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Kinetics of Antimicrobial Drug Ion Transfer at a Water/ Oil Interface Studied by Nanopipet Voltammetry Surendra Raj Puri, and Jiyeon Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03593 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Analytical Chemistry

Kinetics of Antimicrobial Drug Ion Transfer at a Water/Oil Interface Studied by Nanopipet Voltammetry Surendra Raj Puri, Jiyeon Kim* Department of Chemistry, The University of Rhode Island, Kingston, RI, 02881

Abstract Effective delivery and accumulation of antimicrobial agents into the microbial organism is essential for the treatment of bacterial infections. Transports of hydrophilic drug molecules, however encounter a robust barrier of hydrophobic double membrane cell envelope, thus leading to drug-resistance in Gram-negative bacteria. Accordingly, deeper understanding about a transit of charged molecules through bacterial membrane is needed to remediate the antibacterial resistance. Herein, we apply a steady-state voltammetry using nanopipet-supported interfaces between two immiscible electrolyte solutions (ITIES) to quantitatively study transport-kinetics of antimicrobial drug-ions (quinolones and sulfonamides) at a water/oil interface. Importantly, ITIES can mimic cellular membrane system, thus being employed as insightful surrogates for kinetic study of drug entry through bacterial cytoplasmic membranes. This approach enables us to voltammetrically and amperometrically detect redox-inactive drug-ions as pristine under physiological conditions. Considerably slow kinetics of drug-ion transports are successfully measured by nanopipet voltammetry and theoretically analyzed. This analysis reveals that the drug-ion transport is ~3 orders of magnitude slower than tetrabutylammonium ion transport. In addition, the extreme hydrophilicity of drug-ions in comparison to ClO4– is quantitatively assessed from half-wave potentials of obtained voltammograms. The high hydrophilicity exclusively attributed to localized negative charges on carboxylate or amide group of deprotonated quinolone or sulfonamide, respectively may play a dominant role in sluggish kinetics due to the increase in energy barriers upon interfacial ion transfer. Notably, this study using nanopipet voltammetry provides physicochemical insights on the correlation between structural properties of pristine drugions and their transfer-kinetics at a water/oil interface in lieu of biological membranes. Keywords: Nanopipet voltammetry, ITIES, Antimicrobial ions, Ion transfer kinetics, Effective hydrophilicity. 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Bacterial drug resistance has been a global health issue.1,2,3,4,5,6 A major challenge we are facing in Gram negative (GN) antibacterial research, is to thoroughly understand and control membrane permeation of antibiotics in clinically relevant microbial organism. In fact, penetrating the membrane barrier to reach a critical concentration inside microbial organisms is an essential step for most antibiotics7. GN bacteria, however have a hydrophobic double-membrane cell envelope, serving as a major barrier for hydrophilic molecules to enter the cell.8 Accordingly, in a fight with resistant GN organisms, primary obstacle confronting the discovery and development of new antibiotics is the lack of a rational strategy to understand the transit of charged molecules through bacterial membrane, and equip antimicrobial agents with requisite properties to permeate bacterial membrane.9 Hence it is crucial to understand antibiotic permeation and accumulation in the cell, and a better method to quantify antibiotic concentration and kinetics of its permeation through the cell membrane is urgently required. If these measurements are correlated with an intrinsic structural property of respective antibiotics, a deeper and more relevant insight about the physicochemical behavior can be achieved for the battle with bacterial drug resistance. In this regard, ion transfer processes across a polarized interface between two immiscible electrolyte solutions (ITIES) can be a powerful analytical tool fit for this demand compared to other types of either electrochemical10,11,12,

13,14,15

or nonelectrochemical approaches7,16,17.

Specifically, ITIES supported by pipet can selectively and directly probe charged molecules transferring across the various types of liquid/liquid interface.18,19 The interfacial ion transfer studied by amperometry and (or) voltammetry gives an electrical current signal, enabling to quantify the concentration and transfer kinetics of respective ions.18,19,20,21 Importantly, ITIES can mimic a cell membrane system,22 thus being an insightful surrogate for kinetic study of drug entry through the bacterial cytoplasmic membrane. Moreover, this analysis can be used as an excellent alternative for understanding lipophilicity (or, hydrophilicity) of transferred ions.23,24 Considering these aspects, the ITIES would be a proper model system to (1) mimic a cell membrane, (2) study the transfer process of differently charged drug molecules at different pH values, and (3) provide insightful information related to physicochemical behavior and drug delivery, thus assisting drug design and synthesis.

