Nanostructured TiN-Coated Electrodes for High-Sensitivity

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Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Nanostructured TiN-Coated Electrodes for High-Sensitivity Noninvasive Characterization of in Vitro Tissue Models Tobias Schmitz,†,‡ Matthias Schweinlin,†,‡ Robin T. Kollhoff,† Lisa Engelhardt,† Christian Lotz,† Florian Groeber-Becker,§ Heike Walles,†,§ Marco Metzger,†,§ and Jan Hansmann*,†,§ †

Department of Tissue Engineering and Regenerative Medicine, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg, Germany § Translational Center Regenerative Therapies, Fraunhofer Institute for Silicate Research ISC, Roentgenring 11, 97070 Wuerzburg, Germany S Supporting Information *

ABSTRACT: Because of a rising use of in vitro models as an alternative to animal models, the precise assessment of tissuespecific parameters of such in vitro test systems has become a critical part of ensuring predictive results. Impedance spectroscopy as a noninvasive method serves as a reliable and efficient tool for quality control because it only minimally interferes with the system during investigation. In this study, we present a refined impedance measurement system using nanostructured titanium nitride (TiN) electrodes. This advanced material was used to investigate tissue maturation and changes in the barrier integrity of an intestinal Transwellbased in vitro model. The reduction of noise facilitated a more detailed data extraction and biological interpretation. Compared to standard stainless steel electrodes, at a typical measurement frequency of 12.5 Hz, the maximum electrode impact on the signal could be reduced from over 75% to less than 5%. This allowed the accurate determination of transepithelial electrical resistance values from Caco-2 in vitro tissue models without a further mathematical analysis based on a computer simulation. The novel design of a 3D-printed measurement attachment equipped with nanostructured TiN electrodes was used to continuously monitor the barrier integrity of the Caco-2 cells during a permeability assay. Moreover, because of low process temperatures, the TiN coatings for enhanced impedance measurement sensitivity could also be deposited onto several other materials, e.g., commercially available cell culture equipment such as standard disposable multiwell plate dishes. In conclusion, we developed a novel method to improve the electrode properties for impedance spectroscopy, which can be easily implemented into standardized end-point measurement to qualify a variety of in vitro test systems. KEYWORDS: impedance spectroscopy, nanostructured electrodes, titanium nitride, in vitro, test systems, Caco-2, noninvasive validation systems

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INTRODUCTION

preferable, allowing continuous experimental online monitoring and therefore minimizing costs and human resources. Measurement of the transepithelial electrical resistance (TEER) is a standard noninvasive method to analyze the barrier integrity of both two-dimensional (2D) cell cultures and three-dimensional (3D) in vitro tissue models such as blood brain barrier, gastrointestinal tract, or skin.5−7 In the standard experimental setup, measurements can be performed by using handheld chopstick electrodes that determine the TEER value at a single frequency, usually 12.5 Hz. To assess the electrical resistance of the respective tissue, the resistance of an empty insert is measured and subtracted from that of a generated cell

The EU legislation for chemicals REACH represents the largest investigation of consumer product safety of all time, which entails high costs and animal use.1 Following the 3R principle, the application of alternative in vitro methods is strongly supported by the authorities. In particular, in vitro models of the outer epithelia including the skin, intestine, and lung have found to be valid applications in both research and industrial settings as attractive alternatives to animal testing.2 Although there is increasing evidence that these models have a good in vitro−in vivo correlation, a lack of nondestructive analytical tools for reliable end-point measurements and model qualification still leads to hesitant regulatory acceptance.3 To promote acceptance, the development of highly sensitive and standardized tools to assess the quality of tissue models is crucial.4 In addition, the use of noninvasive methods is © XXXX American Chemical Society

