Medical & Biological Engineering & Computing (2021) 59:1709–1721 https://doi.org/10.1007/s11517-021-02382-2 ORIGINAL ARTICLE Gradient‑based impedance synthesis for breast and lung cancer cell screening deploying planar and nano‑structured electrodes Muhammad Awais Aslam1 · Kashif Riaz1 · Muhammad Mubasher Saleem2 Received: 3 December 2019 / Accepted: 8 May 2021 / Published online: 8 July 2021 © International Federation for Medical and Biological Engineering 2021 Abstract World Health Organization articulated 9.8 million casualties globally in 2018 due to cancer. Cancer, as the world’s second most fatal disease, can be recuperated well if diagnosed at an early stage. In this work, a gradient-based impedance synthesis of normal and cancerous cells of breast and lungs, is demonstrated numerically for early-stage cancer detection. Low-voltage single-cell level examination is employed for indomitable diagnosis. MCF-7 and MCF-10A are utilized as breast cancer and breast normal cells, respectively; likewise, SK-MES and NL-20 are utilized as lung cancer and lung normal cell. Preexamination numerical setup validity ensured with multiple test regimes. Micro-scaled planar and nano-structured electrodes are employed individually to witness the effect of the electrode’s structure during electrical impedance examination of cancer and non-cancer cell. Frequency range, at which differential impedance effect is found detectable, for breast and lung cancer cell pairs is determined to be 1­ 07 Hz and 1­ 08 Hz, respectively. By surpassing the conventional impedance spectroscopy with tedious data fitting formalities, the gradient synthesis technique for cancer detection is introduced. The gradient synthesis for cancer detection is found independent of electrode shape effect. Gradient for breast cancer cell is found to be 2 times greater than the normal breast cell while for lung cancer cell it is found to be 1.5 times greater than the normal lung cell. Our results suggest that as the frequency of applied electrical stimulus increases, impedance of cancerous cell falls at the rate almost double than its counterpart normal cell. This work provides a theoretical basis for further experimental exploration of gradient-based impedance synthesis in cancer therapy and serves as a design tool for performance optimization. Keywords Breast cancer · Gradient impedance synthesis · Lung cancer · Nanospike electrodes · Single-cell analysis (SCA) 1 Introduction Improvement in diagnostics results an increase in life expectancy, as more accessible and accurate way of blight detection leads towards rescuing millions of people all around the world. Carcinogenesis is one of the exponentially growing disorder as one out of 5 men and one out of 6 women is diagnosed with cancer globally in 2018, reported by World Health Organization (WHO). Early-stage cancer prognosis can be a way forward to its successful annihilation. * Kashif Riaz kashif.riaz@itu.edu.pk 1 Electrical Engineering Department, Information Technology University, Lahore 54000, Pakistan 2 Department of Mechatronics Engineering, National University of Science and Technology, Islamabad 44000, Pakistan Most of the current cancer detection techniques are invasive, vacillating, non-reproducible, expensive, with labelled detection, and have a very high false detection rate with values up to 20–50% [1]. So non-invasive, explicit, less expensive, reproducible, and label-free cancer detection techniques are present day requirements [2–4]. Single-cell analysis (SCA) has proved to be a very efficient way to detect cancer. Instead of cellular assays, which are accompanied with a large number of cells under examination and take the average values of results at the end, single-cell-based examinations are more realistic and accurate against cellular malfunctioning which can efficiently be interpreted afterwards [5]. This is because when a cell is being disseminated through blight, i.e., viruses, bacteria, or infection, its morphology changes and its electrical, chemical, biological, and adhesive properties also change [6]. If one could manage to interpret electrical changes in a cell like its impedance, capacitance, or conductance which are directly related to the cell health, clear biomarkers could be 13 Vol.:(0123456789) 1710 obtained for diagnosis in comparison to other delayed and inexplicit biological detection techniques [7]. Electrical impedance spectroscopy (EIS) of single cell gives a detail insight into the cell, i.e., one can interpret cellular morphological, physiological, and pathological changes inside the cell since the change in the cell health results in corresponding change in impedance [8]. On the other side, the effect of these electrophysiological variations takes time to become prominent symptoms or detectable at tissue or organ levels [9]. Moreover, the permittivity and conductivity variation with respect to frequency are not identical at tissue level and cell level which ultimately results in contrast behavior in their impedance results [10]. Moreover, multiple cellular assays are more prone towards wrong or less accurate detection [11]. Thus, by single-cell impedance analysis, cancer can be diagnosed at its incubation period. Electrode shape is an important constraint in the impedance measurement of a single cell as impedance results are different using different electrode shapes [12, 13]. In singlecell applications, effect of micro planar, circular, and rectangular electrodes along with electrodes with nano-protrusion on cell has been discussed in detail in [12, 14, 15]. The