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Herein, we apply a steady state voltammetry using an ITIES to sense antibacterial drugs and determine their ion transfer kinetics under a physiological condition. Specifically, we employ nanopipet based ITIES as an artificial membrane system and quantitatively assess the ion transfer kinetics of common antibacterial agents, quinolones and sulfonamides. At ~pH 8.00, all quinolones and sulfonamides undergo deprotonation with a net charge, −1, thus being detectable as pristine with ITIES (Figure 1).25 In comparison to metal electrodes, a nanopipet supported ITIES not only allows to sensitively detect redox inactive ions,23,26,27,28 but also provides a selectivity for diverse ions,29,30 physical robustness,31 and an extremely small size of active area for a high spatial resolution measurement32. In this study, a nanopipet supported ITIES is formed at the 70~120 nm radius pipet tip filled with a water-immiscible 1,2-dichloroethane (DCE) solution to voltammetrically and amperometrically detect an aqueous drug ion (Figure 2). As a potential is applied to the organic phase against aqueous reference electrode, a target ion is driven to transfer from water to organic phase across the interface, giving electrical currents. Importantly, this study reveals that the kinetics of interfacial ion transfer for both quinolone and sulfonamides significantly slows down compared to a typical hydrophobic cation, TBA+. Also, a considerably high hydrophilicity of drug ions is evaluated quantitatively against a hydrophobic anion, ClO4−. We attribute the observed sluggish kinetics exclusively to a high hydrophilicity of respective drug ions (see Result and Discussions). In fact, this high hydrophilicity is likely ascribed to unshielded, localized negative charges on the carboxylate or amide group for quinolones or sulfonamides respectively. Here, we argue that the presence of these localized negative charges could play a dominant role on increasing a hidden barrier relevant to formation/breaking of a water-finger upon interfacial ion transfer. Hence, this structural property could mainly induce a retarded rate of interfacial ion transfer. It should be noted that this study suggests a close correlation between transfer-kinetics and the effective hydrophilicity related to the molecular structure of drug ions during ion transfer, thereby leading to a great insight into the physicochemical behavior of drug ions upon the permeation through an artificial membrane system.



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(C) Compound

pKa

Nalidixic acid (NA) Difloxacin (DFX) Flumequine (FMQ) Sulfadimethoxine (SDM) Sulfamerazine (SMR) Sulfamethazine (SMT)

5.95 5.66, 7.24 6.35 2.13, 6.08 2.22, 6.80 2.37, 7.49

Figure 1. Acid dissociation equilibria of (A) sulfonamides (SDM, SMR, SMT), (B) acidic quinolones (top, NA, FMQ) and piperazinyl quinolones (bottom, DFX). The negatively charged group after deprotonation is highlighted. (C) pKa values for the quinolones and sulfonamides.25 Detailed structures of each drug molecules are shown in Figure S1.


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Figure 2. (A) Schematic illustration of an ion transfer of target drug ions at a water/1,2-DCE interface measured by nanopipet voltammetry. (B) SEM image of an orifice of a silanized nanopipet with 3 nm thick Au layer coated by Au sputter coater. SEM image was obtained using an accelerating voltage 5 kV to minimize charging-up.

Experimental Section Chemicals. The following chemicals were used as received: Boric acid, N, Ndimethyltrimethylsilylamine (≥ 99%), tetrabutylammonium (TBA+) chloride, Nalidixic acid (NA), Flumequine (FMQ), Sulfadimethoxine (SDM), Sulfamethazine (SMT), Sulfamerazine (SMR), and Difloxacin (DFX), sodium perchlorate, 1,2-dichloroethane (DCE), and hydrochloric acid from Sigma

Aldrich.

The

tetrakis(pentafluorophenyl)borate

salt

of

tetradodecylammonium

(TDDA+·TFAB–) was obtained by metathesis and used as organic supporting electrolytes.23 Deionized nanopure water (18.2 MΩ·cm, TOC 2 ppb; Milli-Q Integral 5 system, Millipore) was used to prepare all the aqueous electrolyte solution.