Received: March 5, 2018 Accepted: April 24, 2018 Published: April 24, 2018 A

DOI: 10.1021/acsanm.8b00345 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials or tissue model.8 This procedure represents a fast and easy method without the need to transfer the insets to a specific measuring device or chamber, thereby disturbing the cell integrity. However, the chopstick electrodes need to be cleaned thoroughly. Otherwise, contaminations could be easily distributed throughout several tissue models. Moreover, the positioning of the handheld electrode can vary, and measuring at an open well plate not only makes it mandatory to ensure sterile conditions but also entails that the tissue models are exposed to an uncontrolled atmosphere regarding the CO2 concentration, humidity, and temperature. Thus, the accuracy of this standardly used measurement procedure is limited to 10−30 Ω·cm2.9 Because some tissues possess electrical resistance of the same magnitude, the applicability of this method to determine TEER values is limited. An alternative to the handheld electrode that provides high accuracy is an EndOhm system, where inserts are transferred to a measurement chamber and a special electrolyte is harnessed during analysis.10 With both tools the handheld electrode and the EndOhm systemit is difficult to perform parallel measurements with short measurement intervals (minutes) over a longer time period (hours), as is required, for example, in standardized penetration and transportation assays.11 A measuring system that is capable of performing transportation assays and shows higher accuracy is the so-called Ussing chamber.12 However, this device represents a complex setup that is designed for analyzing only a small number of samples. Although the two advanced systemsEndOhm and Ussing chamberfacilitate a high accuracy, a major drawback is the need to transfer the samples one by one into the measuring devices; thus, it is not possible to measure directly in well plates.9 Alternatively to standard TEER measurements, electrical impedance spectroscopy (EIS) can be employed to characterize a biological sample. By recording a frequency spectrum over a defined frequency range and subsequently extracting biologically relevant data using a simulation software, EIS represents a highly reliable technique that also allows one to obtain TEER values.13 Current systems harness stainless steel chambers, in which in vitro models are cultured and directly measured.14 Because these systems are not working in standard cell culture equipment, e.g., well plates, there are limitations especially in handling and scalability. These limitations of currently available measurement devices were the driving force to design and develop a novel measurement system. The aim of this study was to investigate the feasibility of a measuring system that is capable of analyzing large numbers of in vitro models together with a high measurement accuracy and the opportunity to use it for continuous measurements. To overcome the drawbacks of the current measuring setups, the simplicity of the well-known chopstick electrode setupmeaning measurements directly in the well platesshould be combined with a sophisticated electrode material that minimizes the unwanted effects of electrode polarization on the impedance measurement. Thus, enhanced electrodes should facilitate measurements at low frequencies by limiting deviations between measurements, thereby leading to a higher accuracy of the measurements and possibly increasing information content.15 Coating by physical vapor deposition (PVD) presents a suitable method to improve the surface characteristics of various substrate materials such as metals or polymers.16,17 Titanium nitride (TiN) PVD coatings have widely been used as

protective coatings in many industrial branches because of their excellent corrosion resistance, high hardness, good wear resistance, and biocompatibility but also found application in microelectronics as layers to inhibit atomic diffusion.18−20 These dense protective layers were produced by sputtering, ARC deposition, or evaporation, with thicknesses ranging from 100 nm to several micrometers. However, TiN can also be generated by reactive sputtering with a fractal surface structure, a suitable surface morphology for stimulating and sensing electrodes because it has shown to be a material with low impedance.21,22 In addition, the resistance of sputtered TiN against corrosion and mechanical damage represents favorable characteristics in long-term measurements or use and for repetitive cleaning procedures after use of the electrodes. Furthermore, for the generation of TiN coatings by PVD, a titanium target and gases such as argon and nitrogen are necessary, so there is no need for more expensive raw materials such as noble metals. Therefore, the advantages of nanostructured, fractal TiN regarding its generation and performance as an electrode material made it a highly promising material for the fabrication of a tailored impedance measurement system for easy scalability and long-term application in standard cell culture equipment. In this study, we describe the fabrication of four sets of electrodes that present different surfacessmooth and nanostructured TiN, stainless steel, and goldand investigate their performance in a custom-made impedance measurement system. On the basis of the electrical characteristics, from the four sets of electrodes, we select two sets for monitoring the maturation of intestinal Caco-2 in vitro models over the course of 21 days. These intestinal test systems represent one of the most commonly used systems in preclinical in vitro screenings for perorally administered drugs.23,24 The measurements are compared to measurements performed with a standard handheld electrode. Subsequently, we use a system equipped with nanostructured TiN electrodes to continuously monitor the barrier integrity of in vitro models during an ethylene glycol−bis(β-aminoethyl ether)−N,N,N′,N′-tetraacetic acid (EGTA) treatment. Finally, TiN electrode coatings were deposited on polymeric cell culture equipment to support transfer of the electrode technology into a cell culture routine.

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EXPERIMENTAL SECTION

Caco-2 in Vitro Models. Intestinal Transwell in vitro models were set up by seeding Caco-2 cells (DSMZ, ACC-169) on collagen-I (rattail)-coated, 24-well-plate cell culture inserts with a pore size of 1.0 μm (662610, Greiner Bio-One GmbH, Frickenhausen, Germany). Inserts were coated with 100 μL of a 100 μg/mL collagen−acetic acid solution for 15 min at room temperature. After evaporation of the remaining acetic acid, 100000 cells were seeded into each insert. Inserts were cultured in 24-well plates (782880, Brand GmbH & Co. KG, Wertheim, Germany) with an apical volume of 300 μL and a basolateral volume of 1200 μL of the medium [Dulbecco’s modified Eagle medium (DMEM), 1% NEAA, 1% sodium pyruvate (all Gibco− Thermo Fisher Scientific, Waltham, MA), 10% FCS (Bio&SELL, Nuernberg, Germany)], which was changed every 2 days. In vitro models were used for experimental assays after 21 days of static culture at 37 °C and 5% CO2. Preparation of Electrodes. The bases of all used electrodes were stainless steel screws of 3 or 5 mm diameter, respectively, which were cut and subsequently polished to a mirrorlike surface by using grid paper ascending from #80 to #4000. Prior to the coating process, the electrodes were cleaned in ultrasonic baths (Bandelin Electronic, Berlin, Germany) of isopropyl alcohol and ultrapure water for 10 min each and afterward dried by means of nitrogen gas. The samples were B