potential distribution across the cell is found best and almost equal in the case of planar and circular electrodes than the other shapes mentioned. This uniform potential distribution across the cell leads to a better penetration of current into the cell and hence accurate impedance measurements. The nano-structured electrodes have multiple other advantages over conventional micro/macro electrodes as they do not require surface functionalization to effectively capture the cell in between, which results in economy of time and less parasitic noises [16–18]. The major advantage of nanospike type electrodes, presented in this paper, is their high current densities so that one can operate them at really low voltage to get desired results with maximum cell viabilities. This makes nanospike electrodes economical and power efficient as well [14, 18]. For SCA, the application of high voltages may lead to several chemical reactions in cell solution which lead to sample contamination, bubble production, and morphological and chemical changes in cell and sometimes cell lysis [16, 19–22]. Moreover, most of such high-voltage setups are not portable. In comparison to high-voltage-based cell analysis, low-voltage methods of anomaly detection in cells are more flexible and highly reproducible since the impact of voltage on cell is low and cell can release itself to the original formation after the removal of voltages which allows to perform multiple experiments on the same cell. Kesse et al. [23] demonstrated impedance spectroscopy by culturing cells on electrodes and observed the impedance change as the cell grows. The sensing arrangement was used for the detection of cancer but it had high response time due to the attachment process. Cho et al. [17] performed an impedance analysis on normal and abnormal red blood cells 13 Medical & Biological Engineering & Computing (2021) 59:1709–1721 and reported differences on the basis of impedance magnitude and phase variations. In these two studies, no equivalent circuit model for impedance data fitting is reported which was a passive approach. Jang et al. [24] introduced a microfluidic setup for HeLa cell capture and impedance spectroscopy. In [24], a simple equivalent circuit model, close to experimental setup of impedance spectroscopy, for the impedance data fitting is presented and variation in cell impedance ­Zcell is used as an indication parameter for cancer characterization. Wang et al. [25] performed an impedance analysis on the human cervical cancer cell lines for frequency range up to 100 kHz, and introduced empirical relation with result validation up to 0.6 V only, as discrepancies in impedance results were observed at 1 V. A simplistic equivalent circuit is also used to fit data and for the calculation of overall impedance magnitude and phase. Ren et al. [11] performed an impedance spectroscopy to track single-cell properties. A mesh of resistor–capacitor-based equivalent circuit is introduced to make a database of cell properties after fitting. Membrane capacitance is utilized as one of the identification marks along with three other nonelectrical parameters, i.e., cell adhesion, cell diameter, and cell-electrode distance. Electrochemical impedance spectroscopy is reported by Siddiqui et al. [13] for biosensing applications. Detailed investigation is done for the electrode shape effect on impedance spectrum. The impedance data is fitted in Randle’s model for parameter extraction and electron transfer resistance is used as a parameter of identification. Tabasi et al. [26] demonstrated impedance spectroscopy for the detection of human epidermal growth factor receptor 2 protein (HER2). Modified randle’s model is used for the data fitting and parameter extraction and charge transfer resistance parameter is reported as an identification mark. One of the major drawbacks of conventional impedance spectroscopies is to fit the impedance data after impedance spectroscopy in the electrical circuit configuration to get the identification mark [11, 12, 14, 25]. Data fitting is itself a tedious and vague formality to indicate biomarker. It is mostly non-redundant because it is not sure that impedance data will be fitted to already develop circuit model. Most of the times, one has to design the circuit independently according to impedance data trend. Though there can be many circuits to which same data can be fitted, but it requires physical equivalence to experimental setup to get the marker of identification. Moreover, impedance data may fit exactly to an electrical circuit but the circuit itself cannot be justified as an equivalent of experimental setup. This results in a trade-off between some parameter values, e.g., membrane capacitance and electrode surface to cell capacitance values, to get a better fitting circuit configuration. Thus, there are many variations including multiple circuit models, multiple possible parameters of identification, and parameter Medical & Biological Engineering & Computing (2021) 59:1709–1721 trade-off in conventional impedance spectroscopy that uses data fitting. In this paper, a gradient synthesis method is introduced which allows to clearly identify the differential behavior of cancer and non-cancer cell without data fitting. One must observe gradient behavior as impedance trends from midrange frequency to high-frequency range. It is because of change in dielectric properties of biological cell due to alpha and beta dispersion and at higher frequencies impedance decrease more rapidly due increase in ionic opening [14, 24, 25]. The proposed method is applicable on the impedance data of conventional impedance spectroscopies, implemented either using Nyquist or bode plots of impedance. In the present work, impedance analysis is performed on cancerous and non-cancerous cell pairs of two particular organs, i.e., the breast and lungs, for early cancer detection. The whole impedance analysis is done using two different types of electrodes, i.e., planar and nanospike, to investigate the electrode shape effect on impedance values of cancer and non-cancer cells. Moreover, a gradient synthesis technique is introduced for clear cancer characterization which does not require any extensive and inexplicit data fitting formalities. 