Nanopipet Preparation and Characterization by SEM. Tapered nanopipets with an inner tip radius of 70~120 nm were obtained by puling 10 cm long quartz capillaries (outer/inner diameter ratio of 1.0/0.7; Sutter Instrument Co., Novato, CA) using a CO2-laser capillary puller (model P2000, Sutter Instrument). In details, A quartz capillary was cleaned by compressed-air blowing and pulled in CO2 laser puller with pulling parameters (heat= 710, filament= 4, velocity= 30, delay= 130 and pull= 130). The pipets were then cleaned with UV plasma cleaner for 3 min under pursing with Ar gas before the silanization. The pulled nanopipets were dried for 1.5 hours under vacuum (~70 mTorr) in a desiccator (Mini-vacuum desiccator, Bel-Art Products, Pequannock, NJ) and then silanized by introducing 50 μL of N, N-dimethyltrimethylsilylamine into round bottom flask connected to the desiccator. Silanization was performed for 40 min under a constant relative humidity 16 % at 20 °C controlled in a glove bag purged with N2. After silanization, the desiccator was vacuumed for 10 min followed by purging with N2 for 5 min to remove extra silanization reagent. The silanized pipets were further filled with 10 uL of a 1,2-DCE solution containing 0.1 M of the organic supporting electrolytes (TDDA+·TFAB–) right before electrochemical measurements. The silanized nanopipets were characterized by a field emission SEM (SIGMA VP Field EmissionScanning Electron Microscope, Zeiss) after coating 3~4 nm thick Au layer by Au sputter coater (108 auto sputter coater with thickness controller MTM-20, Cressington). A nanopipet was vertically observed, and its orifice was visualized with 5 kV accelerating voltage (Figure 2B). Solution Preparation for the Electrochemistry. 10 mM boric acid solution with 50 mL was prepared with the nanopure water. By adding ~0.5 mL of 0.1 M NaOH with drop by drop, a pH of boric acid buffer was adjusted to pH 8.20 ~ 8.40 using pH meter. Using this boric acid buffer solution, 5 mM of drug molecule solution with 10 mL was prepared, of which the final pH was adjusted to pH 8.30 ± 0.1. 10 mM TBA+ solution with 10 mL using the boric acid buffer solution was separately prepared as well. Electrochemical Measurements. All electrochemical experiments were carried out at room temperature (20 °C) in a two electrode cell: The silanized nanopipets were filled with organic solution from the rear using a 10 µL syringe. Cyclic voltammograms (CVs) were obtained with a bipotentiostat (CHI 760E, CH Instrument, Austin, TX). The voltage was applied between Ag/AgCl reference electrode in the boric acid buffer solution and an electrochemically etched Pt wire as an 6 ACS Paragon Plus Environment

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electrode in the nanopipet. For the voltammetry measurements, sequential experiments were performed in (1) aqueous boric acid buffer only, (2) with adding drug molecules, and finally (3) adding TBA+ in the solution. Briefly, first, a voltammogram was obtained in 9 mL of an aqueous boric acid buffer solution. Subsequently, 2 mL of 5 mM drug ion solution containing respective quinolones or sulfonamides was added into the aqueous buffer solution, and the voltammogram of a target drug ion was recorded with the final concentration, 0.9 mM of drug ions. Finally, 1mL of solution was taken out, and 1mL of 10 mM TBA+ solution was further added. A voltammogram of TBA+ ion transfer with final concentration, 0.9 mM was monitored to characterize the pipet size. This sequential measurement of voltammeteric response clarifies a wellresolved ion transfer of a target drug ion as well as an accurate pipet characterization. All voltammograms were recorded with a scan rate at 25 mV/s. cell 1 (buffer only in aqueous phase): Pt | 0.1 M TDDATFAB + 1,2-DCE || 10 mM boric acid || 1 M KCl | AgCl | Ag

cell 2 (buffer + drug ions in aqueous phase): Pt | 0.1 M TDDATFAB + 1,2-DCE || 0.9 mM drug ions + 10 mM boric acid || 1 M KCl | AgCl | Ag

cell 3 (buffer + drug ions + TBA+ in aqueous phase): Pt | 0.1 M TDDATFAB + 1,2-DCE || 0.9 mM TBA+ + 0.9 mM drug ions + 10 mM boric acid || 1 M KCl | AgCl | Ag

