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Letter

Superior Robust Ultra-thin Single Crystalline Silicon Carbide Membrane as a Versatile Platform for Biological Applications Tuan-Khoa Nguyen, Hoang-Phuong Phan, Harshad Kamble, Raja Vadivelu, Toan Khac Dinh, Alan Iacopi, Glenn Walker, Leonie Hold, Nam-Trung Nguyen, and Dzung Viet Dao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15381 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 17, 2017

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ACS Applied Materials & Interfaces

Superior Robust Ultra-thin Single Crystalline Silicon Carbide Membrane as a Versatile Platform for Biological Applications Tuan-Khoa Nguyen,

Toan Dinh,

†

∗,†

†

Hoang-Phuong Phan,

Alan Iacopi,

†

Glenn Walker,

†

Harshad Kamble,

Leonie Hold,

Dzung Viet Dao

†

†

†

Raja Vadivelu,

Nam-Trung Nguyen,

†

†, ‡

Queensland Micro-Nanotechnology Centre, Grith University, Nathan QLD 4111, Australia ‡School of Engineering, Grith University, Gold Coast QLD 4217, Australia E-mail: khoa.nguyentuan@grithuni.edu.au Abstract Micro-machined membranes are promising platforms for cell culture thanks to their miniaturization and integration capabilities. Possessing chemical inertness, bio compatibility and integration, silicon carbide (SiC) membranes have attracted great interest towards biological applications. In this paper, we present the batch fabrication, mechanical characterizations, and cell culture demonstration of robust ultra-thin epitaxial deposited SiC membranes. The as-fabricated ultra-thin SiC membranes, with an ultrahigh aspect ratio (length/thickness) of up to 20,000, possess high a fracture strength up to 2.95 GPa and deformation up to 50

µm.

A high optical transmittance of above

80% at visible wavelengths was obtained for 50 nm membranes.

The as-fabricated

membranes were experimentally demonstrated as an excellent substrate platform for

1

ACS Paragon Plus Environment

†

and


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bio-MEMS/NEMS cell culture with the cell viability rate of more than 92% after 72 hours. The ultra-thin SiC membrane is promising for

in vitro

observations/imaging of

bio objects with an extremely short optical access.

Keywords Cell culture, membrane, LPCVD, silicon carbide, ultra-thin

Introduction Culturing cells in articial environments is an indispensable approach for the research of drug, tissue engineering and stem cell. 1 Commercial plastic-wares such as polystyrene Petri dishes, asks and well plates or polymer-supported membranes 2 are commonly employed as the culturing substrates. Current cell culture techniques typically rely on bulky Petri dishes and micro-well plates which have volumes in the milliliter and microliter scales. The technical challenge is that the thick bottom of the Petri dish and well plate limits the optical access for investigations of

in vitro cells. Thanks to the developments of micro/nano-electromechanical

systems (MEMS/NEMS), tissue engineering and cell culture have been being driven towards integrated lab-on-chip models utilizing the miniaturization and integration capabilities of MEMS platforms. 3 As such, the application of micro-machined membranes for cell culture has greatly scaled up the surface-to-volume ratio and mass transfer capabilities and allows the precise applications at the nanoliter scale in cell culture processes. 4 Owing to their unique properties including thin and transparent substrate and bio-compatibility, micro-machined membranes have been widely deployed for bio applications in the last decade, facilitating on-chip culturing and observation. Additionally, the use of micro-machined membranes as the growth substrate also enables the direct application of mechanical stimuli for the study of cell stretching devices and cell micro environment. 5 In terms of semiconductor materials, silicon has been early used as the growth substrate 2


