Redox Potential Measurements in Red Blood Cell Packets Using

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Redox Potential Measurements in Red Blood Cell Packets Using Nanoporous Gold Electrodes Rezaul Karim Khan,† Shanmuka P. Gadiraju,†,# Megh Kumar,†,# Grace A. Hatmaker,‡ Bernard J. Fisher,§ Ramesh Natarajan,∥ Joseph E. Reiner,‡ and Maryanne M. Collinson*,† †

Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284-2006, United States Department of Physics, Virginia Commonwealth University, Richmond, Virginia 23284, United States § Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia 23298, United States ∥ Clinical Investigation Department and Department of Emergency Medicine, Combat Trauma Research Group, Naval Medical Center Portsmouth, Portsmouth, Virginia 23708-2197, United States

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ABSTRACT: The redox potential of packed red blood cells (RBCs) was measured over a 56-day storage period using a newly developed potentiometric methodology consisting of a nanoporous gold electrode and a silver chloride coated silver reference electrode. Both milliliter- and microliter-sized volumes were separately evaluated. The addition of Vitamin C (VitC) in differing doses to the packed RBCs was also assessed as a means to improve redox stability and prolong storage duration. For RBCs containing only saline, the open-circuit potential (OCP) was ∼ −80 mV vs Ag/AgCl and drifted slightly with time; greater differences were also noted between different electrodes. The addition of exogenous VitC to the RBC shifts the OCP to more negative values, stabilizes the redox potential, and improves reproducibly between different electrodes due to the poising of blood. Over the 56-day storage period, the redox potential of the RBCs increased slightly, which can be attributed to change in pH and/or increasing oxidative stress during storage. Cyclic voltammograms acquired after open-circuit potential measurements showed a characteristic peak attributed to the oxidation of VitC. This peak decreased during storage with a time constant of 20.8 days. Likewise, the intercellular concentration of VitC increased with a time constant of 20.2 days as measured using a fluorescence assay. Collectively, these results demonstrate the usefulness of electrochemical measurements in the study of stored blood products. KEYWORDS: potentiometry, blood redox potential, oxidation−reduction potential (ORP), open-circuit potential (OCP), vitamin C, biofouling

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of assessing a patient’s condition.10−14 Recently, it has also been used as a correlate to male infertility.15 However, the measurement of the redox potential of blood, albeit promising, is not as straightforward as the measurement of redox potential in water samples due to the presence of numerous proteins that are known to irreversibly adsorb and unfold on planar metal surfaces.16−20 The measurement challenges introduced by such biofouling include reduced electron exchange rates, reduced sensitivity, and increased response times. In recent work, we have reported an important development in the area of electrochemical redox sensing in complex biologic solutions.21 We showed that a properly nanostructured electrode (e.g., nanoporous gold (NPG))22,23 can make accurate and reliable redox measurements in the presence of high concentrations of proteins known to passivate planar electrodes.21,24 The electrode must have nanopores with

otentiometric measurements provide a relatively simple and cost-effective approach to measure the concentration of an ion in solution (e.g., H+ via a pH electrode) or the redox potential (e.g., oxidation−reduction potential (ORP)) of a sample.1 In the latter case, a metallic electrode is used and the open circuit potential (OCP) measured with respect to a reference electrode using a high impedance voltameter.1 The redox potential thus measured reflects the redox state of the solution or the balance of oxidants and reductants present in solution at appreciable concentrations that are able to quickly exchange electrons with the electrode surface.2 Redox potential measurements have been made in a broad range of samples including soil, water, sludge, and milk.3−9 In soil systems, for example, the redox potential provides a means to evaluate the interplay of complex redox reactions taking place in such environments.3−5 In water systems, the redox potential has been used to estimate its redox status and antioxidant properties, thus allowing for proper water treatment.6−8 Redox potential measurements have also been made in more complex solutions such as blood and/or plasma with the goal © XXXX American Chemical Society

Received: June 15, 2018 Accepted: July 20, 2018

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DOI: 10.1021/acssensors.8b00498 ACS Sens. XXXX, XXX, XXX−XXX

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Figure 1. Schematic illustration of the RBC redox potential study using nanoporous gold electrodes. (A) Cascade of the blood extraction and preparation process. (B) Bulk-based measurements with four NPG electrodes in 7 mL of RBCs and a single Ag/AgCl reference electrode. (C) Microbased measurements utilize a micropipette reference electrode with the different blood droplets (5 μL) deposited onto the nanoporous gold electrode. A, B, C, and D refer to the different blood samples studied and “REF” refers to Zobell’s solution used to calibrate the reference electrode.