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ACS Applied Nano Materials mounted on a substrate holder and, in a final cleaning step, treated in a plasma chamber (Diener Electronic, Ebhausen, Germany) with pure oxygen plasma (100 kHz, 300 s, 210 W, 0.3 mbar). Three different types of electrode coatings were prepared using a custom-made vacuum chamber equipped with a radio-frequency (RF) magnetron sputtering system with a titanium target (12 cm diameter and 10 mm height) and a custom-made substrate heating. After substrates were placed in the vacuum chamber, the chamber was evacuated to a base pressure of 5 × 10−7 mbar. Nanorough TiN coatings were prepared in a gas mixture of Ar (137 sccm) and N2 (2.0 sccm) that was set and controlled by a multi gas controller (MKS Instruments, Andover, MA), resulting in a pressure of 4.0 × 10−3 mbar during deposition. Magnetron sputtering was carried out using a 13.56 MHz RF generator (RF 1000, Huettinger, Freiburg, Germany), which was operated at a sputtering power of first 800 W for 3 min and then 500 W for 42 min. For the preparation of smooth TiN coatings, the substrate holder was heated to a temperature of 200 °C. Using the same process gas mixture as before, samples were coated with a sputtering power of 800 W (2 min) and subsequently 600 W (18 min). Gold electrodes were prepared in a two-step process, where samples were first coated with a titanium layer (Ar 137 sccm, sputtering power 500 W, 20 min) and then transferred to another sputter coater (EM ACE600, Leica, Wetzlar, Germany) and coated with 50 nm of gold. Preparation of the Measuring System. The lid that could be placed on standard 24-well plates (Brand GmbH & Co. KG, Wertheim, Germany) was produced using a 3D printer (Xeed; Leapfrog, Alphen aan den Rijn, The Netherlands) with an autoclavable filament (HTPLA; Protoplant, Vancouver, Canada). The electrodes were screwed into the lid, so the bigger electrodes of 5 mm diameter were positioned in the center of the well and the smaller electrodes of 3 mm diameter fit into the slot on the side of the wells. Material Characterization. Ultrastructural images of the electrode surfaces were taken using high-resolution scanning electron microscopy (SEM; CB 340, Zeiss, Oberkochen, Germany). For determination of the layer thickness, coated glass slides were broken and the coating thickness was determined by SEM analysis of the cross sections. The surface morphology of smooth and nanorough TiN coatings was scanned by atomic force microscopy (AFM; Nanosurf FlexAFM, Nanosurf GmbH, Langen, Germany). Image processing of AFM data was done by freeware software Gwyddion 2.47. The crystallographic properties of the coatings were analyzed by X-ray diffraction (XRD) with control samples on glass substrates in grazingangle geometry under an angle of incidence of 2° using a Siemens D5005 X-ray diffractometer (Bruker AXS, Karlsruhe, Germany) and Cu Kα radiation with a voltage of 40 kV and a tube current of 40 mA. The diffraction patterns recorded in a 2θ range from 30 to 80° were evaluated with the software Dif f racPlus EVA (Bruker AXS, Karlsruhe, Germany) and compared with reference patterns from the JCPDS database.25 Adhesion of the deposited TiN coatings of control samples on glass substrates were investigated by scratch tests with a hardness tester of type 3212B (Zwick, Ulm, Germany) equipped with a Rockwell C diamond with a conical angle of 120° and a tip radius of 200 μm, as was described in a previous study.26 Impedance Spectroscopy. Impedance spectra of the electrodes were recorded using an impedance spectrometer (LCR HiTESTER 3522-50, HIOKI E.E. Corp., Ueda, Japan) with a custom-made user interface, programmed in LabVIEW (National Instruments, Austin, TX). The frequency range of the measurements ranged from 1 to 100 kHz with a voltage amplitude of 2 mV. The characterization of different electrode materials was performed using empty inserts at room temperature with phosphate-buffered saline (PBS; SigmaAldrich, St. Louis, MO) as the electrolyte. In the apical compartment of the inset, 0.3 mL were used, which was balanced by 1.1 mL of PBS in the basolateral compartment to avoid hydrostatic pressure. All simulations of the impedance data were performed using the software NOVA 2.1 (Metrohm Autolab, Utrecht, The Netherlands). Parameters for the electrical equivalent circuit were determined by the program via a nonlinear least-squares optimization method.