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owning to its wide availability and MEMS compatibility. 6 However, there are existing limitations that hinder its usage in biological applications. For instance, the intrinsic crystal structure and bonding of Si atoms leads to potential breakage due to the presence of bio objects and the reaction of Si with organic tissue could result in a chronic astroglial eect, reducing the longevity of the devices. 7 In many studies, nitrogen-containing materials, such as GaN, AlN, AlGaN, and especially Si 3 N4 , have been reported as versatile and compatible materials for bio applications. 8 Nano-thin Si3 N4 membranes have been also tailored for a highly sensitive nanopore sensing platform with sub-microsecond temporal resolution 9 and low noise solid-state nanopore substrate. 10 Furthermore, the use of thin micro-machined membrane enables the on-chip integration including molecular-level imaging/probes/observation and 3D cultures. 11 As such, ultra-thin silicon nitride membranes have been employed as a substrate platform for nano-machined solid-state nanopores using focused transmission electron microscopy (TEM) beam, for DNA/protein/virus sequencing and translocation. 1214 However, the presence of nitrogen in those nitrogen-based materials may cause unexpected interactions with nitride-containing objects, 15 which typically exist in bio cells. Possessing chemical inertness, good bio-MEMS compatibility and integration, 6,1622 SiC materials have attracted a great deal of interest towards biological applications. 2325 The use of SiC-based devices yields long-term stability for

in vitro studies, enabling the development of integrated bio

lap-on-chip while minimizing side eects. However, to date, due to diculties in the deposition and fabrication processes, there are limited reports on sub-hundred nanometers SiC membranes which are employed for cell culture. In this paper, we present robust, transparent, and bio-compatible SiC membranes which are ultra-thin and have an ultra-high aspect ratio of up to 20,000. The membranes were fabricated from low pressure chemical vapor deposition (LPCVD) SiC-on-Si wafers with the large deformation when pressurized, allowing the application of strain/stress to grown cells. The ultra-thin SiC membranes possess high fracture strength of up to 2.95 GPa, facilitating the direct growth of mouse 3T3 broblasts without any additional supporting layer. Fur-

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thermore, this cell culturing platform also allows the observation/imaging within only one substrate device using inverted microscopy and uorescence spectroscopy. The observation of cell growth in periods up to 72 hours shows a high cell viability rate of 92.7 %, indicating the excellent bio compatibility of the as-developed SiC membranes. Additionally, we demonstrated the self-integrated heater in the SiC membrane, which enables the application of the thermal therapy/treatment to the bio objects. The as-fabricate SiC membranes, which are only tens of nanometers thin, can hold the weight of culture media, providing the extremely short working distance for cell imaging and observation.

Results and discussion Fabrication of ultra-thin SiC membranes The process ow of the membrane fabrication is briey shown in Fig. 1(a). We optimized the batch fabrication of SiC membranes with dierent sizes and thicknesses which can be used in a wide range of biological applications. The double sided LPCVD using an alternatively supply epitaxy technique of SiC on Si wafer is presented in the Supporting Information, Section 1. Subsequently, a thin deposited SiC lm of up to 50 nm on Si wafers with a diameter of up to 300 mm were achieved. A standard semiconductor lithography technique was used to transfer patterns to one side of the SiC coated wafer. Subsequently, inductive coupled plasma (ICP) etching by STS etcher was performed to pattern SiC on the backside of the wafer, forming a windows mask for the subsequent wet etching of the Si by KOH. Si was wet etched along specic crystal planes creates square holes and the process is almost self-limiting. Following KOH etching, the membranes were decontaminated using an RCA 2 clean (HCl: H2 O2 : H2 at 70◦ C). Fig. 1(c) shows an atomic force microscopy (AFM) image of the SiC membrane roughness. Evidently, surface roughness of the SiC membrane is in the range of sub 4 nm peak to valley roughness with the root mean square roughness below 1 nm. 4


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a)

Si SiC

Mask Photoresist

b)

Si wafer Epitaxial deposition of 3C- SiC on both sides Exposing photoresist nm

4000 pxl/div

5

8

Developing photoresist

4 0

4

2.5 5 (nm)

