Engineering of Neuron Growth and Enhancing Cell-Chip

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Biological and Medical Applications of Materials and Interfaces

Engineering of Neuron Growth and Enhancing Cell-Chip Communication via Mixed SAMs Aleksandr Markov, Vanessa Maybeck, Nikolaus Wolf, Dirk Mayer, Andreas Offenhäusser, and Roger Wördenweber ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02948 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

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ACS Applied Materials & Interfaces

Engineering of Neuron Growth and Enhancing CellChip Communication via Mixed SAMs Aleksandr Markov*, Vanessa Maybeck, Nikolaus Wolf, Dirk Mayer, Andreas Offenhäusser and Roger Wördenweber Institute of Complex Systems (ICS-8), Forschungszentrum Jülich, Jülich 52425, Germany ABSTRACT: The interface between cells and inorganic surfaces represents one of the key elements for bioelectronics experiments and applications ranging from cell cultures and bioelectronics devices to medial implants. In the present paper, we describe a way to tailor the biocompatibility of substrates in terms of cell growth and to significantly improve cell-chip communication, and we also demonstrate the reusability of the substrates for cell experiments. All these improvements are achieved by coating the substrates or chips with a self-assembled monolayer (SAM) consisting of a

mixture

of

organic

molecules,

(3-aminopropyl)-triethoxysilane

(APTES)

and

(3-

glycidyloxypropyl)-trimethoxysilane (GLYMO). By varying the ratio of these molecules, we are able to tune the cell density and live/dead ratios of rat cortical neurons cultured directly on the mixed SAM as well as neurons cultured on protein-coated SAMs. Furthermore, the use of the SAM leads to a significant improvement in cell-chip communications. Action potential signals of up to 9.4± 0.6 mV (signal-to-noise ratio up to 47) are obtained for HL-1 cells on microelectrode arrays. Finally, we demonstrate that the SAMs facilitates a reusability of the samples for all cell experiments with little re-processing. KEYWORDS: self-assembled monolayer, APTES, GLYMO, molecular layer deposition, mixed molecular monolayers, brain cell, HL-1 cell

INTRODUCTION 1 ACS Paragon Plus Environment

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In the past few decades, many devices for biological and biomedical applications have been developed that require direct contact between biological objects (e.g. cells) and non-biological materials . This is especially the case for bioelectronics applications where not only the 1–3

mechanical but also the galvanic properties of the contact (bioelectronics interface) are vital for the performance of the device. The success of the numerous extracellular interface concepts, such as surface modification with protein-resistant functionalities or protein active groups or adhesion 4

of cells to biocompatible rigid or flexible substrates , is usually limited by various problems, 5–7

which determine for instance the sensing efficiency of bioelectronics devices . To overcome such 8,9

limitations, different approaches such as 3D nanoelectrodes

10,11

or graphene-based devices are 12

being investigated. Another very promising way to improve such interfaces is the use of organic layers. In particular, self-assembled monolayers (SAMs) of organic molecules allow almost any kind of surface of existing samples or devices to be modified . These layers are not only ultrathin, 13,14

but they may also be quite robust and the functionality of the resulting surface can be varied by the choice of molecule. Moreover, using a mixture of different molecules with different properties (e.g. with different functional groups) for the SAM allows the properties of the resulting surface to be fine-tuned. In previous research work, it was shown that it is possible to precisely control surface properties such as the surface energy and surface potential via the ratio of two different silanes, (3-aminopropyl)triethoxysilane (APTES) and (3-glycidyloxypropyl)-trimethoxysilane (GLYMO), forming the mixed SAM . Here, we demonstrate the impact of the APTES/GLYMO ratio on the (i) protein 15

coating, (ii) cell growth and life/death ratio of cortical neurons, and (iii) cell-chip communication for cardiomyocyte-like cells (HL-1 cell line ). We explicitly chose two different types of cells for 20

the different experiments, since the growth mechanism can be analyzed better via cortical neurons, whereas the cell-chip communication is less positional dependent for HL-1 cells. We demonstrate, among other things, a significant enhancement of the cell signal (action potential) detection for HL-1 cells on SAM-coated microelectrode arrays (MEA) and show that these MEAs are reusable in cell experiments.

RESULTS AND DISCUSSIONS

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We previously introduced a method of engineering the surface of SiO -terminated Si by means of 2

in-situ-controlled gas-phase deposition (MLD) for the deposition of mixed APTES – GLYMO SAMs . Since APTES has an amino functional group (positively charged in biocompatible 15

electrolyte), whereas GLYMO has an epoxy functional group (neutral in biocompatible electrolyte), the properties of the resulting mixed molecular monolayers (e.g. effective thickness, hydrophobicity and surface potential) depend on the composition of the molecular layer. Moreover, the molecular ratio can affect, for instance, the successive coating of the SAM with proteins or polycations (e.g. poly(L-lysine) (PLL)). This thus indicates a route for optimizing device-to-cell interfaces for bioelectronics applications. Therefore, in the present work we analyze the potential of mixed APTES-GLYMO SAMs for biological applications.