For cell culture experiments, the same impedance parameters were applied, but those were directly recorded in a cell culture medium. The medium was replaced 1 h before the measurements to ensure comparable test conditions. For the measurements, stainless steel and nanorough TiN electrodes were cleaned with 70% ethanol and placed for 15 min in a laminar-flow cabinet for the evaporation of ethanol. Prior to placing the 3D-printed lid with fixed electrodes on the well plate, the electrodes were shortly dipped into PBS to wash away any remnants of alcohol. Then, the electrodes were positioned in the wells and the setup was stored in an incubator for 60 min. Finally, the well plate was placed on a heating plate at 37 °C, and the electrodes were connected to the impedance spectrometer. Permeability Assay. After determination of the initial impedance characteristics of the Caco-2 in vitro models, 1.5 μL of a 4 M EGTA stock solution (Carl Roth, Karlsruhe, Germany) was added to the apical compartment of the inset. Subsequently, impedance measurement was repeated at several time points (5/15/25/35/45/55 min). In addition, permeability changes of treated and nontreated models were confirmed by 4 kDa fluorescein isothiocyanate (FITC)−dextran permeability assay (10 μM stock solution, Sigma) as described before.27 Immunohistochemistry. To investigate the barrier integrity and differentiation of the Caco-2 models on the protein level, whole samples were fixed after an experimental procedure with 4% paraformaldehyde (Roti-Histofix 4%, Carl Roth, Karlsruhe, Germany) for 1 h at 4 °C. The poly(ethylene terephthalate) membrane with the fixed-cell monolayer was cut out and transferred to a 24-well plate. First, samples were treated with a blocking solution containing 5% bovine calve serum (PanReac AppliChem, Darmstadt, Germany), 5% donkey serum (Biozol, Eching, Germany), and 0.3% Triton-X (SigmaAldrich, St. Louis, MO) for 1 h at room temperature. For specific staining of the tight junctions, fixed models were incubated with a primary antibody for zonula occludens (ZO-1, 1:100, Proteintech, Manchester, U.K.) overnight at 4 °C. The next day the samples were washed three times, incubated with the secondary antibody AlexaFluor-555 (rabbit, 1:400, Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature, and washed again three times. Samples were fixed on a glass slide using a cover glass and Fluoromount-G with 4′,6diamidino-2-phenylindole (Thermo Fisher Scientific, Waltham, MA) to visualize the cell nuclei. Fluorescence imaging was performed on an inverted microscope (Keyence BZ-9000, Japan). Statistics. Statistical analysis was performed with an unpaired t test with Welch’s correction. All statistical tests were performed with GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA). Results are given as mean ± standard deviation. The level of statistical significance is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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RESULTS AND DISCUSSION

Electrode Material: Properties and Characterization. Figure 1 presents the design of the measurement attachment with implemented electrodes as a CAD model in open (Figure 1A) and closed (Figure 1C) states. The PVD-coated electrodes that are shown in the macroscopic image in Figure 1B were implemented in the 3D-printed lid that exactly fit on a 24-well cell culture plate (Figure 1C). The design of the measurement attachment renders the complete system suitable for measuring also under nonsterile conditions. In cell-free impedance measurements using the chopstick setup with electrodes inserted into the apical and basolateral compartments of a cell culture insert, the pair of stainless steel electrodes exhibited the highest impedance and phase shift (Figure 2A,B). The polished surface, and thus a comparably small electrically active surface area (ESA) of those uncoated electrodes, was also reflected in the low coefficient of the constant phase element (CPE; Table 1).22 C


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ACS Applied Nano Materials

However, gold-coated electrodes were later discarded from the cell culture experiments because of their weak mechanical resistance, which caused failure of the coatings after only a few cleaning processes. In contrast, smooth TiN electrodes, which macroscopically appeared shiny gold, were mechanically extremely resistant and exhibited lower impedance values than stainless steel. Heating of substrates during the PVD process achieved an increased motility of the adatoms on the stainless steel surfaces, resulting in a smooth surface morphology of the heated TiN coatings (Figure 2C,E). However, smooth TiN electrodes showed much higher impedance and phase shift than nanorough coatings of TiN. SEM and AFM images demonstrated a significant difference in surface roughness and thus a significantly different ESA between a smooth surface morphology (Figure 2C,E) and a nanorough fractal surface morphology (Figure 2D,F) of TiN coatings. The highly increased ESA was also expressed in a comparably high coefficient of the CPE (Table 1), leading to the lowest impedance and phase shift of all tested electrode materials. The electrical equivalent circuit used for the simulation of electrode parameters consisted of a resistance Rmed representing the media, whereas the electrodes were described by a series connection of a CPE, rendering a blocking electrode with a parallel connection of a capacitor Ci and a second resistor Rp (Figure 2E). Rp and Ci can be described as the inversely coupled polarization resistance and interfacial capacity, which together with the CPE define the impedance response of the electrodes, so higher ESA leads to lower Rp and

Figure 1. CAD model of the measuring attachment with implemented electrodes for analyzing in vitro models in a 24-well plate (A). Stainless steel electrodes after coating with nanostructured TiN by PVD (B). Closed lid equipped with electrodes on a 24-well plate, suitable for measuring under nonsterile conditions (C).