3

Plasma etched back side 3C SiC

0 2 -4

7.5

1

Wet etching Si to form SiC membranes

-8 0

Histogram

c) Mask

-2.5

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1

2

3 (µm)

4

5

Figure 1: a) Process ow of the SiC membranes fabrication. b) Arrays of square SiC membranes on the wafer with various sizes. Inset: SEM image of 1x1 mm 2 square membranes. Scale bar, 20 mm. c) AFM image of 5x5 µm2 area on the SiC membrane surface, surface roughness of SiC membranes are in the range of sub 4 nm peak to valley roughness with the root mean square roughness below 1 nm.

Large deection of ultra-thin SiC membrane Figure 2(a) presents an approximation modal shape of the membrane 's deection with applied pressure. The as-manufactured SiC membranes are intended as a cell growth substrate, facilitating further studies of cell stretching and mechano-transduction. Therefore, the deformation of ultra-thin SiC membrane under pressure was characterized using the Von Karman model for non-linear-large-deection of rectangular membranes subjected to a uniformly distributed load q (see Supporting Information, Section 2). To simplify the solution for the non-linear problem, the rst order approximation can be used to derive the out-of-plane

5



̟x

̟y

c)

w=W0cos L cos L

50

4t C σ δ + 4Ef(v) δ3 L2(1-v) L2 1 0

50 nm

40 30

L

L/2

b)

q= 60

1 0.8 0.6 0.4 0.2 0 L L/2 0 1

Center deflection (μm)

Deformation (a.u)

a)

Total tensile stress (a.u)

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0.5

σ0 =0 σ0 =200MPa σ0 =500MPa σ0 =1000MPa

20 10 0 50

100 nm

40 30 20 10 0 40

150 nm

30

0

20

L

L

L/2

L/2

10 0

0

0

40

80

120

160

Pressure (kPa)

Figure 2: a) First order approximation of the large deection of square SiC membranes under dierential pressure. b) Von Mises stress distribution on the SiC membrane. c) Theoretical analysis of the center 's deection of 1x1 mm 2 membranes with thicknesses of 50 nm, 100 nm, and 150 nm and residual stresses of 0, 200, 500, 1000 MPa. The residual stress minimizes the amplitude and the non-linearity of the membranes 's deection. deformation of the thin membrane:

w(x, y) = W0 cos

πy πx cos L L

(1)

where W0 is the maximum deection at the center of membrane, L is the side length of the square membrane. 26 The solution for membrane deection would be signicantly dierent when considering membrane residual stress σ0 . 27 The energy minimization method has been utilized to solve the general deection formula of square membranes. However, the resulting non-linear equations of total potential energy must be solved numerically, depending on specic value of pressure q and residual stress σ0 . To obtain the solution for Eq. 1, the deformation of the membrane 's center W0 versus the applied external load q should be solved. The diculty of considering the membrane 's residual stress can be simplied by 6


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b)

a) Autofocus

Initial height

SiC membrane Computer Deformed height

Center point SiC membrane

Pressure controller

Specimen stage

Marker

Re-focused height

d) fitting line measured data

50 40

50 nm

30 20 10 0

0

10 20 30 40

50 60

e)

40

fitting line measured data

30

100 nm

20 10 0

0

Pressure (kPa)

20

40

60

80


c)


XYZ stage


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pressure (kPa)

40

fitting line measured data

30

150 nm

20 10 0

0

20 40 60 80 100 120

Pressure (kPa)