Figure 1. Schematic of a SiO2-terminated Si sample covered with mixed molecular SAM, partially coated with PLL and finally cultured with cortical neurons on the surface. Cell density and live/dead ratio are analyzed on the Si/SiO2 area covered only with the SAM (a), on the border of the PLL-coated area (b) and inside the PLL-coated area (c), the size of the images is 580 µm x 580 µm. The schematics of the molecular layers in (a) and (c) are out of scale in order to visualize the assumed molecular arrangement of the positively charged APTES and neutral GLYMO in the SAM and the PLL, which seems to adhere to the GLYMO.

In a first step, we analyzed the impact of APTES-GLYMO SAMs on primary cortical neurons. To this end, we prepared APTES-GLYMO SAMs on SiO -terminated Si substrates. These were then 2

partially coated with PLL (see fig. 1). In this way, we can simultaneously analyze the direct (fig. 1a) and indirect (with an additional PLL coating, fig. 1c) impact of the SAMs on the cell growth. The resulting cell density and the live/dead ratio were obtained via fluorescence microscopy and 3 ACS Paragon Plus Environment


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live/dead staining (see Methods). Images were recorded and evaluated for the different areas, i.e. on the SAM (fig. 1a), on the PLL-covered surface area (fig. 1c), and at the border between the two areas (fig. 1b). Please note that in the following the APTES/GLYMO ratio represents the mixture of the molecules defined by the ratio of APTES with respect to the total amount of molecules, i.e. “APTES/(APTES+GLYMO)”.

Neuronal growth on mixed APTES-GLYMO SAMs: We first consider the cell cultures directly immobilized on the SAM (fig. 1a). The detailed analysis of this area is summarized in Fig. 2. The fluorescence images (fig. 2b-f) show exemplary images of living (green) and dead (red) neurons cultured directly on the SAMs with different APTES/GLYMO ratios. There are no cells on the GLYMO SAM (fig. 2b). However, the density of cells increases strongly with increasing amounts of APTES in the SAM (fig. 2i). Finally, for the pure APTES SAM (fig. 2g), cell densities of ~0.8´10 µm are obtained, which is comparable to standard values for protein-coated substrates . -3

-2

11

The APTES/GLYMO ratio also affects the live/dead ratio (fig. 2h). Obviously, there is no live/dead ratio for the pure GLYMO SAM, which does not host any neurons. However, starting with 20% APTES a live/dead ratio of ~0.5 is observed, which increases nearly linear with the APTES content and finally reaches approximately 1.5, which again is similar to values observed for neuronal culture in the literature. In summary, the neuron population and live/dead ratio 11

strongly depend on the APTES/GLYMO ratio. No cells adhere on GLYMO, the cell density and live/dead ratio increase more or less linearly with the amount of APTES in the SAM and finally reach values comparable to reference cell cultures on PLL-coated substrates . In addition to the 11

individual cells, a few huge clusters (neurospheres) are observed on APTES. These neurospheres float above the surface itself and use neurites to fix themselves to a small number of points either directly on the surface or on other cells (fig. 2f-g). Since the total number of neurons in each neurosphere is difficult to determine and many are not in contact with the test surface, these cells are not are not considered in the evaluation (fig. 2h-i).


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Figure 2. (a) Schematic of rat cortical neurons directly immobilized on the SAMs with different APTES/GLYMO ratios. (b-g) Examples of fluorescence microscope images (580 µm x 580 µm) of the cultures for different ratios ranging from pure GLYMO (b) to pure APTES (g), the numbers provide the percentage of APTES in the mixed SAMS (c-f), living cells are marked with calcien-AM (green) and dead cells are marked with ethidium homodimer (orange). (h) and (i) display the resulting live/dead ratio and the density of live and dead neurons as a function of APTES concentration. The values on the bars in (i) represent the total amount of cells (live and dead) averaged over several areas of size 580 µm x 580 µm (0.3364 mm2).

