Keywords: sickle cell disease, cell sickling, electrical impedance, microfluidics, cyclic hypoxia, biomarker, biosensor

Sample Preparation.

Five blood samples (ss1, ss2, ss3, ss4, and ss5) from individuals with SCD were collected following Institutional Review Board (IRB) approvals from Florida Atlantic University and the University of Miami. A normal blood sample (aa) was obtained from a local blood bank and used as a control in this study. All samples were stored at 4 °C and tested within 2 weeks of collection. Blood samples were washed twice with phosphate-buffered saline (PBS) at the speed of 2000 rpm at room temperature for 2 min. The hematocrit of each tested sample was 0.6% by resuspending 6μL red blood cell pellet into 1 mL PBS.

Microfluidic Chip for Impedance Measurement.

The microfluidic chip consists of a PDMS double-layer microchannel and interdigitated electrode array (IEA) that is printed on a glass substrate (Figure 1). The upper gas channel is 1500μm wide and 75μm deep and the lower cell channel is 500μm wide and 150μm deep. A 150-μm-thick PDMS film is used to separate the gas channel from the cell channel, allowing gas molecules to permeate through easily but preventing cells from drying out. Four openings, with 1.5-mm diameters, are created, allowing separate gas and medium exchanges using standard 0.02 in. ID/0.06 in. OD microbore tubing. The IEA consists of 16 pairs of fingers with 20μm bands and 20μm gaps. Each finger has a 100-nm-thick Au layer atop a 10-nm-thick Ti layer that are deposited on the 700-μm-thick glass substrate using E-beam vaporization and patterned by standard photolithography techniques. The PDMS microchannel was obtained by casting a microfabricated SU-8/Si master with a degassed PDMS mixture (10:1 of base to agent ratio) and curing under 70 °C for 2 h. A permanent bonding was accomplished between the microchannel and the IEA chip by air plasma treatment. Two-inch-long, 22-gauge copper-based wires were soldered to the IEA pads and connected to the impedance spectroscope. The cell channel was washed with 70% ethanol followed by deionized (DI) water, then primed with PBS. The cell suspension was then injected gently into the liquid channel of the device using a 1 mL syringe. Cells were allowed to sediment to the bottom of the channel before impedance measurement was performed.

Cyclic Hypoxia Control.

Oxygen concentration in the cell channel was controlled by the oxygen concentration in the supplied gas mixture to the gas channel. Cyclic hypoxia was created by switching between an oxygen-rich gas mixture (17.5% oxygen, 5% carbon dioxide with the balance of nitrogen) and an oxygen-poor gas mixture (5% carbon dioxide with the balance of nitrogen) using a programmable 3-way valve (LabSmith., CA, USA) (Figure 1). The programmable 3-way valve was switched between the oxygen-rich mixture and the oxygen-poor mixture at a time interval of 60 s. The cyclic hypoxia lasted for 8 min in each testing. To verify the effectiveness of the gas control from the gas channel and calibration of the oxygen concentration in the cell channel, a fluorescence dye, tris (2,20-bipyridyl dichlororuthenium) hexahydrate (Ru(bpy)) (Sigma-Aldrich) was used. Its fluorescence intensity is inversely proportional to the dissolved oxygen concentration.³⁵ The cell channel was filled with DI water containing Ru(bpy). Gray values (red curve) converted from the fluorescence intensity of Ru(bpy) were plotted as a function of time (Figure 2). The corresponding oxygen concentration in the gas channel (blue curve) was shown as a reference. The delay in the transition from one oxygen concentration level to the other in the cell channel (gray value) was diffusion limited, depending on the thickness of PDMS film. The current setting resulted in a hypoxia (O₂% near zero) duration of approximately 44.5 s and a normoxia (O₂% near 17.5%) duration of about 42.5 s.

Electrical Impedance Measurement.