a diameter of ∼5−20 nm and a specific morphology that resembles the surface of a nanoporous filtration membrane.21,25 Biofouling agents, being similar in size to the nanopores, cannot reach the inner surfaces while much smaller redox species can.21,25 The inner surfaces thus stay “clean”, providing a pristine surface for the much smaller redox species to freely exchange electrons.21 Since the measured redox potential depends on concentration and electron exchange rates, having an electrode that can detect redox species with relatively slow electron exchange rates is an asset.2,26 Nanoporous electrodes are able to also improve electron transfer rates of traditionally slow redox couples.27 We have recently measured the redox potential of both blood and plasma at both nanoporous and planar gold and have shown a difference that ranges from 40 to 60 mV between the two.24 We have also recently showed the promise nanoporous gold electrodes have as a direct measurement of oxidative stress in blood.28 In this work, we demonstrate the ability of NPGs to quickly assess the redox state of red blood cells (RBC) solutions during a 56 day storage period at 4 °C collectively in both microliter volumes and milliliter volumes. RBC units, prepared by plasma removal and leukocyte depletion, are stored in specialized additive solutions at 4 °C to improve long-term stability.29 Even in the presence of these additive solutions, however, stored RBCs undergo deleterious metabolic, biochemical, and molecular changes collectively referred to as “storage lesions” (RCSL).30 A major contributor to RCSL is oxidative stress, which progressively increases over the storage period due to consumption of endogenous antioxidants. The goal of this study is twofold: to demonstrate the use of NPG electrodes as a potentially new measurement tool to evaluate the redox potential of RBC solutions over a 56 day storage period in both milliliter and microliter volumes of blood and to explore the role that increased concentrations of Vitamin C (VitC) have on the long-term stability of stored blood. Recent studies have indicated that optimal concentrations of VitC have the potential to mitigate oxidative injury and thus could provide a means to improve the long-term storage of

RBCs.31−33 Our measurements show consistent results between the micro- and bulk-based measurements and that redox potential measurements may provide a rapid and useful means to evaluate the redox stability of stored blood.



EXPERIMENTAL SECTION

Preparation and Storage of RBCs. The preparation and storage of RBCs was performed as described previously.34 Briefly, freshly donated whole blood units from 5 deidentified donors were purchased from Virginia Blood Services (Richmond, VA, USA). RBC concentrates were prepared by Virginia Blood Services using standardized protocols of plasma removal and leukocyte depletion, followed by the addition of SAGM (saline-adenine-glucose-mannitol) to RBCs. Each RBC unit was equally divided into four pediatric storage bags (volume of ∼75 mL per aliquot) and supplementations for the study were made to each aliquot at the supplier facility as described below using aseptic techniques. Prior to supplementation, an initial baseline sample was collected at the blood supplier facility and transported to the laboratory for analysis. RBCs that passed standard screening tests were transported 2 days later to the Virginia Commonwealth University Transfusion Medicine Center and stored at 4 °C for 56 days. All subsequent collections were made at the Virginia Commonwealth University Transfusion Medicine Center on Days 7, 21, 42, and 56. Study Design. RBCs in pediatric storage bags were treated with one of four additives: (a) normal saline (saline); (b) 0.3 mmol/L reduced VitC (Lo VitC); (c) 3 mmol/L reduced VitC (Hi VitC); or (d) 0.3 mmol/L oxidized VitC (dehydroascorbic acid, DHA) as final concentrations and gently mixed. The reduced VitC additive was preservative-free buffered ascorbic acid in water (Ascor L500, McGuff Pharmaceuticals, Santa Ana, CA, USA), pH 5.5−7.0 adjusted with sodium bicarbonate and sodium hydroxide. Oxidized VitC (DHA) was procured from Sigma-Aldrich (St. Louis, MO, USA). DHA was dissolved in saline at 60 °C for 30 min. It was filter sterilized and injected into the pediatric bags using a sterile technique. RBC’s were pelleted by centrifugation (10,000 g, 10 min at 4 °C) and the supernatant plasma saved for pH determination.34 An overview of the study design is shown in Figure 1a. Electrode Fabrication. Nanoporous gold (NPG) electrodes were prepared as previously described.21 In brief, commercially available 12 Karat gold−silver alloy leaf (50:50 wt % Au:Ag; loose leaf, Fine Art Store) was dealloyed in concentrated nitric acid for 14 min followed B