These observations indicate that neurons attach and survive at the amino functional group of APTES but not at the epoxy functional group of GLYMO. In the case of the APTES, the cells 26

seem to bind non-specifically to the positively charged surface formed by the amino group, whereas in the case of GLYMO, hydrolysis of the epoxy group in the basic solution during cell culturing (pH = 7) leads to the formation of trans-diols , which are known to have a cytotoxic 16

effect on neurons. Therefore the cell population density and also the live/dead ratio depend 17,18

strongly on the APTES/GLYMO ratio of the SAM. For the pure GLYMO SAM, all the cells are eliminated, not even dead cells from the primary neuron isolation stick to the surface. For small amounts of APTES cells are observed, although most of them are dead, and finally for large amounts of APTES in the SAM not only the cell population but especially the proportion of living cells increases strongly. This offers an option for guided cell growth for example via introducing local variation or gradients in the APTES/GLYMO concentration of SAM-coated carriers. 5 ACS Paragon Plus Environment


Neuron growth in the PLL-coated area: In order to analyze the neuron growth on the PLL-coated part of the substrate (see fig. 1c), we first checked the adhesion of PLL on the mixed APTESGLYMO SAMs. For this purpose, SiO -terminated Si substrates with mixed SAMs of different 2

APTES/GLYMO ratios were coated with fluorescently labeled PLL (PLL with fluorescein isothiocyanate marker (FITC)). The complete preparation including deposition and rinsing is described in the ‘Methods’ section below. The fluorescent marker is added for optical inspection of the adhesion of the PLL. During the final preparation step, rinsing with GBSS, the PLL+FITC that is not bound to the molecular layer is removed. The resulting fluorescence images (fig. 3a) reflect the amount (density) of PLL bound by the SAM. (a)

(b)

GLYMO

60

(b) 22.5% 0%

47.5%

22.5% 22.5%

75%

37.5% 0% 10 µm APTES

GLYMO 100%

Intensity [a.u.]

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40

20

0 0 GLYMO

20

40

60

80

APTES ratio [%]

100

APTES

Figure 3. (a) Fluorescence microscope image of samples with mixed SAM (the percentage given in the image refers to the fraction of APTES in the layer) coated with PLL+FITC (exposure time 600ms) and (b) resulting intensity of PLL+FITC fluorescence as a function of the APTES/GLYMO ratio.

Fig. 3 shows a clear tendency. The brightness of the fluorescence image decreases linearly with the APTES/GLYMO ratio. Due to its amino group, the PLL only binds to the epoxy functional group of GLYMO and not to the amino group of APTES. As a consequence, we can assume that the GLYMO functional groups that are detrimental to the cells adhesion and survival are now covalently bond and thus ‘covered’ by PLL, similar to the schematic in fig. 1c. As a result, we see different cell population densities and live/dead ratios inside and outside the PLL-coated area, as


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shown in the fluorescence image of the border of the PLL-coated area in fig. 1b. With this knowledge, we can now move to the cell cultures on PLL-coated mixed SAMs.

Figure 4. (a) Schematic of neurons immobilized on PLL-treated SAMs with different APTES/GLYMO ratios. (b-g) Examples of fluorescence microscope images (580 µm x 580 µm) of the cultures for different ratios ranging from pure GLYMO (b) to pure APTES (g), the numbers provide the percentage of APTES in the mixed SAMS (c-f), living cells are marked with calcien-AM (green) and dead cells are marked with ethidium homodimer (orange). (h) and (i) display the resulting live/dead ratio and the density of live and dead neurons as a function of APTES concentration. The values on the bars in (i) represent the total amount of cells (live and dead) averaged over several areas of size 580 µm x 580 µm (0.3364 mm2).

In the PLL-coated area, the surface should consist of APTES and PLL, the latter being covalently bonded to the epoxy functional group of GLYMO. The possibility should also be considered that not all of the unbound PLL is removed during rinsing. Since neurons tend to adhere to areas of positive charge, they should bind to the amino groups of PLL and APTES, which define the surface of the PLL-coated area. This can be seen in fig. 4. The cell density is generally high for all APTES/GLYMO ratios. It is significantly higher than for the GLYMO-containing SAMs without PLL coating (compare fig. 2i and fig. 4i). This confirms that direct contact with GLYMO seems to be detrimental for the cells and that the PLL helps to avoid this direct contact. The amount of cells surviving on the APTES with and without PLL coating (fig. 2i vs. fig. 4i) is not significantly 7 ACS Paragon Plus Environment


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different (p>0.5, t-test). However, the number of dead cells on the surface drastically reduces the live/dead ratio. This suggests small amounts of PLL on APTES do not support cell growth, but are sticky enough to prevent dead cells from the preparation from being washed away. Moreover, cells on PLL tend to grow in a homogeneously distributed manner (fig. 4b-e) and on APTES they form neurospheres similar to the behavior observed for the cell cultures on APTES without PLL (fig. 2f-g). Finally, the live/dead ratio of neurons in the PLL-treated area decreases with increasing amounts of APTES – i.e. a decreasing amount of GLYMO – in the underlying mixed layer (see fig. 4h). This plot shows that the largest live/dead ratio is not at either end of the concentration scale. There seems to be an optimal value for the molecules at 22.5% of APTES. However, the total amount of both live and dead cells on this sample is smaller than on all other PLL-treated SAMs. This suggests a slightly less adhesive surface overall, where both live but weakly adhering and dead cells may be washed away when the medium is replaced.