Results and Discussion Drug Ion Transfer at a Water/1,2-DCE Interface and Kinetic Analysis of Nanopipet Voltammograms. A boric acid buffer at pH 8.30 was particularly used, where a neutral acid form is dominant (pKa 9.24)33, thus minimizing interference from ionic species in the aqueous bulk solution, and better resolving a response of a target drug ion transfer during the voltammetric 7 ACS Paragon Plus Environment


measurements. Particularly, pH 8.30 is within a physiological pH range in human digestive track e.g. duodenum (pH 7.0 ~ 8.5)34. More importantly, bacteria have shown the highest activity at pH 8.0~9.0 range.35 Given these two factors, all the electrolytes with drug ions were prepared at pH 8.30 ± 0.10. At pH 8.30, drug molecules are deprotonated, and negatively charged on the carboxylate or amide group of quinolones (NA−, DFX− and FMQ−) or sulfonamide derivatives (SMR−, SMT−, SDM−), respectively (Figure 1), which enables to be sensed as pristine by ITIES. To clarify the ion transfer of a target drug as well as a characterization of the corresponding nanopipet geometry simultaneously, a series of electrochemical measurements were sequentially performed. First, we recorded a voltammetric response of the bulk solution and confirmed the available potential window. Once we defined an available potential window in aqueous buffer solutions, a stock solution of drug molecules (5 mM) was further added in the bulk solution to make a final concentration 0.9 mM. Then, voltammetry was carried out in the presence of drug ions in the bulk solution within the potential window determined by the above bulk experiment. Any voltammetric current in this potential range is solely attributed to a drug ion transfer across an interface. After either well or poorly resolved voltammograms of a drug ion transfer were obtained, 1 mL of 10 mM TBA+ solution was finally added, thus being a final concentration 0.9 mM TBA+ used to characterize a nanopipet size and the intactness of ITIES. In this case, the direction of a potential scan is opposite to the case of anionic drug molecules, i.e. from positive to negative polarity, thereby the obtained current from the TBA+ ion transfer has an opposite sign as well, showing a positive limiting current (Figure 3). Experimental voltammograms of TBA+ and drug ions (SDM−, SMR−, SMT−, NA−, FMQ−, and DFX−) are shown in Figure 3 a-f and Figure S4. The ion transfer of tested drug-ions gave wellresolved voltammetric responses against a response from the bulk solution in a given potential window. In addition, nearly well-defined voltammograms for SDM−, SMR−, SMT−, NA− and FMQ− could be obtained with distinct inflection points, whereas DFX− showed a poorly defined voltammogram without any noticeable inflection point (see Figure S5 in supporting information). Although most of recorded voltammograms were fairly well defined, we could not obtain a clear/stable steady-state diffusion limiting current (iT∞, i). It is because a drug ion transfer occurred in highly positive potential window, thereby not only partially overlapping with a bulk solution response, but also increasing a resulting current due to a deformed geometry of an interface 8 ACS Paragon Plus Environment

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induced by extreme potential36. Except DFX−, the inflection points observed from other drug ion transfers were distinct enough to allow for extrapolation of voltammograms to estimate a steady state limiting current as explained below. A steady-state diffusion limiting current and mass transfer coefficient (mi) based on the ion transport are given by19, 37 (1)

𝑖𝑇∞, 𝑖 = 4𝑥𝑧𝑖𝐹𝐷𝑖𝐶𝑖𝑎 𝑚𝑖 =

4𝑥𝐷𝑖

(2)

𝜋𝑎

where F is the Faraday constant (96485 C/mol), and zi, Di, and Ci are the charge (−1) of the transferred drug ion i, its diffusion coefficient, and bulk concentration (0.9 mM) in the aqueous boric acid buffer solution, respectively, and x is a function of outer radius (rg) / inner radius (a) of a nanopipet. Silanized pipet with rg/a = 1.4 determined by SEM measurements has x = 1.18.38 a was determined from the limiting current of a TBA+ transfer using eq 1 with z TBA+ = 1, C TBA+ = 0.9 mM, and DTBA+ = 6.0 × 10−6 cm2/s.23 The inner radii from this electrochemical characterization and SEM measurements are nearly identical indicating an inlaid-disk shaped geometry of ITIES owing to a well-controlled silanization of a nanopipet. With using parameters, a nanopipet radius (a) determined from TBA+ voltammogram and an extrapolated steady state limiting current from drug ion voltammogram, a diffusion coefficient of each drug ion could be calculated (see in Table 1). Herein, In order to not only extrapolate the limiting current, but also theoretically analyze experimental voltammograms for extracting the kinetic information, we performed the finite element analysis using COMSOL Multiphysics version 5.3a (COMSOL, Inc., Burlington, MA). In this analysis, we assume Butler-Volmer model with the heterogeneous rate constants, kf and kb given by37