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Figure 2. (A) OCP vs time for RBCs doped with saline (black), Hi-VitC (blue), Lo-VitC (red), or DHA (green) at NPG electrodes. Error bars represent the standard deviations of four NPG electrodes. (B) Representative open-circuit potentials of 5 μL droplets on nanoporous electrodes. Data are from unit 2, day 21. As with the bulk measurement case, increasing the VitC concentration in RBCs reduces and stabilizes the potential. Stabilization time constant is consistent with the bulk measurements. by thorough rinsing and finally captured on a precleaned rectangular gold slide (evaporated metal films (EMF)). After visual confirmation of complete dryness, the substrate with the dealloyed gold leaf was placed under a UV lamp (254 nm, 20 W) at a distance of 5 cm for ∼24 h. Top-down and cross-sectional scanning electron microscope (SEM) images shown in prior work provide evidence of pore size.21,24 Unfortunately, not enough material is present to make isotherm measurements from which a pore size distribution can be obtained.35 To create the NPG electrode for the bulk redox potential measurements, the substrate was appropriately covered with Teflon tape containing a 1/8 in. hole to define the electrode area. Electrodes were used within a 1−3 h after taping. Bulk Redox Measurements. Electrochemical measurements were performed in a one-chamber three-electrode cell containing up to four NPG electrodes as the working electrodes (WE), a platinum wire as a counter electrode, and a single AgCl coated silver wire as a reference electrode (RE). The volume of solution was 7 mL. By using a single AgCl coated Ag wire as a reference, no liquid junction potential is present in the cell. The reference is still poised as the RBC solutions contain chloride. The electrochemical measurement were undertaken using a Metrohm Autolab PGSTAT128N that allows the open circuit potential (OCP) to be measured simultaneously at four working electrodes in a single solution with a single reference electrode (Figure 1b). After measuring OCP for 10 min, cyclic voltammetry was undertaken simultaneously at the four electrodes. A Pt wire was used as the auxiliary electrode in these experiments. After completion of each set of experiments, the AgCl coated silver wire reference electrode was thoroughly rinsed and was calibrated using Zobells solution (potassium ferro/ferricyanide in KCl)36 and redox potential values were corrected as needed. The NPG electrodes were soaked in MaxiZyme enzymatic detergent, rinsed, and discarded. A new set of electrodes was used for each blood sample. Microdroplet Blood Redox Potential Analysis. A series of redox potential measurements in parallel with the bulk measurements were undertaken using microcapillary reference electrodes. Figure 1c shows a schematic illustration of this approach where silicone supports with several 5 μL holes were adhered onto a nanoporous gold leaf that itself was adhered to a gold-coated microscope slide. Blood of various VitC concentrations was received on the day of each measurement and stored at 4 °C and 5 μL volumes were pipetted into a particular well minutes before measurements commenced. The end of a microcapillary reference electrode was positioned down within the blood and held fixed there throughout the measurement period. Measurements lasted anywhere from 5 to 30 min and were typically 10 min in duration. Microcapillary reference electrodes were formed with a laser-based puller (P-2000, Sutter Instruments) using borosilicate glass capillaries (Sutter, OD = 1.0 mm, ID = 0.78 mm) and preset program #11 with the following parameters (HEAT = 350, FIL = 4, VEL = 30, DEL = 200, PULL = 0). These capillaries were backfilled with 0.1 M KCl solution and a Ag/AgCl electrode was

placed into the capillary. The capillary was mounted to an Axopatch 200B headstage amplifier and Axoclamp (Molecular Devices) software was used to operate the headstage in current clamp mode (I = 0) while the OCP redox potential was digitized (Digidata 1220) and recorded with software (pClamp 10). Data was collected using a low-pass filter of bandwidth 1 kHz and sampled at 5 kHz. Each reference capillary was discarded and replaced with a new reference electrode after each measurement. The NPG electrode was attached to the ground side of the headstage amplifier. The NPG electrode was also used to measure a small volume of Zobell’s solution to allow comparisons with bulk blood data measured on the same day. All data for the microelectrode experiments was analyzed with IGOR Pro 6 software. Measurement of Intracellular Ascorbate Concentration. For VitC quantification, RBCs were pelleted by centrifugation (10,000 g, 10 min at 4 °C). Pelleted RBCs were washed with cold saline; deproteinized in 200 μL of cold 20% trichloroacetic acid (TCA), followed by the addition of 200 μL of cold 0.2% dithiothreitol (DTT) to prevent oxidation. RBC lysates were vortexed and centrifuged at 10,000 g for 10 min at 4 °C. The supernatants were stored at −80 °C for batch vitamin C analysis. Total vitamin C content was assessed using a Tempol-OPDA-based fluorescence end-point assay as previously described.37



RESULTS AND DISCUSSION Background. Nanoporous gold (NPG) electrodes provide a valuable means to make redox measurements in complex biological solutions due to their unique biosieving capabilities21,24 and their ability to improve electron exchange rates via nanoconfinement.27 In this study, we demonstrate that NPG electrodes can be used as a tool to quickly assess the redox state of RBCs during storage and evaluate the use of the antioxidant VitC as an additive to RBCs to stabilize the redox potential. The RBCs were spiked with different doses of the reduced form of VitC (0.3 and 3 mM) as well as the oxidized form of VitC (0.3 mM). In the latter case, RBCs possess GLUT receptors that allow uptake of oxidized VitC [dehydroascorbic acid, DHA], which is subsequently reduced intracellularly and stored as VitC. These samples are referred to as Lo-VitC, Hi-VitC, and DHA. For comparison, one bag of RBCs was spiked with an equal volume of sterile saline (Figure 1a illustrates the design study). The potentiometric measurement of the redox potential itself is simple: a high impedance voltmeter measures the zerocurrent potential (or open-circuit potential, OCP) of nanoporous gold (ENPG) with respect to the reference electrode (Eref) such that ERedox = ENPG − Eref. Since redox potential is an C