Cell-chip communication: Finally, in order to check whether our mixed SAMs can affect the growth of cells on an electronic device and thus influence cell-chip communication, we recorded action potential signals of HL-1 cells that were immobilized on microelectrode arrays (MEA) of chips coated with mixed APTES-GLYMO SAMs and a protein (in this case fibronectin). We explicitly chose HL-1 cells for this type of experiment. In contrast to cortical neurons, the cellchip communication can be dominated by the passivation-SAM-cell interaction because the HL-1 cells form a more or less continuous tissue and thus automatically cover the recording electrodes of the MEAs. In order to provide a comprehensive statistical analysis of the impact of the mixed monolayer on extensive cellular recordings, MEAs fabricated in house combined with a 64-channel amplifier system (BioMAS ) were used. The MEAs were fabricated on a borosilicate wafer with 10 nm of 19

Cr as an adhesive sublayer, with electrodes made of 200 nm Pt and covered with 3µm of polyimide HD8820. The polymer cover was opened down to the Pt structures at 64 positions forming Pt electrodes with a diameter of 24μm. The MEAs were covered by mixed APTES/GLYMO monolayers of different ratios using the same parameters as for the Si/SiO samples. In order to 2

15


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demonstrate the suitability of the devices for bioelectronics applications, the devices were encapsulated and prepared for cell culture (see Methods). Subsequently, HL-1 cell line was cultured on the chip surface (see fig. 5a). The changes in cell potential were measured after maturation of the cells on the custom-built amplifier system, BioMAS, against a silver/silver chloride reference electrode placed directly in the cell culture medium.

Figure 5. Schematic of the investigated MEA structure (a), image of the MEA structure and electrode array (b), and action potential (time trace and single action potential) of HL-1 cells on MEAs coated by APTES/GLYMO mixed monolayers and fibronectin for 10% APTES (c-d) and 75% APTES (e-f).

A typical time trace recording from a MEA covered by mixed films with HL-1 action potentials is shown in fig. 5c and 5e, for samples with 10% and 75% APTES, respectively. The cells beat (producing repetitive action potential (AP) that propagates through the whole cellular layer, accompanied by cell contraction to visually confirm cell activity) at a rate about 23 bpm and an



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optimal recorded AP amplitude on the 10% APTES sample of 2.8±0.2 mV, representing the poorest performance of the mixed monolayer chips (fig. 5d), and an optimal recorded AP amplitude of 9.4±0.6 mV for the best performing monolayer with 75% APTES (fig. 5f). Considering the noise level of 200 µV, the overall signal-to-noise ratio (SNR) is better than 14±1 and reaches 47 ± 0.3, which is considerably superior to that reported elsewhere for MEAs . The 21,22

shape of the APs is shown in fig. 5d and 5f. The shape of the action potential, in agreement with previous work, represents a very good sealing between the cell and the electronic device . The 12,23

overall results of the action potential measurements exceeded our expectations by far. Signals obtained from samples with 75% and 100% APTES display values higher by at least an order of magnitude than those previously observed from protein-coated MEAs with planar electrodes. Even samples with the lowest amplitude of signal show AP values of 2 mV. The AP signal dependence on the APTES/GLYMO ratio represents a particular point of interest (see fig. 6). Initially, it was expected that the behavior would be similar to the neuronal dependence on the Si/SiO samples, where higher fraction of GLYMO resulted in a better cells attachment to 2

the surface. Here, we have fibronectin instead of PLL as a protein linker between the surface and the HL-1 cells. In contrast to neurons, HL-1 cells attach and spread on APTES molecules without forming semi-detached cell clusters. Therefore, HL-1 behaved similarly on both fibronectin and APTES layers. According to previous work, the thickness of the fibronectin layer is about 8.7nm . 24

Thus, this distance from the metal electrode to the cell is greater when fibronectin is present than for the mixed film alone. This increased distance may increase the electrical leak current between the cell and the device during the action potential measurement. This could explain the probability of a decreased signal level obtained from samples with higher GLYMO ratio (higher fibronectin ratio as a result). Another explanation could be that the mixed molecular SAM protects the Pt electrodes from activation and coating with fibronectin. This would lead to an improved coupling of the cells’ electronic signal to the electrodes. More extensive research is required for an exact explanation. In order to demonstrate the ability to reuse samples with mixed films, all MEAs were cleaned by ultrasonication in the surfactant 2% Helmanex III followed by 5 min rinsing in bidest water. During the cleaning process, the HL-1 cell culture was removed from the samples, while the mixed films and the covalently bound protein remained. After cleaning, the devices were re-sterilized prior to 10 ACS Paragon Plus Environment

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cell culture in 70% ethanol (see Methods). Next, a new round of HL-1 cells was cultured on the surface and the devices were re-measured according to the previous experiments.