[

0

𝑘𝑓 = 𝑘 𝑒𝑥𝑝

[

𝑘𝑏 = 𝑘0𝑒𝑥𝑝

―𝛼𝑧𝑖𝐹(∆𝜑 ― ∆𝜑0𝑖 ′) 𝑅𝑇

]

(1 ― 𝛼)𝑧𝑖𝐹(∆𝜑 ― ∆𝜑0𝑖 ′) 𝑅𝑇


(3)

]

(4)


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where 𝜑0𝑖 ′is a formal transfer potential of ion i, α is a transfer coefficient, R is a gas constant, k0 is a standard heterogeneous ion transfer rate constant, and ∆𝜑 is the Galvani potential difference between the aqueous and DCE phases. Simply, the corresponding ion transfer with a charge of zi is defined as below kf 𝑧𝑖

𝑖 (𝑎𝑞𝑢𝑒𝑜𝑢𝑠)

kb

𝑖𝑧𝑖 (1,2 ― 𝐷𝐶𝐸)

(5)

As a result, theoretical currents of an interfacial ion transfer were simulated by solving diffusion problems for ion transfer voltammetry with COMSOL Multiphysics, and plotted together with the experimental voltammograms (Figure 3). For the precision of kinetic analysis, background currents measured in aqueous buffer solution only (grey solid curves in Figure 3) were numerically subtracted from the experimental voltammograms of respective drug ions. The forward waves of each experimental voltammogram were fitted with theoretical curves. From these fittings, we could assess the kinetic parameters for both the drug ions and TBA+ transfer. In Table 1, we summarize all the geometric and kinetic parameters determined from nanopipet voltammograms with drug ions and TBA+. Notably, we could determine k0 = 3.61 ± 0.02 cm/s and α = 0.45 for the interfacial transfer of TBA+, showing a quasi-reversible behavior. These obtained values are nearly close to k0 = 6.1 ± 0.9 cm/s and α = 0.49 ± 0.09 reported for the transfer of tetraethylammonium (TEA+) at the water/DCE interface using nanopipet voltammetry.19 This result implies a good quality of the experimental conditions for our nanopipet voltammetry, thus guaranteeing a reliability of our kinetic study with drug ion transfer under the given system. In this analysis, the determined k0 values of the ion transfer for target drugs are markedly lower than the rate constant of TBA+ ion transfer. For instance, they range from 0.0011 to 0.0034 cm/s, thus being ~3 orders of magnitude smaller compared to the TBA+ ion transfer at a water/1,2-DCE interface, and indicating an obvious kinetic limitation. This clear kinetic effect on the drug ion transfer could be ascribed to structurally relevant properties such as a hydrophilicity of the drug molecules. We will discuss more details about this kinetic issue related to the hydrophilicity of drugs in the following section. The values of α = 0.41 ± 0.04 showed a good agreement with the experimental voltammograms, nearly close to 0.5. α = 0.5 has been predicted in the ion transfer


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models considering either the slow diffusion of the transferred species through the mixed interfacial layer39,40or assuming an activation barrier41,42.

Figure 3. Steady-state voltammograms of (A) TBA+ (B) NA− (C) FMQ− (D) SDM− (E) SMR− (F) SMT− transfers across the DCE/water interface obtained with nanopipets. Inset shows a molecular structure of each ion. The best theoretical fitting (red open circles) to the experimental curves (black solid curves) were calculated from simulations with COMSOL Multiphysics using parameters in Table 1. Scan rate is 25 mV/s. Voltammograms of blank buffer solutions (grey solid curves) are plotted together.



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ions

pipet radius, a (nm)

D in aq. phase (cm2/s)

m (cm/s)

k0 (cm/s)

′ 𝜑0𝑖 (V)

