21 The ability to transport and store a large human blood inventory for transfusions is an 22 essential requirement for medical institutions. Thus, there is an important need for 23 rapid and low-cost characterization tools for analyzing the properties of human red 24 blood cells (RBCs) while in storage. In this study, we investigate the ability to use 25 dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical 26 properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant 27 and stored in a blood bank refrigerator for a period of 20 days. Cells were removed 28 from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction 29 to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage 30 during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity 31 DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to 32 measurements taken with a 2D quadrupole electrode array. The DEP analysis 33 confirms that RBC electrical property changes occur during storage and are only 34 discernable with the use of the cell crosslinking reaction above a glutaraldehyde 35 fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed 36 to decrease by more than 75% while the RBC membrane conductance was observed 37 to increase by more than 1000% over a period of 20 days. These results show that 38 the presented combination of chemical crosslinking and DEP can be used as rapid 39 characterization tool for monitoring electrical properties changes of human red blood 40 cells while subjected to refrigeration in blood bank storage. 41 42 43 1 Introduction 44 Subcellular components such as the lipid bilayer membrane, cytoskeleton, and 45 cytoplasm play essential roles in maintaining cellular function and cell health. 46 Environmental influences such as disease, exposure to pathogens, and 47 pharmacological interventions can impact the physiological characteristics and 48 structural organization of these subcomponents, which can influence their 49 biomechanical and bioelectrical properties. Electrical property changes have been 50 correlated with many physiological processes, including the differentiation state of 51 neural and embryonic stem cells [1,2]. During cell invasion and development, the 52 malaria inducing parasite, Plasmodium falciparum induces electrical changes in 53 human red blood cell (RBC) cytoplasm and membrane conductance [3]. 54 Pharmacological interventions, such as cell exposure to ion channel blockers and anti-55 cancer treatments have been shown to induce changes in cancer cell electrical 56 properties. Membrane potential has also shown to be an important indicator in 57 biological function, including during the progression of cancer [4]. Similarly, recent 58 studies demonstrate the significance of membrane conductance and cytoplasmic 59 conductivity in contributing to the regulation of the circadian rhythm in human red blood 60 cells [5] and in HL-60 cells undergoing apoptosis [6,7]. A significant and continuing 61 growing body of work therefore demonstrates that cellular electrical properties have 62 the potential to serve as label-free diagnostic markers in cell identification, disease 63 detection, cell separation and for health monitoring [1,8,9]. 64 One important diagnostic marker is the quality of stored human blood for 65 transfusion [10]. Modern blood storage systems enable the donation and transfusion 66 of blood to be dynamically separated in time and space. Large medical centers can 67 use hundreds of units of RBCs a day, and the ability to transport and build large blood 68 storage inventory is an important requirement for medical institutions. However, such 69 readily available blood storage often raises the question: how long can blood be stored 70 before it is no longer safe and effective for blood transfusions? 71 RBCs show clear signs of degradation during storage, and there are many 72 known physiological changes associated with RBC storage, including changes in cell 73 morphology and loss of metabolism [11]. A number of RBC quality measures exist, 74 including measuring hemoglobin concentration in RBC storage and measuring the 75 potassium ion concentration in suspension solution, however, no such methods 76 quantify the electrical properties of the RBCs themselves. One metric related to cell 77 electrical properties is the diffusive loss of cytoplasmic cations, such as K+, Na+ and 78 Ca2+ via the cell membrane while the blood is stored under refrigeration in a blood 79 bank. In particular, the activity of the sodium-potassium ion pump is highly sensitive to 80 temperature, and refrigerated RBCs have been shown to leak potassium into their 81 suspending buffer during storage [12]. When reinfused, cells have the potential to 82 reabsorb potassium which can alter the local extracellular potassium concentration. In 83 cases with an infant or when a large volume of blood relative to the patient’s circulating 84 volume is required, such ion reabsorption can lead to death [12]. Development of low-85 cost and rapid methods to dynamically track the electrical properties of human red 86 blood cells in storage could therefore play an important role in dynamically monitoring 87 the health of stored blood. 