2 Methodology Figure 1 shows the layout of impedance spectroscopy for cancer detection using gradient synthesis. Initially, a rectangular setup with a spherical cell enclosed in electrodes is created in a finite element modelling tool followed by testing of simulation setup for simulation validations. After the simulation validations, impedance examination is performed for breast normal and cancerous cell detection. Then, the 1711 electrode shape is changed from planar to nanospike and impedance examination for breast cancer cell pair is performed. After this, the same numerical analysis is performed on more blood-perfused organs, i.e., lung cancer and lung normal cell with planar and nanospike electrodes. After getting all impedance results, the next part is to deploy data synthesis. The proposed gradient detection technique results for normal and cancerous breast and lung cell pair with both types of electrodes are obtained. In gradient synthesis, the percentage impedance drop of all types of cell under examination is determined and gradients are calculated. This data synthesis is performed for range of frequency up to detectable frequency range. Details for detectable frequency range can be found in Section 4.1. After the completion of all these numerical examinations and synthesis, the impedance results are compared for detection (Fig. 1). 3 Simulation setup for impedance analysis Two simulation setups are considered for analysis; one is macro setup of 0.7 × 1 m rectangular setup with two planar electrodes of 0.7 m length having 0.3 m distance between them. This setup suggested here is for macro level examination. This setup is used for initial simulation validity test and for recording the electrode shape variation effects on electric potential distribution between the electrodes. After the initial simulation validity, the whole setup is shifted to microlevel for real-sized cell examination, circumscribed by electrodes, and remaining testing and cancer impedance spectroscopy examinations are carried out on this setup. Microlevel simulation setup is selected as we are intended to conduct single-cell level examination. This micro simulation setup consists of a Fig. 1 The layout of impedance spectroscopy for cancer detection using gradient synthesis 13 1712 27 × 31 µm rectangular setup with two planar electrodes (le) of 23 µm length. The average cell diameter (dav,c) used is 16 µm [18] and distance between electrodes (de) is 10 µm distance between the electrodes. The reason behind the selection of this microlevel dimension is that the cell should be truly encapsulated between electrodes and the effect of cell and electrode surface interaction can be analyzed. There are multiple physics modules for examination in commercially available FEM (finite element method) solvers, like electromagnetic, electric current, and streamline flow. The numerical simulation trials are performed in electric current physics. This physics interface solves a current conservation equation based on Ohm’s law using the scalar electric potential as the dependent variable. Stationary, frequency-domain, small-signal analysis, and time-domain modelling could be supported domains/solvers in Fig. 2 The results of simulation validation tests with and without cell encapsulated between the electrodes are shown. a Surface electric potential variation at 1 V for simulation validation, where σe and εr,e are conductivity and permittivity of PbS electrolyte. b 1-D electric potential distribution at 1 V according to electric potential theory E = -∇V (i.e., potential variation only in y coordinate position according to Poisson equation) which validates the simulation. c Surface plot of electric field distribution having normal breast cell enclosed in 13 Medical & Biological Engineering & Computing (2021) 59:1709–1721 all space dimensions. We used frequency domain for impedance examination (stationary domain was also employed in one of simulation validation test, Fig. 2b). The current conservation equation for electric potential employed during numerical examination is given as: ∇ ∙ J = Qj (1) J = 𝜎E + j𝜔D + Je (1a) E = −∇V (1b) where J is the current density, Qj is the charge, E is the electric field, D is the displacement field, Je is the electron current density, 𝜎 is the electrical conductivity, and V is planar electrodes, where σnb and εr,nb are conductivity and permittivity of normal breast cell, respectively. σe and εr,e are conductivity and permittivity of PbS electrolyte respectively and their values can be found in Table 1. d Electric field distribution between two electrodes, a normal cell enclosed, shows permittivity and conductivity difference of inner cell and outer electrolyte and shows the higher strength of electric field at the boundary of cell and electrodes Medical & Biological Engineering & Computing (2021) 59:1709–1721 the applied voltage. Gold is