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ACS Sensors integrated measure of the total oxidants and reductants in a biological system able to exchange electrons with the electrode surface,2 a change in the redox state of the biological system will lead to a change in potential. In these measurements, it is imperative that the Eref does not change, which can be verified via calibration with Zobell’s solution. Large Volume Measurements. Figure 2A shows representative potential−time traces of NPG immersed in RBCs for 10 min. The trace shown represents the average of 4 NPG electrodes in each blood sample and the error bar represents the standard deviation. As expected, the most negative redox potential corresponds to the samples containing VitC. Blood samples containing saline or DHA always have a more positive redox potential than those that contain added VitC. As can be seen in this data set, the redox potential drifts to more negative values over the 10 min measurement period. This drift is always smaller for the samples containing added VitC. The addition of VitC helps to poise the redox system, which results in a more stable potential as previously described in a classic book by Zieglar.38 This poising effect is analogous to pH.39 When both the conjugate acid−base pair is present in viable concentrations, the pH of the solutions is very stable. Small Volume Measurements. Alongside the large volume measurements, the redox potential was also measured in microliter-sized samples using microcapillary reference electrodes. Figure 2B shows time traces and longterm values of the OCP redox potential for the four conditions listed above (saline, Lo-VitC, Hi-VitC, and DHA treated samples). This data, typical of all the samples measured, shows that stabilization appears steadier for the higher VitC concentrations with similar time constants to those seen in the bulk measurement. It can also be noted in both the bulk and droplet experiments shown in Figure 2 that there is significantly more variability in the value of the redox potential as judged by the standard deviations obtained from the 3−4 NPG electrodes for the bulk measurement case (Figure 2A) and the different traces shown over 2 runs performed in the microdroplet approach (Figure 2B). Considerable variability is observed in both cases for the saline and DHA additive cases, but this is not surprising given that these systems are unpoised.39 The slower drift in the redox potential for samples doped with saline or DHA reflects changes in the oxidant/ reductants as the sample equilibrates to room temperature and this could result from slight changes in oxygen or settling of blood cells on the electrode surface. In any case, we address this drift here by reporting the average OCP value over the last 10 s of each 10 min run. In Figure 3 (left side), the average redox potentials obtained at the 3−4 NPG electrodes over the 56 day period are reported for three RBCs units collected from different patients over a 5month period corrected for drifts in the reference electrode via Zobells solution. The first point is the redox potential of the RBCs on day 0 upon receipt and before addition of the additive. Upon addition of VitC, the redox potential lowers to ∼ −80 mV. This lowering is expected because a reducing agent has been added to the sample. Also noteworthy, and as explained earlier, the standard deviation is larger (and hence there is more variability) in the value of the OCP between the four NPG electrodes in the as-received samples that only contain saline as the additive. For the microdroplet measurement, similar trends are observed. As expected, the redox potential is more negative for both the Lo-VitC and Hi-VitC

Figure 3. OCP of three different units of RBCs (A, B, C) over a 56 day storage period at 4 °C for both the bulk measurements (left) and the micro measurements (right). The potential was measured on the day of collection and is shown as the solid black point at day = 0.

compared to the saline doped RBCs. In all cases except for unit C day 42 to day 56 and microdroplet unit A day 7 to day 21, a positive shift in the redox potential is noted, although the absolute values are slightly different between the large and small volume setup. The small differences in the magnitude of the OCP noted between the microdroplet and large volume measurements could result from a number of factors. These include the presence of a junction potential in the capillary reference electrode or offset in the microdroplet experimental setup as well as subtle differences in the composition between the large (mL) and small (μL) sample volumes due to sampling and/or temperature variations. Further work is needed to tease out these differences. Nevertheless, the similarities between the two methodologies and the clearly increasing trends for most of the measurements suggests that small volume measurements of the blood’s redox potential could provide an effective means for characterizing the state of blood under long-term storage. A number of important results can be noted from Figure 3. First, the magnitude of the OCP becomes more positive with storage time and the extent at which the potential increases to more positive values depends on the additive and also varies among the three different units obtained from different donors. Second, the degree of uncertainty as parametrized by the ±1 std dev error bars (bulk measurements) increases with storage time, and finally, the degree of uncertainty decreases with the addition of more VitC due to the poising of the blood.38 Collectively, these results show that long-term storage of blood leads to a slightly positive shift in the redox potential and the fact that both methodologies yield similar trends suggests a means for characterizing blood-based redox potential without the need for an intravenous blood-draw. Because the absolute value of the OCP depends on the blood donor, for this method to be realized in a clinic, a redox potential measurement will need to be taken initially and then this value compared to the value acquired at a later time. D

