Chemicals and materials
Chemicals for surface preparation
Octadecyltrichlorosilane (OTS, 97%) and (11-mercaptoundecyl)trimethoxysilane (MUTMS, 95%) were purchased from Gelest. Toluene (HPLC grade) was purchased from Fisher Scientific. Ultraflat silicon (100) wafers (N-type) were purchased from Sigma-Aldrich Corporation. Sulfuric acid and hydrogen peroxide were purchased from Sigma-Aldrich Corporation.
Materials for CaM expression, purification, and reaction
Luria–Bertani (LB) broth, used to grow the cell culture, and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) disulfide reducing agent were purchased from Sigma-Aldrich Corporation. Calcium chloride (CaCl2) was purchased from Flinn Scientific. CaM was purified using chitin beads from New England Biolabs. 2-anilinonaphthalene-6-sulfonic acid (ANS) used for fluorescence experiment and SDS-PAGE were obtained from Invitrogen Corporation. Calmodulin—dependent protein kinase I (299–320) binding domain, which is a putative CaM-binding region, was obtained from AnaSpec. All the solution was prepared with water from a Millipore Direct-Q UV water purification system.
Protein expression and purification
Purification and expression of genetically engineered CaM with cysteine on N-terminus is based on instructional manual prepared by New England Biolabs [20]. In order to prevent dimer formation, TCEP was applied in protein solution. SDS-PAGE was used to confirm the CaM purity (see Additional file 1).
In our experiment, we used 2,6-anilinonaphthalene sulfonate (ANS) fluorescent probe to test the bio-activity of the purified solution-state Ca2+/CaM. It is well established that solvent-exposed hydrophobic surfaces are formed upon Ca2+ binding to CaM, and ANS binds to the hydrophobic parts of proteins through polar interactions and can be monitored by the increase in fluorescence emission intensity, which demonstrates the activity of Ca2+/CaM indirectly [21]. When EDTA is added to the solution, Ca2+ is removed from Ca2+/CaM, and the hydrophobic binding pocket disappears. This conformational change causes the release of bound ANS from CaM to the aqueous solutions, leading to a decrease in fluorescence intensity. Therefore, by monitoring the fluorescence intensity variation we can confirm the conformational change in CaM, which is an indication of CaM viability [22].
During the experiment, the protein was labeled with a 1:1 ratio of ANS overnight at room temperature followed by dialysis against the same buffer. 1 µL increments 0.5 mmol L−1 EDTA was added into the 400 µL CaM solution each time. The solution was excited at 310 nm, and emission spectra in the range from 400 to 500 nm were obtained with a Perkin Elmer LS-55 fluorescence spectrometer. Figure 1 shows a sigmoidal shape of the binding curve which was observed by adding EDTA solution into CaM solution accumulatively. As expected, the increase of EDTA amount led to a decrease in fluorescence signal intensity due to the release of ANS caused by EDTA-induced CaM conformational change. The fluorescence intensity change indicates that our purified CaM was capable of changing its conformation properly in the solution state.
Surface fabrication
The fabrication and characterization of the chemical pattern were performed with an Agilent PicoPlus 3000 AFM in an environmental chamber. AFM can provide atomic-level resolution in z axis. The Si (100) wafer was cut into 1 cm × 1 cm pieces. Then, the wafer was boiled in the piranha solution (two parts of 98% sulfuric acid and one part of 30% hydrogen peroxide) at 170 °C for 30 min. At high temperature, the H2O2 was decomposed; O· and OH· were generated to remove all organic contaminants and also help to grow a thin oxide layer of silanol (Si–OH) on the surface. After that, the wafer was dipped into 5 mmol L−1 OTS toluene solution for a pinhole-free OTS-coated wafer fabrication, which was capable of being used for the follow-up experiment [23,24,25,26].
The experimental scheme was shown in Fig. 2. Chemical patterns on the OTS coated Si wafer were fabricated using local oxidation lithography first (Fig. 2a). With the help of the chemical patterns, we are able to modify surface with defined chemistry and create topography with references in positions and height. A detailed description of the OTS partially degraded pattern (OTSpd) fabrication has been demonstrated in Additional file 1, and an OTSpd pattern fabrication set-up was demonstrated in Additional file 1: Figure S2 [27].
From the AFM topography histogram (Additional file 1: Figure S3b), we can know the depth of the OTSpd pattern is 10.60 ± 0.01 Å lower than the OTS background. The depth of the OTSpd chemical pattern provides a height reference for calculating the thickness of other parallel layer on itself. Although some studies applied AFM cross-section profile to analyze the height of object [28,29,30], it is believed that AFM topography histogram can better represent the average height change of pattern areas in the present work due to the protein film, which is immobilized on the chemical patterns, exhibiting an “unflat” surface. Histograms of the corresponding heights were fitted to two Gaussian functions by using MicroCal Origin software in order to enable a quantitative comparison. The distance between these two peaks is the height of the disk pattern [31].
After the OTSpd patterns were fabricated, the substrate was rinsed in 10% hydrochloric acid for 10 min and cleaned with the super-critical carbon dioxide snow jet cleaner from Applied Surface Technologies. The possible electrostatic charges and contaminates were completely removed as a result of above procedures. Then, the pattern was soaked in a 10 mmol L−1 MUTMS toluene solution overnight to convert the carboxylic acid-terminated OTSpd surface pattern to a thiol-terminated surface pattern (Fig. 2b). The structure and formation of MUTMS layer on OTSpd pattern is illustrated in Fig. 3. MUTMS molecules react with the trace amount of water in the solution, forming silanols in the first step. Then the silanols cross-linked and selectively anchored on the hydrophilic OTSpd surface. The pattern in Additional file 1: Figure S4 is a representative MUTMS silane monolayer self-assembled on top of the OTSpd pattern. From AFM characterization, the height of the MUTMS pattern over the OTS background is 10.62 ± 0.02 Å.
Then, the sample with MUTMS patterns was incubated into 10 mmol L−1 HgCl2 solution for half an hour to form SH-Hg coupling, as shown in Fig. 3c, which will be used to immobilize cysteine-mutated CaM. 5 μg mL−1 CaM with buffer solution (25 mmol L−1 Tris–HCl, 1 mmol L−1 CaCl2, pH 8.0) was deposited onto the pattern area for one hour in refrigerator at 4 °C (Fig. 3d) [32]. Then the sample surface was wiped with a piece of ChemWipe paper, in a typical force of 1 N [33], to remove the nonspecifically adsorbed protein on the OTS background, while those specifically bind to substrate surface remained.
Surface characterization
Because AFM imaging in liquid environment provides a less accurate measurement [34], and it is difficult to interpret the AFM phase image taken in liquid environment [35]; CaM patterns were imaged at 75% relative humidity environment (at 25 °C) in air in ac mode with MikroMasch NSC-14 tips. The imaging set point was maintained at 99% of the tip free oscillation amplitude so that the tip tapped the CaM immobilized surface under a minimal force. Because the tip touched the protein surface in the humid environment, a possible electrostatic charge from the sample was dissipated after the tip touched the sample. Hence, the height measurement was not affected by the protein’s electrostatic charge. All AFM images were processed using WSxM [36].