Electrical impedance measurements were taken at 100 kHz and 500 kHz, which provided favorable conditions for the measurements of both cellular membrane and interior with current setup, as well as to avoid the adverse effects from electrolysis at lower frequencies or parasitic inductance effects at higher frequencies. Feasibility of the selected frequencies was verified by numerical simulation of the electrical field magnitude and electric current streamlines in the testing zone using COMSOL Multiphysics 5.2 (COMSOL, Inc., Burlington, MA). The numerical model consisted of a pair of IEA fingers where a cell suspending in the proximity, similar to that described in a previous publication.³⁶ As shown in theFigure 1b, at 100 kHz, near-cell current streamlines are blocked by the capacitive cell membrane and take their paths through the medium, suggesting that the impedance signal will be dominated by the shape and size of the cell; at 500 kHz, most near-cell current streamlines pass through cell membrane and the electric field strength of cell interior increases, suggesting the role of intracellular material in the impedance signal increases. In the experiments, sinusoidal voltages of 100 mV_pk at frequencies of 100 kHz and 500 kHz were applied simultaneously to the IEA chip. The collected electrical current signals were converted to voltage signals using a current amplifier (HF2TA, Zurich Instrument, AG, Switzerland) and then fed back to the impedance spectroscope (HF2IS, Zurich Instrument, AG, Switzerland). A sampling rate of 57 points of data per second was used to capture the variations in the cell suspension in response to gas exchange through the valve. This allows a measurement, in real time, for the electrical impedance of the cell suspension. The raw data recorded by the impedance spectroscope was in the format of complex current, and it was converted into complex impedance based on Ohm’s Law.²⁹ To remove the effect of residuals between the instrument calibration plane and the specimen under test, the calculated impedance value was corrected by the standard open/ short/load compensation method.³⁷

Cell Morphology Analysis.

The IEA chip contains electrode-free viewing strips to facilitate observation using an inverted microscope (Olympus IX-81, Olympus America, PA, USA). Microscopic videos were recorded through a digital camera (The imaging Source, NC, USA) simultaneously with the impedance measurement. Cell morphological change, quantified by the overall area blocked by the cells in the viewing strips, was analyzed from the time lapse video using ImageJ.³⁸ For each specimen, the relative cell area was normalized to that of the initial frame, and averaged between 6 and 10 microscopy views. Cell morphological change was then mapped to the impedance data of the cell suspension for each individual sample in time domain. The percentage of cells that exhibited characteristics of morphological sickling upon hypoxia was defined as sickled fraction, following a previous publication.¹³ The sickled fraction of each sample was expressed as mean ± SD, averaged from 8 microscopy views.

Electrical Impedance as a Marker of Cell Sickling and Unsickling Events.

The measurement starts from normoxia and the changes of both components of the impedance signals, resistance,R, and reactance,X, relative to the start pointsR₀ andX₀ were calculated separately for each blood sample

Figure 3 shows the calculated data at both measured frequencies. Both resistance and reactance parts have high sensitivities in response to variations in the oxygen level in the cell channel. They follow closely the profile of the oxygen level. When the valve shifts its position for gas exchange, it exerts a dynamic load to the cell suspension, which can be identified by a small brief peak in the recorded impedance signal. Subtle variations in the impedance signals were observed for medium without cells (m, black curves) and normal cell suspension (aa, pink curves) in response to cyclic hypoxia treatment, indicating the content of dissolved oxygen does not lead to a detectable signal as being due to the cell sickling–unsickling events by electrical impedance. Remarkable changes in electrical impedance were observed in all the sickle cell samples during the hypoxia-normoxia cycles. In response to deoxygenation, electrical resistance of sickle cell suspension decreases, and reactance increases. In response to reoxygenation, the two measures change oppositely. At 500 kHz, the resistance is slightly noisier than that of 100 kHz. As frequency increases, more current passes through the cell membrane into the cell interior. Each sickle cell sample showed a slightly different response among the consecutive hypoxia–normoxia cycles.