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Figure 4. CVs acquired at 50 mV/s at NPG in RBCs spiked with either saline, Hi-VitC, Lo-VitC, or DHA. The RBCs were stored at 4 °C and measurements were acquired on day 7 (black), 21 (red), 42 (blue), and 56 (green).

seen in the RBC solutions containing either saline or DHA. As the RBC solutions age, the VitC voltammetric peak decreases in intensity. Over a two week period (day 7 to day 21) the Faradaic current drops by a factor of 2 indicative that the concentration of VitC has dropped by half. After the 56 day storage period is over, the oxidation of VitC cannot be detected by CV as the concentration is below the detection limit. To obtain a more quantitative assessment of the drop in VitC concentration, the faradaic peak current was measured from the CVs collected after the OCP measurements for the Hi-VitC sample. Figure 5A shows the result for one of the units. A drop in the faradaic current can be readily seen and a fit to an exponential decay curve yields a time constant of 20.8 days. The decrease in VitC noted in the CVs during the 56 day storage indicates that VitC is being lost from the solution surrounding the blood cells due to uptake by GLUT receptors located on the RBCs. Because RBCs do not have transporters for reduced VitC, the VitC must become oxidized during the storage period. To assess this hypothesis, the intracellular VitC concentrations in RBCs were measured over the 56 day storage period as described in the Experimental Section. The results (average of 5 units) are shown in Figure 5B. In saline, the concentration of ascorbate is 104 μM on day 0 and drops ∼14% over the 56 day storage period indicative of oxidative stress and subsequent uptake by the GLUT receptors, which are plentiful in RBCs.41 The addition of exogenous VitC results in a rapid increase in RBC VitC content (Figure 5B) from 104 μM to 710, 159, and 519 μM for the Hi-VitC (3 mM), Lo-VitC (0.3 mM), and oxidized VitC (dehydroascorbic acid, DHA, 0.3 mM), respectively. The intracellular RBC VitC content for Lo- and Hi-VitC continues to increase with storage time again due to oxidation, which then allows for its uptake into RBCs. This happens gradually with Lo-VitC and much more rapidly with Hi-VitC. For the RBC-DHA sample, the intracellular ascorbate concentration decreased with time from day 7 to day 56. With DHA, the peak occurs at the first time point analyzed (day 7) after baseline since it is already in a form that is transported into RBCs. Once inside the RBCs, VitC is converted back to

The nature of the electrode potential measured at a redox electrode (e.g., gold, platinum) particularly in biological solutions is complex and not straightforward to understand.2 This is because the measured potential is a mixed potential and depends on the redox species present, their electron transfer rates, and their concentrations.2 A positive shift in the redox potential with storage time could be attributed to ongoing oxidative stress; it could also result from a change in pH of RBC changes during storage. Measurement of pH of the RBCs over the 56 day period shows that the pH changes from ∼7.5 to 6.5. Because the redox potential of blood is likely determined from redox reactions involving protons (e.g, ascorbic acid), the redox potential is expected to shift positive (e.g., 30−60 mV) during storage because the pH decreases. At this point in time, it is not possible to correct for the shift in redox potential with pH because the particular reactions that contribute to the measured redox potential of blood/RBCs are not fully understood. More detailed experiments need to be undertaken to fully apprehend the significance of positive shift in redox potential observed. To obtain more information about the redox process taking place, at the end of each experiment, cyclic voltammograms (CVs) were collected. The goal was to semiquantitatively evaluate the presence of ascorbic acid and/or uric acid (UA) in the samples, which can be seen in the CVs in blood.24,40 The concentration of VitC and UA in blood is ∼80 μM and 300 μM, respectively. Figure 4 shows the CVs collected at 50 mV/s at a NPG electrode over the course of the 56 day storage period. In the RBC solutions that contain either saline or DHA, a small peak can be observed near 0.35 V. Based on control experiments in buffer, this peak is believed to be due to the oxidation of uric acid. The magnitude of the faradaic current stays approximately the same suggesting the concentration of uric acid does not change much over the storage period within the uncertainty of the measurement technique. In the samples containing Lo- and Hi-VitC, a large peak at ∼50 mV is observed. This peak is attributed to the oxidation of VitC. As expected, the peak is significantly higher in the CV acquired in the high dose VitC RBC than in the low dose VitC RBC solutions. Furthermore, this particular peak cannot be E

