Electrochemical Substrate and Assay for Esterolytic Activity of Human

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Electrochemical Substrate and Assay for Esterolytic Activity of Human White Blood Cells Douglas Hanson, Travis Menard, Stanton F McHardy, Andrew Fleischman, and Waldemar Gorski Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01858 • Publication Date (Web): 13 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 2017

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Analytical Chemistry

Electrochemical Substrate and Assay for Esterolytic Activity of Human White Blood Cells Douglas Hanson,1 Travis Menard,1 Stanton McHardy,1 Andrew Fleischman,2 and Waldemar Gorski1,* Corresponding Author *Email: [email protected] 1 Department of Chemistry, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, USA; 2The Rothman Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA Abstract The ester 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (TAPTA) was synthesized and tested as a substrate for leukocyte esterase (LE), an enzyme produced by leukocytes (white blood cells). In the presence of LE, TAPTA released a redox-active fragment whose oxidation at an electrode provided a direct numerical measure of LE activity. The assays showed that LE recognized TAPTA as its substrate with the Michaelis constant Km and Imax equal to 0.24 mM and 0.13 mA cm-1, respectively. The esterolytic activity of leukocyte suspensions was determined by using the internally calibrated electrochemical continuous enzyme assay (ICECEA). One activity unit (U) of LE catalyzed the hydrolysis of 1.0 µmole of TAPTA per minute in a pH 7.40 phosphate buffer saline solution containing 10 % DMSO at 21 oC. The measured units were directly proportional to the number of leukocytes in the range of 0.028- 4.2 U L-1 (9 - 690 µg L-1 LE protein). One white blood cell displayed the average esterolytic activity of 0.86 and 1.4 nU when the ultrasonic and chemical cytolysis was used, respectively. The ICECEA is an electrochemical alternative to optical assays for the determination of LE activity as an inflammatory biomarker and proxy for the presence of leukocytes. Introduction The activity of enzyme leukocyte esterase (LE) has been commonly used as a proxy for the presence of active leukocytes in the diagnosis of infections. The LE has been typically analyzed by using commercial ELISA kits and kinetic assays to determine its concentration and activity, respectively. The literature on the development of LE assays is scarce.1-4 Dominant are the studies of clinical utility of existing commercial LE kits and strips,5-7 which are all based on the optical detection. Most of LE assays are qualitative or semi-quantitative and rely on reactions that yield products, which subsequently react with additional chemicals to produce a color change proportional to the activity of esterase. For example, a commercial test kit for leukocytes is based on a strip, which displays four color intensity zones that correlate with a white blood cell count from trace, 1+, 2+, to 3+ covering the range of 30-1500 µg L-1 LE protein. The test uses an amino acid ester that is hydrolyzed by LE to a product, which then reacts with a diazonium salt yielding a purple color. While such test strips have been fairly useful, they may have a limited utility in color/opaque solutions. In addition, with only four available color intensity increments, the resolution of differences in LE is often uncertain. 1 ACS Paragon Plus Environment