The relative change in the impedance of cell suspension between normoxia and hypoxia was quantified separately during each cycle, by calculating the relative difference between the resistanceR_r or reactanceX_r values at the two transition points where the valve switched between hypoxia and normoxia (Figure 4a)

The subscripts “hypo” and “norm” denote hypoxia and normoxia, respectively. This resulted in four pairs of data for each sample at a specific frequency. The cycle-averaged values of resistance/reactance relative changes for the normal red blood sample are negligible at 100 kHz with 0.04%/0.08% and at 500 kHz with 0.025%/0.044%. The corresponding values of the cycle-averaged impedance relative change for ss1 are greatest with changes of 3.61%/0.71% at 100 kHz and 1.10%/1.85% at 500 kHz. These values are more than 1 order of magnitude higher than those of sample aa at both frequencies.

Significant variation exists among the tested sickle blood samples as quantified by the relative changes in the magnitudes ofR_r andX_r. Both intersample and intercycle variations can be observed (Figure 4). Even though the intercyclic variations are obvious at both frequencies, all the tested samples can be successfully differentiated with a minimum overlap using a combination ofR_r andX_r at both frequencies. Permanent cell damage due to exposure to electrical field³⁹ and repeated sickling–unsickling process⁸ was not observed during the four cycles (8 min in total). This allowed us to probe the processes of HbS polymerization–depolymerization and the consequent shape change using impedance without complications from other factors. The intercycle variations can be detected by the relative reactanceX_r at 100 kHz and the relative resistanceR_r at 500 kHz. Such variation may be caused by the uncertainty in the orientation and structure of the formed HbS fibers⁴⁰ in each hypoxia session, delay time in the HbS polymerization–depolymerization, as well as the associated variations in cell shapes. The intersample variations among the five sickle cell samples showed a consistent trend inR_r between the two frequencies, as ss1 > ss2 > ss3 > ss5 > ss4, while no such trend was identified inX_r. This suggests that the relative resistance is a more robust assessment for intersample variations than the relative reactance, regardless of the electrical frequency.

Cell Shape Change Associated with Sickling–Unsickling Process.

Both extracellular and intracellular parameters associated with the process of cell sickling can influence the electrical impedance of cell suspension. Under normoxia, the majority of sickle cells exhibited a biconcave disk shape similar to normal cells (Figure 5a). Under hypoxia, sickle cells became darker and the majority of them exhibited obvious shape change, specifically, the sickled friction of each sample is at least 91.6 ± 3.6% (ss3) (Table S-1). The relative cell area,A, corresponding resistance,R, and absolute value of reactance, |X|, were normalized separately for each sickle cell sample

whereP stands forA,R, or|X|. Normalized data per sample during the first hypoxia–normoxia cycle were compared at each frequency (Figure 5b, seeFigure S-1 for the other three cycles). The gray shading represents the hypoxia session during which cells experience the sickling process and the bright area represents the normoxia session where cells go through the unsickling process. It was observed experimentally that the rate of cell unsickling was higher than the rate of cell sickling. This was reflected in the cell area, which changes slowly in response to deoxygenation but abruptly in response to reoxygenation.

There is an obvious delay before the area starts to decrease after the application of hypoxia, especially for ss1, ss2, ss3, and ss5. However, the change of impedance begins almost immediately after the start of hypoxia. Furthermore, cell area decreases quickly compared to the rate of impedance change once it gets started, so that after the impedance change lags far behind the cell area change at both frequencies. This suggests intracellular polymerization begins before and continues after cell morphological sickling. In ss2, ss3, and ss5, recovery of impedance lags far behind the cell area recovery at both frequencies; suggesting the rate of intracellular HbS fiber dissolution is much lower than that of membrane recovery. In contrast, no obvious delays were found in ss1. These findings indicate that the five sickle cell samples in the current study have significantly different kinetics of morphological cell sickling and intracellular polymerization and depolymerization. In addition, there is a delay from resistance to reactance in both the sickling and unsickling processes. This is probably due to the fact that resistance is essentially a friction while reactance is an inertia against the motion of electrons; therefore, resistance reacts faster than reactance.⁴¹

Correlation between Electrical Impedance and Hematological Parameters.

