This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.
The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.
Owing to its efficient and less labor-intensive experimental process, label-free cell-based technology has witnessed rapid growth over the past decade for analytical as well as screening purposes such as in the aspect of proteomics1,2, drug delivery3, etc.4,5 Compared with traditional biochemical methods aimed at cell analysis, label-free real-time cell assay with the prototype developed by Giaever and coworkers previously6 is based on the principle of recording electric signal changes on the surface of cell-attached microchips, which permit a continuous measurement of cell growth or migration in a quantitative manner. Following this strategy, a real-time cell electronic sensing (RT-CES) system using electric impedance-based detection principle was introduced7,8 and more recently a commercial real-time cell analyzer (RTCA) was launched for laboratory research9.
The commercial real-time cell analyzer mainly reads out the cells' evoked signals of electric impedance, which result from the physiological changes of incubated cells including cell proliferation, migration, viability, morphology, and adherence on the surface of microchips10,11. Such electric signals are further converted by the analyzer into a dimensionless parameter named the Cell Index (CI) to assess the cell status. The change of the impedance of microchips mainly reflects the local ionic environment of covered cells at the electrode/solution interface. Therefore, the analytical performance of the cell analyzer relies heavily on the core sensing unit, the disposable microchips (i.e., so-called electronic plates, e.g., 96/16/8-well), which are made of arrayed gold microelectrodes lithographically printed at the bottom of incubation wells. The gold microelectrodes assemble in a circle-on-line format (Figure 1) and cover most of the surface area of the incubation wells, which allow for dynamic and sensitive detection of attached cells3,12,13,14. The CI will increase in the case of more surface coverage of cells on the chip, and decrease when cells are exposed to a toxicant resulting in apoptosis. Although the real-time cell analyzer has been frequently used to determine cytotoxicity11 and neurotoxicity15 and provide more kinetic information than classical endpoints method, the disposable electronic plates are the costliest consumable.
Until now, there have been no available methods for the regeneration of the electronic plate, which is probably due to the fact that harsh regeneration conditions, such as piranha solution or acetic acid are involved16,17,18, which may alter the electric status of gold microchips. Therefore, a mild and efficient method to remove the adherent cells and other substances from the surface of gold chips will be desirable for the electronic plate regeneration process. We have recently developed a protocol aimed at the regeneration of disposable electronic plates using non-corrosive reagents and the regenerated chips were characterized by electrochemical as well as optical methods19. By using readily available and moderate laboratory reagents including trypsin and ethanol, we have established a general method to regenerate the commercial electronic plate without adverse effects, which is successfully applied to regenerate the two main types of electronic plate (both 16 and L8) used for RTCA.
NOTE: In general, the regeneration process includes trypsin digestion and rinsing step with ethanol and water. The digestion time changes according to the number of cells used, and the type and number of cells used may differ depending on the experimental purposes. It is advised to check the regenerated microchips using optical and electrochemical methods to optimize the regeneration conditions. During the experiment, soluble and insoluble chemicals may be involved, and here these two typical cases of the regeneration procedures are detailed.
1. Preparation of Regeneration Solutions
NOTE: Prepare and handle all the solutions under sterile conditions.
2. Incubation and Proliferation of A549 Cells in Electronic Plates
NOTE: Electronic plate types 16 and L8 are both made of arrayed gold microelectrodes lithographically printed at the bottom of incubation wells. Electronic plate 16 has 16 wells while electronic plate L8 has 8 wells. The wells of electronic plate 16 are round while the wells of electronic plate L8 are rounded rectangles. The volume of each electronic plate 8 well is about 830 µL and each electronic plate 16 well is about 270 µL.
3. Regeneration of Electronic Plates
NOTE: For each rinsing step, the solution in the electronic plate was mixed thoroughly (5-10 times) using a pipette.