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The present paper describes the development of a quantitative electrochemical LE assay as a high-resolution alternative to optical LE assays. The concept of assay is based on a new type of LE substrate that releases a redox-active fragment whose oxidation at an electrode provides a numerical measure of LE activity. While the release of products from LE reactions for optical monitoring is known (vide supra), such release for electrochemical monitoring has not been reported. In this proof-of-concept study, we designed and synthesized a new LE substrate, determined its properties, and used it for the determination of esterolytic activity of leukocyte suspensions in buffers and saliva samples via the internally calibrated electrochemical continuous enzyme assay (ICECEA).8,9 EXPERIMENTAL SECTION Reagents and Solutions. The milky white suspension of human white blood cells in 154 mM NaCl solution was purchased from MyBioSource (cat. # MBS173116, 0.0867 mg mL-1 LE protein, 4 x 108 WBC mL-1). The commercial test strips for leukocyte esterase (Siemens Multistix 10 SG) were purchased locally. The NaH2PO4.H2O, Na2HPO4, dimethylsulfoxide (DMSO), trimethylamine (99%, 157008), Na2CO3, CH3COOH, HCl, and NaOH were from Fisher. The hydroquinone (>99%, WXBC2331V), HCl in dioxane (4.0 M, SHBF6951V), and 4toluenesulfonyl chloride (>98%, STBC7571V) were purchased from Sigma Aldrich. The (tertbutoxycarbonyl)-L-alanine was from Combi Blocks (99%, B79606). The 4dimethylaminopyridine was purchased from Acros (99%, B0132458A) and N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride from AK Scientific (99%, LC37743). All aqueous solutions were prepared using 18-MΩ-cm deionized water that was purified with a Synergy Millipore cartridge system. The 8.8 mM TAPTA stock solutions were prepared in 1.00 mL of DMSO. Their proton and carbon NMR spectra recorded after 1 h, 5 days, and 2.5 weeks of storage at either room temperature or in a refrigerator did not show any noticeable changes documenting good stability. The 50-mM pH 7.40 phosphate buffer saline (PBS) solution containing 154 mM NaCl was used as a background electrolyte in electrochemical measurements. The pH studies were performed by using buffers that were prepared by substituting the pH 7.40 phosphate in PBS with pH 5.00 acetate, and pH 9.20 carbonate buffers. Preparation of Leukocyte Suspensions. Two preparations were studied. In the first one, the original suspension of leukocytes was diluted 10 times with a PBS solution and ultrasonicated for 15 s (Q125 Qsonica, 60 % power) to lyse the leukocytes and release the LE into a solution. The sonication was done in 5-s intervals with 5-s rest periods in between to minimize the overheating of solution and deactivation of enzyme. In the second preparation, the original suspension of leukocytes was diluted 10 times with a PBS solution that contained 10 vol.% DMSO and left for 10 min to lyse the leukocytes chemically. These protocols yielded suspensions that had a constant esterolytic activity for at least 5 days. The ultrasonicated or chemically lysed leukocyte suspensions were subsequently used as a solution C (vide infra) for the ICECEA. 2 ACS Paragon Plus Environment

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Electrochemical Measurements. All electrochemical measurements were performed in the three-electrode cell with a 3.0-mm-dia. glassy carbon (GC) working electrode (BAS), Pt wire counter electrode, and Ag/AgCl/3M NaCl (BAS) reference electrode. The cyclic voltammograms and amperograms were recorded by using a CHI 601B workstation (CH Instruments, Inc.). The working electrode was wet polished on an Alpha A polishing cloth (Mark V Lab) with successively smaller particles (0.3 and 0.05-µm diameter) of alumina. The slurry that accumulated on the electrode surface was removed by a 30-s sonication in deionized water and methanol. All electrochemical experiments were done at room temperature (21 oC). They were repeated at least three times to report the precision of results with the relative standard deviation. Principle of ICECEA. The internally calibrated electrochemical continuous enzyme assay (ICECEA) is a flexible analytical protocol that can be adapted to a variety of enzymes.8,9 In the form relevant to LE, the ICECEA measures the esterolytic activity as the initial rate of enzymatic reaction

(1) by monitoring the formation of redox-active product with the help of a working electrode, which is poised at a constant detection potential that is selected based on the cyclic voltammogram of product.

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b

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Figure 1. Theoretical ICECEA trace recorded at a working electrode in a stirred assay solution A to which three aliquots of calibration solution B (arrows “a”) and an aliquot of enzyme solution C (arrow “b”) were added. Solution B contains a product of reaction 1 and solution C contains an assayed enzyme. The ICECEA is performed by using the three solutions: (A) assay solution, (B) calibration solution, and (C) enzyme solution. Figure 1 shows a theoretical shape of ICECEA trace, which results from a sequential addition of a calibration solution B (arrows a) and an assayed enzyme 3 ACS Paragon Plus Environment


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solution C (arrow b) to a stirred assay solution A. The calibration aliquots of solution B yield the current-time (I-t) steps that are used to calculate the calibration slope CS. The aliquot of enzyme solution C triggers the reaction 1, which yields a product that is subsequently detected at a working electrode resulting in a rising linear I-t segment with an assay slope AS. The slopes AS and CS are used in equation 2 to calculate the activity unit (U) of assayed enzyme ( ) =