88 One method to measure cell electrical properties is to use the electrokinetic 89 technique, dielectrophoresis (DEP) [13]. Conventionally, DEP is performed using two-90 dimensional (2D) thin-film electrode arrays with quadrupole, interdigitated, comb-tooth 91 or castellated structures that are typically fabricated atop a glass substrate and 92 observed under an optical microscope. Cells suspended in a buffer of known electrical 93 conductivity and permittivity are deposited on the electrode array and subjected to a 94 sweeping electric-field frequency. The electric field from the electrodes polarizes the 95 cells and dielectrophoretically drives them to spatially assemble within the confines of 96 the electrode array. The specific geometric location of the DEP cell assembly is based 97 on cell and buffer electrical properties and the AC field frequency; cells will experience 98 a different DEP force - both in direction and magnitude - depending on the frequency 99 applied. The resulting frequency dependent DEP force curve, or DEP spectrum, will 100 consist of finite field frequency ranges where cells are either attracted towards 101 (positive DEP) or repelled away from (negative DEP) the high electric field regions as 102 defined by the array’s electrodes. The frequency in which this attraction/repulsion 103 reverses and cell DEP motion ceases is defined as the DEP crossover frequency 104 (COF). The frequency dependent DEP behavior can be measured optically and 105 combined with an appropriate Maxwell-Wagner cell polarization model to empirically 106 deduce cell electrical properties. In this manner, the entire cell suspension is utilized 107 to measure the COF and therefore the resulting DEP method yields electrical 108 properties reflective to the mean value of the entire cell suspension under observation. 109 While the 2D electrode array method is effective and responsible for many 110 impactful contributions within the DEP community, it does suffer from several 111 limitations. First, this method typically requires one to manually observe DEP-induced 112 cell trajectories. This process requires a highly trained operator and can be tedious for 113 large numbers of samples. Further this manual method is vulnerable to human error 114 and bias. Second, very often this method is only useful in measuring cell COFs which 115 represents single data points within the entire DEP spectrum. Therefore, significant 116 experimental data and DEP behavior in the positive and negative DEP frequency 117 ranges of the spectrum are often not taken into consideration. Such a constraint places 118 a limit on the ability to accurately correlate DEP behavior to DEP polarization theory 119 and determine the cell electrical properties. In an effort to overcome the drawbacks of 120 conventional 2D experiments and to realize DEP as a more formal analytical tool, 121 Hughes et al. developed the 3DEP dielectrophoretic cytometer [14]. The 3DEP is 122 capable of measuring a more complete DEP force spectrum and empirically calculate 123 the cell suspension electrical properties. 124 In this work, we demonstrate the use of DEP to quantify the storage-induced 125 electrical property changes of human RBCs subjected to blood-bank refrigeration. In 126 order to minimize ion leakage across the RBC membrane during DEP measurements, 127 we utilize a glutaraldehyde crosslinking reaction prior to cell washing and suspension 128 in a low conductivity-high permittivity zwitterion DEP buffer [15–17]. The 129 glutaraldehyde crosslinks aminated protein groups and serves to inhibit ion loss 130 through the membrane, while the zwitterion buffer reduces the second high frequency 131 DEP COF to within the operating range of most standard function generators. We 132 analyzed the influence of varying concentrations of glutaraldehyde on the DEP 133 behavior of human RBCs over a period of 20 days and used the 3DEP to calculate the 134 cell electrical properties. We also analyzed and compared the DEP spectrum obtained 135 by the 3DEP with that of a 2D quadrupole electrode array in order to ensure these two 136 methods do indeed provide comparable experimental COFs. Results suggest that the 137 3DEP method is capable of discerning storage-induced electrical property changes of 138 human RBCs. With further development, we believe this glutaraldehyde-3DEP 139 approach can potentially offer a rapid low-cost method for electrically quantifying the 140 status of blood stored for transfusions and for detecting autologous blood transfusions 141 in doping/cheating endurance athletes. 142 2 Theory 143 2.1 DEP-Shell Model 144 We utilize DEP to investigate the dynamic changes of RBC electrical properties 145 when subjected to storage at 4 oC in a blood bank refrigerator over a period of 20 days. 146 The DEP force and polarization equation can be derived for a homogeneous 147 axisymmetric spherical particle and then modified for a RBC. The DEP force on a 148 spherical particle suspended in an electrolyte media is given as [18,19]: 149 F"⃗!"#=2π𝜀$ε%r&Re[K(ω)]𝛻E""⃗' , (1) 150 where 𝜀$, ε%, r and 𝛻E""⃗', is the permittivity of free space, dielectric constant of the 151 media, the radius of the particle and the gradient of the electric field squared, and Re[∙152 ] represents the real part of a complex variable. K(ω) is the Clausius-Mossotti factor 153 (CMF), 154 𝐾(𝜔)=8(!∗)(#∗(!