Normalized impedance of all the five samples during the first hypoxia session were compared, allowing us to identify intersample variations (Figure 6a; seeFigure S-2 for the other three cycles). The difference in sickling behavior is substantial. Both normalized resistance and reactance of ss4 decrease most rapidly within the first 30 s and tend to flatten afterward, while those of ss2 decrease at a relatively lower speed along the deoxygenation process. To quantify these variations, we introduced a new parameter, decreasing rate of resistanceν, which was defined as the inverse of the area under the$\overline{R}-t$ curve within a hypoxia session using the trapezoidal method

wheren is the index of each measured data point. By plotting the value ofν against %HbF, a moderate linear correlation was found slowly decreasing the rate of resistance corresponding to higher concentration of HbF at 500 kHz (Figure 6b2). At 100 kHz (Figure 6b1), the resistance decreases at a lower rate ofν ≈ 0.045 when %HbF is higher than 17.4%, in contrast to the significantly increased rate ofν ≈ 0.059 when %HbF is as low as 14.2%. These correlations agree with the previous report⁴² that increased levels of HbF retard the polymerization of HbS. Resistance relative changeR_r was plotted against the %HbS of individual samples. We noticed that there is a strong linear relationship betweenR_r and %HbS at both 100 kHz (Figure 6b3) and 500 kHz (Figure 6b4); the sample with higher concentration of HbS showed a greater change in impedance resistance. These results indicate that electrical impedance can potentially be used as a supplementary detection method for hematological assay in SCD. HbS% and HbF% are important parameters used clinically for assessment of disease severity⁶ and SCD associated complications,⁴³ which are measured from cell lysate by electrophoresis technique. As electrical impedance is a label-free, sensitive marker of cell sickling and unsickling processes, establishment of its correlations with HbS % and HbF% may offer a new opportunity for rapid and label-free assessment of SCD in conjunction with the clinical measures.

Modeling of Electrical Impedance.

Equivalent circuit modeling was adopted to describe the cell suspension system and identify the contribution of each component to the measured impedance change. In general, an equivalent electrical circuit model analogous to the physical model is utilized to describe the electrical properties of cell suspension, including the electrical double layer as a capacitor, the conductive PBS solution as a resistor, and each individual cell as a series of capacitors and resistors (Figure S-3a). Considering the significant variations in cell size and shape of sickle cells, a simplified circuit model was used to interpolate the overall electrical impedance of cell suspension in response to changes in oxygen tension (Figure S-3b). In this model, all the cells were simplified as an entity consisting of a resistor,R_c, in series with a capacitor,C_c. The surrounding medium was simplified as a single resistor,R_m. Similarly, the electrical double layer from all the IEA fingers has been modeled as a single capacitor,C_edl. Specifically, the total complex impedance of the circuit becomes

Correspondingly, the total resistance and total reactance are

whereω is the angular frequency. Impedance valuesR_norm,X_norm,R_hypo, andX_hypo at 100 kHz and 500 kHz were used to fit the above circuit model.

Three cell suspension parameters, medium resistance,R_m, cell capacitance,C_c, and cell resistance,R_c, that are associated with cell sickling/unsickling processes, as well as electrical double layer capacitanceC_edl were listed inTable 1. Values ofR_m andC_c decreased, andR_c increased in response to hypoxia for all five samples, suggesting the reasonable accuracy of the equivalent circuit model in the description of sickle cells in suspension. The sickling process of cells yield more space for electrical current going through the conductive medium, resulting in a decreasedR_m. Along with the HbS polymerization, individual sickle cells become dehydrated and more solid; thus, the suspended sickle cells become more resistive and less capacitive. These results demonstrated that a multifrequency electrical impedance spectroscopy is useful in measurements of extra- and intracellular compartments during the cell sickling and unsickling processes.

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Supplementary Materials