Figure 3: a) Conguration of the bulging test of SiC membranes under controlled pressure through Eveow OB1 pressure controller with a resolution of 0.1 kPa. b) Optical image of the membrane's center, the height was measured using the focus indicator of an Olympus MX50 . Load versus deection of center points of 1 ×1 mm2 square SiC membranes with thicknesses of c) 50 nm, d) 100 nm, and e) 150 nm. Scale bar, 300 µm. dividing applied pressure q into two components: q1 to oset the eect of residual stress and

q2 to deform the membrane, then the relationship between applied pressure and deection of the membrane's center δ can be deduced by: 28

q = q1 + q2 =

4t 4f (ν) E 3 C σ δ + δ 1 0 L2 L2 1 − ν

(2)

where L, E and ν are the edge length, Young 's modulus and Poisson 's ratio of the SiC membrane, respectively; C1 = 3.41 and f (ν) = 1.37(1.446-0.427 ν ) are the empirical constant and empirical function for square membranes, respectively. Figure 2(c) plots the theoretical analysis for the load-deection relationship when considering the built-in residual stresses of 0, 200, 500, and 1000 MPa in the 1 ×1 mm2 SiC membranes with thicknesses of 50, 100, and 150 nm. It can be seen that the deection of SiC membranes is non-linear with respect 7



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to the thickness and applied dierential pressure. Additionally, the membrane 's residual stress decreases the amplitude and the non-linearity of the membrane 's deection, which is described by the rst term in the right-side of Eq. 2. Based on the solution for the membrane deformation, the normal stress σx , σy , and the Von Mises stress σv of the deformed SiC membranes can be given by (see Supporting Information, Section 2):

1 2πy ∂ 2Φ 1 2πx ∂ 2Φ ∗ ∗ + cos σy = + cos =σ =σ σx = ∂y 2 1−ν L ∂x2 1−ν L q 2 π Et(1 + ν)δ 2−νδ σ∗ = 1+ σv = σx2 − σx σy + σy2 2 2L (1 − ν) 4 t

(3)

It should be noted that in ultra-thin membranes, the total tensile stress is much greater than the shear stress as the eect of bending stress can be negligible throughout the membrane, except the region close to the edges. 29 Additionally, the middle of the four edges experienced the maximum total stress as shown in Fig. 2(b). In many bio applications, the thin membranes are used as a micro vacuum chamber, where they are expected to withstand a dierential pressure of one atmosphere (100 kPa). 30 The SiC membranes with thicknesses of 50, 100, and 150 nm in 5 ×5 mm2 chips were prepared for the load-deection measurement. Figure 3(a) illustrates the experimental bulging apparatus, a typical technique for characterizing the mechanical properties of thin membranes. 27,28 A stainless-steel specimen stage has a designated inner tunnel where air, the pressurized medium, was pumped at precisely controllable pressures using the Eveow OB1 Mk3 pressure controller. The SiC membranes supported by outer Si frames were axed to the stage and sealed by a soft O-ring, creating an enclosed chamber for the pressurizing test. The specimen stage was placed on an XYZ stage, which has a precision of 1 µm for z-axis, in order to adjust the position of the SiC membranes. The deection of the SiC membrane 's center δ under applied pressure q was measured using the auto focus tool of the Olympus MX50 (Fig. 3(b) and Supporting Information, Section 3). It should be pointed out that in the present measurement, we employed

8


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air as the pressurized medium to eliminate the unexpected eect of surface tension in the interface of the membranes when using water. Figure 3(c), (d), and (e) show the measured deections of the membrane 's center versus applied pressures of 1 ×1 mm2 SiC membranes with thicknesses of 50 nm, 100 nm, and 150 nm, respectively. Fitting the experimental data with the aforementioned theoretical model, the built-in residual stresses in the 1 ×1 mm2 SiC membranes with thicknesses of 50 nm, 100 nm, and 150 nm could be estimated as 200, 300, and 350 MPa, respectively.