∗*'(#∗9 . (2) 155 Physically, Eq. (2) describes the frequency dependent particle dipole moment, and is 156 based on a complex permittivity of both the particle, ε+∗ and media, ε%∗. This factor 157 describes the frequency dependent polarization, and ultimately the direction a particle 158 will move in a field gradient under DEP. The DEP COF, for example occurs at a 159 frequency such that Re[K(ω)] = 0, while the particle exhibits positive DEP (pDEP) and 160 negative DEP (nDEP) when Re[K(ω)] > 0 and Re[K(ω)] < 0, respectively. 161 Electrically, each domain – the particle and surrounding electrolyte - is modelled 162 as a leaky dielectric which behaves as an equivalent resistor and capacitor in series. 163 For a harmonic AC field with an angular frequency, 𝜔 this circuit exhibits a complex 164 permittivity, 𝜀∗as 165 𝜀∗= ε−𝑖 -. , (3) 166 which has a real part composed of the permittivity, 𝜀 and an imaginary part (i2 = -1) 167 that is a function of the angular field frequency, 𝜔, and the electrical conductivity, 𝜎. 168 Here, we consider the DEP force acting on a dilute suspension of human RBCs. 169 The DEP particle force equation must be modified to account for the outer RBC 170 membrane and inner cytoplasmic domain. Based on previous DEP work, we assume 171 each individual human RBC is modelled as sphere of cytoplasm with an interior radius, 172 r1 surrounded by a shell of cell membrane with an outer radius, r2 as determined by 173 the cell membrane thickness. The polarization characteristics of both domains are 174 captured with an effective complex permittivity term, 𝜀!""∗ and can be shown to be 175 [18,19]: 176 𝜀!""∗ = 𝜀$!$∗%!"!#&$' )*%&'()∗+%,-,∗%&'()∗."%,-,∗+%!"!#&$, %&'()∗+%,-,∗%&'()∗."%,-,∗ , (4) 177 where 𝜀!""∗ replaces 𝜀/∗ equation (Eq. 2). This effective term is a function of the electrical 178 properties of both cell domains with a complex permittivity of the cytoplasm, 𝜀012$∗ and 179 the cell membrane, 𝜀343∗. 180 It is worth briefly justifying our assumption to use a spherical polarization model 181 as an approximation for capturing the DEP behavior of an inherently non-spherical 182 biconcave shaped human RBC. An alternative single-shelled DEP model utilizes an 183 oblate spheroid to capture the RBC disc shaped geometry. In some instances, this 184 oblate model is necessary to properly fit DEP polarization data, as was shown 185 previously using bovine RBCs [21]. Using the opensource DEP spectrum software, 186 myDEP [20] one can show that a single-shelled spherical DEP model for this study 187 largely captures the DEP spectrum – including both low and high COFs – produced 188 from an oblate spheroidal polarization model to within ~90% accuracy. It should be 189 noted that the 3DEP commercial instrument software is not yet capable of performing 190 regression analysis and data fitting with a non-spherical model. Therefore, while it is 191 the only existing option within the instrument, it is still important to validate the use of 192 a spherical assumption for any given cell system. Based on our DEP spectra 193 comparisons, we believe a spherical assumption is a valid DEP approximation for 194 human RBCs (experimentally measured mean radius, r2 = 4.2 μm + 0.4; membrane 195 thickness = 7 nm) for our DEP experimental system. 196 3 Materials and methods 197 We now describe the fabrication and operation of the 2D and 3DEP devices, the high 198 permittivity zwitterion DEP buffer and the RBC glutaraldehyde chemical crosslinking 199 workflow. 200 3.1 Dielectrophoresis Devices 201 DEP experiments were performed using two different experimental methods. 202 First, a more complete DEP spectrum (up to a field frequency of 45 MHz) was 203 determined using the commercial 3DEP instrument (DEPtech, Heathfield, UK). 204 Second, a 2D quadrupole electrode array was used to measure the cellular DEP COF 205 and subsequentaly compared to that of the 3DEP spectrum. 206 3.2 3DEP Analysis 207 A detailed analysis of the operating theory and data analysis for the 3DEP has 208 been described elsewhere [14]. The 3DEP system consists of a printed circuit board 209 (PCB) chip which is inserted into a reader (Fig. 1A). The reader contains twenty 210 independent DDS-signal generators each capable of delivering a 20 Vpp waveform 211 source at frequencies up to 45 MHz. Each PCB 3DEP chip contains 20 small pinhole 212 wells capable of initiating DEP. The chip is illuminated using a collimated light source 213 and monitored using an integrated CMOS camera. When an electric field is applied to 214 each well, the resulting DEP force drives cell motion towards the well edges by pDEP, 215 or into the well center by nDEP depending on the frequency applied. The resulting 216 light intensity of each well is then used to compute a DEP force spectrum dataset (Fig. 217 1C). This process is repeated multiple times to increase the number of spectrum data 218 points and improve experimental accuracy (Fig. 1D). The 3DEP makes it possible to 219 measure the CMF of cells by monitoring the evolution of optical intensity passing 220 through each chip well. Each resulting 3DEP spectrum is a relative DEP force 221 spectrum and relates the CMF to the light intensity measured in each well through a 222 mathematical factor that remains unchanged during the course of the DEP experiment 223 [14]. The resulting spectrum is then not the complete DEP force, but instead is a 224 relative DEP force, or CMF, which can be used to deduce RBC electrical properties 225 by fitting these relative force spectra to the polarization model defined previously in 226 Eqns. 2 – 4. 