Sensor Coating with ORDYL SY 300
Gold interdigitated electrodes (IDEs) were purchased from DropSens (Asturias, Spain), (cat. N.: PW-IDEAU50). Each IDE has a finger width and spacing of 50 μm, with a total number of 70 fingers, a total electrode length of 7 mm, and electrode surface area of 8.45 mm2. Initially, the IDEs were immersed in an acetone solution, then in 100% isopropanol and finally, they were washed with distilled water. (Chemicals were purchased from Sigma-Aldrich (St. Louis, CA, USA).)
For sensor coating, it was chosen to use dry resin, specifically ORDYL SY 300, which is a permanent dry film with a very high possible aspect ratio and excellent high thermal and chemical stability. The experimental procedure is shown in the following Figure; The IDEs were placed in a hot plate at 110 °C. An ORDYL SY 300 piece, slightly larger than the IDE sensitive area, was cut, ORDYL protective liner was removed from the one side and the tape was coated at the IDE surface. A photolithography mask was designed and printed. The sample was exposed at UV for 48 sec (0.8 min The white dark areas of the mask protected the photosensitive layer from UV, whereas the dark transparent areas were exposed. The ORDYL protective liner was removed from the other side, revealing its photosensitive surface. Then, it was immersed in its developer solution, in an ultrasonic bath, for another 5 min. As a result, the ORDYL SY 300, being negative photoresist, was removed from the areas protected from the UV exposure. After the developing process, the sample was immersed again in 100% isopropanol solution and washed with distilled water. At the end, it was placed at a hot plate at 120 °C for 45 min, in order to harden the ORDYL layer. The thickness of the ORDYL layer before the post baking process was 90 μm.
Grafting Antibody on Interdigitated Gold Surface
To clean the electrodes' surface, interdigitated capacitors prepared with ORDYL SY 300 were immersed in a solution of 100% isopropanol for 5 min and then rinsed in Milli-Q water. 30 μL (25 mM) of L-Cysteine were placed on the electrode surface and let to form bonds between gold and the amino acid’s thiol groups. The reaction was stopped when all L-cysteine solution dried out and three washes were performed using PBS to remove unbound material. Subsequently, a solution containing 5 μL (50 ng/ml) of ERK antibody, 5 μL (0.877 g/ml) of EDC and 10 μL (0.1 M) of MES was placed on top of the L-Cysteine layer. Specifically, EDC and MES activate the carboxylic terminus (-COOH) of ERK antibody. Once active the carboxylic terminus of ERK antibody reacts with the amine group (-NH2) of L-Cysteine forming a covalent peptide bond. Due to the cross reactivity between C- and N- termini of both ERK antibody and L-Cysteine, unwanted covalent bonds were formed also among the C-terminal of L-Cysteine and the N-terminal of ERK antibody. After each step, the biosensor was rinsed 3 times in Milli-Q water, in order to remove unbound molecules from its surface. The IDE with immobilized ERK antibody or L-Cysteine alone was covered with 20 μl of PBS and kept wet until its use. Some of the biosensors were kept standby for 7 days at 4 ◦C in order to be used for the biosensor stability calibration.
Cell Culture
The Α453 cell line was maintained in DMEM medium, containing 10% fetal bovine serum (FBS), 1X penicillin, 1X streptomycin, and 2 mM L-glutamine. Cells were incubated at 37°C, 5% CO2 in a humidified atmosphere. A453 cell lines used in the study have been purchased from American Type Culture Collection (ATCC).
Western Blot and Antibodies
Total protein was extracted with 60 mL RIPA lysis buffer and Western blotting was performed according to standard protocols. Blots were incubated overnight at 4°C with the appropriate primary antibodies. Protein extracts have been prepared from at least two independent experiments and blots repeated at least twice.
To realize a bioassay for detection of protein phosphorylation we first used an in vitro cell model to test the antibodies that will be used to construct the biosensor. The cell model was A453, a human skin cancer cell line. This cell can be grown in the lab using specific media and instrumentation. We kept cells in (Dulbecco-Modified Egle Medium) DMEM supplemented with 10% (Fetal Bovine Serum) FBS, and antibiotics. At the time of the experiment 2 identical flask (75cm2) where seeded with roughly 2*106 cells in 18 ml of DMEM containing FBS and antibiotics. Both flasks were incubated for 3 days at 37°C and 5% CO2. After 3 days both flasks were washed 3 times with (Phosphate-buffered saline) PBS solution in order to remove dead cells, debris and previous medium components.
Subsequently, in order to reduce as much as possible protein phosphorylation at global level, in one flask it was added only DMEM with antibiotics and no FBS (FBS starvation), whereas the second flask was kept in normal condition (DMEM+FBS+antibiotics). Both flasks were incubated for another day at 37°C and 5% CO2, and then proteins were extracted from both flasks.
FBS contains growth factors, hormones, cytokines and other active molecules that stimulate cell growth. Such molecules usually bind extra- or transmembrane receptors that, upon activation, start signaling cascades from the plasma membrane to the nucleus. Basically, such signaling cascades are sustained by kinase proteins that phosphorylate other target proteins (in some cases kinases as well).
Therefore, the absence of FBS in the medium causes a generalized reduction of phosphorylation activity of kinases and consequently a reduction of phosphorylated proteins.
To verify the reduction of protein phosphorylation we tested the phosphorylation status of a marker protein, ERK. Such protein is usually phosphorylated when cells grow at high rate, therefore is a good candidate to verify if the FBS starvation caused any reduction in kinase activity.
We used western blot (WB) assay to test the phosphorylation status of ERK. To perform the test 2 different antibodies were used, one recognizing ERK protein independently by its phosphorylation status, and one that specifically recognizes the phosphorylated form of ERK (pERK).
The WB assay was conducted as following:
- 20 micrograms of total proteins extracted from cells grown in DMEM+FBS+antibiotics were loaded in two consecutive WB wells. These wells will be the control sample for phosphorylation
- 20 microgram of total proteins extract from cells grew in DMEM+antibiotics were loaded in two consecutive WB wells next to the control wells
Two gels with these arrangements were run at the same time and subsequently transferred on nitrocellulose membranes resulting in 2 membranes with 4 protein strips.
At this point one membrane was incubated with the antibody recognizing ERK native protein (independently from phosphorylation), and the second gel was incubated with the antibody recognizing the pERK.
Since the quantity of protein is equal in each gel the signal from ERK should be the same in all wells of both gels, whereas the signal from pERK should be strong in cells grown in normal condition and absent or very poor in cells grown in FBS starved condition.