used as an electrode material and PBS (phosphorous buffer saline) as an electrolyte. Low voltages are employed to get desired results with maximum cell viabilities. Additionally, increasing the operation voltage reduces the magnitude of the cell impedance because a strong electric field may promote the exchange of ions between the cytoplasm and the isotonic solution. So, 0.1 V dc is chosen to minimize the effect of this signal during impedance measurements [24]. Voltages are applied for frequency sweep ­(105–109 Hz). Upper electrode is applied with potential and lower electrode is at ground potential. All simulations are performed in commercially available finite element method software. Cell dielectric parameters for lungs are taken from [27]. In [27], Egot et al. performed seven experiments to analyze permittivity and conductivity for the range of 200 MHz to 2 GHz. After fitting the data mean values of conductivity and permittivity for lung cell, the pair was retrieved. Similarly, for breast cell, parameters are taken from [28]. In [28], An et al. scanned the frequency range from 0.1 MHz to 0.1 GHz and calculated conductivity values. Permittivity assumed to be near water’s permittivity and assumed constant throughout the frequency range. The frequency range used in the current work is ­105 to ­109 Hz which is within the scanned range of the above discussed literature. In this frequency range, biological cell parameters (conductivity and permittivity) show less variations [29] which also authenticate the use of average approximation on cell parameters in this targeted range. That is why dielectric cell parameters used are assumed to be effective for our case study. Details about parameter selection can be found in [27, 28]. All the values of constants and average value of cell parameters used in the calculations and simulations are given in (Table 1). Table 1 Values of constants and average value of cell parameters used in normal and cancer cell models for impedance spectroscopy References [28] [28] [12, 25] [28] [30] [12, 25] [27] [27] [27] [27] [12, 18] [12] 1713 3.1 Simulation validity tests 3.1.1 Without cell inclusion Several tests are conducted to confirm the validity of simulation and physics applied according to the electrostatics and electric potential theory. First, the simulation is checked for the basic electric field equation, i.e., E = V/d. The distance between the electrodes varied to measure the normalized electric field and then the results are compared with the calculated ones. At an applied voltage of 1 V, the electric field values are obtained, through FEM (finite element method) simulations, with distances between electrodes of 5.5 µm, 13 µm, and 23 µm are 1.82 × ­105 V/m, 7.69 × ­104 V/m, and 4.34 × ­104 V/m respectively (supporting Fig. 1) which showed a close agreement with the analytically calculated values. Initially, the effect of corner-shaped planar electrodes on electric field shape is analyzed through FEM simulations. The results showed that the electric field shape at the corner is too distorted and very non-uniform (supporting Fig. 2a, b) which is undesired for biological experiments where a uniform or concentrated electric field is required. Moreover, corner-shaped planar electrodes may undergo degradation with frequency of utilization. Thus, the corner edges of electrodes are then replaced with round ends. The results show that with round edges, a uniform and streamlined electric field is obtained (Supporting Fig. 2c, d) along with limiting the degradation effect. The electric potential variation is checked as predicted by the electrical theory, i.e., in 2D the electric potential in the center between two electrodes changes only in the y coordinate position. The results obtained through FEM simulations are found to be in good agreement with the predicted theories in literature [25] (Fig. 2a, b). Parameters Description Values 𝜎 nb 𝜎 cb 𝜎e 𝜀0 𝜀r,nb 𝜀r,cb 𝜀r,e 𝜎 nL 𝜎 cL 𝜀r,nL 𝜀r,cL dav,c de le Conductivity of interior cell for MCF-10A Conductivity of interior cell for MCF-7 Conductivity of exterior cell medium Dielectric permittivity of free space Permittivity of interior cell for MCF-10A Permittivity of interior cell for MCF-7 Permittivity of exterior cell medium Conductivity of interior cell for NL-20 Conductivity of interior cell for SK-MES Permittivity of interior cell for NL-20 Permittivity of interior cell for SK-MES Average diameter of cell Distance between electrodes Length of electrodes 0.3 S/m 0.23 S/m 2 × ­10−5 S/m 8.85 × ­10−12 As/Vm 80 78.69 136 0.89 0.77 48.1 45.6 16 µm 10 µm 23 µm 13 1714 To select the electrolyte/extracellular material/cell culture, medium pure water, saline 0.9%, and PBS are tested numerically for their impedances at higher frequency. Saline 0.9% showed low impedance and nonlinear behavior at high frequency and water showed high impedance and nonlinear behavior with high frequency. PBS showed intermediate impedance values which are slightly influenced by higher frequencies and showed almost linear behavior (Fig. 3a). Thus, PBS is chosen as an electrolyte for further testing and impedance spectroscopy. On the other hand, as we have to deal with higher frequencies in biological manipulations and at higher frequencies, the morphology of biological setup gets affected (bubble production, evaporation, degradation) [18]. 