( ) ∗ 60 ( ) (2) # ( )

where L (liter) is a total volume of solution in the electrolytic cell. The ICECEA traces shown in this paper are shortened to show only the linear portion of rising I-t segment. The ICECEA has several inherent benefits and advantages. It is rapid (minutes), uses small amounts of enzyme (ng), and does not require enzyme (often expensive) for calibration. It integrates the calibration and actual assay in one continuous experiment using the same electrode and assay solution. This eliminates the need for transferring electrodes between the calibration and assay solutions, which is often a source of error in electrochemical measurements. It provides consistent results regardless of the surface activity of working electrode because a change in such activity impacts both slopes CS and AS equally (equation 2). In addition to quantifying enzyme activity, the ICECEA can quickly screen for potential enzyme substrates and inhibitors. The unique shape of its trace with rapid and slow changes in current (Figure 1) allows for the selective determination of enzyme activity even in the presence of interfering redox active species as long as their concentration stays constant. General Synthetic Procedures. Starting reagents were commercial compounds of the highest purity available and were used without purification. Solvents used for reactions were of commercial dry, extra-dry, or analytical grade. Analytical thin layer chromatography was performed on aluminum plates coated with Merck Kieselgel 60F254 and visualized by UV irradiation at 254 nm or by staining with a solution of potassium permanganate. Flash column chromatography was performed on Biotage Isolera One 2.2 using commercial columns that were pre-packed with Merck Kieselgel 60 (230– 400 mesh) silica gel. Final compound was of ≥95% purity as determined by HPLC-MS and 1H NMR. 1H NMR experiments were recorded on Agilent DD2 400 MHz spectrometer at room temperature. All samples were analyzed on Agilent 1290 series HPLC system (UV detector) that was equipped with an auto-sampler coupled with Agilent 6150 mass spectrometer. Purity was determined via UV detection with a bandwidth of 170 nm in the range from 230-400 nm. The liquid chromatography was performed by using the Zorbax Eclipse Plus C18 column (2.1 x 50 mm), solvent A (aqueous solution of 0.10 % formic acid), solvent B (acetonitrile), flow rate of 0.70 mL min-1, gradient of solvent B from 5 % to 95 % in 5 min followed by a 2-min constant 95 % of solvent B, and UV detector channels at 254 nm. Mass detector was the Agilent Jet Stream – Electron Ionization (AJS-ES). The high-resolution mass spectrometry (HRMS) was conducted by using the MaXis plus QTOF mass spectrometer equipped with an electrospray ionization source (Bruker Daltonics). 4 ACS Paragon Plus Environment