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without the addition of VitC via measurement of redox potential is described. Nanoporous gold electrodes (NPG) known to help reduce the effect of biofouling on the electrochemical response and improve electron exchange rates were used. By measuring the redox potential of up to four NPG electrodes at the same time using a single reference electrode, both averages and standard deviations can be obtained in as little as 10 min. Further reduction in analysis time and sample volumes can be achieved using a small volume cell and microcapillary reference electrodes. Collectively the results in both large and small volumes indicate that the redox potential of the RBC becomes increasingly positive with time. The addition of VitC helps improve the stability of the redox potential measurement itself by poising the redox potential as noted by Zeigler in 1965.38 In addition, it is clear from the cyclic voltammetric studies and a Tempol-OPDA-based fluorescence end-point assay that the VitC is being taken up by the RBCs with a time constant of ca. 20 days. The addition of VitC may help to stabilize the redox potential over the 56 day storage period; however, more studies are needed. For this method to be realized in a clinic as a means to assess blood quality, an initial value of OCP will be needed and a clinician would focus on changes in OCP with storage rather than absolute values.

Figure 5. (A) Average drop in Faradaic current for the oxidation of ascorbic acid in the Hi-VitC sample over the 56 day storage period. The solid line represents an exponential fit to the experimental data (F = A exp(−t/τ), A = 60.6 μA, τ = 20.8 days, R2 = 0.970) (B) Average increase in the ascorbate concentration inside the cells over the 56 day storage period. Blue triangles: Hi-VitC; Red circles: LoVitC; Green triangles: DHA; Black circles: Saline. The solid blue line represents an exponential rise fit to the experimental data (Hi-VitC, blue line). As = As0(1 − exp(−t/τ)) with As0 = 2400 μM, τ = 20.2 days, and R2 = 0.990.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 804-828-7509. ORCID

Rezaul Karim Khan: 0000-0002-7736-3278 Bernard J. Fisher: 0000-0002-3273-2025 Ramesh Natarajan: 0000-0002-3431-9971 Joseph E. Reiner: 0000-0002-1056-8703 Maryanne M. Collinson: 0000-0001-6839-5334

the reduced form at the expense of glutathione, which is also very abundant in RBCs. The Hi-VitC and Lo-VitC data were fit to an exponential growth curve and a time constant of 20.8 day was obtained. The time course for the decrease of VitC extracellularly as evaluated from the CV curves is consistent with the time course for the uptake of VitC as measured intracellularly. The time constant (τ) is 20.2 and 20.8 days from the CV data and fluorescence assay data, respectively. Since RBC have GLUT receptors that only allow the uptake of oxidized VitC, VitC must first be oxidized to DHA intercellularly, which allows for the initial increase in RBC VitC content in the cells as observed in Figure 5B. What is particularly interesting is that the redox potential of the Hi-VitC and Lo-VitC RBCs does not change significantly even though it is clear from the voltammetry that the VitC is being taken up by the RBCs. If VitC was the only redox species contributing to the ERedox, then according to the Nernst equation, a 10-fold change in concentration will lead to a ca. 29 mV change in OCP. The lack of a large change in ERedox suggests that the value of redox potential is not strictly determined by ascorbic acid. Rather, as indicated earlier, it is a mixed potential whose value is determined by other redox molecules present in blood as eloquently described by Peiffer.2 Based on concentration and heterogeneous rate constants, for blood, these could potentially include uric acid and cysteine among others. Future studies will be needed here.

Author Contributions #

Students (Shanmuka P. Gadiraju and Megh Kumar) contributed equally.

Notes

Disclaimer: The views expressed in this work are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the United States Government. Some authors are military service members or employees of the United States Government. This work was prepared as part of their official duties. Title 17 U.S.C. 105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. 101 defines a United States Government work as a work prepared by a military service member or employee of the United States Government as part of that person's official duties. The authors declare the following competing financial interest(s): MMC is a co-author on a patent application related to a bioelectrochemical sensor incorporating nanoporous gold.



ACKNOWLEDGMENTS We gratefully acknowledge support of this work by the Commonwealth Transfusion Foundation (formally known as the Virginia Blood Foundation). We also thank Dr. Kimberly Sanford for her assistance with the acquisition of the RBCs and Dr. Christopher J. Freeman for his initial assistance with this project.



CONCLUSIONS In this pilot study, a new methodology to evaluate the longterm stability of red blood cells (RBCs) packets with and F