4. Assessment of Regeneration Effect
Surface properties of gold microchips: The regeneration procedures used in this protocol were outlined in Figure 1. Figure 2 shows the microscopic surface pictures of fresh and regenerated electronic plates by the optical microscope. As shown in Figure 2A and Figure 2C, microscopic observations indicated that there was virtually no difference in the optical properties of the microchip surface between fresh and treated electronic plates. Using the spontaneous fluorescence of doxorubicin molecules included into the cells convenient for assessment of cell status, we observed a similar homogeneity of cultured cells on the gold microchips after the regeneration (Figure 2B and Figure 2D). Additionally, the incubated cancer cells displayed a well-defined cellular morphology in the electronic plate demonstrating the reusability of chips. Figure 3 shows the Raman spectra of fresh and regenerated electronic plates. The virtually identical Raman peak distributions between the fresh and treated electronic plates indicated a successful regeneration process of the gold microchip surface. The Raman signals of electronic plates covered with A549 cells were distorted to some extent, probably due to the interference from the spontaneous fluorescence of A549 cells.
Regenerated microchips for the cytotoxicity assay using RTCA: We also applied the regeneration protocol for a test of cell proliferation and material cytotoxicity evaluation (Figure 4). A549 cells of optimal density were seeded onto the regenerated electronic plate 16 and growth kinetic curves were recorded by real-time cell analyzer. As shown in Figure 4A, all the resulting growth curves of parallel experimental wells showed adequate consistency, which indicated that the surface electric status of electronic plate 16 is greatly retained after the regeneration. The regenerated electronic plate L8 was also tested for cytotoxicity using mesoporous silica materials, as shown in Figure 4B. Compared with the control experiment, the CI of mesoporous materials-treated samples decreased to some extent, indicating a slight cytotoxicity of the materials. Further encapsulation and release of the anti-cancer drug of Dox by mesoporous materials caused a dramatic decrease of the growth curve of A549. These experimental results showed consistency with using fresh electronic plates and with previously reported work19, which verified the efficiency of this proposed regeneration protocol.
Regeneration efficiency assessed by electrochemical methods: Commercial Au electrodes and glassy carbon electrodes were used as test platforms for the evaluation of regeneration efficiency. Figure 5 shows the EIS of Au electrodes and GCE that were modified with cancer cells. It was shown that after incubation of A549 cells on the electrode, an evident increase of electron transfer resistance (Ret) was observed compared with that of bare electrodes. The Ret was decreased significantly after the Au electrode and GCE were treated according to the protocol in Figure 5. The similar changes of Ret of both Au electrode and GCE indicated an effective and universal regeneration process of microchips regarding their surface electric properties.
Figure 1: The regeneration process of electronic plates. In a typical regeneration run, four main steps were involved including incubating cells as well as their detachment on the surface of electronic plates followed by the rinsing, spinning, and ultraviolet sterilization steps. Please click here to view a larger version of this figure.
Figure 2: The surface properties of electronic plates. (A) New electronic plates (B) A549 cells were plated on A. (C) Regenerated electronic plates (D) A549 cells were plated on C. In the case of cells attached on gold microchips, DOX was used. Figure 2A and Figure 2C have been modified from Xu, Z. et al.19 Please click here to view a larger version of this figure.
Figure 3: Raman spectra of different treated electronic plates. (A) New electronic plate. (B) New electronic plate seeded with A549 cells. (C) Regenerated electronic plate. Please click here to view a larger version of this figure.
Figure 4: A549 cell growth curves on three-time regenerated electronic plates. (A) Parallel growth curves of cells seeded on electronic plate 16. (B) Cytotoxicity test of mesoporous silica materials on electronic plate L8 with error bars (standard deviation, STD) of recorded curves of each incubation well. Figure 4A has been modified from Xu, Z. et al.19 Please click here to view a larger version of this figure.
Figure 5: Electrochemical impedance spectra of different modified gold electrodes and glassy carbon electrodes (GCE) in 10 mM KCl electrolyte solution containing 1 mM K3[Fe(CN)6]. (A) Different treated Au electrodes. (B) Different treated GCE. Figure 5A has been modified fromXu, Z. et al.19 Please click here to view a larger version of this figure.