Fracture strength Figure 4(a) shows the recorded data of the burst test to evaluate the fracture strength of the as-fabricated SiC membranes with the same conguration as used for the bulging test, except the membranes were pressurized until fractured. The breaking pressure of each membrane was precisely recorded by the Eveow software. The fracture pressures of 50 nm and 100 nm thick SiC membranes were found to be 98 kPa and 128 kPa, respectively. It should be noted that the fracture pressure of the 50 nm thick SiC membrane is comparable with that of standard 100 nm thick Si 3 N4 membranes, while 100 nm SiC membrane exhibited fracture pressure of approximately 30 % higher than that of Si 3 N4 membranes with the same dimensions. Additionally, the SiC membranes with a surface roughness below 1 nm have a higher fracture strength of approximately 10 % in comparison to those with a surface roughness above 1 nm. Combining the initial residual stress of the membranes, the total fracture stresses of 50 nm, 100 nm and 150 nm thick 1 ×1 mm2 SiC membranes were derived and shown in Table 1. The fracture strength of the as-fabrication SiC membranes, with an aspect ratio of up to 20,000, is comparable with other micro-machined membranes which have much lower aspect ratios (e.g. well below 10,000). This indicates that the SiC membranes could retain the high fracture strength despite their sub-hundred nanometer thickness.

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b)

150

Transmittance (%)

a) Burst pressure (kPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

100 50 0

c)

Si N

10

Si C 0n

m

50

*S SiC iC 10 10 nm 0n 0n m m

Cells

100

50 nm 100 nm 150 nm

90 80 70 60

400 450 500 550 600 650 700

Wavelength (nm)

d)

Imaging

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5V

3V

SiC membrane Electrode

Integrated heater

25

100

Temperature (oC)

Figure 4: a) Fracture strength of 1 ×1 mm2 SiC membranes with thicknesses of 50 nm, 100 nm, in comparison with standard 100 nm thick Si 3 N4 membranes. *SiC membranes with surface roughness above 1 nm have 10 % lower fracture strength. b) Optical transmittance of the 50 nm, 100 nm, 150 nm SiC membranes for visible wavelengths. c) Demonstration of SiC integrated heater. d) Infrared images of the integrated heater at applied voltages of 3V and 5V, a relatively high temperature of 82 ◦ C was obtained when supplying 5V. Scale bar, 300 µm.

Optical transmittance Being developed as a transparent substrate for cell growth and imaging, high transparency is an important characteristic for the as-manufactured SiC membranes. Therefore, the optical transmittance of 50 nm, 100 nm, and 150 nm thick SiC membranes was evaluated at visible wavelengths as shown in Fig. 4(b). In the measurement, the intensity of the transmitted light through the membranes was recorded by a photo detector (Nanospec AFT 210). As can be seen from the result, a high transmittance of above 80 %, in the wavelength ranging from 350 to 700 nm, was obtained for 50 nm thick membranes while the 100 nm and 150 nm membranes exhibited good transmittances well above 60 %. Additionally, 100 nm and

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Table 1: Fracture strengths of 1 ×1 mm2 SiC membranes in comparison with standard Si 3 N4 membranes; ∗ SiC membranes with the surface roughness above 1 nm. Type A B C D

Name 100 nm Si3 N4 50 nm SiC 100 nm SiC∗ 100 nm SiC 150 nm SiC

Fracture strength (GPa) 1.85 2.37 2.75 2.95 > 3.0

150 nm membranes possess high transmittances at the wavelengths of 510 nm and 410 nm, respectively. It should be noted that the interference of the certain wavelength with the SiC layer, resulting in a lower transmittance (i.e. ∼400 nm with 100 nm membrane, ∼600 nm with 150 nm membrane). The excellent optical transmittance of the as-fabricated membranes conrms the capability of either conventional top-down or inverted bottom-up microscopy.