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Synthesis of Redox Substrate of Leukocyte Esterase (LE). The substrate 4-((tosyl-Lalanyl)oxy)phenyl tosyl-L-alaninate (TAPTA) was synthesized in three steps. Step 1. Synthesis of 1,4-phenylene (2S,2'S)-bis(2-((tert-butoxycarbonyl)amino)propanoate). A flame-dried vessel purged with nitrogen was charged with hydroquinone (1.04 g, 9.5 mmol) and acetonitrile (120 mL) at room temperature. The (tert-butoxycarbonyl)-L-alanine (3.62 g, 19 mmol) and 4-dimethylaminopyridine (0.117 g, 0.96 mmol) were added to this solution and stirred for 5 minutes before adding N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.9 g, 20 mmol) and triethylamine (2.8 mL, 20 mmol). This reaction mixture was stirred at room temperature for 12 hours. Next, the mixture was poured into ice-cold 1.0 M HCl (300 mL) and stirred for 30 minutes. The resulting precipitate was filtered and dried under reduced pressure. The crude solid was recrystallized in a mixture of hexanes and ethyl acetate (4:1). The solid was filtered and dried under reduced pressure to give 2.57 g (64% yield) of white solid 1,4-phenylene (2S,2'S)-bis(2-((tert-butoxycarbonyl)amino)propanoate) that was used in the next synthetic step; 1H NMR (400 MHz, CDCl3) δ 7.12 (s, 4H), 5.07 (br s, 2H), 4.52 (br m, 2H), 1.51 (d, J=7.7 Hz, 6H), 1.47 (s, 18H). Step 2. Synthesis of 1,4-phenylene (2S,2'S)-bis(2-aminopropanoate)-bis hydrochloride. A 4 M HCl solution in 1,4-dioxane (60 mL) was slowly added to 1,4-phenylene (2S,2'S)-bis(2-((tertbutoxycarbonyl)amino)propanoate) (1.87 g, 4.4 mmol) and stirred for 30 min at room temperature. The resulting precipitate was filtered, washed with cold diethyl ether, and dried under reduced pressure to yield a white solid of 1,4-phenylene (2S,2'S)-bis(2-aminopropanoate)bis hydrochloride (1.31 g, 93% yield), which was used in the step 3 without further purification; 1 H NMR (400 MHz, CD3OD) δ 7.30 (s, 4H), 4.42 (q, J=7.2 Hz, 2H), 1.72 (d, J=7.3 Hz, 6H). MS (m/z) 505.3 (2M + H+), 253.2 (M + H+). Step 3. Synthesis of 4-((tosyl-L-alanyl)oxy)phenyl tosyl-L-alaninate (TAPTA). The 1,4phenylene (2S,2'S)-bis(2-aminopropanoate)-bis hydrochloride (0.222 g, 0.7 mmol) was dissolved in an anhydrous dichloromethane (3.5 mL) under a nitrogen atmosphere, mixed with 4toluenesulfonyl chloride (0.394 g, 2.1 mmol), 4-dimethylaminopyridine (0.0087 g, 0.07 mmol), and triethylamine (0.40 mL, 2.9 mmol) and stirred for 4 hours at room temperature. The crude reaction mixture was treated with equal portions (~5 mL) of water and ethyl acetate and transferred to a separatory funnel. The layers were separated and the aqueous layer was extracted with ethyl acetate (3 x 2 mL). The combined organic layers were dried with anhydrous sodium sulfate, decanted, and concentrated under reduced pressure. The crude product was dissolved in dichloromethane and precipitated out with hexanes. The solid was filtered, washed with hexanes, and dried under reduced pressure to yield 4-((tosyl-L-alanyl)oxy)phenyl tosyl-Lalaninate (TAPTA, 0.255 g, 70% yield) as a white solid; 1H NMR (400 MHz, DMSO) δ 8.47 (d, J=7.6 Hz, 2H), 7.72 (d, J=8.1 Hz, 4H), 7.39 (d, J=8.2 Hz, 4H), 6.92 (s, 4H), 4.14 – 4.05 (m, 2H), 2.37 (s, 6H), 1.33 (d, J=7.1 Hz, 6H). 13C NMR (101MHz, CDCl3) δ 170.89, 147.79, 144.02, 136.87, 129.94, 127.42, 122.02, 51.72, 21.65, 19.69. RESULTS AND DISCUSSION 5 ACS Paragon Plus Environment


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Design, Synthesis, and Voltammetry of Substrate for LE. The hydrolysis of synthetic substrates by leukocyte esterase (LE) requires a small amino acid (e.g. alanine) in their structure at the P1 site as defined by Schechter and Berger.10,11 In addition, the presence of structural sulfonyl group has been shown to enhance the rate of their enzymatic hydrolysis.4,12 These observations were considered in the design of new LE substrate. To make the substrate suitable for electrochemical assays, the redox active motif (hydroquinone) was also incorporated in its primary structure. Scheme 1 summarizes steps taken to synthesize the LE substrate in line with this design.

Scheme 1. Synthesis of TAPTA

Treatment of hydroquinone 1 with (tert-butoxycarbonyl)-L-alanine in the presence of EDCI and TEA, cleanly produced the desired bis-ester 2 in 64% yield, following a single recrystallization.13 It is worth noting that the use of the N-BOC (tert-butoxycarbonyl) protecting group, which could be removed under acidic conditions, was critical as other N-carbamate protecting groups were unsuccessful in the subsequent deprotection step to produce compound 3.14 Acid catalyzed (4N HCl) removal of the N-BOC group cleanly produced compound 3 as the di-HCl salt in 93% yield. Finally, treatment of compound 3 with 4-toluenesulfonyl chloride and TEA provided the desired bis-sulfonamide TAPTA in 70% yield. The structure of TAPTA was confirmed by proton and carbon NMR (Experimental Section). The molecular weight of TAPTA was equal to 560.136 Da based on HRMS.