Although the film thickness of gold is approximately 7-fold lower than the thickness of the smooth TiN film (Table 1), because of a nanorough surface structure and a higher surface area, which was facilitated by the PVD process, the impedance of gold-coated electrodes was lower than that of stainless steel or smooth TiN. Because we could confirm in preliminary experiments (data not shown) that a smooth bulk sample of gold shows impedance values comparable to those of stainless steel and therefore much higher than that of the deposited gold film, a pure material effect as a cause for lower impedance compared to smooth TiN or stainless steel was excluded.

Figure 2. Characterization of electrode materials. Amplitudes (A) and phase angles (B) of impedance spectra from four different electrode materials: uncoated stainless steel, steel coated with a double layer of titanium and gold, and coatings of smooth and nanorough TiN. The inset in part A is a small scheme of the chopstick setup as well as an electrical equivalent circuit for simulation of the electrode parameters in this setup. SEM and AFM images of smooth (C and E) and nanorough (D and F) TiN coatings produced by PVD. The small insets in the SEM images depict highmagnification images of 1 μm × 1 μm area. Macroscopic images of glass slides coated with smooth (E) or nanostructured (F) TiN are shown in the background of the AFM images. D


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ACS Applied Nano Materials Table 1. Simulated Electronic Parameters, Coating Thickness, and Critical Load of Characterized Materialsa material stainless steel gold TiN smooth TiN nanorough a

exponent of CPE N 0.88 0.90 0.81 0.65

± ± ± ±

0.01 0.01 0.01 0.01

coefficient of CPE Y0[×10−5 Ω−1 sN cm2] 1.8 9.3 5.4 128.7

± ± ± ±

0.1 0.1 0.1 2.0

polarization resistance Rp [Ω·cm2] 83.0 62.2 89.9 2.6

± ± ± ±

interfacial capacity CI [×10−6 F cm−2]

coating thickness d[nm]

critical load LC[N]

± ± ± ±

50 ± 10 370 ± 10 840 ± 10

18 >18

3.4 2.2 5.4 0.9

6.0 35.3 11.1 309

0.3 1.6 0.8 166

Impedance measurement was performed in PBS at room temperature using an empty inset in a two electrode chopstick setup.

higher Ci, respectively.22 The assumption of infinitely large counter electrodes, which is usually made, did not exactly fit in this setup. Because the working (WE) and counter (CE) electrodes are comparable in geometrical size, the parallel circuit of Ci and Rp works as a correction factor for the inadequately small counter electrode. An exception was the TiN−nano electrode, which could be adequately described by a single CPE. Because of its high ESA, the corresponding CE could be regarded as infinite. The redundancy of the parallel circuit in this case leads to high relative errors for Rp and Ci when it was still implemented in the electrical equivalent circle. Further material characterization of the coatings included XRD measurements. The diffraction patterns of both TiN coatings are included in the Supporting Information (Figure S1). In the diffraction pattern of the smooth coatings, two shifted signals could be identified because of the change of the process parameters during the coating procedure. Only one signal could be identified for the TiN−nano films, which showed high crystallinity, as indicated by well-defined diffraction peaks. Although the diffraction patterns for both coatings exhibited a shift of the diffraction peaks due to distortions of the TiN lattice, they could be clearly attributed to a δ-TiN lattice with face-centered-cubic crystal structure.17 In order to quantify the adhesion strength and mechanical stability of the coatings, scratch tests were performed. The adhesion strength of the coatings revealed that TiN coatings could endure loads up to 18 N without signs of delamination (Table 1). SEM images of the scratches made with a load of 18 N are presented in the Supporting Information (Figure S2). The adhesion strength of the coatings is therefore sufficiently high to ensure their use as electrode coatings, especially in this application, where they are not exposed to strong mechanical loads. As was already mentioned above, the gold coatings were mechanically fragile and could not withstand the minimum load of 2 N (Table 1). Impedance Measurement. Because gold coatings were mechanically not resistant enough to ensure long-term performance and smooth TiN coatings only moderately improved the impedance signal compared to uncoated stainless steel electrodes, further experiments were conducted with nanostructured TiN and steel electrodes. In order to assess the measurement stability and robustness, in total 24 independent measurements were performed with both the manufactured electrodes, i.e., TiN and stainless steel, and the handheld electrode, using empty inserts on different days. The estimation of the standard deviation due to multiple independent measurements of empty inserts (Rblank) in the cell culture medium at 37 °C is given in Table 2. The comparison between columns 1 and 2 should highlight how the handheld electrode is prone to systematic error. From the values given in Table 2, it can be concluded that the smallest deviation of all applied electrode systems can be