Chip application | Regeneration buffer | Regeneration strategy |
Surface plasmon resonance (SPR) | Piranha solution (concentrated H2SO4/30% H2O2 = 3:1, v/v)17 | strip both the analyte and probe based on strong oxidization |
Piezoelectric Microcantilever Sensors (PEMS) | Glycine-HCl mixture23,24 | dissociate the target-probe complex and remove the analyte |
High salt concentrations solution (2M MgCl2 + 1.5 M Tris)25 | ||
Gold film | 50mM KOH+25% H2O226 | Oxidized organics |
Table 1: A brief summary of reported regeneration strategies.
We summarized several available methods17,23,24,25,26 for regenerating microchips in Table 1. Basically, these methods involved relatively harsh experimental conditions to achieve the complete regeneration of chips because of the presence of strong molecule-molecule interactions such as that of the immune complex used for SPR chips. However, these harsh experimental conditions may be extremely harmful for the surface properties of arrayed gold microchips used in RTCA. Therefore, a mild and efficient method were the major considerations in the development of the presented regeneration strategy. Other factors that are equally important are environmental concerns, since these disposable microchips are frequently used together with targeted cancer cells.
In this protocol, we introduce a general method for regenerating commercial arrayed gold microchips used for RTCA. Our protocol was developed based on trypsin-digestion and rinsing with ethanol and water. Extra spinning steps are considered to be effective in removing the insoluble particles used for the assay for the regeneration process. Compared with the reagents used in previously reported methods, the regenerating reagents trypsin and ethanol are readily available for daily laboratory use and these biological products/chemicals are of low toxicity, and are thus considered to be environmentally friendly, which is desirable for purposes of regeneration.
In this protocol, the critical step is the trypsin digestion to ensure the regeneration performance of gold microchips. The optimization of trypsinization, including concentration and time, should be the first priority to achieve regeneration. Besides the digestion time that is rationalized in our previous report19, a good assessment for the optimization process is simply to check the surface properties using an optical microscope.
Another concern is the handling of microchips during the regenerating process. As a pipette is frequently used in the experiments, attention should be paid by personnel to avoid scratching the tip on the surface of microchips. Furthermore, it is beneficial to pre-sterilize the tips, gauze, and spoon to ensure regeneration efficiency. Last but not least, the regeneration process should be conducted in a biosafety hood to avoid any contamination.
It should be noted that the rinsing processes involved in the regeneration protocol are manually conducted, which will inevitably cause bias due to differing personnel. To overcome this limitation, automatic or semi-automatic equipment for rinsing purposes could be custom designed, considering the modular electronic plates used. In addition, the regeneration protocol has been tested to be effective for cell-based assays. However, in the presence of strong molecular interactions, such as antibody-antigen formation at the interface of chips, the proposed regeneration conditions may be limited to a great extent and relatively strong buffer conditions should be considered.
Overall, the current regeneration protocol has been tested to allow the repeated usage of commercial gold microchips, as well as other conducting electrodes such as Au and glassy carbon electrodes. We expect that the application of this protocol will save on the high running cost of real-time chip-based cell assays. In the long run, microchip-based assays are becoming more frequently used for proteomic and cellular analysis and the proposed protocol in this study can offer an option for the regeneration, in which automatic or semi-automatic equipment for rinsing could be designed and applied in cases of accumulated disposable chips.
The authors have nothing to disclose.
The work was supported by National Natural Science Foundation of China (U1703118), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Shuangchuang Program, Open Funds of the State Key Laboratory for Chemo/Biosensing and Chemometrics (2016015) and the National Laboratory of Biomacromolecules (2017kf05) and Jiangsu Specially-Appointed Professor project, China.