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-0.5 0.0 0.5

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Figure 2. Cyclic voltammograms recorded in a solution of (a) saturated TAPTA (light blue), (b) 520 µg L-1 LE (green), (c) saturated TAPTA + 520 µg L-1 LE (red), (d) 38 µM hydroquinone (dark blue), and (e) background electrolyte (black). Each solution was stirred for 15 min before recording its voltammogram. Background electrolyte, pH 7.40 PBS solution with 10 vol.% DMSO. Scan rate, 50 mV s-1. The reactivity of TAPTA with LE was investigated by voltammetry. The expectation was that LE would cleave ester bonds of TAPTA releasing the hydroquinone, which would then react at an electrode yielding voltammetric current peaks. To this end, a series of cyclic voltammograms was recorded at a GC electrode in pH 7.40 PBS solutions that contained different components of the TAPTA + LE system. Figure 2 shows that voltammograms recorded in a solution of only TAPTA (line a) or LE (line b) were featureless indicating no redox activity of these species at a GC electrode. On the other hand, a pair of current peaks was recorded in a solution that contained both LE and TAPTA (red line c). In a control experiment, a pair of similar current peaks was recorded in a solution that contained original hydroquinone (blue line d). Thus, one can conclude that currrent peaks seen in the voltammogram c were due to the redox of hydroquinone that was released from TAPTA by LE proving the presence of enzymatic reaction between these two species (equation 3). TAPTA as a Substrate for LE. The properties of TAPTA as an enzyme substrate were studied at a constant concentration of LE by using amperometry. The expected hydrolysis of TAPTA by LE

%&% ℎ() (3) was investigated by monitoring the released hydroquinone (C6H6O2) with a GC electrode held at a potential of 0.40 V that was sufficient for the heterogeneous oxidation of C6H6O2 @01 232456782

#+ ,+ -. 9::::::::: #+ ,; -. + 2, = + 2 (4) 7 ACS Paragon Plus Environment


triggerring the anticipated increase in current flowing through the electrode. The experiments were conducted by adding known aliquots of TAPTA (0-260 µM) to 5.00 mL of a stirred solution of sonicated leukocyte suspension that contained 3.33 mg L-1 LE protein. The background electrolyte was a PBS solution containing 10 vol.% DMSO. The DMSO was necessary to solubilize a sparingly soluble TAPTA. While adding TAPTA to a LE-free solution caused no change in faradaic current, the addition of TAPTA to a leukocyte suspension containg LE yielded extra current. This corroborated the voltammetric study (Figure 2), which showed that LE recognized TAPTA as its substrate and cleaved its ester bonds releasing the hydroquinone whose oxidation at a GC electrode (equation 4) yielded extra current. Figure 3 shows that this current was directly proportional to the concentration of TAPTA up to ∼40 µM; the limit of detection was 5 µM TAPTA (S/N=3). At higher concentrations, the current was gradually progressing toward a plateau indicating that the enzymatic reaction was becoming less dependent on substrate concentration. The simplest model explaining the shape of plot in Figure 3 is that of Michaelis-Menten kinetics with one substrate and no cooperative binding or inhibition. The corresponding Lineweaver-Burk plot of I-1 vs. CTAPTA-1 (inset) was linear (R2, 0.999; N=8) providing the kinetic parameters Michaelis constant Km and Imax equal to 0.24 (± 5%) mM and 0.13 (± 11%) mA cm-2, respectively. This Km value is lower than that for some of the optical LE substrates (e.g. Km=0.9-1.5 mM for indoxyl ester)3 indicating more efficient catalysis with TAPTA substrate.

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1

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Figure 3. Calibration plot for TAPTA based on amperometry (E=0.40 V) performed in a stirred solution of 3.33 mg L-1 LE protein. Inset: Corresponding Lineweaver-Burk plot. Background electrolyte, pH 7.40 PBS containing 10 vol.% DMSO.

pH and Interference Effects on ICECEA of LE. The assays of LE were conducted at a constant concentration of TAPTA by using ICECEA to quantify the enzymatic activity of LE. The activity was determined as the initial rate of reaction 3 by using the three solutions: (A) an 8 ACS Paragon Plus Environment