Integrated heater for cell thermal stimulations The as-fabricated ultra-thin SiC membranes can be a versatile growth substrate owing to the MEMS miniaturization and integration capabilities. As such, an integrated heater could be formed in the SiC membrane by conventional MEMS processes, as shown in Fig. 4(c). Aluminum electrodes were sputtered using SNS Sputterer with a thickness of 100 nm, creating a SiC heater in the middle of the parallel electrodes since the SiC thin lms deposited on Si were unintentionally n-type doped with a conductivity of 114.8 Sm −1 . Utilizing the Joule heating eect and the good conductivity of the SiC lm, a relatively high temperature above 82◦ C was obtained using Xeneth IR camera when applying a relatively small applied voltage of 5V. This is suitable and sucient for the bio cell thermal stimulation. Since the SiC membranes were released from the Si substrate, the leakage current and heat lost due to conduction are completely eliminated. This is favorable for the thermal stimulation of cells since the required voltage and power consumption are relatively small. Additionally, thanks to the resistive characteristic of the thin SiC membranes, the temperature of the integrated heater is controllable by varying the applied voltage, which is versatile for the 11



a)

SiC membranes 0h

e)

12h

24h

48h

72h

12h

24h

48h

72h

Standard dishes 0h

f)

on standard dishes

on SiC membranes 100

100

24 hours

95

Cell viability (%)

d)

c)

b)

Cell viability (%)

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90 85 80 75 70

1

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72 hours

95

1

2

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Sample No.

Sample No.

Figure 5: a) Experimental apparatus for mouse 3T3 broblasts cells cultured on the SiC membranes and standard dishes located in arrays of PDMS reservoirs. Fluorescent images of the cell attachment on b) the SiC membranes and c) the standard dishes after 72 hours. Cell attachment and growth after 12, 24, 48, and 72 hours on d) the SiC membranes and e) standard dishes. Scale bar, 100 µm. f) Measured cell viability of 5 dierent SiC membranes with an average viability rate of more than 92.7 % after 72 hours which is comparable with that of the standard dishes. thermal stimulation of bio cells.

Cell culture Prior to cell culturing, to remove residual organic, the SiC membranes were sterilized by 80 % ethanol and cleaned 3 times by 1 × phosphate-bueredsaline (PBS) solution. They were then inserted in an ultra violet chamber and treated under low-intensity exposure for 20 minutes. Subsequently, the SiC membranes were immersed with a fresh medium and incubated at 12


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37◦ C and 5% CO2 for 1 hour before used for seeding, ensuring its cytological compatibility. Mouse 3T3 broblast cells were cultured in T75 asks using Gibco dulbecco's modied eagle medium nutrient mixture F-12 (DMEM/F-12) as the basal medium. Subsequently, the medium was enriched with 10 % fetal bovine serum (FBS) and supplemented with 1 % penicillin before culturing cells. The cell cultivation was maintained at the temperature of 37◦ C and 5% CO2 atmosphere in a standard incubator to achieve 80 % conuency. Afterwards, 3T3 broblast cells were trypsinized from the asks using TrypLE Express enzyme. The cells were subsequently suspended in a fresh medium with a concentration of 10 4 cells per milliliter. Finally, the 3T3 broblasts cells were cultured on SiC membranes with the seeding density of 5 × 103 cells per milliliter, all reservoirs were then continuously incubated at 37◦ C and 5% CO2 atmosphere in 18 hours, facilitating the attachment and growth of the cells. To track the cells growth, the SiC membranes and benchmarking dishes were imaged using a phase contrast microscope (e.g. Nikon Eclipse Ts2). Figure 5(a) shows the cell culturing apparatus using polydimethylsiloxane (PDMS) reservoirs attached to the surface of 10 ×10 mm2 Si chips, forming open wells for the culturing process. Mouse 3T3 broblasts cells were then grown on the SiC membranes after sterilization and cleaning of the substrates. Taking advantage of ultra-thin, chemically inert and highly transparent SiC membranes, both conventional microscopy and uorescence microscopy protocols for cell mapping and imaging can be directly carried out on the growth platform without the need for harvesting and re-depositing cells. For benchmarking, the cells were simultaneously cultured on standard cell culture dishes with the same preparation and growth conditions used for the SiC membranes. The uorescent images of the attachments of the as-grown cells after 72 hours on the SiC membranes and the standard dishes are illustrated in Fig. 5(b) and (c), respectively. The growth progress of the cells was monitored and shown after periods of 12, 24, 48, and 72 hours using a Nikon Eclipse Ts2-S-SM microscope (Fig. 5(d)). The cell vitality test, xing and immunouorescence staining for imaging procedures can be found in the Supporting Information, Section 4 and 5. Healthy

