Electrochemical Assays and Immunoassays of the Myeloperoxidase

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Electrochemical Assays and Immunoassays of the Myeloperoxidase/SCN-/H2O2 System Michael Bekhit, and Waldemar Gorski Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05855 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Analytical Chemistry

Electrochemical Assays and Immunoassays of the Myeloperoxidase/SCN-/H2O2 System Michael Bekhit and Waldemar Gorski* Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249 ABSTRACT: Strategies to detect and characterize myeloperoxidase (MPO) are needed, given that this “split personality” enzyme kills harmful microorganisms but also damages a host tissue. Here, we describe electrochemical approaches to measure MPO by using the pseudo-halogenation (MPO/SCN-/H2O2) and catalase-like (MPO/H2O2) cycles. Their kinetics were determined by monitoring the consumption of H2O2 with a nitrogen-doped carbon nanotubes (N-CNT) electrode, which could detect 0.50 M H2O2 at -0.20 V. The unique design of internally calibrated electrochemical continuous enzyme assay (ICECEA) and electrode stability allowed to use one N-CNT electrode for over half a year to reliably determine MPO. The kinetic measurements showed that (a) SCN- did not affect the affinity of MPO for H2O2, (b) catalase-like cycle was slower, and (c) MPO retained enzymatically active conformation after complexation with its antibody Ab both in a solution and on the surface of antibody dipstick (d/Ab). The homogenous assays could detect 5.2 g L-1 MPO (35 pM) via a faster cycle. The heterogeneous immunoassays with the capture of MPO on d/Ab could detect 60 g L-1, which was suitable for the accurate detection of MPO in human saliva (101 % recovery). Replacing a detection antibody of ELISA with ICECEA as a signal transducer for immunoassays offers a rapid method for the selective determination of enzymes, e.g. time of MPO quantification was cut from 3-4 h (sandwich ELISA) to 20 min (ICECEA-dipstick). Introduction The myeloperoxidase (MPO) is a leukocyte-derived indiscriminate enzyme that displays a variety of activities.1 In the first step of enzymatic process, the MPO is oxidized by H2O2 to an intermediate compound I. The latter, depending on the local environment, can be involved in the oxidation of various organic substrates (peroxidase cycle), oxidation of Cl-, Br-, and I- (halogenation cycle), oxidation of SCN- (pseudo-halogenation cycle), and disproportionation of H2O2 (catalase-like cycle). Such a broad reactivity yields a variety of oxidants and radical species in vivo that kill the invading microorganisms but can also cause a damage to host cells and tissues. In this context, the extracellularly released MPO has been detected in a wide range of inflammatory and autoimmune diseases and has become an object of intense medical research as a biomarker of inflammation and oxidative stress.2-6 At present, there is no consensus on

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how to measure and report the activity of MPO, in a large measure due to its substrate promiscuity. The demand for faster, simpler, and selective MPO assays continues. Traditionally, the enzymatic activity of MPO has been determined by using assays based on the peroxidation and halogenation cycles with a spectrophotometric detection. While commercially available, the kits for such assays have a poor selectivity and often require a long sample incubation. Another commonly used method for MPO is the sandwich ELISA.6,7 This is a powerful but long assay, which involves several incubation and washing steps and requires detection antibodies to generate an analytical signal. The alternatives to optical are electrochemical measurements of MPO, which attract a growing interest. The recent examples include an immunomagnetic assay based on a peroxidation cycle8 and indirect immunosensors based on the changes in the impedance9 or faradaic current10 caused by MPO binding to its antibody on the surface of a working electrode. While these are compelling developments, they often require extensive modifications of electrodes, and complex protocols and cell assembly. Here, we explore a different approach, namely, the rapid MPO assays based on the pseudo-halogenation and catalase-like cycles using a simple electrode and a conventional cell. The kinetics of the two cycles were determined using the internally calibrated electrochemical continuous enzyme assay (ICECEA)11,12 to guide the assay optimization. Two advances were pursued including a short assay with no incubation steps and no antiMPO antibody (Ab), and a fast immunoassay with ICECEA providing a transduction of Ab-MPO binding into a readily observable electronic signal. To this end, an amperometric H2O2 sensor with a sub-micromolar detection limit at a negative potential was developed. EXPERIMENTAL SECTION Reagents and Chemicals. Mouse anti-MPO monoclonal antibody (Isotype: IgG2b, clone: 4A4, 2 mg/mL) and Na2CO3-NaHCO3 buffer (0.2 M, pH 9.4) were from Thermo Fisher Scientific. The myeloperoxidase (MPO, from human leukocytes-lyophilized powder, EC 1.11.1.7, ≥ 50 U/mg, ∼150 kDa, purity index > 0.68 (A430/A280)), chitosan (MW ∼1 × 106 Da, ~80% deacetylation), NaOH, HCl, NaH2PO4.H2O, Na2HPO4, sinapinic acid, H2O2 (30 wt. %), and catalase (from bovine liver, lyophilized powder, EC 1.11.1.6, ≥ 20,000 U/mg, ∼250 kDa) were from Sigma-Aldrich, and NH4SCN was from Avantor (Phillipsburg, NJ). The multi-walled carbon nanotubes doped with 1-2 at. % of nitrogen (N-CNT, 20-40 nm diameter, 50 𝜇m average length, 8% at. Fe) were purchased from NanoTechLabs (Yadkinville, NC) and used as received. Solutions and Suspensions. All solutions were prepared by using 18.2 MΩ ∙ cm water that was purified with a Synergy cartridge system (Merck Millipore). The concentration of MPO and H2O2 solutions was determined by using absorbances at 430 nm (, 89,000 M-1 cm-1) and 240 nm (, 39.4 M-1 cm-1), respectively. The chitosan solution (0.10 wt. %) was prepared by dissolving chitosan flakes in a hot (80–90 °C) 2