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assay solution of 88 µM TAPTA in PBS + 10% DMSO, (B) a calibration stock solution of 25100 µM hydroquinone in PBS, and (C) a suspension of sonicated human leukocytes in PBS. The detection potential for ICECEA was determined based on the cyclic voltammogram of hydroquinone at each pH. In a typical ICECEA experiment, three 20-125 µL aliquots of a solution B and one 5.0-200 µL aliquot of solution C were sequentially added to the 5.00 mL of stirred assay solution A. The assays were performed in the linear range of a calibration plot for the hydroquinone. Figure 4A shows that the ICECEA trace recorded at pH 7.40 displayed a theoretical shape with three I-t steps (calibration phase) and the ascending I-t segment (assay phase). The I-t steps were due to the oxidation of added hydroquinone at a GC electrode (equation 4) and the ascending I-t segment was due to the oxidation of hydroquinone that was enzymatically released from TAPTA by LE (equation 3). The height of I-t steps and the angle of ascending I-t segment yielded the calibration slope CS and assay slope AS, respectively, which were used in equation 2 to calculate the activity units of LE. The flat I-t trace in Figure 4A (red line), which was recorded in a LE-free solution of TAPTA, documents the absence of non-enzymatic hydrolysis of TAPTA futher corroborating the stability of TAPTA seen in voltammetric studies (Figure 2, line a). The ICECEA trace in Figure 4A was not affected by the pH of assay solution in the range from 6.90 to 7.90. However, larger pH changes had a negative impact on ICECEA.



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3 2 1 0 -1 0

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Figure 4. ICECEA current-time (I-t) traces recorded in stirred assay solutions of different pH (A) 7.40, (B) 5.00, (C) 9.20. Arrows show the moment of addition of a 15-s sonicated leukocyte suspension to generate 87 µg L-1 LE protein in a solution. Each current step is due to the addition of hydroquinone aliquot to generate 0.48 µM in a solution. Flat current trace in panel A (red line) was recorded in an assay solution. Assay solution, 88 µM TAPTA in a buffer solution that contained 10 vol.% DMSO. Detection potential, (A) 0.40, (B) 0.50, (C) 0.10 V. At pH 5.00 (Figure 4B), while the hydroquinone aliquots generated the expected current steps, the anticipated current rise was not observed when the sonicated leukocyte suspension was added to an assay solution. This could be the result of a decomposition of TAPTA and/or deactivation of LE at this pH. The HPLC/MS experiments did not show any degradation of 10 ACS Paragon Plus Environment

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TAPTA at pH 5.00 during a few days of testing. Apparently, the lower pH negatively impacted the active conformation of LE. Indeed, the activity of leukocyte esterase has been shown to decrease at lower pH.2 At pH 9.20 (Figure 4C), instead of expected current steps, only a rapidly decaying small current was recorded after each hydroquinone addition to an assay solution. This could be ascribed to the competing oxidation of hydroquinone by oxygen in alkaline solutions. However, the LE retained some of its activity as indicated by the ascending I-t segment that was triggered by the addition of leukocyte suspension to an assay solution. Considering these pH effects, the remaining ICECEA experiments were conducted at pH 7.40. The ubiquitous vitamin C (ascorbic acid, AA) is notorious for interfering with electrochemical measurements because it is readily oxidized at conventional electrodes. Often, the lack of interference from AA is a good predictor that another redox-active species may not interfere too. The AA was tested as a possible interference by using two samples of leukocyte suspension with and without AA. The addition of sample containing AA yielded an instant jump in current due to the oxidation of AA at an electrode (Figure S1). This was followed by the expected ascending I-t segment. The activity of LE in both samples was statistically the same (t test, 95% probability level). The lack of interference from AA demonstrated that the redoxactive interfering species should not impact the assay as long as their concentration remains constant and they do not react with the components of assay solution during the ICECEA. In general, the adsorption of different species on the surface of working electrode can negatively impact electrochemical measurements, including the ICECEA. Such issues can be minimized/eliminated by coating a working electrode with a polymeric film that limits the access of species to the electrode surface based on their charge (e.g. Nafion) or size (dialysis membrane). ICECEA of Original and Lysed Leukocyte Suspensions. Figure 5A (trace a) shows that the addition of original un-sonicated suspension of leukocytes to an assay solution triggered a rise in current that was delayed by ∼2 min (circle vs. arrow). Apparently, the delayed onset of current rise was due to a slow release of LE from leukocytes in the presence of DMSO in an assay solution. In contrast, no such delay was observed when the leukocyte suspension that was sonicated before the assay was used (Figure 5A, trace b). This indicated that the LE was released from leukocytes into a solution during the sonication and reacted with TAPTA with no time lag during the ICECEA. Figure 5B shows that 15-s sonication provided leukocyte suspensions with maximum esterolytic activity.