227 3.3 The Quadrupole Electrode Array 228 2D quadrupole electrodes were fabricated using vapor deposition and wet 229 chemical etching techniques atop of thin glass coverslips (#1, 50 x 30 mm Fisher 230 Scientific). Each slide was coated with 20 nm of chromium and 30 nm of gold using 231 electron beam evaporation and subsequently patterned using a positive photoresist 232 (Shipley 1318). The exposed metal was then etched using gold and chromium 233 chemical etchant (Transene Company, Inc) to create a quadrupolar electrode array 234 geometry with four individual pointed electrodes, each with a separation lengthscale 235 of 50 µm and electrically accessible by a rectangular pad (Fig. 1B). A 20 Vpp voltage 236 over a varying frequency range was then applied to every other electrode using a 237 function generator (Rigol DG1022Z) while the other two non-active electrodes were 238 grounded. The voltage generated a well-defined high field region in the viscinity of the 239 electrode edges and a low field region in the center of the array. The DEP COF was 240 measured manually under brightfield microscopy by determining the field frequency in 241 which cell assembly transitioned from pDEP to that of nDEP (Fig. 1B). 242 243 Figure 1. Dielectrophoresis Devices and Spectrum Analysis. (A) 3DEP chip and 244 reader for measuring RBC DEP spectrum. (B) 2D quadrupolar array for DEP COF 245 measurments. RBCs collect at the electrode edges by pDEP or in the center by 246 nDEP depending on the AC frequency applied. (C) Relative DEP force spectrum 247 from a single experiment is fit to a single-shell spherical model. The circled 248 minima and maximum regions correspond to the DEP cell distributions within 249 the pictured 3DEP pinholes. (D) Multiple 3DEP sample repeats (N = 7) 250 superimposed and fit to the polarization model using regression analysis (R = 251 0.9487). 252 253 254 3.4 Overview of Buffer and RBC Electrokinetic Optimization 255 In this section we describe the preparation of our DEP buffer and the chemical 256 treatment of human RBCs for DEP analysis. Our overall strategy is to minimize as 257 much as feasible cytoplasmic leakage and interaction of a RBC interior with that 258 associated with resuspension in a high permittivity-low conductivity DEP buffer 259 immediately prior to DEP analysis. Therefore, in order to effectively capture the 260 storage related electrical property changes occuring in the RBCs we devised a 261 chemical crosslinking scheme to diffusively “seal” the interior cell cytoplasmic ionic 262 distribution during analysis in DEP buffer to more accurately elicidate RBC electrical 263 property changes that occured while in refrigerated storage. Our previous work has 264 demonstrated that chemical crosslinking by the fixation agent, glutaraldehyde is an 265 effective approach to prevent cell leakage when cells are resuspended from 266 physiological buffer into a low conductivity DEP buffer [15,21]. Without a crosslinking 267 protocol, previous DEP studies with bovine RBCs showed that any storage induced 268 electrical changes were not resolvable by DEP [15,21]. After glutaraldehyde treatment 269 at the appropriate concentration, the chemically crosslinked cells are capable of 270 handling DEP analysis in low conductive DEP buffers without cytoplasmic leakage or 271 hemolysis. Therefore in this work, human RBCs were first treated with varying 272 concentrations of glutaraldehyde in order to chemically crosslink the aminated proteins 273 of the RBC and to determine the most effective concentration in which this effect is 274 observed with human blood. While we could attempt to instead use an osmotically 275 balanced DEP buffer as demonstrated in other DEP studies with human blood [5], 276 previous work with storage related DEP analysis has shown that it is important to 277 minimize the degree to which cells are capable of being osmotically influenced by a 278 change in buffer composition. Mass transport across the membrane during DEP 279 analysis has been shown to reduce DEP resolution and lead to dynnamically changing 280 DEP behavior. Therefore, in this work we utilize chemical crosslinking to maximize 281 DEP resolution for detecting age related electrical changes in human RBCs. 282 In addition to chemically treating the RBCs, we also optimize our DEP buffer 283 with a polarizable zwitterionic salt in order to increase the buffer permittivity. In doing 284 so, the upper high frequency portion of the DEP spectrum associated with the second 285 DEP COF is shifted into a lower frequency space and within the full working range of 286 the 3DEP instrument. As such, a DEP spectrum which would normally require a > 60 287 MHz bandwidth to fully observe only requires an upper frequency of ~ 20 MHz. The 288 following sections below describe the chemical cell crosslinking and DEP buffer 289 preparation protocols. 290 3.5 High Permittivity DEP Buffer 291 The DEP buffer used in this work was a 0.8 M 6-aminohexanoic acid (AHA) 292 (Millipore Sigma) dissolved in deionized water. The final buffer electrical conductivity 293 was controlled by first polishing the 0.8 M AHA solution for 10 min in 5g/mL Dowex 294 MR-3 ion exchange resin (Millipore Sigma) to remove trace salts, and to lower the 295 solution’s electrical conductivity to a baseline value. The buffer solution electrical 296 conductivity was then adjusted to a final target value using 1X phosphate buffered 297 saline (PBS). The final DEP buffer possessed an electrical conductivity of 100 μS/cm 298 and a dielectric constant of 110, as verified in our previous electrokinetics work using 299 fluidic dielectrophoresis (fDEP) [22,23]. 