0.9% saline solution is pure salt solution (having salt PH) which might hamper cell or denature the cell in biological manipulations [31]. On the other hand, PBS is a buffer saline in which disodium hydrogen phosphate is also added with sodium chloride and this buffer helps to maintain constant PH [25, 32]. Due to that, most of the cell do not undergo denaturing. As we know at high frequencies the current density will be higher (supporting Fig. 3). So, we prefer such electrolyte whose response to higher frequencies should be minimum so that if we note any abruption or change it will be assumed that it is not due to electrolyte and solemnly due biological matter under observation (in our case it is cell). This is the reason linear behavior of PBS (Fig. 3b) was taken into account as superior at higher frequencies. Medical & Biological Engineering & Computing (2021) 59:1709–1721 setup discussed in the last section, are performed before our actual cancer and non-cancer impedance examination. At first, electric field intensity variation is investigated in between the electrodes. Highest electric field is observed at the boundary of cell and electrodes (Fig. 2c) (greater rate of increase of intracellular electric field relative to extracellular) which advocate the conductivity and permittivity of both intracellular and extracellular space are not identical (Fig. 2d). To analyze the effect of increasing the frequency on current density, a frequency vs. current analysis is carried out in the frequency range of 10 to 300 MHz. The results showed that with an increase in frequency, the current density increases (Fig. 3b), which is in agreement with the previous studies [15]. Detailed electric potential and current variations at very high-frequency range are shown in (supplementary Fig. 3). This test helped us to select suitable frequency sweep under examination considering two points, suitable current passage and avoiding cell lysis [18]. As cell lysis is also a constraint while biological manipulation which should be taken under account. Secondly, to observe the passage of current through the cell w.r.t to frequency employed as we are dealing with impedance of cell so higher will be the current passage through the cell lesser will be the impedance of cell [24, 25]. 4 Results and discussion An elliptical-round cell is sandwiched in between the electrodes and again simulation validity tests, with simulation MCF-10A is used as the normal breast cell and MCF-7 is used as breast cancer cell. NL-20 is used as lung normal cell and SK-MES is used as lung cancer cell in the following impedance analysis. Fig. 3 a Cell enclosed, current, and frequency analysis for the range of 10 to 300 MHz (only five instances and shown here) which clearly shows as the frequency increases current density increases (for detail visualization, see supporting Fig. 4). b Impedance analysis of three extracellular material or electrolytes, i.e., water, saline 0.9%, and PBS, for higher frequencies. 3.1.2 With cell inclusion 13 Medical & Biological Engineering & Computing (2021) 59:1709–1721 4.1 Impedance spectroscopy The impedance of normal breast cell MCF-10A was found higher in detectable frequency range than that of cancerous counterpart MCF-7, as previous studies [23, 25, 33–35] showed that cancer cell has higher dielectric properties than normal cell. For lung cancer cell’s SK-MES and lung normal cell’s NL-20 impedances, the same behavior was observed but in the lung’s case the difference in the impedance of normal and its cancerous counterpart is less. The impedance of lung normal cell is slightly higher than the lung cancerous counterpart. These results were in good agreement with the results presented previously in the literature [11, 14, 25]. Impedance spectroscopy was performed for three ranges of frequencies, i.e., low-frequency range ­105 to ­107 Hz, intermediate frequency range 1­ 0 7 to 1­ 0 9 Hz, and highfrequency range ­109 + Hz (up to 100 GHz). This is because biological cells behave differently in different range of frequencies which is a result of variation in the dielectric properties of biological cell from alpha dispersion to beta dispersion [36]. So, the range of frequency in which differential behavior of biological cells is very clear usually annotated as detectable frequency range. We found detectable results of impedances for both organ cell pairs in intermediate frequency range. According to Wang et al. [25], in low-frequency range, the conductivity and permittivity of cell increase which results in low impedance values with rapid change in this frequency range. This concludes in favor of our results that in low-frequency range the difference between normal and cancerous cell impedance is not detectable. Due to rapid change in impedance, the constant comparable impedance values are not obtained. In mid-range or intermediate frequency range, the conductivity increases and permittivity is reduced slowly according to Wang et al. [25]. Thus, one will get much smoother and less variable or almost constant values for impedance as a result. In this range, the difference between the impedance of the both cell pairs (breast normal and breast cancerous and lung normal and cancerous) is observable and detectable. For higher frequency range, the conductivity increases and permittivity reduces sharply to zero which may cause the cell break down. This results in equal impedances of both normal and cancerous cells Table 2 Comparative analysis of the reasons behind the determination of detectable frequency range 1715 which is almost equal to zero (Table 2). The particular detectable frequency range of impedance spectroscopy for breast cancer detection was observed around 1­ 07 Hz. And for lung cancer detection, it was found to be around ­108 Hz (Fig. 4). The starting