10

Action potential [mV]

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8 1st run

6 4

2nd run

2 0

0 GLYMO

20

40

60

80

APTES ratio [%]

100 APTES

Figure 6. Action potential as a function of the molecular ratio of the mixed monolayers in terms of APTES content for the 1st (solid black) and 2nd (open blue) cell culturing. The 1st and 2nd runs were performed on one and the same MEA for each concentration, for the second culturing only the cells were removed.

The typical time trace recordings of HL-1 APs from reused MEAs covered by mixed films were similar to those measured in the first run. The tendency towards increasing AP values from GLYMO to APTES samples is still present (fig. 6). However, the amplitude of the AP peaks became considerably smaller. The smallest signal (average over all measured channels) is ~0.8mV for the sample with 25% APTES and the highest averaged signal is ~2.6mV, which is still comparable to or even better than previously reported results . This reproducibility of results is 21,22

applicable for all cell experiments in this report, i.e. neuronal cultures on Si/SiO and HL-1 cells 2

on MEAs show reproducible results with merely overall smaller amount of cells attached to the surface (neurons on Si/SiO ), or lower amplitudes for AP (for HL-1 cells on MEAs). This 2

underlines the significance of this work, because the mixed films do not only seem to enable a 11 ACS Paragon Plus Environment


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tailoring of surface properties in terms of controlling the growth of cell cultures on the surface or enhancing AP values measured from these cells, but also provide the ability to reuse a device with minimal re-processing (only cleaning via ultrasonication with 2% Helmanex and rinsing with bidest water), thereby increasing productivity and reducing the cost of experiments. This is particularly relevant for substrates with patterned adhesive regions, since patterning methods such as microcontact printing, optical lithography, and laser-writing are all very time- and resourceconsuming.

SUMMARY In the present paper, we describe a way to tailor the biocompatibility of substrates and, thus, significantly improve cell-chip communication by coating the substrates or chips, respectively, with SAMs consisting of a mixture of organic molecules APTES and GLYMO. By varying the ratio of the two different silanes we can tune (i) the cell density in a controlled and continuous way from 0 to values comparable to standard cell densities for protein-coated substrates and (ii) the live/dead ratio from ~0.4 to ~1.5. The tuning is observed for rat cortical neurons cultured directly on the organic SAMs as well as neuron cultures on PLL-coated SAMs. In the latter case, the GLYMO, which proved to be detrimental for cell adhesion and cell survival, is ‘covered’ with PLL. Furthermore, we achieved a significant improvement in cell-chip communications using the coating with the organic SAM. AP signals of up to 9.4± 0.6 mV (SNR of up to 47) for the action potential of HL-1 cells on MEAs covered with the mixed molecular SAMs are measured. Finally, we demonstrated the ability to reuse the samples with mixed films for all cell experiments, i.e. the neuronal cultures on Si/SiO and HL-1 cells on MEAs. 2

In conclusion, the mixed molecular SAMs seem to (i) enable a tailoring of surface properties in terms of controlling the growth of cell cultures on the surface, (ii) lead to a significant improvement in cell-chip communication, and (iii) offer re-usability of bioelectronic devices with minimal reprocessing. Whereas the first possibility offers options for patterning adhesive regions for cell growth and guided cell growth, the second is of general importance for bioelectronic devices, and the third could lead to increasing productivity and also reductions in the cost and time of cell12 ACS Paragon Plus Environment

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culture and bioelectronic experiments. This study indicates that mixed molecular SAMS might be a very powerful tool for the improvement of cell experiments, bioelectronic devices, and even biological applications ranging from medical implants to biosensors.