300 3.6 Human Red Blood Cells 301 Single donor O- human blood (ZenBio, Inc) was drawn intravenously in 10 mL 302 volumes and stored in DB vacutainer K2EDTA which contains di-potassium K2EDTA 303 which blocks the coagulation cascade. All human donors passed required FDA 304 screening and provided informed consent prior to blood collection. Collected blood 305 was stored in a blood bank refrigerator (Jewett) at a temperature of 4oC. Upon arrival, 306 and prior to each use, blood hematocrit was measured using pocH-100i hematology 307 analyzer (Sysmex). A depiction of the RBC washing and crosslinking DEP workflow is 308 shown in Fig. 2. For each DEP experiment, a 20µL volume of blood was removed from 309 the vacutainer and suspended in 1X PBS. The cell suspension was then gently mixed 310 by inverting the micro-centrifuge tube 15 times and rotating it gently for 30 seconds. 311 The diluted whole blood suspension was then centrifuged at 2000 rcf for two minutes 312 to pellet the RBCs and remove blood components. Then supernatant was discarded 313 and replaced with 1X PBS and the sample was further washed for a total of three 314 times. Cells were then either crosslinked in a dilute glutaraldehyde solution or used in 315 DEP experiments. Prior any DEP experiment, cells were washed twice in the 0.8M 316 AHA DEP buffer and centrifuged at 6000 rcf for two minutes to remove trace amounts 317 of 1X PBS prior to dielectrophoretic characterization. The final washed RBCs were 318 then immediately used in the DEP experiments. 319 320 321 Figure 2. Human RBC glutaraldehyde crosslinking workflow. Whole human 322 blood is washed three times in 1X PBS and subjected to chemical fixation for 1 323 hour. The cells are washed three times again in 1X PBS, washed twice in DEP 324 buffer, and suspended in fresh DEP buffer for immediate DEP analysis. 325 326 3.7 Glutaraldehyde Crosslinking 327 Following the RBC wash in 1X PBS, cells were chemically crosslinked in 328 glutaraldehyde in a similar manner as described previously [15,21]. As shown in the 329 experimental workflow in Fig. 2, glutaraldehyde (EM Grade, 25%; Polysciences, Inc) 330 was performed by first adjusting RBC concentration to 106 cells/ml in a 1X PBS 331 solution. A 25 w/v% glutaraldehyde stock solution was used to produce varying RBC 332 crosslinking concentrations (0, 0.05, 0.1, 0.5, 1.0, and 2.5 w/v %). Crosslinking 333 reactions were performed atop a shaker (Standard Analog Shaker 3500, VWR) for 1 334 hour at 250 rpm. To quench the crosslinking reaction, samples were centrifuged at 335 2000 rcf for 2 minutes and the supernatant was replaced with 1X PBS. After three 1X 336 PBS wash cycles, the samples were washed twice with 0.8M AHA DEP buffer and 337 then suspended in fresh 0.8M AHA DEP buffer for immediate DEP analysis. 338 339 3.8 DEP Experiments 340 Blood aliquots were collected after fixed storage time points (1, 5, 10, 15 and 20 341 days) after the blood draw and crosslinked. DEP analysis with the 3DEP and 2D 342 electrodes were performed immediately after PBS and AHA washing. For 3DEP 343 experiments, a gel loading pipette tip was used to load 75 µL of a RBC suspension 344 into a 3DEP 806 chip (DEPtech, Heathfield, UK). The top surface of the chip was then 345 covered with a 20x20 mm glass coverslip and loaded into the 3DEP reader. The pin 346 connections between the 3DEP reader and the DEP chip then delivered an AC 347 potential to each of the 20 chip pinholes for 30 seconds. This experiment resulted in a 348 point-wise dataset for a relative DEP force spectrum (Fig. 1C). To improve accuracy 349 the experiment was repeated, for example N = 7 times, and the combined dataset was 350 regressively fit to the single-shell polarization model and used to deduce the RBC 351 electrical properties (Fig. 1D). This process was then repeated across varying RBC 352 storage time and glutaraldehyde crosslinking concentrations. 353 354 Figure 3. DEP Spectrum and electrical properties of fresh donor RBCs. (A) 3DEP 355 model spectra of RBCs for varying glutaraldehyde concentrations, n=5 for 356 crosslinked, and n=10 for no crosslinked RBCs (B) RBC membrane 357 conductance, (C) RBC membrane capacitance, and (D) RBC cytoplasm 358 conductivity (b-d represent mean ± SEM, n=5 for cross-linked, n=10 for no 359 crosslinking, and p<0.0001 by one-way ANOVA and compared to w/v% control 360 using an unpaired t test with Welch's correction (****, ***,**, and * and 361 correspond to p values < 0.0001, 0.001, 0.01, 0.05 and ns denotes p > 0.5). 362 363 4 Results and discussion 364 4.1 3DEP Spectra of Fresh Donor Cells 365 The 3DEP spectra of fresh single donor human RBCs was first measured at 366 different glutaraldehyde crosslinking concentrations. Shown in Fig. 3A., the DEP 367 spectrum of fresh donor cells is plotted for varying glutaraldehyde concentrations. For 368 fresh RBCs, the DEP spectrum of non-crosslinked cells closely mimics the spectrum 369 measured for fresh cells crosslinked at 2.5 w/v%, as indicated by the two overlapping 370 spectra. Each fresh RBC spectrum then was used to calculate the cell membrane 371 conductance (Fig. 3B), membrane capacitance (Fig. 3C), and cytoplasm conductivity 372 (Fig. 3D). For each 3DEP spectrum fitting experiment, the mean value of the cell radius 373 was measured using the sample population. Shown in Fig. 3B, the cell membrane 374 conductance initially increased and then decreased with continued increases in 375 glutaraldehyde concentrations, and finally plateaued above a glutaraldehyde 376 concentration greater than 0.5 w/v%. We observe an inverse trend with membrane 377 capacitance above concentrations of 0.05 w/v%, as capacitance steadily increased 378 with glutaraldehyde concentration from 19 mF/m2 to 41 mF/m2 between 0.1 w/v% and 379 2.5 w/v% and was observed to stabilize at concentrations above 0.5 w/v% (Fig. 3C). 