Table 2. Estimation of the Resistance Measurement Accuracy for 24 Independent Experiments Using 24 Empty Insets (Rblank) in the Cell Culture Medium at 37 °C with One Measurement per Experimenta Rblank,12.5 Hz

material handheld electrode stainless steel TiN−nano

single measurement at 12.5 Hz [Ω·cm2]

triplicate measurement at 12.5 Hz [Ω· cm2]

90 ± 10

88 ± 8

517 ± 11 138 ± 3

after baseline correction using R100 kHz [Ω·cm2]

simulation [Ω·cm2]

392 ± 9

2±2

6±2

1±1

a

Triplicate measurements refer to 24 independent experiments, with each measurement repeated three times.

achieved by using TiN−nano electrodes, which is important to facilitate reproducible measurements and a suitable signal-tonoise ratio. This advantage can be attributed to the characteristics of the interface. By a comparison of columns 1 and 3 of the TiN−nano measurements, it can be clearly seen that the impedance at 12.5 Hz was particularly caused by the resistance of the electrolyte, which could be excluded by subtracting the impedance value at 100 kHz. In the high-frequency range (100 kHz), the elements of an electrical equivalent circle (e.g., Figure 2A) that exhibit in parts capacitive behavior show very high conductance. In this case, other resistances are bypassed with only one exception, Rmed, the resistance caused by the medium. This resistance is influenced, among other things, by not only the type of electrolyte and its temperature but also the geometrical surface area (GSA) of the electrodes and in the high frequency range dominates the overall measured electrical resistance of the system.9,13 The deviation could be even further limited because these deviations were mainly attributed to small temperature variations or the inhomogeneous insertion depth of the electrodes in the electrolyte. Moreover, comparing the composition of the two signals at 12.5 Hz shows that, upon measurement of an empty insert, the TiN−nano electrode causes approximately 5% of the total signal (6 Ω·cm2/138 Ω· cm2), whereas the signal of the stainless steel electrodes is to approximately 76% composed of information on the electrode characteristics (392 Ω·cm2/517 Ω·cm2), thereby overlaying the biological readout. The low signal-to-noise ratio measured for the stainless steel electrodes leads to uncertainties in the measured values that can have a significant influence on the results of the experiment. This is especially the case when the system under investigation has low resistance, as is the case at early time points of the cell culture or for cells with generally low TEER values. Application: Biological Readout. In order to test the effect of electrodes with significantly lower impedance in cell culture experiments, the TEER values of Caco-2 models were E


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Figure 3. Impedance spectra from cell culture experiments. The positioning of the electrodes in a well with an inset and a histological image (H&E) of a Caco-2 layer after 21 days of culture on a synthetic semipermeable membrane of the inset (A). Scheme of the electric circuit that was used for simulation of the impedance spectra (B). Representative amplitudes and phase angles of two Caco-2 in vitro models over the time course of 21 days and impedance spectra recorded with stainless steel (C and E) or nanorough TiN (D and F) electrodes, respectively. Electrical equivalent circuit used for simulation of the TEER values, with predefined simulation parameters for the parts describing the electrodes (G). TEER determined from measured impedance spectra, by simulation, the selection of 12.5 Hz values from the spectra or handheld electrode measurements (H). Error bars result from the standard deviation between three independent tests including two biological replicates each and the measurement uncertainty estimated in Table 2 caused by the respective measurement system.

Caco-2 models were also determined using the well-known standard chopstick electrodes. Between day 2 and day 4, a decrease in the electrical resistance of the models was observed. On the basis of the postulations of Claude and Goodenough (1973)28 and Claude (1978),29 we assume that this effect is caused by the following

determined by impedance measurements with nanorough, fractal TiN electrodes (TiN−nano), and uncoated stainless steel electrodes over the course of 21 days. These intestinal in vitro models represent one of the standard test systems used in preclinical in vitro screenings for perorally administered drugs.23,24 For further evaluation, the TEER values of the F


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Figure 4. Permeability assay. Mature Caco-2 models showing a confluent monolayer expressing tight-junction proteins (zonula occludens 1, ZO-1) (A). Permeability for FITC−dextran with and without EGTA treatment after 60 min (B). Amplitude and phase angle of impedance measurements of Caco-2 models, which had been cultured under static conditions for 21 days after treatment with EGTA. Error bars indicate standard deviations between three independent tests including two biological replicates each (D and F) and nontreated controls (C and E). Impedance spectra for a Caco-2 model measured with uncoated steel electrodes were inserted for a comparison of the measured signals of different electrode materials.