impedance (impedance at low frequency) of normal cells in frequency spectra was observed lower than that of cancerous counterpart. This is because of two reasons, the first one is that the normal cell contains much higher ions and charged particles in them [37–39] in contrast to the cancerous cell whose major constituent is water [37], since cancer cell loses almost all of its mineral which are the source of ion production under the influence of electric field. The second reason is that at the turn from lower frequencies to intermediate frequencies, there is high permittivity and conductivity of cell start increasing rapidly which ultimately cause reduction in impedance [25]. For lung cancer and normal cells, the impedance results were found in good agreement with the findings presented by Egot-Lemaire et al. [27]. In [27], it was shown that either of both due to high blood profusion and extracellular matrix or normal and malignant lung cell shows almost same behavior for permittivity and conductivity. In the current work, we are particularly targeting the impedance of normal and cancer lung cells instead, which on the other hand is function of their permittivities and conductivities. This is also confirmed by the slight impedance difference in our results for both normal and malignant lung cell. In [27], for intermediate frequency range, the dielectric properties of normal lung cells were reported to be a bit higher than that of its counterpart malignant cell, which is consistent with our results. So, our results of impedance for lungs (which is the function of conductivity and permittivity) are in good agreement with the previous work presented in the literature. 4.2 Electrode shape effect The whole numerical analysis of impedance spectroscopy for cancer detection was again done with different shapes of electrode to analyze the electrode shape effect on impedance. This time, some nano protrusions (nanospikes) were included on the planar electrode surface. Impedance results Frequency Low ­105 to ­107 Hz Intermediate ­107 to ­109 Hz High ­109 + Hz Conductivity [25] Permittivity [25] Impedance [25] Increases Increases Reduces Increases Slow reduce Slow reduce (almost constant) Increases Reduces sharply Almost zero (cell breakdown) Equal Our detectable results Low (detectable @boundary (of impedance) of intermediate f range) High detectable 13 1716 Medical & Biological Engineering & Computing (2021) 59:1709–1721 Fig. 4 a Results of impedance spectroscopy for breast cancer cell MCF-7 and breast normal cell MCF-10A in its detectable frequency range, i.e., 1­ 07 Hz using planar and nanospike electrodes. b Results of impedance spectroscopy for lung cancer cell SK-MES and lung nor- mal cell NL-20 in its detectable frequency range, i.e., ­108 Hz using planar and nanospike electrodes (zoomed section shows clear differentiation between impedance results for lung cell pair) for normal and cancerous breast cell pair and for normal and cancerous lung cell pair were analyzed in the detectable frequency range. As nanospike electrode was proved to be an efficient shape with multiple advantages, i.e., can capture cell better, economical, to fabricate, etc., as discussed in the above section. Design details of nanospike electrodes can be found in [18]. The results obtained were in good agreement with our previous results of impedance spectroscopy using planar electrodes. But the overall impedances for all four cell models were 1 to 2 unit lesser using nanospike electrodes than the impedances obtained using the planar electrodes. This was because of high aspect ratio of nanospikes [18] or sharp edges which results in concentrated electric field (Fig. 5a). This concentrated electric field increased the overall current density (Fig. 5b) resulting in less impedances. Surface plots of electric field and current density at 100 kHz are shown (Fig. 5b) to analyze differential effect of electrode shape. The nanospike electrodes are found better over planar electrode. They have multiple other advantages over conventional micro/macro electrodes and found best during examination, as they do not require surface functionalization to effectively capture the cell in between, which results in economy of time and less parasitic noises [14, 17, 18]. The major advantage of nanospike type electrodes is their high current densities (due to spike shape) so that one can operate them at really low voltage to get desired results with maximum cell viabilities. This makes nanospike electrodes economical and power efficient as well. Nanospike electrodes are economical to fabricate as they can be fabricated on aluminum foil with scalable and controllable electrochemical anodization and etching processes [14, 18]. 13 4.3 Gradient synthesis for detection In this section, an effective method of cancer detection is proposed, which includes some statistical analysis after achieving impedance data and provides a much better differentiation between the impedances of normal and cancerous cells. In this method of cancer cell detection, the gradient calculation of impedance drop for all frequency range up to the detectable frequency range is proposed (Fig. 6). For further synthesis, the initial and final values of impedance gradient up to detectable frequency range were utilized. This impedance data for all types of cells under examination and for both the electrode types (planar and nanospike electrodes) used in the numerical analysis was plotted for clear understanding (Supporting Fig. 5). Impedance plot with sweep of complete frequency range depicts a