380 Finally, we observed an initial drop in cytoplasm conductivity at a low 0.05 w/v% 381 crosslinking concentration followed by a steady increase with increasing crosslinking 382 concentrations (Fig. 3D). As observed from the measured spectra, the use of the high 383 permittivity DEP buffer enabled both the low and high frequency COFs and a 384 significant portion of the high frequency DEP spectrum to be measured within the 45 385 MHz bandwidth of the 3DEP. These experiments demonstrate that a glutaraldehyde 386 crosslinking concentration of 2.5 w/v% is likely to fully crosslink the RBC, as indicated 387 by the observation that cell membrane electrical properties stabilize and plateau with 388 crosslinking concentrations between 1.0 and 2.5 w/v%. These results also suggest 389 these higher concentrations serves to prevent membrane ion leakage during DEP 390 analysis as indicated by the cytoplasm conductivity at 2.5 w/v% approaching to within 391 5% of the original non-crosslinked RBC values. It is therefore anticipated that a 392 crosslinking concentration of 2.5 w/v% provides the most optimum crosslinking 393 concentration for our DEP analysis. 394 395 4.2 DEP Analysis of Stored RBCs 396 RBCs were stored and subsequently washed and crosslinked according to the 397 workflow presented in Fig. 2. The DEP spectrum was measured after 5, 10, 15 and 20 398 days of refrigeration. Select spectra obtained for storage times of 5 and 15 days across 399 five glutaraldehyde concentrations are plotted in Fig. 4A and 4B, respectively, to 400 illustrate the 3DEP data collection workflow. As shown, the spectra are dependent on 401 both the storage time and the glutaraldehyde concentration. In order to validate the 402 3DEP method with that of the DEP quadrupole method that was used in our previous 403 bovine RBC work, we measured the high frequency COF for both fresh and stored 404 RBCs at storage time points of 5, 10 and 15 days for all crosslinking concentrations. 405 DEP motion within the quadrupole was observed under brightfield microscopy. The 406 AC field frequency for when the first RBC “crossed over” from the quadrupole high 407 field region to the low field region, and later the frequency where the last cell crossed 408 over, was optically determined and measured. The average of these two values was 409 plotted as the high frequency COF and compared with the COF measured by the 410 3DEP. As shown in Fig. 5 for fresh RBCs (day 0), and cells stored for 5 and 15 days, 411 the 3DEP COFs agree well with the values obtained by the 2D quadrupole across all 412 measured glutaraldehyde concentrations. As such, we believe the 3DEP instrument is 413 a more complete DEP measurement method when compared to point-wise COF 414 measurements obtained with 2D electrode arrays. 415 416 Figure 4. 3DEP spectra and electrical properties of stored human RBCs. (A) 417 3DEP spectrum for RBCs stored 5 days. (B) 3DEP spectrum for RBCs stored 15 418 days. (C) RBC cytoplasm conductivity, (D) RBC membrane conductance and (E) 419 RBC membrane capacitance showed statistical significance from a ordinary 420 two-way ANOVA analysis with p < 0.0001 for both the row and column 421 interactions; average +/- SEM, and n=10 for 0 w/v% otherwise n=5. 422 423 The 3DEP was then utilized to deduce the RBC cytoplasm conductivity, 424 membrane conductance and membrane capacitance across the varying 425 glutaraldehyde concentrations and storage times. As shown in Fig. 4C-D, and as 426 observed in previous work with bovine RBCs, no discernable different in RBC electrical 427 properties was observed with human RBC storage age at a 0 w/v% glutaraldehyde 428 concentration. However, the discernment between storage-induced RBC cytoplasm 429 conductivity and membrane conductance increased with glutaraldehyde concentration 430 (Fig. 4C-D). As observed, the optimum crosslinking concentration for human RBC 431 DEP resolution occurs at a glutaraldehyde concentration between 1.0 and 2.5 w/v% 432 and is very similar to what was observed in previous crosslinking DEP experiments 433 with bovine RBCs [15,21]. 434 435 Figure 5. Human RBC Crossover comparison between 3DEP spectrums and 2D 436 quadrupole array for RBCs at day 1, 5 and 15 of storage. 