± 3

(F0/F)/(τ0/τ) vs [Q]c 1200 ± 100 520 ± 30

[MnCl42−] F0/F vs [Q] 1 ± 2

τ0/τ vs [Q] 0.2 ± 0.1

(F0/F)/(τ0/τ) vs [Q]d 1 ± 1

[FeCl4−] F0/F vs [Q] 40 ± 2

τ0/τ vs [Q] 6.0 ± 0.2

(F0/F)/(τ0/τ) vs [Q]d 30 ± 2

aThe relationships are given in eqs 2−8. [Q] is the quencher concentration. bFor [P66614+]2[CoCl4

2−], the F0/F vs [Q] plot shows a large deviationfrom linearity with an upward curvature. cThe plot of (F0/F)/(τ0/τ) vs [Q] is nonlinear and fit to eq 8. dThe plot of (F0/F)/(τ0/τ) vs [Q] is linearand fit to eq 5.



7752

Figures S4−S7). The quenching of C153 and Cy5 by MnCl2 ispurely dynamic, and the values of the quenching constants areKD = 2.1 ± 0.1 and 0.1 ± 0.3 M−1 for C153 and Cy5,respectively. Therefore, the extent of quenching by MnCl2 isnegligibly small. On the other hand, FeCl3 has a significanteffect on the fluorescence of C153 (KD = 15 ± 1 M−1, KS =1700 ± 60 M−1, and V = 730 ± 9 M−1). It also quenches thefluorescence of Cy5 considerably (KD = 3.0 ± 0.2 M−1 and KS

= 11.9 ± 0.6 M−1).Fluorescence Quenching of Cy5 by Metal-hfacac

MILs. Steady-state spectra and Stern−Volmer plots for thequenching of Cy5 in methanol are presented in Figures S8 andS9 and in Figure 6 for the [P66614

+][Co(hfacac)3−], [P66614

+]-[Ni(hfacac)3

−], and [P66614+][Mn(hfacac)3

−] MILs, respec-

tively. The quenching parameters obtained from eqs 2 to 8 aresummarized in Table 3. One of the most striking differencesbetween the hfacac MILs with respect to the metal-halideMILs is that hfacac solvents are all well described by dynamicquenching, eq 2, whereas it is necessary to describe the halidesolvents by static quenching mechanisms as well. This suggeststhat hfacac is effective in preventing the formation of ground-state complexes. For example, the quenching by [P66614

+][Co-(hfacac)3

−] is quite different from that of [P66614+]2[CoCl4

2−],the chloride analog. Figure S8a indicates that its absorptionspectrum is not as significantly altered by the quencher, as inthe case of the chloride analog. Unlike its chloride analog, theplot of F0/F against the concentration of [P66614

+][Co-(hfacac)3

−] is linear within experimental error (Figure S8c),yielding the Stern−Volmer quenching constant, KD = 69 ± 2M−1. For [P66614

+][Ni(hfacac)3−], a plot of F0/F against its

concentration is best fit, within experimental error, to a line(Figure S9c), yielding KD = 73 ± 4 M−1. It can thus beconcluded that [P66614

+][Ni(hfacac)3−] quenches Cy5 to a

similar degree as does [P66614+][Co(hfacac)3

−]. The absorptionspectra of Cy5 in the presence of [P66614

+][Mn(hfacac)3−]

change the least for the three hfacac-based MILs. The plot ofF0/F against its concentration (Figure 6c) yields KD = 1.9 ±

0.3 M−1, indicating a very small degree of quenching,comparable to that of [P66614

+]2[MnCl42−]. The viscosities of

the hfacac-based MILs containing Ni(II), Co(II), and Mn(II)were 927.9, 575.8, and 401.8 cP, respectively,20 demonstratingthat viscosity has little to no influence on the quenchingconstants for this series of MILs and that the identity of themetal ion primarily dictates this parameter. These resultssuggest that among the paramagnetic ions we investigated,Mn(II) is the optimum choice for fluorescence-based analyticaltechniques.Finally, to illustrate the suitability of Mn(II)-MILs for

fluorescence-based assays of DNA, we compared the labeledoligonucleotide, Cy5-DNA, in neat: [P66614

+][FeCl4−] and

[P66614+]2[MnCl4

2−]. The absorption and fluorescence emis-sion of Cy5 are greatly modified and the fluorescence intensitywas quenched significantly in [P66614

+][FeCl4−] as compared to

that in [P66614+]2[MnCl4

2−] (Figure 7). The fluorescencelifetime of Cy5-DNA in neat [P66614

+][FeCl4−] is <100 ps,

whereas in neat [P66614+]2[MnCl4

2−], it is 1.56 ns and single

Figure 5. Fluorescence quenching of Cy5 in methanol as a function of[P66614

+][Cl−] concentration. (a) Absorption spectra. (b) Fluores-cence emission spectra at λex = 600 nm. Intensities are corrected forthe absorption at the wavelength of excitation. (c) Stern−Volmer plotfor the integrated fluorescence intensity ratio (F0/F) as a function ofthe IL concentration.