METHODS Substrates. In this study, p-doped silicon (Si (111)) with a 90 nm thick SiO termination layer was 2

used for the PLL+FITC and neuronal cell experiments and MEAs fabricated in house for the cellchip communication experiments. The MEAs were fabricated on borosilicate wafers with electronic leads and electrodes consisting of 10 nm of Cr (adhesive sublayer) and 200 nm of Pt, an approximately 3µm thick electronic isolation of polyimide HD8820, and an array of 64 electrodes provided by 24μm diameter openings in the polyimide down to the Pt. Both types of substrates (silicon and MEA) are equally suited for the deposition of organic layers and biological applications. Mixed monolayers. Mixed SAMs were deposited from the gas phase using a specially developed MLD device with in situ control of the deposition . In this study, two types of molecules were used 15

to engineer the properties of the resulting SAMs – (3-aminopropyl)-triethoxysilane (APTES) and (3-glycidyloxypropyl)-trimethoxysilane (GLYMO). The deposition ratio of APTES and GLYMO onto substrates was precisely controlled during the fabrication of mixed SAMs via in situ control. Deposition takes place in two steps. First, a submonolayer of APTES is deposited, then the monolayer is completed by the deposition of GLYMO. A detailed description of the technique is provided in previous publications . The APTES/GLYMO ratio that represents the mixture of the 15,25

molecules is defined by the ratio of APTES with respect to the total amount of molecules, i.e. “APTES/(APTES+GLYMO)”. PLL and FITC coating. PLL (poly(L-lysine)) is a synthetic polymer, a homopeptide (cationic polypeptide with amino acid lysine as a repeat unit) commonly used to coat substrates as an attachment factor that improves cell adhesion. FITC (fluorescein isothiocyanate), a fluorescein molecule functionalized with an isothiocyanate reactive group replacing a hydrogen atom on the bottom ring of the structure, is used as an optical marker. It is reactive with respect to nucleophiles 13 ACS Paragon Plus Environment


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including amine and sulfhydryl groups on proteins and thus binds to PLL. The FITC-labeled PLL (P3069, Sigma-Aldrich) was mixed in solution with Gey's Balanced Salt Solution (GBSS, SigmaAldrich) to a final concentration of 10 µg/mL (i.e. 10 µg of PLL+FITC mixed in 1 mL of GBSS). The samples were coated with a drop (45 µl) of the solution and kept at room temperature for 1 hour avoiding any exposure to light. The remaining drop was then removed from the surface by rinsing with pure GBSS and washing away the residual protein that had not bonded to the SAM. The sample was subsequently rinsed in Milli-Q water for 5 minutes and purged in a N flow. After 2

the entire process had been completed, it was assumed that PLL+FITC had bonded to GLYMO but not to APTES as shown in fig. 3. The fluorescence imaging was performed via a Zeiss Apotome microscope using Zen software. In each case, 3 images were acquired with an exposure time of 600 ms and an illumination intensity of 3.04 V using a Zeiss HXP light source. The resulting fluorescence intensity values were determined by averaging the measured intensity values taken from a 30 µm × 30 µm area using ImageJ software. Neuronal culture. Cortical neurons were obtained from E18 Wistar rat embryos. Briefly, the cortex was dissected from the embryonic brain tissue and digested with trypsin-EDTA at 37°C, 5% CO , 100% humidity for 15 min. In order to remove trypsin, the cortex was washed 5 times 2

with Neurobasal medium (Life Technologies GmbH, Germany) supplemented with 1% B27 (Life Technologies, Germany), 0.5 mM L-glutamine, and 50 μg/mL gentamicin. Then the cortex was dissociated gently with a 1mL pipette. Cell clumps were allowed to settle for 2 min at room temperature. The supernatant was diluted in supplemented neurobasal medium and 75k cells per substrate per well were plated in a 12 well culture dish. The medium was replaced completely 4 hours after plating. In the following days, half of the medium was changed twice per week. Animal work was carried out with the approval of the Landesumweltamt für Natur, Umwelt und Verbraucherschutz

Nordrhein-Westfalen,

Recklinghausen,

Germany,

number

84-

02.04.2015.A173. Neuronal cultures were performed with primary cells pooled from siblings and repeated with non-sibling primary cells to avoid effects from a single individual. Multiple areas of each surface condition were imaged and the total cells analyzed (in the hundreds for each condition) is greater than many evaluations of cell-surface interactions. HL-1 cell culture. The cardiomyocyte-like cell line HL-1 was cultured in T25 flasks. Prior to seeding on MEAs, the chips were cleaned with 70% ethanol and coated with fibronectin (5 µg/mL). 14 ACS Paragon Plus Environment