437 438 439 The RBC cytoplasm conductivity and membrane conductance exhibited 440 observable trends with increasing storage time at a glutaraldehyde concentration of 441 2.5 w/v% (Fig. 4C-E). Here, the cytoplasm conductivity was observed to steadily 442 decrease from an initial value of 0.036 S/m to 0.026, 0.023, 0.011, and 0.012 S/m with 443 increasing storage times of 5, 10, 15 and 20 days, respectively. Conversely, the 444 membrane conductance was observed to increase with increasing storage time, 445 increasing from an initial value of 500 S/m2 to 3000, 5000, 10,000, 12,000 S/m2, at 446 storage times of 5, 10, 15, and 20 days, respectively. Interestingly, the membrane 447 conductance appears to have an inverse relationship with the cytoplasm conductivity, 448 as shown in Fig 6. While this trend presented is from a single human donor, this 449 inverse relationship between cytoplasm conductivity and membrane conductance was 450 been consistently observed with 9 total blood donors that were studied using this DEP 451 method. However, a more detailed investigation into the DEP variability in electrical 452 properties and COF across the human donors will be the subject of a future study. The 453 inverse correlation between decreasing conductivity and increasing conductance has 454 also been observed in other DEP work. In Henslee et al. for example, the authors 455 showed an anti-phase relationship between these two electrical parameters [5]. We 456 speculate here that this inverse trend is driven by the single-shelled relationship 457 between the cell cytoplasm conductivity and the surrounding cell membrane. In the 458 absence of cell hemolysis, a main driver for ion transport and a decrease in RBC 459 cytoplasm conductivity during refrigerated storage is diffusive ion flux from the 460 cytoplasm and across the cell membrane through the physical migration of ions across 461 this domain. Ionic concentration within the cell membrane would therefore increase 462 during this process and subsequently lead to an increase in membrane conductance. 463 Further storage experiments using both dynamic DEP measurements and inductively 464 coupled plasma mass spectrometry (ICP-MS) are needed to better understand the 465 relationship between RBC DEP and ionic composition overtime and will be the subject 466 of a future study. While a clear trend was observed between cytoplasm conductivity 467 and membrane conductance with increasing storage time, no storage time 468 dependence was observed with membrane capacitance for any experimental 469 glutaraldehyde crosslinking concentration. It is worth noting that our 3DEP measured 470 cytoplasm conductivity values (~10 – 40 mS/cm) are lower than values reported 471 elsewhere (~100 mS) for human RBCs [5]. While our results demonstrate that 472 glutaraldehyde is capable of minimizing dynamic ion leakage across RBCs, the cell 473 handling conditions may also contribute to the electrical state of the RBCs. These 474 conditions were held constant when RBCs were washed and crosslinked for this work, 475 however, an understanding of how varying centrifuge conditions and incubation 476 temperatures ultimately influence RBC electrical properties is likely warranted to better 477 understand this observed electrical. Previous reported values, for example, were 478 acquired after RBCs had been incubated at 35 – 37 oC for 48 h [5], while this DEP 479 work maintained cell samples at a continuous refrigerated temperature of 4 oC. We 480 therefore believe that cell handling and incubation conditions can strongly influence 481 the RBCs electrical state and can give rise to measured differences such as what is 482 observed here. 483 484 Figure 6. RBC cytoplasm conductivity decreases with increasing membrane 485 conductance with increasing storage time. 486 487 The 3DEP was able to resolve the electrical property changes in single-donor 488 human RBCs with increasing storage time using a chemical crosslinking method. The 489 cell crosslinking reaction served to prevent ionic leakage across the cell membrane 490 during DEP analysis and therefore the cellular ionic composition more accurately 491 represented that which existed in the original storage buffer prior to crosslinking. It is 492 well known that RBC ion channels are sensitive to changes in temperature and human 493 red blood cells leak potassium ions during storage [12]. Ion leakage such as this would 494 likely lead to a reduction in the cytoplasm conductivity and an increase in ionic species 495 within the cell membrane. If, however, glutaraldehyde was not utilized in this work, the 496 ion depleted cytoplasm and ion accumulated membrane would dynamically respond 497 to a resuspension in the low conductivity DEP buffer and dynamically reach a new 498 ionic equilibrium with their surroundings. Such communication with the surrounding 499 cell buffer would ultimately lead to an internal cytoplasm conductivity that is not 500 representative of the leakage that occurred during the storage process. 501 Glutaraldehyde is a well understood cell fixation agent which crosslinks the 502 aminated protein groups of the RBC [24]. These results suggest that the 503 polymerization of the cell membrane significantly reduces the rate of ion leakage 504 across the cell membrane during DEP analysis if a large enough crosslinking 505 concentration is utilized. In this work, DEP resolution occurred at crosslinking 506 concentrations above 1.0 w/v%. Below this concentration a trend emerged 507 dynamically and became discernable in a manner that was dependent of the age of 508 the RBC sample. In particular, the initial observable trend for the cytoplasm 509 conductivity was strongly influenced by RBC storage time. No age-related trend was 510 observable in RBCs without crosslinking, but a measurable difference in cytoplasm 511 conductivity was first observed with the youngest RBC populations. Shown in Fig. 4C, 512 a crosslinking concentration of 0 and 0.05 w/v% showed no observable change in 513 conductivity with storage time. However, at a concentration of 0.1 w/v% a difference 514 in cytoplasm conductivity was observable for a single sample: fresh day 1 cells. 