α

TBA+

75

6.0 × 10–6

1.20

3.6

0.0675

0.45

NA−

100

7.0 × 10–6

1.05

0.0011

0.110

0.36

FMQ−

121

1.35 × 10–5

1.69

0.0034

0.190

0.45

SMT−

68

1.5 × 10–5

3.22

0.0016

–0.060

0.37

SMR−

77

8.0 × 10–6

1.50

0.0030

0.070

0.40

SDM−

89

8.5 × 10–6

1.42

0.0028

0.001

0.40

DFX−

77

1.1 × 10–5

2.15

NA

NA

NA

Table 1. Geometric, transport and kinetic parameters determined from nanopipet voltammograms with target drug ions and TBA+. Note that a pipet radius, a for all the drug ions was determined by subsequently measuring a limiting current of TBA+ ion transfer. More details about determining the above parameters are given in SI. Effective Hydrophilicity of Drug Ions. Herein, we quantitatively assess the effective hydrophilicity of tested drug molecules in comparison to a typical hydrophobic anion, ClO4−. For the comparison among the different ions, the experimental voltammograms were normalized against the theoretically predicted or experimentally obtained limiting currents, iT,∞ for drug ions or TBA+, respectively, and plotted together with respect to Ag/AgCl reference electrode in the aqueous phase. The voltammogram of a more hydrophilic anion appears at the more positive potentials, showing an order of FMQ− > NA− > SMR− > SMT− > SDM− > ClO4−. From the half-wave potentials of the experimental voltammograms, the effective hydrophilicity of FMQ−, NA−, SMR−, SMT− and SDM− was quantitatively evaluated given by23,24 𝑃𝑖 𝑃𝐶𝑙𝑂4 ―

[

= 𝑒𝑥𝑝 ―

𝑧𝐹(𝐸𝑖,1/2 ― 𝐸𝐶𝑙𝑂4 ― , 1/2) 𝑅𝑇

]

(6)

where Pi and PClO4− are the partition coefficients of drug ion, i and ClO4− between the aqueous buffer and 1,2-DCE phases, z is a net charge (z = −1), and Ei,1/2 and EClO4−,1/2 are the half-wave potentials of the corresponding ions as determined from the experimental voltammograms in Figure 4. 12 ACS Paragon Plus Environment

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For instance, FMQ− is approximately 105 times more hydrophilic than ClO4− with a difference of ~300 mV between their half-wave potentials. Likewise, SMT− showed ~120 mV difference in halfwave potentials, thereby showing ~102 times higher hydrophilicity than ClO4−. Quantitatively assessed effective hydrophilicity for all other drug ions with respect to the corresponding halfwave potential differences is summarized in Table 2. Notably, the resulting hydrophilicity of deprotonated drug ions are extremely high with about 2 ~ 5 orders of magnitude compared to a hydrophobic anion ClO4−. From the given structure of drug molecules (see insets in Figure 3 and S1), one could intuitively expect a high lipophilicity because of the presence of aromatic ring with delocalized π-electrons. These drugs, however, have isolated negative charges on the carboxylate or amide group once deprotonated, which are unshielded in their molecular structures as well.43,44,45,46 Such localized negative charges could dominate the effective hydrophilicity of the drug ions, especially when they transfer across a water/DCE interface. In fact, nanopipet voltammetry in this study revealed an extremely high value of effective hydrophilicity for deprotonated quinolone and sulfonamide ions. So then, how does the high hydrophilicity of drug ions affect the kinetics of ion transfer? Recently, a hidden barrier of ion passage has been proposed during ion transfer across the interface between a water/dichloromethane (DCM) by the study of the molecular dynamic simulation.47 In this theoretical approach, a barrier accompanied with a formation/breaking of water-finger explicitly elucidates the retarded rate of interfacial ion transfer. For example, upon the formation of water-finger structure, the excessive free energy barrier of ΔG‡ = 4 kcal/mol is estimated.47 According to Arrhenius equation (k = A exp(–ΔG‡/RT), this energy barrier leads to decrease in transfer kinetics with three orders of magnitude, which is consistent with our observation of ~3 orders of magnitude slower kinetics of drug molecules in interfacial transfer than typical hydrophobic cation, TBA+ interfacial transfer. In fact, higher charge densities localized on the carboxylate or amide group could cause stronger interactions between the ion and water,48 thereby increasing an energy barrier upon the water-finger formation and breaking during the interfacial ion transport. Correspondingly, a higher barrier leads to slow kinetics of drug ion transfer across the interface.



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Figure 4. Normalized voltammograms of various ions transferred across a water/1,2-DCE interface supported at a nanopipet. Reference electrode is Ag/AgCl in the aqueous phase. Potential sweep rate, 25 mV/s.