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Upon reaching 100% confluency, the cells were passaged and seeded on top of the MEAs at 10k cells per chip. The chips were then placed in an incubator (37 °C and 5% CO ) for the cells to 2

mature. Claycomb medium, supplemented with 10% fetal bovine serum, 100U/ml-100µg/ml penicillin-streptomycin, 0.1mM norepinephrine, and 2 mM L-glutamine was replaced every day (100%) and two hours before the measurements. Live/dead imaging. Live/dead staining was performed using 1 µg/ml calcein-AM and 2 µM ethidium homodimer (both Life Technologies) in supplemented cell growth medium to stain live and dead cells green and red, respectively. Cells and dyes were incubated for 15 minutes in a 37 °C incubator or on a 37 °C hot plate (if performed after the electrical measurements). The samples were observed via a Zeiss Apotome microscope using Zen software. Three positions for each condition (the protein treated area, the untreated SAM, and the border between these two regions) were imaged in 2 separate cultures. ASSOCIATED CONTENT Supporting Information Available: Water contact angle of mixed monolayers of APTES and GLYMO as a function of molecular ratio (Figure S1). Surface potential of mixed monolayers of APTES and GLYMO as a function of molecular ratio (Figure S2) and AFM images of cleaned Si/SiO substrates and Si/SiO substrates covered by mixed monolayer (Figure S3). This material 2

2

is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest

ACKNOWLEDGMENTS



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The authors would like to thank Rolf Kutzner and Stephan Trellenkamp for their cooperation and discussions.

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(10) Santoro, F.; Dasgupta, S.; Schnitker, J.; Auth, T.; Neumann, E.; Panaitov, G.; Gompper, G.; Offenhäusser, A. Interfacing Electrogenic Cells with 3D. ACS Nano 2014, 8 (7), 6713–6723. (11) Santoro, F.; Panaitov, G.; Offenhäusser, A. Defined Patterns of Neuronal Networks on 3d Thiol-Functionalized Microstructures. Nano Lett. 2014, 14 (12), 6906–6909. (12) Kireev, D.; Brambach, M.; Seyock, S.; Maybeck, V.; Fu, W.; Wolfrum, B.; Offenhäusser, A. Graphene Transistors for Interfacing with Cells: Towards a Deeper Understanding of Liquid Gating and Sensitivity. Sci. Rep. 2017, 7 (1), 6658. (13) Ulman, A. Formation and Structure of Self-Assembled Monolayers. Chem. Rev. 1996, 96 (4), 1533–1554. (14) Schreiber, F. Self-Assembled Monolayers: From “Simple” Model Systems to Biofunctionalized Interfaces. J. Phys. Condens. Matter 2004, 16 (04), 881–900. 16 ACS Paragon Plus Environment

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(15) Markov, A.; Wolf, N.; Yuan, X.; Mayer, D.; Maybeck, V.; Offenhäusser, A.; Wördenweber, R. Controlled Engineering of Oxide Surfaces for Bioelectronics Applications Using Organic Mixed Monolayers. ACS Appl. Mater. Interfaces 2017, 9 (34), 29265−29272. (16) Heinz G.O. Becker In Organikum: Organisch-Chemisches Grundpraktikum; VEB Deutshcer Verlag der Wissenschaften, 1986; pp 257-258. (17) Morshed, K. M.; Jain, S. K.; McMartin, K. E. Acute Toxicity of Propylene Glycol: An Assessment Using Cultured Proximal Tubule Cells of Human Origin. Fundam. Appl. Toxicol. 1994, 23 (1), 38–43. (18) Regulska, M.; Pomierny, B.; Basta-kaim, A.; Starek, A.; Filip, M.; Lasoñ, W. Effects of Ethylene Glycol Ethers on Cell Viability in the Human Neuroblastoma SH-SY5Y Cell Line. 2010, 1243–1249. (19) Maybeck, V.; Schnitker, J.; Li, W.; Heuschkel, M.; Offenhäusser, A. An Evaluation of Extracellular MEA versus Optogenetic Stimulation of Cortical Neurons. Biomed. Phys. Eng. Express 2016, 2 (5), 055017. (20) Claycomb, W. C.; Lanson, N. A.; Stallworth, B. S.; Egeland, D. B.; Delcarpio, J. B.; Bahinski, A.; Izzo, N. J. HL-1 Cells: A Cardiac Muscle Cell Line That Contracts and Retains Phenotypic Characteristics of the Adult Cardiomyocyte. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (6), 2979–2984. (21) Eschermann, J. F.; Stockmann, R.; Hueske, M.; Vu, X. T.; Ingebrandt, S.; Offenhäusser, A. Action Potentials of HL-1 Cells Recorded with Silicon Nanowire Transistors. Appl. Phys. Lett. 2009, 95 (8), 1–4. (22) Blaschke, B. M.; Lottner, M.; Drieschner, S.; Calia, A. B.; Stoiber, K.; Rousseau, L.; Lissourges, G.; Garrido, J. A. Flexible Graphene Transistors for Recording Cell Action Potentials. 2D Mater. 2016, 3 (2), 025007. (23) Schottdorf, M.; Hofmann, B.; Kätelhön, E.; Offenhäusser, A.; Wolfrum, B. FrequencyDependent Signal Transfer at the Interface between Electrogenic Cells and Nanocavity Electrodes. Phys. Rev. E - Stat. Nonlinear, Soft Matter Phys. 2012, 85 (3), 1–7. (24) Nelea, V.; Nakano, Y.; Kaartinen, M. T. Size Distribution and Molecular Associations of Plasma Fibronectin and Fibronectin Crosslinked by Transglutaminase 2. M.T. Protein J 2008, 27(4), 223. (25) Markov, A.; Greben, K.; Mayer, D.; Offenhäusser, A.; Wördenweber, R. In Situ Analysis of the Growth and Dielectric Properties of Organic Self-Assembled Monolayers: A Way to Tailor Organic Layers for Electronic Applications. ACS Appl. Mater. Interfaces 2016, 8 (25), 16451–16456. (26) Gilles, S.; Winter, S.; Michael, K. E.; Meffert, S.; Li, P.; Greben, K.; Simon, U.; Offenhäusser, A.; Mayer, D. Control of cell adhesion and neurite outgrowth by patterned gold nanoparticles with tunable attractive or repulsive surface properties. Small 2012, 8 (21), 3357–3367



