Name: regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer
Uploaded: 2018-03-12T12:30:00
Description: Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

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.

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 pr...

<ol>
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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, t...

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&#39; 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.

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			<td style="width:317px;">Southern Biotech</td>
			<td style="width:263px;">Cat# 0100-20&#160;</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Poly-D-Lysine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A-003-E</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Primaria Plates&#160;</td>
			<td style="width:317px;">VWR</td>
			<td style="width:263px;">Cat# 62406-456</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Stock reagents&#160;</td>
			<td style="width:317px;">reconstitution&#160;</td>
			<td style="width:263px;">Concentration used</td>
			<td style="width:192px;">Storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin</td>
			<td style="width:317px;">10 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">N-acetyl cysteine</td>
			<td style="width:317px;">5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">2.5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:25,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Collagen IV</td>
			<td style="width:317px;">200 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">TGF-b2</td>
			<td style="width:317px;">20 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:10,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">IL-34</td>
			<td style="width:317px;">100 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ovine wool cholesterol</td>
			<td style="width:317px;">1.5 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Heparan sulfate</td>
			<td style="width:317px;">1 mg/mL in H2O</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Oleic acid/Gondoic acid</td>
			<td style="width:317px;">Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:317px;">50 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Solutions&#160;</td>
			<td style="width:317px;">Recipe&#160;</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Comments</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Perfusion Buffer</td>
			<td style="width:317px;">50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +)</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Douncing Buffer</td>
			<td style="width:317px;">200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Panning Buffer</td>
			<td style="width:317px;">2 mg/mL of peptone from milk solids in DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Microglia Growth Medium (MGM)</td>
			<td style="width:317px;">DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Coating</td>
			<td style="width:317px;">MGM containing 2 &#956;g/mL collagen IV</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Myelin Seperation Buffer</td>
			<td style="width:317px;">9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr height="235">
			<td height="235" style="height:235px;width:293px;">TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td style="width:317px;">MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/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.&#160;</td>
		</tr>
	</tbody>
</table>

regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer

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.

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 diffe...

Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

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 ...