Hydrophilicity factor

ions

ΔE1/2 (mV)

SDM−

108

1.8

SMT−

126

2.1

SMR−

184

3.1

NA−

218

3.7

FMQ−

336

5.7

(Pi/PClO4−) (10x)

Table 2. Estimated effective hydrophilicity of drug ions against a typical hydrophobic anion, ClO4− determined from Figure 4. ΔE1/2 is a difference in half-wave potentials between corresponding drug ions and ClO4−. DFX− was not applicable in this analysis.


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Stability in Amperometric Response of Drug Ion Transport. Amperometry was also performed to probe a stability of steady-state current of drug ion transfer over time. A constant potential at which a maximum steady state current was obtained, was applied, and the resulting currents were monitored over 800~1000 s. Stable currents over the measurement time have been observed for all the drug ions maintaining 80~100 % of initial currents (Figure S5 in supporting information). Notably, DFX− also showed remarkably stable currents with time, whereas an accurate kinetic analysis was hampered by a poorly defined voltammogram. These results imply that nanopipet supported ITIES can be employed as a nanoprobe to sense drug ions for the high spatial steady state measurement, such as scanning electrochemical microscopy (SECM).32 For instance, a sufficiently stable steady-state current over ~1000 s promises a reliable mapping of local drugpermeation across biological membranes of microbial organisms at a high spatial resolution.

Conclusions This work successfully employed nanopipet supported ITIES in lieu of a biological system for studying the ion transfer of antimicrobial drugs, quinolones and sulfonamides as pristine. The high selectivity of nanopipet tips enabled us to quantitatively monitor drug ion transports across the interface under a physiological condition. Using theoretical analysis, we quantitatively evaluated kinetics of interfacial ion transfer of various target drugs, and revealed ~3 orders of magnitude slower kinetics compared to a typical hydrophobic cation, TBA+. In addition, extremely high effective hydrophilicity of target drug ions was quantitatively assessed from the nanopipet voltammograms. Notably, we could estimate about 2 ~ 5 orders of magnitude higher effective hydrophilicity of deprotonated drug ions than a typical hydrophobic anion, ClO4−. Herein, we ascribed such a high hydrophilicity exclusively to negative charges localized on the carboxylate or amide group of deprotonated quinolones or sulfonamides, respectively. Moreover, this localized charge density may play a dominant role in an increase of the energy barrier upon interfacial ion transfers, since stronger interaction between a high density of negative charges and water is expected during a water-finger formation/breakage near the interface, thereby leading to sluggish transfer kinetics. Our finding based on the study of nanopipet voltammetry gave an insight on the physicochemical behavior of pristine drug ions in the permeation through artificial cell membranes. Finally, a high stability of amperometric current response over time promises an application of 15 ACS Paragon Plus Environment


nanopipet to a nanoprobe of high spatial steady-state measurements, e.g. SECM to spatially resolve a drug-permeation in the biological system. Author Information Corresponding Author *Phone: +1 (401) 874-2143. E-mail: [email protected]. ORCID: 0000-0002-7624-6766 Author Contributions All authors have given approval to the final version of the manuscript Notes The authors declare no competing financial interest. Supporting Information Nanopipet voltammogram of DFX−. Amperometric responses of drug ions over time. Geometry and parameters used for finite element analysis with COMSOL Multiphysics. A summarized simulation report. Acknowledgements This work was supported by the Research Bridge Funding Initiative and Medical Research Fund (20174373) from Rhode Island Foundation. References 1. Munita, J. M.; Ariasa, C. A., “ Mechanisms of Antibiotic Resistance”, Microbiol. Spectrum., 2016, 2, 1-24. 2. Cantas, L.; Shah, S. Q. A.; Cavaco, L. M.; Manaia, C. M.; Walsh, F.; Popowska, M.; Garelick, H.; Bürgmann, H.; Sørum, H., “A brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota”, Front. Microbiol., 2013, 4, 1−14. 3. McKenna, M., “Antibiotic resistance: The last resort”, Nature, 2013, 499, 394−396. 4. Davies, J.; Davies, D. Microbiol. and Mol. Biol. Rev. 2010, 74, 417−433. 5. Rice, L. B., “Progress and challenges in implementing the research on ESKAPE pathogens”, Infect. Control Hosp. Epidemiol., 2010, 31 Suppl 1, S7–S10. 6. Boucher H.W., Talbot G.H., Bradley J.S., Edwards J.E., Gilbert D., Rice L.B., Scheld M., Spellberg B., Bartlett J., “Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America”, Clin. Infect. Dis., 2009, 48, 1–12.


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