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solution of 0.10 M HCl. The solution was cooled to room temperature, adjusted to pH 3.5, filtered with a 0.45 μm Millex-HA syringe filter unit (Millipore Sigma), and stored at 4 °C when not in use. The 0.10 wt. % suspension of N-CNT in a 0.10 wt. % chitosan solution was prepared by a 15-min sonication. Electrochemical Measurements. The measurements were performed by using a CHI 832B workstation (CH Instruments) and a conventional cell composed of a 3.0-mm diameter glassy carbon disc working electrode coated with N-CNT, a Pt wire counter electrode, and a Ag/AgCl/3MNaCl reference electrode (BASi, West Lafayette, IN). Before coating with a N-CNT/chitosan suspension, the glassy carbon electrode was wet polished with 0.3 and 0.05 μm diameter alumina particles on an Alpha A polishing cloth (Mark V Lab, East Granby, CT) and cleaned by a 30 s sonication in water and methanol. All experiments were performed at room temperature (21 ± 1 °C). The pH 7.40 phosphate buffer (0.050 M) was used as a background electrolyte solution. The experiments were repeated at least three times and the means of measurements are presented with the relative standard deviation (RSD). N-CNT Electrode. The electrode was prepared by covering a glassy carbon disk of inverted working electrode with a 5.0-μL aliquot of N-CNT/chitosan suspension. After 2 h, a well-adhering surface film was formed that contained N-CNT trapped in the matrix of chitosan chains. The chitosan served as an electrochemically-silent dispersant of NCNT and the adhesive holding the nanotubes on the surface of electrode. The good adhesion of chitosan chains to the surface of glassy carbon contributed to a long-term stability of N-CNT electrode (> 7 months). Before its first use, the N-CNT electrode was soaked in a pH 7.40 phosphate buffer solution for 2 h to hydrate the chitosan matrix and remove any loosely bound material. Afterward, the electrode was just rinsed with water before use and stored capped at 4 °C. Homogeneous Assays. The homogenous enzymatic reactions of MPO were studied by using the ICECEA and a N-CNT electrode held at -0.20 V in a stirred buffer solution (5.0 mL). The measurements were done in the presence (pseudo-halogenation cycle) and absence (catalase-like cycle) of SCN- in a solution. The current flowing through a NCNT electrode was measured continuously while the solution was sequentially spiked with three 50-L calibrating aliquots of H2O2 and one 50-L aliquot of MPO. This yielded the amperograms (Figures 2, 4, 5) featuring three current-time (I-t) steps due to the reduction of H2O2 at a N-CNT electrode and a descending I-t segment that was caused by the consumption of H2O2 in an enzymatic reaction triggered by the addition of MPO to a solution. The activity unit (U per liter) of MPO was calculated from equation UL ―1(𝜇M min ―1 ) =

AS (𝜇A s ―1 ) × 60 (s min ―1 ) CS (𝜇A 𝜇M ―1 )

3


(1)