Table 3. Parameters for the Quenching of Fluorescence ofCy5 by [P66614

+]-Based MILs with hfacac Ligands

anion KD or KS (M−1)a

[Co(hfacac)3−] 69 ± 2

[Ni(hfacac)3−] 73 ± 4

[Mn(hfacac)3−] 1.9 ± 0.3

[Cl−]b −1 ± 1aThe data that are fit well to eqs 2 and 3 are formally identical and canonly be distinguished by measuring fluorescence lifetimes, which wasnot possible for this series of samples. Because, however, there was nochange in the absorption spectrum as a function of quencherconcentration (Figure 6a), it is appropriate to conclude that the[Mn(hfacac)3

−] solvent quenches by a dynamic process. There isevidence of some ground-state complexation for the [Co(hfacac)3

−]and a considerable amount for [Ni(hfacac)3

−] solvents (Figures S8and S9, respectively) but not to that extent as observed for the halides.In all instances, then, it appears that the hfacac ligand suppresses orsubstantially mitigates ground-state complexation. b[P66614

+][Cl−] is a“conventional” ionic liquid with no magnetic moment and serves as acontrol.



7753

exponential, demonstrating the effective homogeneity of thenonquenching environment to which the Cy5 is exposed.These data confirm the utility of Mn(II)-based MILs.

■ CONCLUSIONS

We have investigated MILs comprised of metal-based anions inthe form of chloride or hfacac complexes with phosphonium([P66614

+]) cation counterparts using absorption and emissionspectroscopy to identify MILs that render them compatiblewith downstream fluorescence assays for DNA biopolymers.This entailed the evaluation of the quenching efficiency, withrespect to the commonly used fluorescent tag, Cy5, of theparamagnetic ions, Mn(II), Fe(III), Co(II), and Ni(II) both

isolated and associated with various components of the MILs,and the MILs themselves.Although MILs containing metal ions such as Fe(III) may

be advantageous for magnet-based extraction applications dueto their higher magnetic moments, Fe(III)-MILs stronglyquench (probably by intersystem crossing or possibly byexcited-state electron transfer) fluorescence that couldpotentially be used for emission-based techniques. Similarly,Co(II)-MILs are strong quenchers that preclude the use offluorescence. Mn(II)-MILs, however, balance a high magneticmoment with low fluorescence quenching.A summary and assessment of the nature and magnitude of

the quenching processes follows. Co(II) is the only component

Figure 6. Fluorescence quenching of Cy5 in methanol as a function of[P66614

+][Mn(hfacac)3−] concentration. (a) Absorption spectra. (b)

Fluorescence emission spectra at λex = 600 nm. Intensities arecorrected for the absorption at the wavelength of excitation (c)Stern−Volmer plot for the integrated fluorescence intensity ratio (F0/F) as a function of the quencher concentration.

Figure 7. Absorption and emission of Cy5-DNA (∼1 μM) in neat[P66614

+][FeCl4−] (gray) and in neat [P66614

+]2[MnCl42−] (red). (a)

Absorption spectra. (b) Fluorescence emission spectra, λex = 600 nm.Note that the spectra are normalized to the same average peakintensity but that the intensity in the [FeCl4

−] solvent is roughly 200times less than in the [MnCl4

2−] counterpart. (c) Time-resolvedfluorescence decay traces confirming the results of panel (b). IRF isthe instrument response function of the apparatus. The lifetimes ofCy5-DNA are <100 ps in neat [P66614

+][FeCl4−] and 1.56 ns in neat

[P66614+]2[MnCl4

2−].



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of the MILs that can significantly quench by nonradiativeForster resonant energy transfer (Table 1). In contrast, Fe(III)is a quencher, as can be determined from Table 2, but eitherfunctions via intersystem crossing or excited-state electrontransfer as the overlap between the Cy5 emission spectrumwith its absorption spectrum is small. Hfacac keeps the Co(II)farther away from the Cy5 than does the halide and thuschanges the quenching interaction. Specifically, R0 is smallerwith hfacac than with the halide anion (Table 1). Furthermore,there seems to be little or no static quenching with hfacac. Thisis suggestive of the role hfacac may play with the other metalsas well. The metal hfacac anion is preferable to the metal-halide because it provides “cleaner” spectra by reduced ground-state complexation (and quenching) with the fluorescent tag,Cy5, as indicated by the relatively smaller or absent changes ofthe Cy5 absorption spectrum with hfacac concentration (Table3). The Stern−Volmer quenching constants for Mn(II)-basedMILs are one order of magnitude smaller than those of Co(II)or Ni(II)-based MILs. Mn(II) does not obfuscate the spectraof the fluorescent label, as do Fe(III) and Co(II). Finally, andmost importantly, our results demonstrate that Mn(II)-basedMIL extraction solvents such as [P66614

+]2[MnCl42−] and


−] are ideal for direct spectroscopicanalysis of extracted DNA and possibly other biologicalmaterials, such as RNA or proteins.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jpcb.8b05580.

Parameters for fluorescence quenching of C153 and Cy5by metal chloride salts (Table S1); fluorescencequenching of C153 and Cy5 by metal chloride saltsand metal hexafluoroacetylacetonate salts (Figures S1−S9) (PDF)

■ AUTHOR INFORMATION

Corresponding Author*E-mail: [email protected]. Phone: +1 515 294 9422. Fax: +1515 294 0105.

ORCID

Kalyan Santra: 0000-0002-2556-1889Kevin D. Clark: 0000-0002-0576-7716Jacob W. Petrich: 0000-0001-9527-6832Jared L. Anderson: 0000-0001-6915-8752

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

J.L.A. acknowledges support from the Chemical Measurementand Imaging Program of the National Science Foundation(CHE-1709372). We thank Matthew Klyman for technicalassistance.

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