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Figure 5. (A) ICECEA traces for (a) original, and (b) 15-s sonicated suspension of leukocytes. (B) Effect of sonication time on the esterolytic activity of leukocyte suspensions. Arrows show the moment of addition of leukocyte suspension to generate 87 µg L-1 LE protein in an assay solution. Circles show the onset of current rise triggered by the addition of leukocyte suspension. Each current step is due to the addition of hydroquinone aliquot to generate 1.9 µM in a solution. Assay solution, 88 µM TAPTA in pH 7.40 PBS containing 10 vol.% DMSO. Detection potential, 0.40 V. In addition to ultrasonic, the chemical lysing of leukocytes in DMSO was tested by ICECEA. The 90 vol.% DMSO totally eliminated the esterolytic activity of leukocyte suspensions (Figure S2). However, 10 vol.% DMSO proved to be a good alternative to the ultrasonic lysing (Figure 6). In this scenario, the DMSO played a double role of facilitating the dissolution of TAPTA and inducing a chemical lysing of leukocytes. Esterolytic Activity of Lysed Leukocyte Suspensions. The ultrasonically and chemically lysed leukocyte suspensions were studied by ICECEA to determine the relationships between their esterolytic activity (U L-1) and the content of LE protein (µg L-1) and white blood cell count (WBC µL-1). Figure 6 shows that such relationships were linear (R2, 0.994) up to at least 690 µg L-1 LE protein (3,200 WBC µL-1, 4.2 U L-1 LE) with the limit of detection equal to 9 µg L-1 LE protein (40 WBC µL-1, 0.028 U L-1 LE) for both ultrasonically and chemically lysed leukocyte suspensions. However, the DMSO-induced cytolysis of leukocytes yielded suspensions with 12 ACS Paragon Plus Environment

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higher esterolytic activity as indicated by the higher slope of plot 1 in Figure 6 (red line). Based on the slopes of plots in Figure 6B, one white blood cell displayed the average esterolytic activity equal to 0.86 and 1.4 nU when the ultrasonic and chemical cytolysis was used, respectively. In addition, smaller error bars in the case of plot 1 indicated that the chemical lysing yielded a better precision of measurements (±1-10%) than the ultrasonication (± 6-61 %). These results show that the chemical lysing provided a more efficient and consistent release of LE from leukocytes in conjunction with less enzyme deactivation.

Figure 6. Plots of esterolytic activity (A) vs. LE content, and (B) vs. LE content and white blood cell count for (1) chemically (red line), and (2) ultrasonically (black line) lysed leukocyte suspensions. Four panels of increasing color intensity represent a response of commercial colorimetric LE test strips to increasing esterolytic activity from a trace to 1+, 2+, and 3+, respectively. Assay solution, 88 µM TAPTA in pH 7.40 PBS containing 10 vol.% DMSO. Hydroquinone, 0.42-3.3 µM. Detection potential, 0.40 V. 13 ACS Paragon Plus Environment


Figure 6 also shows that the activity units of LE determined by the proposed electrochemical assay correlate well with the changes in the color intensity of commercial LE test strips. The deeper the color of a strip the higher activity units were measured by ICECEA. The advantage of ICECEA was that it provided a higher resolution and allowed to distinguish differences in esterolytic activity within one color zone. The correlation between the ICECEA and LE test strips further supported the hypothesis that ICECEA with TAPTA as a LE substrate detected the esterolytic activity of leukocyte suspensions. ICECEA of LE in Saliva. The LE assays in saliva are typically performed to evaluate the presence or severity of gingival and periodontal diseases.7 The performance of ICECEA of LE in saliva was evaluated by conducting spike-and-recovery experiments with DMSO-lysed leukocyte suspensions. Figure 7 (trace a) shows the ICECEA trace for a healthy uninfected saliva. The addition of the 100-µL aliquot of such saliva to a 5.0-mL assay solution at 400 s caused an instant jump in current, which was due to the electro-oxidation of redox-active species present in a saliva (e.g. vitamin C). This was followed by a current plateau instead of current rise, which indicated that the esterolytic activity of healthy saliva was below the detection limit of ICECEA.