515 Interestingly, the sample storage age in which a measurable difference was first 516 observed increased with increasing crosslinking concentration. At a concentration of 517 0.5 w/v%, for example, day 0 and day 5 RBCs exhibited measurable differences in 518 cytoplasm conductivity, while the remaining cell sample storage times showed no 519 discernable electrical differences at this crosslinking concentration. Further increases 520 in crosslinking concentration (>1.0 w/v%) then produced observable differences in the 521 cytoplasm conductivity across the entire experimental time course. Given that the 522 RBCs shed membrane proteins during storage, fewer available crosslinking sites are 523 available as storage age increases. We observe here that a smaller crosslinking 524 concentration is required to influence ionic leakage in “fresh” cells than what is 525 required for cells stored at the longer 5-, 10- and 20-day timepoints. This interplay 526 between storage age and minimum crosslinking concentration for preventing cell 527 leakage is therefore likely due to the age-dependent availability of RBC aminated 528 crosslinking sites. While clear statistically significant differences in RBC cytoplasm 529 conductivity and membrane conductance are observable over a refrigeration period of 530 20 days at crosslinking concentrations > 1.0 w/v%, no such difference was detected 531 in the membrane capacitance over this period for any concentration. As such, it is 532 likely refrigerated RBC storage over a period of 20 days did not significantly alter the 533 membrane permittivity, thickness or cell surface area to the extent to which a change 534 in membrane capacitance could be detected by the 3DEP. 535 5 Concluding remarks 536 In this work, we have presented a glutaraldehyde chemical crosslinking method 537 for the detection of human RBC electrical properties by DEP. Single donor type O- 538 human RBCs were stored in a blood bank refrigerator over a period of 20 days. Blood 539 samples were withdrawn from refrigerated storage at varying time points 0, 5, 10, 15, 540 and 20 days and chemically crosslinked in varying concentrations of glutaraldehyde 541 ranging from 0 w/v% - 2.5 w/v%. Crosslinked cells were then suspended in a high 542 permittivity 0.8M AHA buffer in order to increase the buffer permittivity and 543 subsequently reduce the frequency bandwidth required to obtain a full DEP spectrum. 544 Using this chemical crosslinking high permittivity buffer system, we performed human 545 RBC DEP analysis using a commercial 3DEP instrument. The high frequency COF 546 obtained using the 3DEP method was compared and shown to agree well with the 547 COF values obtained using a conventional 2D quadrupolar electrode array. DEP 548 spectra from 3DEP experiments were measured and fitted using nonlinear regression 549 to a spherical single-shell polarization model in order to determine the RBC cytoplasm 550 conductivity, membrane conductance and membrane capacitance over a period of 20 551 days of refrigerated storage. We show that at chemical crosslinking concentrations 552 above 1.0 w/v%, 3DEP analysis yielded discernable differences in cell electrical 553 properties with increasing storage time. In particular, the RBC cytoplasm conductivity 554 was observed to increase while the membrane conductance decreased with 555 refrigerated storage time. No trend was observed in RBC membrane capacitance with 556 increasing storage time. Further, no RBC electrical differences were resolvable by 557 DEP without glutaraldehyde crosslinking. The chemical crosslinking approach serves 558 to eliminate ionic diffusion across the cell membrane when cells are resuspended in 559 DEP buffer for analysis. Therefore, this chemical crosslinking method enables the 560 discernment of electrical property changes during storage and prevents cells from 561 reestablishing ionic equilibrium with the low conductivity DEP buffer. This assay can 562 successfully detect differences in blood storage, or starvation age, which is currently 563 challenging to perform using patch clamp or single cell electrophysiology techniques. 564 Interestingly, an inverse relationship between cytoplasm conductivity and membrane 565 conductance is observed. This work offers a robust method for characterizing the 566 quality of blood electrically and has applications in blood transfusion storage logistics 567 and for detecting age related electrical property changes in blood samples from 568 endurance athletes which have undergone illegal autologous blood transfusions. 569 Future efforts will focus on better understanding the species involved with ionic 570 leakage and understanding how these measurements vary across human donors. 571 Acknowledgements 572 The authors acknowledge funding and support from the Partnership for Clean 573 Competition (PCC) and the World Anti-Doping Agency (WADA). 574 Conflict of interest 575 The authors declare no financial interest. 576 Data Availability Statement 577 The data that support the findings of this study are available from the corresponding 578 author upon reasonable request. 579 6