linear gradient up to detectable frequency range for each cell type as shown in Fig. 6. First of all, percentage drop in impedance was analyzed which itself an indication to differentiate between cancer and non-cancer cells. As shown in Fig. 7, the percentage drop in impedance for breast cancer cell MCF-7, for complete range of frequencies up to detectable one, was around 16%, while the percentage impedance drop of normal breast cell MCF-10A was just 9% using planar electrode. All the results obtained using nanospike electrodes were also in good agreement with the results obtained for planar electrodes. Medical & Biological Engineering & Computing (2021) 59:1709–1721 1717 Fig. 5 Surface and contour plots of electric field and current density respectively, for a planar and b nanospike electrodes at 100 kHz which shows that due to high aspect ratio and sharp edges, nanospike electrodes have higher current density than planar electrodes, where σnb and εr,nb are conductivity and permittivity of normal breast cell, respectively. σe and εr,e are conductivity and permittivity of PbS electrolyte respectively and their values can be found in Table 1 Percentage impedance drop is a clear indication for breast cancer but it has also shown very good results for the lung cancer. For lung cancer cell, the percentage impedance drop was around 28% and for normal lung cell, it was observed to be 22% using planar electrode. As discussed in the previous section, using conventional impedance spectroscopy, the difference in impedance of lung normal and cancer cells was very less. Those results were perfectly in accordance to the previous work done on lung cancer detection using dielectric properties [27] with the valid reason of higher blood profusion. But the fact is that conventional impedance spectroscopy results for lungs cannot be use for cancer detection effectively. On the other hand, percentage drop and gradients method showed a clear differentiation for breast cancer but performed exceptionally well for the lung cancer also. Percentage impedance drop was calculated using formula Eq. 2: Fig. 6 a Impedance variation plot of breast normal cell MCF-10A and breast cancerous cell MCF-7, throughout the range of frequencies (starting from lower to higher frequency) using planar and nanospike electrodes. Zi and Zf are initial and final impedance points used in Eq. (2) and Table 3. b Impedance variation plot of lung normal cell NL-20 and lung cancerous cell SK-MES, throughout the range of frequencies (starting from lower to higher frequency) using planar and nanospike electrodes Zdrop = (1 − (Zf ∕Zi )) × 100 (2) 13 1718 Medical & Biological Engineering & Computing (2021) 59:1709–1721 Fig. 7 With reference to Fig. 6, the percentage drop in impedance is plotted here. The bar plot is plotted for both types of electrodes. And the results from both electrodes are in good agreement with each other, i.e., the percentage drop in impedance for cancer cells of both organs is prominently higher than their normal counterpart where Zf is the final value of impedance and Zi is the initial value of impedance, their values can be found in Table 3. The next part of quantitative analysis of impedance data is gradient calculation (Fig. 8) Although the percentage change in impedance is higher for planar electrodes as compared to nanospike electrodes, yet these nano-structured electrodes are preferred over planar ones in biological cell impedance spectroscopy. The planar electrodes have relatively longer response time as cells take time to attach themselves on planar electrode surfaces. Surface chemistry techniques are used to modify electrode surfaces to enhance cell-electrode adhesion process. This will minimize the parasitic signals from gaps between cell membrane and planar electrode surfaces. Modifications through the surface chemistry involve complex chemical and electrochemical preparation steps. This results in undesirable electrochemical reactions at the surface of electrodes which have impact on sensing and electrode stability. Nanostructured electrodes can directly penetrate into cell membrane and enhance the cell-electrode adhesion very quickly without surface modification [18]. The electrical information can be extracted directly from cell membrane and can be used for different applications like cancer cell detection and discrimination. Using the abovementioned gradient method of cancer detection, the impedance gradient obtained would always be negative which proved the fact that as the frequency increases, the impedance decreases [14] (Table 3). The gradient of cancerous cell for both organs cell pairs was found 1.5 to 2 times greater than the gradient of its counterpart normal cell. It depicts that up to detectable frequency range, the impedance of cancer cell decreases more faster than the normal cell which, in fact, is the validation of the reported result in the literature that the dielectric properties of cancer cells are higher than the normal cell [27] and cancerous cells are much diluted than normal cells [37, 40]. Gradient for breast cancer cell MCF-7 was found to be 2 times greater than the gradient of normal breast cell MCF-10A and gradient of lung cancer cell SK-MES was almost 1.5 times greater than the gradient of normal lung cell NL-20 (Table 3). This method of cancer detection was found independent of electrode shape. The behavior of gradient for both the cancer cell pairs was same irrespective of electrode shape, i.e., the gradient of breast cancer cell was double than the normal breast cell for both electrode shapes and