b

50 40 Current (nA)

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a 30 20 10 0 0

100 200 300 400 500 600 Time (s)

Figure 7. ICECEA traces for (a) healthy, and (b) infected saliva. Each current step corresponds to 0.63 µM hydroquinone. The ascending I-t segment starting at 400 s was triggered by the addition of infected saliva to generate 35 µg L-1 LE in an assay solution. Assay solution, 88 µM TAPTA in pH 7.40 PBS containing 10 vol.% DMSO. Detection potential, 0.40 V.

The infected saliva was prepared by spiking a healthy saliva with DMSO-lysed leukocyte suspension to contain 1,730 µg L-1 LE protein, which corresponded to ∼8,000 WBC µL-1 (infected saliva can contain as much as 12,000 WBC µL-1).15 Trace b in Figure 7 shows that the injection of such saliva into an assay solution at 400 s, in addition to the instant jump in current, triggered a rise in current indicative of esterolytic activity. The resulting content of LE protein in an assay solution was equal to 34.6 µg L-1 considering the fifty-fold dilution of saliva sample. 14 ACS Paragon Plus Environment

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The measured activity of such a solution was equal to 0.21 (± 9%) U L-1, which represented ∼97 % recovery of spiked LE protein based on the slope of red line 1 in Figure 6. This documented a good accuracy of ICECEA even in the presence of redox-active species in saliva. CONCLUSIONS The first electrochemical assay was developed for the determination of both the leukocyte esterase (LE) activity and cytolytic effects of external agents on white blood cells. The assay is based on the LE-induced discharge of redox-active fragment from a custom-made substrate and its oxidation at an electrode to provide a quantitative measure of LE activity. This study lays the groundwork for the development of rapid assays for proteins with esterolytic activity in a variety of systems including saliva and other biological fluids. Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENTS Research reported in this publication was supported by The Rothman Institute and National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number G12MD007591. We thank Dr. Wendell Griffith for the high resolution mass spectrometry analysis of TAPTA. References 1. Kotani, K.; Minami, T.; Abe, T.; Sato, J.; Taniguchi, N.; Yamada, T. Clinica Chim. Acta 2014, 433, 145-149. 2. Mastropaolo, W.; Yourno, J. Anal. Biochem. 1981, 115, 188-93. 3. Murthy, V. V.; Karmen, A. Biochem. Med. Metabol. Bio. 1988, 40, 260-268. 4. Johnson, G. M.; Schaeper, R. Bioconjugate Chem. 1997, 8, 76-80. 5. Yadav, P.; Aparna, P.; Sharma, M.; Chaudhary, U. Int. J. Pharm. Bio. Sci. 2015, 6B, 370375. 6. Ducharme, J.; Neilson, S.; Ginn, J. L. Can. J. Emergen. Med. 2007, 9, 87-92. 7. Bimstein, E.; Small, P. A., Magnusson, I. Pediatr. Dentist. 2004, 26, 310-315. 8. Zhang, M.; Karra, S.; Gorski, W. Anal. Chem. 2013, 85, 6026-6032 9. Zhang, M.; Karra, S.; Gorski, W. Anal. Chem. 2014, 86, 9330-9334 10. Bieth, J. G., In Elastin and Elastases, vol. II, pp. 23-31, CRC Press, Inc. Boca Raton (1989), FL 11. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157-162. 12. Jackson, D. S.; Brown, A. D.; Schaeper, R. J.; Powers, J. C. Arch. Biochem. Biophys. 1995, 323, 108-114. 13. Jing, Z.; Chu, G.; Yan, S. Z. Krist. –New Cryst. St. 2013, 228, 132-134. 14. Greene, T. W.; Wuts, P. G. M., In Protecting Groups in Organic Synthesis, 2nd Edition, pp. 315348, John Wiley & Sons, Inc., (1991) New York, NY.

15. Calouius, P. E. B. Oral Surgery, Oral Medicine, Oral Pathology 1958, 11, 43-46. 15 ACS Paragon Plus Environment


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Electrochemical Substrate and Assay for Esterolytic Activity of Human

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