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ACS Sensors



(21) Patel, J.; Radhakrishnan, L.; Zhao, B.; Uppalapati, B.; Daniels, R. C.; Ward, K. R.; Collinson, M. M. Electrochemical properties of nanostructured porous gold electrodes in biofouling solutions. Anal. Chem. 2013, 85 (23), 11610−11618. (22) Seker, E.; Reed, M. L.; Begley, M. R. Nanoporous gold: fabrication, characterization, and applications. Materials 2009, 2 (4), 2188−2215. (23) Collinson, M. M. Nanoporous gold electrodes and their applications in analytical chemistry. ISRN Anal. Chem. 2013, 2013, 21. (24) Farghaly, A. A.; Lam, M.; Freeman, C. J.; Uppalapati, B.; Collinson, M. M. Potentiometric measurements in biofouling solutions: comparison of nanoporous gold to planar gold. J. Electrochem. Soc. 2016, 163 (4), H3083−H3087. (25) Serrano, M. B.; Despas, C.; Herzog, G.; Walcarius, A. Mesoporous silica thin films for molecular sieving and electrode surface protection against biofouling. Electrochem. Commun. 2015, 52, 34−36. (26) Noyhouzer, T.; Valdinger, I.; Mandler, D. Enhanced Potentiometry by Metallic Nanoparticles. Anal. Chem. 2013, 85 (17), 8347−8353. (27) Park, S.; Kim, H. C.; Chung, T. D. Electrochemical analysis based on nanoporous structures. Analyst 2012, 137 (17), 3891−3903. (28) Daniels, R. C.; Jun, H.; Tiba, M. H.; McCracken, B.; HerreraFierro, P.; Collinson, M.; Ward, K. R. Whole Blood Redox Potential Correlates with Progressive Accumulation of Oxygen Debt and Acts as a Marker of Resuscitation in a Swine Hemorrhagic Shock Model. Shock 2018, 49 (3), 345−351. (29) Van de Watering, L. Red cell storage and prognosis. Vox Sang. 2011, 100 (1), 36−45. (30) Chin-Yee, I.; Arya, N.; d’Almeida, M. S. The red cell storage lesion and its implication for transfusion. Transfusion science 1997, 18 (3), 447−458. (31) Vani, R.; Soumya, R.; Carl, H.; Chandni, V.; Neha, K.; Pankhuri, B.; Trishna, S.; Vatsal, D. Prospects of vitamin C as an additive in plasma of stored blood. Adv. Hematol. 2015, 2015, 1. (32) Stowell, S. R.; Smith, N. H.; Zimring, J. C.; Fu, X.; Palmer, A. F.; Fontes, J.; Banerjee, U.; Yazer, M. H. Addition of ascorbic acid solution to stored murine red blood cells increases posttransfusion recovery and decreases microparticles and alloimmunization. Transfusion 2013, 53 (10), 2248−2257. (33) Raval, J.; Fontes, J.; Banerjee, U.; Yazer, M.; Mank, E.; Palmer, A. Ascorbic acid improves membrane fragility and decreases haemolysis during red blood cell storage. Transfusion Medicine 2013, 23 (2), 87−93. (34) Sanford, K.; Fisher, B. J.; Fowler, E.; Natarajan, R. Attenuation of Red Blood Cell Storage Lesions with Vitamin C. Antioxidants 2017, 6 (3), 55. (35) Tan, Y. H.; Davis, J. A.; Fujikawa, K.; Ganesh, N. V.; Demchenko, A. V.; Stine, K. J. Surface area and pore size characteristics of nanoporous gold subjected to thermal, mechanical, or surface modification studied using gas adsorption isotherms, cyclic voltammetry, thermogravimetric analysis, and scanning electron microscopy. J. Mater. Chem. 2012, 22 (14), 6733−6745. (36) Nordstrom, D. K. Thermochemical redox equilibria of ZoBell’s solution. Geochim. Cosmochim. Acta 1977, 41 (12), 1835−1841. (37) Mohammed, B. M.; Fisher, B. J.; Huynh, Q. K.; Wijesinghe, D. S.; Chalfant, C. E.; Brophy, D. F.; Natarajan, R. Resolution of sterile inflammation: role for vitamin C. Mediators Inflammation 2014, 2014, 15. (38) Ziegler, E. The Redox Potential of the Blood In Vivo and In Vitro. Its measurement and Significance; Thomas CC: Springfield, IL, 1965; p 196. (39) Teasdale, P. R.; Minett, A. I.; Dixon, K.; Lewis, T. W.; Batley, G. E. Practical improvements for redox potential (EH) measurements and the application of a multiple-electrode redox probe (MERP) for characterising sediment in situ. Anal. Chim. Acta 1998, 367 (1−3), 201−213. (40) Mittal, A.; Goke, F.; Flint, R.; Loveday, B. P. T.; Thompson, N.; Delahunt, B.; Kilmartin, P. A.; Cooper, G. J. S.; MacDonald, J.;