Rat: Sprague-Dawley | Charles River | Cat# 400 | |
mouse anti-rat CD11b monoclonal (clone OX42) | Bio-Rad | Cat# MCA275R | Panning: 1:1,000; Staining: 1:500 |
Goat polycolonal anti-Iba1 | Abcam | Cat# AB5076 | Staining: 1:500 |
Rabbit polyclonal anti-Ki67 | Abcam | Cat# AB15580 | Staining: 1:500 |
Alexa Fluor Donkey anti-mouse 594 | Invitrogen | Cat# 11055 | Staining: 1:500 |
Alexa Fluor Donkey anti-goat 488 | Invitrogen | Cat# A-21203 | Staining: 1:500 |
Alexa Fluor Donkey anti-Rabbit 647 | Invitrogen | Cat# A-31573 | Staining: 1:500 |
Triton-X (detergent in ICC staining) | Thermo Fisher | Cat# 28313 | |
Heparan sulfate | Galen Laboratory Supplies | Cat# GAG-HS01 | |
Heparin | Sigma | Cat# M3149 | |
Peptone from milk solids | Sigma | Cat# P6838 | |
TGF-β2 | Peprotech | Cat# 100-35B | |
Murine IL-34 | R&D Systems | Cat# 5195-ML/CF | |
Ovine wool cholesterol | Avanti Polar Lipids | Cat# 700000P | |
Collagen IV | Corning | Cat# 354233 | |
Oleic acid | Cayman Chemicals | Cat# 90260 | |
11(Z)Eicosadienoic (Gondoic) Acid | Cayman Chemicals | Cat# 20606 | |
Calcein AM dye | Invitrogen | Cat# C3100MP | |
Ethidium homodimer-1 | Invitrogen | Cat# E1169 | |
DNaseI | Worthington | Cat# DPRFS | |
Percoll PLUS | GE Healthcare | Cat# 17-5445-02 | |
Trypsin | Sigma | Cat# T9935 | |
DMEM/F12 | Gibco | Cat# 21041-02 | |
Penicillin/ Streptomycin | Gibco | Cat# 15140-122 | |
Glutamine | Gibco | Cat# 25030-081 | |
N-acetyl cysteine | Sigma | Cat# A9165 | |
Insulin | Sigma | Cat# 16634 | |
Apo-transferrin | Sigma | Cat# T1147 | |
Sodium selenite | Sigma | Cat# S-5261 | |
DMEM (high glucose) | Gibco | Cat# 11960-044 | |
Dapi Fluoromount-G | Southern Biotech | Cat# 0100-20 | |
Poly-D-Lysine | Sigma | Cat# A-003-E | |
Primaria Plates | VWR | Cat# 62406-456 | |
Stock reagents | reconstitution | Concentration used | Storage |
Apo-transferrin | 10 mg/mL in PBS | 1:100 | -20°C |
N-acetyl cysteine | 5 mg/mL in H2O | 1:1,000 | -20°C |
Sodium selenite | 2.5 mg/mL in H2O | 1:25,000 | -20°C |
Collagen IV | 200 μg/mL in PBS | 1:100 | -80°C |
TGF-b2 | 20 μg/mL in PBS | 1:10,000 | -20°C |
IL-34 | 100 μg/mL in PBS | 1:1,000 | -80°C |
Ovine wool cholesterol | 1.5 mg/mL in 100% ethanol | 1:1,000 | -20°C |
Heparan sulfate | 1 mg/mL in H2O | 1:1,000 | -20°C |
Oleic acid/Gondoic acid | Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol | 1:1,000 | -20°C |
Heparin | 50 mg/mL in PBS | 1:100 | -20°C |
Solutions | Recipe | コメント | |
Perfusion Buffer | 50 μg/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +) | Use when ice-cold | |
Douncing Buffer | 200 μL of 0.4% DNaseI in 50 mL of DPBS++ | Use when ice-cold | |
Panning Buffer | 2 mg/mL of peptone from milk solids in DPBS++ | ||
Microglia Growth Medium (MGM) | DMEM/F12 containing 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 5 μg/mL N-acetyl cysteine, 5 μg/mL insulin, 100 μg/mL apo-transferrin, and 100 ng/mL sodium selenite | Use ice-cold MGM to pan microglia off of immnopanning dish. | |
Collagen IV Coating | MGM containing 2 μg/mL collagen IV | ||
Myelin Seperation Buffer | 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 μL 1 M CaCl2, 5 μL 1 M MgCl2 | Mix well after the addition of CaCl2 and MgCl2 | |
TGF-b2/IL-34/Cholesterol containing growth media (TIC) | MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 μg/mL ovine wool cholesterol, 10 μg/mL heparan sulfate, 0.1 μg/ml oleic acid, and 0.001 μg/ml gondoic acid | Make sure to add cholesterol to media warmed to 37 °C and do not add more than 1.5 μg/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH. |