given in Figure 3H are based on the standard deviation between three independent tests including two biological replicates each (i.e., six individual samples for each material or the handheld electrode, respectively) and the measurement uncertainty estimated in Table 2 caused by the respective measurement system. The impedance spectra of the blank values (Table 2) were also used to predefine the electrode parameters N, Y0, Rp, and Ci, which were necessary for determination of the TEER values by simulation. Rmed was defined by the value of the amplitude at the highest measured frequency 100 kHz.9 For a better comparison, with the TEER values obtained by the handheld electrode, the TEER values were also calculated from the spectra using the difference in the amplitude at 12.5 Hz between cell-seeded and empty inserts. The TEER values extracted from the impedance spectra of TiN−nano electrodes by simulation, using the equivalent

process: (I) The cells reached the initial confluency on day 2, leading to an increased transient barrier function. (II) Proliferation of the cells leads to an increased number of cell borders that could act as pathways for the current signal, resulting in decreasing barrier resistance. (III) During the further differentiation process, the cells build more tight junctions strands, leading to a continuous strengthening of the barrier. Indeed, from day 4 to day 21, we observed a constant increase in the amplitude as well as the phase signal for both sets of electrodes because of the growth and extended barrier formation of the Caco2 monolayer that can be seen in the representative impedance spectra for two in vitro models in Figure 3C−F. For the quantitative evaluation of the barrier formation, additionally only the TEER values were plotted over the culture duration (Figure 3H). The error bars for the TEER values G


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reduced signals of amplitude and phase (Figure 4D,F) can be attributed to a continuously increasing opening of tight junctions.30 Impedance measurements 24 h after the initial application of EGTA could also demonstrate the almost complete regeneration of the disturbed barrier, demonstrating the high sensitivity of this method. However, a comparison with the impedance spectra of the untreated models confirmed that the significant decrease of the amplitude and phase could not be attributed to a variation of the temperature or pH value because of the limited time that the in vitro models were kept out of the incubator. Besides the described analytical advantages, the application of single-use systems in biological experiments would be favorable for a variety of reasons such as the elimination of necessary cleaning processes or a reduced risk of contaminations. Because of the increasing availability of additive manufacturing, there are no obstacles to reproducing the lid and implementing coated electrodes, but ideally the electrodes would be directly integrated into standard cell culture equipment. The material of choice for single-use devices are polymers because of their fabrication costs, which are way lower compared to metals, for instance. Because the deposition of TiN with a fractal nanostructure was possible without increasing the substrate temperature, it was also possible to directly coat low-melting substrate materials, i.e., polystyrene (Figure 5). This opened up

circuit model in Figure 3G, were in the same range but on every day constantly lower by approximately 35 Ω·cm2 than the TEER values measured with the handheld electrode (Figure 3H). This is likely due to a difference in the applied signal waveforms (sinusoidal vs rectangular) used by impedance spectroscopy or the handheld electrode, respectively. The TEER values calculated from the TiN−nano electrodes at 12.5 Hz were in good agreement with the simulated values. In contrast, the TEER values determined at 12.5 Hz from the impedance spectra of the stainless steel electrodes differed the most from all other measured TEER values, which can be attributed to a high influence of the uncoated stainless steel electrodes on the measurement. Using a simulation software to extract the TEER values from the stainless steel spectra allowed one to remove the electrode influence on the measurement and resulted in values that were again in good agreement with the ones obtained by the TiN electrodes. Thus, in this twoelectrode chopstick setup, it is mandatory to use a simulation software to determine the correct TEER values using stainless steel electrodes, whereas it was not necessary when TiN−nano electrodes with low impedance were applied. However, this simplification is not necessarily valid also for other models than the Caco-2 cell line used in this study, investigated at a low frequency of 12.5 Hz, in which the capacity of the cell membrane does not play an important role. In the case of the Caco-2 cell line used in this study and other models that exhibit a negligible impact of the cell membrane at low frequencies, it is possible to extract precise TEER values directly from the impedance data by simple subtraction without the need for further computer simulation. Sensitivity Assay. As proof of concept to measure the sensitivity of our impedance measurement system, we incubated the Caco-2 models with the chelating agent EGTA to bind Ca2+ cations in the medium. This leads to a subsequent disturbance of the calcium-dependent epithelial cell−cell contacts (i.e., tight junctions) and decreasing barrier integrity, which is reversible after the removal of EGTA. Zonula occludens protein-1 poses a prominent tight-junction-associated protein in fully differentiated Caco-2 cells with an intact cell barrier, which can be visualized by immunocytochemical staining (ZO-1; Figure 4A). As functional proof of an increased permeability after EGTA treatment, we first measured the increased paracellular transport of FITC−dextran, which is ∼5 times higher compared to the untreated control (Figure 4B). Interestingly, we could not detect any visible changes in the immunocytochemical staining pattern of ZO-1 (data comparable to Figure 4A), indicating that the moderate disruption of the barrier occurs only on a functional level under these conditions. The impedance spectra indicated with “0 min” correspond to a measurement performed directly before the application of EGTA (Figure 4C−F). Impedance spectra from 1 to 60 min refer to the time intervals after the application. The impedance spectra of control measurements (Figure 4C,E) for every 5 or 10 min, respectively, demonstrated the stability of the measurement signal for the duration of the experiment of roughly 60 min. Small variations in the signal may be due to temperature variation because the models were only heated from below by a heating plate but were not positioned in an incubator under completely defined climatic conditions. The addition of EGTA to the apical site of the insets led to a spontaneous decrease of the barrier function that could already be detected after only 1 min with our system. The significantly