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cell-to-substrate adhesion and growth were conrmed in the phase contrast image after 12, 24, 48, and 72 hours. As can be seen, these cells continuously exhibit attened and elongated morphology as well as strong connection to their neighbors while maintain a strong adhesion to the SiC membrane substrate. At 72 hours, uorescent imaging showed evidence of broblast morphology with prominent stress bers. The obtained results indicate the excellent cytological compatibility of the as-manufactured SiC membranes when compared with the result of the standard dishes shown in Fig. 5(e). High rates of the cell viability for the SiC membranes and the standard dishes are shown in Fig. 5(f). As such, the average viability rate of the cells cultured in the SiC membranes were obtained as high as 86.6 % and 92.7% after 24 and 72 hours, respectively, which are comparable with the gures of the standard dishes (e.g. 90.0 % and 94.3%). The high cell viability rate is correlated to the excellent chemical inertness of the as-fabricated SiC membranes, minimizing side eects of the chemical interaction between the cells and substrate. Furthermore, the use of SiH 4 and C3 H6 in the deposition of SiC in the LPCVD process produced a large number of SiH x and Cx Hy terminal groups on the SiC surface, facilitating the adhesion and attachment of the cells to the SiC surface. 25 In conclusion, the large deection and high fracture strength of the as-fabricated ultrathin SiC membranes were conrmed through the bulging and burst tests. These properties are promising for the direct application of mechanical stimuli, enabling the studies of cell stretching in

in vitro environments.

Additionally, the SiC membrane can be an integrated

heater which is convenient for the application of thermal stimulations to bio cells, with a generated temperature as high as 82 ◦ C. The optical transparency of the SiC membranes was also determined in which 50 nm membranes exhibited a high transmittance of above 80 % at the visible wavelengths while the gures for 100 nm and 150 nm membranes were well above 60%. The excellent bio-compatibility of the SiC membranes as a cell culturing substrate was demonstrated in which mouse 3T3 broblasts cells attened and elongated, indicating the strong attachment to the SiC substrate and neighboring cells. Furthermore, a high cell

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viability rate of more than 92 % of cell cultured in the SiC membranes was obtained. The miniaturization and integration of the culturing platform using the ultra-thin SiC membranes are promising for integrated and miniaturized cell culturing devices in which mechanical and thermal stimuli can be directly applied without the need for external equipment. The capability of cell observation/imaging is also strengthened by the high optical transparency of the ultra-thin SiC membranes. The use of ultra-thin SiC membranes can enable the single cell analysis (e.g. single cell in one SiC well) with conventional optical tools. Furthermore, bio cells/DNA translocation or sequencing, which typically requires very thin substrate, are also possible using the ultra-thin SiC membrane as the culturing platform.

Acknowledgement This work has been partially supported by Australian Research Council grants LP150100153 and LP160101553. This work was also supported by the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and micro-fabrication facilities for Australia 's researchers.

Supporting Information Available The following les are available free of charge.

Supporting Information Detailed SiC-on-Si deposition, theoretical analysis of large deformation of the SiC membrane, methodologies for the measurement of the non-linear deformation of the ultra-thin SiC membrane, cell vitality test, cell xing and immunouorescence staining for imaging.

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Graphical TOC Entry Imaging

Cells

Integrated heater

Large deformation

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72 hours

Cell viability (%)

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Superior Robust Ultrathin Single-Crystalline ... - ACS Publications

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