Fabrication of nanostructures on the surface of SU-8 microprobes
To improve neuronal attachment and neurite outgrowth, we fabricated nanostructures on the surface of SU-8 microprobes, as shown in Fig. 1. Conventional photolithography and nanosphere lithography were used to fabricate the microprobe and nanostructures, respectively. The overall fabrication process is shown in Fig. 2. SU-8 2002 (MicroChem Corporation, Newton, MA, USA) was first spin-coated onto a glass wafer (Borofloat 33; Schott, Jena, Germany) for 30 s at 1500 rpm (Fig. 2a). The wafer was baked for 10 min at 120 °C; the resulting SU-8 film was ~ 1.6 μm thick. Next, a 20-nm-thick titanium layer and 150-nm-thick gold layer were deposited using an e-beam evaporator (SRN-200; Sorona Inc., South Korea). Ti functions as an adhesive layer between the SU-8 and Au layers. Next, AZ5214 photoresist (AZ 5214, Microchemicals GmbH, Germany) was spin-coated at 1550 rpm to a target thickness of ~ 2.2 μm and baked for 45 s at 120 °C. As a mask for electrode patterning, a ~ 100 nm chromium (Cr) layer was deposited using an e-beam evaporator. AZ5214 was removed by sonication in acetone (JT Baker, Phillipsburg, NJ, USA), and Ti/Au layers were patterned using a dry-etching system (FabStar; Top Technology Ltd., South Korea) for 90 s at 2 m Torr with Cl2 and Ar flow rates of 15 and 5 sccm, respectively. The inductively coupled plasma (ICP) source power and bias power with radio frequency (RF) were 500 and 250 W, respectively. After removing the Cr mask layer using a CR-7 etchant (Cyantek, Fremont, CA, USA), the electrode patterns were fabricated as shown in Fig. 2b.
To fabricate nanostructures around the electrodes, a second SU-8 layer of identical thickness to the first was added using the same procedure (Fig. 2c). For the selective generation of nanostructures around the Au electrodes, a Cr layer was deposited and patterned by a lift-off process using a positive photoresist (AZ 5214) (Fig. 2d). For nanosphere lithography, the substrate with SU-8 microprobes was cut into 18 × 18 mm pieces, and coated with a monolayer of 0.3-µm-diameter polystyrene nanoparticles (PS NP suspension, 5.0% coefficient of variation; Spherotech, Lake Forest, IL, USA) by transferring the PS monolayer to the substrate by the water–air interface [22]. The size of the PS nanobeads was reduced by oxygen plasma reactive-ion etching (RIE) (FabStar; Top Technology Ltd., South Korea) for 1 min with an O2 flow rate of 30 sccm and power of 50 W. A second 30-nm-thick layer of Cr was deposited in the spaces among the PS nanobeads using an evaporator (Fig. 2e). The PS nanobeads were dissolved in toluene at 40 °C for 30 min with sonication, and nanostructures were generated by RIE with an O2 flow rate of 30 sccm and power of 40 W for 4 min. The Cr layer on SU-8 functions as an etch mask for the fabrication of nanostructures. Finally, the residual Cr layer (Fig. 2d, e) was removed using CR-7 etchant for 1 min and the substrate was cleaned in deionized water and disinfected in 100% ethanol (Merck, Darmstadt, Germany) for 24 h in the dark. The substrate was washed three times in sterile distilled water and dried in a laminar flow hood.
Characterization of the nanostructured surface
A field-emission scanning electron microscope (FE-SEM) (S-4800; Hitachi, Tokyo, Japan) was used to observe the surface morphology of the nanostructured microprobes. An AFM (NX10; Park System Corp., South Korea) in non-contact mode in air at room temperature was used to measure the surface roughness and topography of the nanostructured surface.
PC12 cell culture
PC12 cells (CRL-1721; ATCC, Manassas, VA, USA) are extensively used in neurobiological and neurochemical studies [23, 24]. In this study, PC12 cells were used to investigate the responses of neurons to the nanostructured and non-nanostructured microprobes in vitro. PC12 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with l-glutamine (Gibco, Grand Island, NY, USA) supplemented with 10% horse serum (Sigma-Aldrich, St. Louis, MO, USA), 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA, and Thermo Fisher Scientific, Rockford, IL, USA) and 1% antibiotic–antimycotic solution (penicillin 10,000 units/mL, streptomycin 10,000 μg/mL, Fungizone (amphotericin B) 25 μg/mL; Gibco Life Technologies, Grand Island, NY, USA) at 37 °C in 5% CO2 and subcultured at least twice weekly. For differentiation, PC12 cells were seeded onto plates at 2.5 × 105 cells/mL and treated with 50 ng/mL nerve growth factor (NGF; BD Biosciences, Bedford, MA, USA) for 7 days. NGF-supplemented medium was changed every 2 days.
Statistical analysis
Neurites longer than 25 μm (i.e., the average cell body diameter) were enumerated. Three areas were randomly selected on each substrate by scanning from top to bottom under a microscope. Experiments were conducted in triplicate. Comparisons were carried out using Student’s t test. Results are presented as means ± standard errors, and a value of p < 0.001 was considered to indicate statistical significance.