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where AS is a (assay) slope of a linear descending I-t segment (initial reaction rate) and CS is a calibration slope calculated from the three I-t steps. Immunoassays with Antibody Dipstick. For the sake of brevity, the abbreviation d/Ab will be used for a dipstick d coated with an anti-MPO antibody (Ab), and d/AbMPO for a dipstick coated with an antibody-bound MPO. In the ICECEA-dipstick immunoassays, the ICECEA was used to determine the amount of MPO immobilized on the d/Ab-MPO via antigen-antibody interactions. The L-shaped dipsticks (5.0 cm length, 2.5 cm2 total surface area) were cut from polystyrene Petri dishes (Thermo Fisher Scientific, Waltham, MA), sonicated in methanol for 5 min, rinsed with deionized water, and air dried before use. To make a d/Ab, a bare dipstick was placed in 300 L of stirred 1.0 g mL-1 Ab solution (pH 9.40 carbonate buffer, 0.2 M) in a low-binding polypropylene tube (Millipore, Bedford, MA). After 2 h, the weakly adsorbed Ab was removed from d/Ab by triplicate 1-min soaking in gently stirred 300 L fresh portions of pH 7.40 phosphate buffer. The random rather than oriented adsorption of Ab on a dipstick was adopted to simplify and shorten the preparation of d/Ab, which was used once and then disposed. To separate the MPO from a sample matrix, the d/Ab was placed in 300 L of gently stirred MPO sample for 15 min to form the d/Ab-MPO. The latter was then washed 3 times with aliquots of pH 7.40 phosphate buffer to remove any weakly bound species. The dipping of d/Ab-MPO into a solution during the recording of amperogram triggered a drop in current IMPO, which was proportional to the amount of immune complex AbMPO on the surface of d/Ab-MPO. The IMPO was standardized by dividing it by the average calibration current step IH2O2 (see Figure 7). The optimization experiments showed that IMPO was not improved by extending a time of Ab adsorption from 2 to 2448 h. The time of immunocapture of MPO on a d/Ab (15 min) was adopted from existing ELISA protocols for MPO. The concentration of MPO was determined by using the immunosorption isotherm. Recovery Experiments. The spike-and-recovery experiments were conducted with dipsticks d/Ab, which were incubated for 15 min in samples of saliva, and saliva and buffer spiked with 352 g L-1 MPO. Such dipsticks (d/Ab-MPO) were then analyzed with ICECEA to obtain a ratio IMPO/IH2O2 for each sample. The % recovery of MPO was calculated according to

% 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 =

(Δ𝐼MPO/𝐼H2O2)(saliva spiked w/ MPO) ― (Δ𝐼MPO/𝐼H2O2)(saliva) (Δ𝐼MPO/𝐼H2O2 )(buffer spiked w/ MPO)

× 100

(2)

The non-stimulated whole human saliva samples (~ 2.0 mL) were collected early in the morning before consuming any food/drink. The samples were centrifuged for 30 min at 10,000 x g RCF, and the clear supernatant was transferred to a 1.70-mL microcentrifuge tube (Corning, NY) and stored in a freezer at -20 oC when not in use. 4


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Mass Spectrometry. The MPO and catalase were analyzed by the matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the sinapinic acid as a matrix. The mass spectra were collected on an ultrafleXtreme MALDI-TOF/TOF mass spectrometer equipped with a smartbeam II laser (Bruker Daltonics) in the positive ionization mode. The instrument was used in a linear mode and calibrated using bovine serum albumin. The acquisition was optimized in the mass range 3000 ≤ m/z ≤ 200000. The 10000 shots were acquired per mass spectrum using 1000 Hz acquisition. FlexAnalysis 3.3 software (Bruker Daltonics) was used for data processing. RESULTS AND DISCUSSION Determination of H2O2 at N-CNT. The N-CNT electrode was selected to monitor the consumption of H2O2 in MPO assays. The platinum electrode, which is a good sensor for H2O2, was not used because it yielded a drifting baseline current in the presence of SCN- in a solution. The inset A in Figure 1 shows cyclic voltammograms that were recorded at a N-CNT electrode in the absence and presence of H2O2 in a solution. The voltammograms crossed at one potential (0.22 V) indicating a significant lowering of the overpotentials for the redox processes of H2O2 at N-CNT. At undoped CNT, such a crossing point has not been observed, which was indicative of the large overpotentials at regular CNT.13 The lowering of a cathodic overpotential for H2O2 has been attributed to the disproportionation of H2O2 at N-CNT doped with 7.4 at. % N.14 Here we show that N-CNT doped at 1-2 at. % N also decrease the H2O2 overpotentials. Given this, the potential of -0.20 V was selected for the amperometric detection of H2O2 at N-CNT electrode. Such a low potential prevented the interferences from redox active species other than H2O2, e.g. ascorbic acid, which is notorious for interfering with electrochemical assays. The amperograms recorded at this potential displayed a stable baseline current.

Figure 1. Amperometric trace recorded at a N-CNT electrode (-0.20 V) in a stirred solution that was spiked with seventeen aliquots of H2O2 (0.7-110 μM) and a related calibration plot (right inset). Inset A: cyclic voltammograms (50 mV s-1) recorded at the 5



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same electrode in a solution of (a) 0, (b) 0.70, (c) 1.4, (d) 2.0 and (e) 2.8 mM H2O2. Background electrolyte, pH 7.40 phosphate buffer. Figure 1 shows a constant-potential (-0.20 V) amperogram that was recorded at a NCNT electrode, which was immersed in a stirred solution that was spiked with the aliquots of H2O2. The sharp rise of current after each aliquot addition demonstrated a fast response (t90%

Electrochemical Assays and Immunoassays of the Myeloperoxidase

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