for lungs it was 1.4 times. The differentiation is clear and justified and using this gradient synthesis, the complex and less efficient data fitting technique can be avoided to get marker of identification. The only limitation of this gradient-based detection is when there is not gradient formation or very minor non detectable gradient is found in impedance spectroscopy results which Table 3 Quantitative analysis and data synthesis of logarithmic impedance plot in Fig. 6 for cancer detection Type of cells Planar electrodes MCF-7 MCF-10A SK-MES NL-20 Nanospike electrode MCF-7 MCF-10A SK-MES NL-20 13 Initial value of impedance, Zi (ohms) Final value of impedance up to detectable range, Zf (ohms) %drop (Zdrop) Gradient Multiple increase (for cancer cell) 3.3450 2.5528 0.9979 0.8629 2.8239 2.3028 0.7237 0.6653 15.578 9.793 27.478 22.899 − 0.1834 − 0.0879 − 0.0760 − 0.0550 2 times 3.1056 2.3802 0.9283 0.8032 2.6760 2.1619 0.6974 0.6369 13.833 9.1714 24.873 20.7046 − 0.1508 − 0.0773 − 0.0641 − 0.0461 2 times 1.4 times 1.4 times Medical & Biological Engineering & Computing (2021) 59:1709–1721 Fig. 8 The histogram shows that for breast cancer cell, the gradient is two times higher than its normal breast cell. And for lungs, the gradient of lung cancer cell is 1.4 times higher than its normal lung cell. And most importantly this quantity we found is independent of electrode shape, i.e., trend of gradient with both electrodes for both cell types is exactly in agreement with each other. Details can be seen in Table 3 is very uncertain to happen. Usually all EIS spectra contain gradient which can be synthesized using the abovementioned technique. The whole experimentation presented in this paper leads towards an effective and non-invasive early detection of cancer at a very low-voltage level. In the light of the above discussion, it is shown that the proposed gradient-based cancer detection technique is easy to perform, economical, and effective as well because it uses a single-cell level characterization. Moreover, the proposed gradient-based impedance detection technique finds utility in all EIS applications to avoid tedious data fitting process. Specifically, it is applicable in DNA biosensors, bacterial contamination analysis, detection of microorganisms by the means of EIS, detection of interfacial behavior of molecules, EIS-based body composition analysis, and assessment of food quality which includes dairy products, fruits, and oils [41, 42]. The proposed detection techniques can certainly find their application in micro total analysis system (µ-TAS) and portable biomedical detector, i.e., lab on chip and smart health care devices. 5 Conclusion A gradient-based impedance synthesis has been presented for early-stage cancer detection. For the first time, cancerous cell and normal cell of same particular organ are taken under numerical examination for cancer detection. Breast and lung cancer cells and their normal counterparts are analyzed for impedance detection using micro-scaled planar and nanospike electrodes independently. Differential 1719 effects of impedance for cancer and its normal counterpart have been found in detectable frequency range which vary organ to organ. Effect of electrode sculpt on normal and cancer cell impedance has been rigorously considered. The results of impedance for both electrode shapes have been found in good agreement with each other. Overall impedance for sharp edge nanospike electrodes is found 1.5 to 2 unit lesser due to the high current density. A technique using gradient synthesis has been proposed and extensively analyzed for cancer detection which is found independent of electrode shape effect since it utilizes gradient calculation. The results depicted that impedance of cancer cell declines with the rate double to its normal counterpart as frequency of applied pulse increases. This assured that under electrical manipulation, cancer cell behaves as divergent unlike healthy cell. 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W: http:// n anot e chlab. i tu. e du. p k/ Kashif Riaz : is an assistant professor in Electrical Engineering Department of Information Technology University (ITU), Lahore, Pakistan. He is one of the supervisors of Nanotech Lab. He did his PhD from Hong Kong University of Science and Technology (HKUST). His current research interests are on design, modelling, simulation, and characterization of nano-structures for biological manipulations and application using electrical methods like electroporation for biomolecules delivery, irreversible electroporation for cancer cell lysis, extraction of proteins from cell using electroporation, and cancer cell detection using electrical method at low voltages. W: http://n anote chlab.i tu.e du.p k/t eam/ dr-kashif-riaz/ 1721 Muhammad Mubasher Saleem : received his B.Sc. Electrical Engg. from University of Engineering and Technology (UET) Lahore (2008), MS in Electronic Engineering from Ghulam Ishaq Khan Institute of Engineering Sciences and Technology (GIKI), Topi (2010) and Ph.D. degree in Mechanical Engineering from Politecnico di Torino, Italy (2015). His PhD research work was related to RF-MEMS design optimization, fabrication, testing, and failure analysis for the space applications, in collaboration with Fondazione Bruno Kessler (FBK), Trento, Italy. He has also worked and published in the field of MEMS inertial sensors (Microgroscopes and Microaccelerometers). W: http://www.nust.edu. pk/INSTITUTIONS/Colleges/CEME/Depar tments/Mechatronics% 20Engineering/Faculty/Pages/Dr-Muhammad-Mubasher-Saleem.aspx 13