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TOC graphic:



Schematic of a SiO2-terminated Si sample covered with mixed molecular SAM, partially coated with PLL and finally cultured with cortical neurons on the surface. Cell density and live/dead ratio are analyzed on the Si/SiO2 area covered only with the SAM (a), on the border of the PLL-coated area (b) and inside the PLLcoated area (c), the size of the images is 580 µm x 580 µm. The schematics of the molecular layers in (a) and (c) are out of scale in order to visualize the assumed molecular arrangement of the positively charged APTES and neutral GLYMO in the SAM and the PLL, which seems to adhere to the GLYMO. 82x46mm (300 x 300 DPI)

ACS Paragon Plus Environment

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(a) Schematic of rat cortical neurons directly immobilized on the SAMs with different APTES/GLYMO ratios. (b-g) Examples of fluorescence microscope images (580 µm x 580 µm) of the cultures for different ratios ranging from pure GLYMO (b) to pure APTES (g), the numbers provide the percentage of APTES in the mixed SAMS (c-f), living cells are marked with calcien-AM (green) and dead cells are marked with ethidium homodimer (orange). (h) and (i) display the resulting live/dead ratio and the density of live and dead neurons as a function of APTES concentration. The values on the bars in (i) represent the total amount of cells (live and dead) averaged over several areas of size 580 µm x 580 µm (0.3364 mm2). 112x85mm (220 x 220 DPI)



(a) Fluorescence microscope image of samples with mixed SAM (the percentage given in the image refers to the fraction of APTES in the layer) coated with PLL+FITC (exposure time 600ms) and (b) resulting intensity of PLL+FITC fluorescence as a function of the APTES/GLYMO ratio. 82x28mm (300 x 300 DPI)


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(a) Schematic of neurons immobilized on PLL-treated SAMs with different APTES/GLYMO ratios. (b-g) Examples of fluorescence microscope images (580 µm x 580 µm) of the cultures for different ratios ranging from pure GLYMO (b) to pure APTES (g), the numbers provide the percentage of APTES in the mixed SAMS (c-f), living cells are marked with calcien-AM (green) and dead cells are marked with ethidium homodimer (orange). (h) and (i) display the resulting live/dead ratio and the density of live and dead neurons as a function of APTES concentration. The values on the bars in (i) represent the total amount of cells (live and dead) averaged over several areas of size 580 µm x 580 µm (0.3364 mm2). 82x62mm (300 x 300 DPI)



Schematic of the investigated MEA structure (a), image of the MEA structure and electrode array (b), and action potential (time trace and single action potential) of HL-1 cells on MEAs coated by APTES/GLYMO mixed monolayers and fibronectin for 10% APTES (c-d) and 75% APTES (e-f). 82x80mm (300 x 300 DPI)


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Action potential as a function of the molecular ratio of the mixed monolayers in terms of APTES content for the 1st (solid black) and 2nd (open blue) cell culturing. The 1st and 2nd runs were performed on one and the same MEA for each concentration, for the second culturing only the cells were removed. 82x57mm (300 x 300 DPI)



TOC - Graphic for manuscript 354x151mm (150 x 150 DPI)


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Engineering of Neuron Growth and Enhancing Cell-Chip

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