REFERENCES

(1) Bakker, E.; Pretsch, E. Modern Potentiometry. Angew. Chem., Int. Ed. 2007, 46 (30), 5660−5668. (2) Peiffer, S.; Klemm, O.; Pecher, K.; Hollerung, R. Redox measurements in aqueous solutionsa theoretical approach to data interpretation, based on electrode kinetics. J. Contam. Hydrol. 1992, 10 (1), 1−18. (3) DeLaune, R. D.; Reddy, K. R. Redox Potential. In Encylopedia of Soils in the Environment, Hillel, D., Ed.; Academic Press, 2004; pp 366−371. (4) Pang, H.; Zhang, T. C. Fabrication of redox potential microelectrodes for studies in vegetated soils or biofilm systems. Environ. Sci. Technol. 1998, 32 (22), 3646−3652. (5) Jang, A.; Lee, J. H.; Bhadri, P. R.; Kumar, S. A.; Timmons, W.; Beyette, F. R.; Papautsky, I.; Bishop, P. L. Miniaturized redox potential probe for in situ environmental monitoring. Environ. Sci. Technol. 2005, 39 (16), 6191−6197. (6) Carlsson, T.; Muurinen, A. Practical and theoretical basis for performing redox-measurements in compacted bentonite − A literature survey; Posiva, Finland; http://www.posiva.fi/files/842/WR_200851web.pdf2008; pp 1−74. (7) Goncharuk, V. V.; Bagrii, V. A.; Mel’nik, L. A.; Chebotareva, R. D.; Bashtan, S. Y. The use of redox potential in water treatment processes. Journal of Water Chemistry and Technology 2010, 32 (1), 1− 9. (8) Schüring, J.; Schulz, H. D.; Fischer, W. R.; Böttcher, J.; Duijnisveld, W. H. M. Redox: Fundamentals, Processes and Applications; Springer-Verlag: New York, 1999. (9) Matia, L.; Rauret, G.; Rubio, R. Redox Potential Measurement In Natural-Waters. Fresenius' J. Anal. Chem. 1991, 339 (7), 455−462. (10) Khubutiya, M. S.; Goldin, M. M.; Romasenko, M. V.; Volkov, A. G.; Hall, P. J.; Evseev, A. K.; Levina, O. A.; Aleschenko, E. I.; Krylov, V. V. Redox Potentials of Blood Serum in Patients wtih Acute Cerebral Pathology. ECS Trans. 2009, 25 (19), 63−71. (11) Rael, L. T.; Bar-Or, R.; Kelly, M. T.; Carrick, M. M.; Bar-Or, D. Assessment of Oxidative Stress in Patients with an Isolated Traumatic Brain Injury Using Disposable Electrochemical Test Strips. Electroanalysis 2015, 27 (11), 2567−2573. (12) Zhi, L.; Hu, X.; Han, C. Biphasic changes (overreduction and overoxidation) of plasma redox status and clinical implications in early stage of severe burns. J. Crit. Care 2014, 29 (6), 1063−1068. (13) Zhi, L.; Hu, X.; Xu, J.; Yu, C.; Shao, H.; Pan, X.; Hu, H.; Han, C. The characteristics and correlation between the ischemiareperfusion and changes of redox status in the early stage of severe burns. American Journal of Emergency Medicine 2015, 33 (3), 338− 343. (14) Zhi, L. Z.; Liang, J.; Hu, X. L.; Xu, J.; Yu, C. H.; Shao, H. W.; Pan, X. L.; Han, C. M. The reliability of clinical dynamic monitoring of redox status using a new redox potential (ORP) determination method. Redox Rep. 2013, 18 (2), 63−70. (15) Agarwal, A.; Bui, A. D. Oxidation-reduction potential as a new marker for oxidative stress: Correlation to male infertility. Investigative and clinical urology 2017, 58 (6), 385−399. (16) Barfidokht, A.; Gooding, J. J. Approaches Toward Allowing Electroanalytical Devices to be Used in Biological Fluids. Electroanalysis 2014, 26 (6), 1182−1196. (17) Moulton, S. E.; Barisci, J. N.; Bath, A.; Stella, R.; Wallace, G. G. Investigation of protein adsorption and electrochemical behavior at a gold electrode. J. Colloid Interface Sci. 2003, 261 (2), 312−319. (18) Roscoe, S. G.; Fuller, K. L. Interfacial Behavior Of GlobularProteins At A Platinum-Electrode. J. Colloid Interface Sci. 1992, 152 (2), 429−441. (19) Wisniewski, N.; Moussy, F.; Reichert, W. M. Characterization of implantable biosensor membrane biofouling. Fresenius' J. Anal. Chem. 2000, 366 (6−7), 611−621. (20) Hanssen, B. L.; Siraj, S.; Wong, D. K. Y. Recent strategies to minimise fouling in electrochemical detection systems. Rev. Anal. Chem. 2016, 35 (1), 1−28. G

DOI: 10.1021/acssensors.8b00498 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors Hickey, A.; Windsor, J. A.; Phillips, A. R. J. The Redox Status of Experimental Hemorrhagic Shock as Measured by Cyclic Voltammetry. Shock 2010, 33 (5), 460−466. (41) May, J. M.; Qu, Z.-c.; Qiao, H.; Koury, M. J. Maturational loss of the vitamin C transporter in erythrocytes. Biochem. Biophys. Res. Commun. 2007, 360 (1), 295−298.

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DOI: 10.1021/acssensors.8b00498 ACS Sens. XXXX, XXX, XXX−XXX