Figure 5. TiN-coated 12-well plates of polystyrene. Low temperature during the PVD process allowed also the implementation of electrodes directly into commercially available well plates (A). Magnification of a single well coated with electrodes (B).

the opportunity of implementing the TiN electrodes into standard polymeric cell culture equipment. Impedance of the TiN coatings deposited directly into the cell culture wells was analyzed with a TiN−nano-coated stainless steel CE, inserted into the associated polystyrene lid. The preliminary examination of this system revealed that the electrical characteristics are comparable to the 3D-printed setup equipped with two TiN− nano-coated stainless steel electrodes. A Bode plot of the impedance spectrum is presented in the Supporting Information (Figure S3). However, these measurements were made without inserts in the wells and should just demonstrate the functionality of the coatings. Further thorough mechanical and electrical characterization of the coatings on this substrate will take place in a future study when the lid in its current form and geometry (as presented in Figure 1) is available completely from injection-molded polystyrene.

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CONCLUSION In this study, we demonstrated the advantage of fractal TiN coatings produced by PVD for noninvasive characterization of in vitro tissue models via impedance spectroscopy. The H


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and Dentistry in Wuerzburg for providing the PVD system used for the electrode coatings. Finally, we acknowledge Judith Friedlein and Dr. Claus Moseke for the assistance with the SEM measurements.

application of TiN−nano coatings reduces the deviation of measurement results associated with the influence of the electrodes to only ±1 Ω·cm2 and thus enables one to assess tissues with a low inherent barrier function. The generation of electrodes via PVD enabled the design of a novel impedance measurement device that is based on the simple measurement setup known from the handheld chopstick electrodes but eliminating most of the shortcomings such as lower accuracy or difficulties in handling during time-consuming transport assays. On the one hand, the new measuring system can be used as a fast and easy-to-apply measurement device, which is applicable in daily routines for different users or parallel experiments. The design of the newly developed device for impedance measuring allowed the analysis of Caco-2 in vitro models directly in well plates with no need to transfer the models, whereby the higher accuracy compared to stainless steel electrodes and a standard handheld electrode could be shown. On the other hand, also the feasibility of continuous online measurement of the barrier integrity could be demonstrated after the treatment of an epithelial in vitro model with EGTA. Furthermore, the low temperature during the coating process allowed deposition of the TiN electrodes directly onto polymeric cell culture equipment. This represents a significant step forward toward single-use cell culture equipment with integrated electrodes for impedance measurements that could even further increase the scalability and availability of impedance spectroscopy for the characterization of in vitro models. Taken together, this could have an important impact on improving the quality and costefficient development of in vitro tissue models, as well as investigating the models’ response to applied chemicals. Thereby, crucial requirements needed for the implementation of large-scale investigations such as REACH are met.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00345. XRD patterns, SEM images, and functional testing of TiN-coated cell culture wells (PDF)

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ABBREVIATIONS CPE = constant phase element EIS = electrical impedance spectroscopy ESA = electrically active surface area GSA = geometrical surface area PVD = physical vapor deposition TEER = transepithelial electrical resistance TiN = titanium nitride XRD = X-ray diffraction

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 931 3181209. E-mail: [email protected]. ORCID

Matthias Schweinlin: 0000-0003-2172-0827 Author Contributions ‡

These authors contributed equally to this work and should be considered cofirst authors. Notes

The authors declare the following competing financial interest(s): The patent for electrode coating was submitted by the Fraunhofer ISC, Patent 102017219425.1, Nanostrukturierte Titan-Mehrschichtelektrode.

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ACKNOWLEDGMENTS This investigation was supported financially by the German Federal Ministry of Education and Research, program NanoMatFutur (Grant 13N12971), and ETface and Free State of Bavaria, BayernFIT program. The authors thank Prof. Jürgen Groll of the Department for Functional Materials in Medicine I


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Nanostructured TiN-Coated Electrodes for High-Sensitivity

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