The overall goal of this procedure is to regenerate commercial electronic plates equipped for a Real-Time Cell Analyzer, or RTCA. This method can help answer key questions in the regeneration of disposable gold-based chips field, including the regeneration of electronic plans used in RTCA. The main advantage of this technique is that the regeneration procedure can be conducted using mild and readily available lab reagents with low toxicity.We first had the idea for this method when we started to use the Real-Time Cell Analyzer which the cost gold-based chips. Begin this procedure with incubation and proliferation of A549 cells in cell-culture dish as described in the text protocol. Add 150 microliters of cell medium to each incubation well of the electronic plate L8.Leave the plate for 30 minutes in the bio-safety hood for equilibration.After 30 minutes, manually insert the electronic plate L8 in the Real-Time Cell Analyzer in the CO2 incubator at 37 degrees Celsius. Next, launch the Real-Time Cell Analyzer operation program on the computer. On the default experiment pattern setup page, select Experiment Model, and name the running assay.On its layout page, select the corresponding wells that are included in the experiment, and input the information such as cell type, number, and drug names in the edit boxes. On its schedule page, add the experimental steps, then select the running time of each step and interval of realtime data acquisition. Click start to measure the background impedance of the media.The experiment file will be saved. The resulting data will be automatically subtracted. On the plot page, add wells, and the graph of time-cell index will be showed.For the cell-proliferation experiment, first click the pause button to pause the program. Then take out the electronic plate, and add 100 microliters of A549 cell suspension buffer per well at room temperature. Leave the plate for 30 minutes in the bio-safety hood to let the cells settle to the bottom of incubation wells.Manually insert the electronic plate in the realtime cell-analyzer station. Then click the start button to continue the experiment. On the plot page, the analyzer continuously records cell index values, or curves, throughout the experiment.When the experiment is complete, enter the plot page, and open the experiment file. Then select all the tested incubation wells and the resulting cell index curves that can be averaged or normalized for the last time before adding pharmaceutical drugs or changing medium to reduce variations between assays. On the data-analysis page, EC50 or IC50 can be calculated.After performing cytotoxicity evaluation of anticancer drug as described in the text protocol, take out the electronic plate containing cells from the Real-Time Cell Analyzer station. Place the used electronic plate in a sterile bio-safety hood at room temperature, and mix the culture medium thoroughly using a pipette. Then use a pipette to aspirate the entire medium.Rinse the electronic plates with 200 microliters of deionized water for three times at room temperature. Digest the remaining cells on the electronic plates with 0.25%freshly-prepared trypsin for one to two hours in an incubator at 37 degrees Celsius. When the digestion is completed, work in the bio-safety hood at room temperature to mix the incubation solution five to 10 times using a pipette, and then remove the solution.Rinse the electronic plates with 200 microliters of deionized water two times. Then wash with 200 microliters of 100%ethanol two times, followed by the same volume of 75%ethanol two times. Finally, rinse with another 200 microliters of deionized water two times.Invert the electronic plate on a sterile gauze or tissue paper to decant the remaining water at room temperature. Then perform ultraviolet sterilization on the regenerated electronic plate for one to two hours in the bio-safety hood at room temperature. After using the plates for drug delivery using mesoporous material as described in the text protocol, digest the remaining cells with trypsin as before.As the pipette is frequently used in the experiments, attention should be taken by the person to avoid scratching of the tip and the surface of the microchips. Rinse the microchips with 200 microliters of deionized water once in the bio-safety hood at room temperature. Then rise the microchips with 200 microliters of absolute ethanol two times at room temperature.Next, rinse the microchips with 200 microliters of 75%ethanol once at room temperature. Add 250 microliters of 75%ethanol in each well, and seal the electronic plate with the sealing film at room temperature. After spinning the electronic plate at 114 times G for two minutes, empty the plate.Invert the electronic plate on a sterile gauze or tissue paper to decant the remaining liquid at room temperature. Repeat the 75%ethanol rinses followed by the spin. Then perform ultraviolet sterilization on the regenerated electronic plate for one to two hours in the bio-safety hood at room temperature.Assess optical characteristics of the regenerated electronic-plate surface by first disassembling the bottom part of the incubation well as described in the text protocol. To evaluate the viability of attached cells after anticancer-drug uptake on the fresh and regenerated electronic plates, use a confocal laser-scanning microscope to monitor the spontaneous fluorescence of DOX molecules absorbed by A549 cells. To record the spontaneous fluorescence of absorbed DOX by the cells, use a 63-times oil objective and 485 nanometers as the excitation wavelength.Next, assess the electrochemical behaviors of regenerated electrodes by first polishing the gold electrode and the glassy carbon electrode to a mirror-like surface with an illuminoslurry on a polishing cloth. Then sonicate the electrodes in water to remove any particles. Next, activate the gold electrode in 0.5-molar sulfuric acid.Obtain Electrochemical Impedance Spectroscopy, or EIS data, of the bare gold electrode and the glassy carbon electrode in 10-millimolar potassium-chloride-electrolyte solution, containing one millimolar potassium pheracyanide. Now, drop 10 microliters of a cell suspension on the surface of the gold electrode in the GCE. After adding the cells, incubate the gold electrode in the GCE in a 37-degree-Celsius incubator for three hours before obtaining EIS data of the electrodes modified with cells, as described in the text protocol.To regenerate the electrodes, immerse the gold electrode in the GCE modified with cells in 0.25%trypsin for 0.5 to two hours. Rinse both electrodes softly with water four times. Then, rinse the electrodes softly with absolute ethanol four times.Next, immerse the gold electrode in the GCE in 75%ethanol for approximately five minutes. After five minutes, rinse the chip softly with 75%ethanol four times before obtaining EIS data of the regenerated electrodes, as described in the text protocol. To evaluate the regeneration efficiency of the arrayed gold electronic plate, microscopic observations were made for both fresh and treated electronic plates.No virtual difference in the optical properties of microchip surface was found. Throescence of doxorubicin molecules is used to assess the cell status on the gold microchips before and after regeneration. The similar homogeneity and well-defined cellular morphology demonstrated the reusability of the chips.The regeneration protocol is applied for the real test of cell proliferation. The growth curves obtained from multiple incubation wells of a tested electronic plate show good consistency, indicating the successful regeneration of the commercial chip. Shown here, the regenerated electronic plate L8 was tested for cytotoxicity evaluation using mesoporous silica materials.The cell index of the mesoporous-materials-treated sample decreased as compared with the control, indicating a slight cytotoxicity of the materials. Further encapsulation and release of the anticancer drug doxorubicin by mesoporous materials caused a dramatic decrease in the growth curve of A549 cells. Once mastered, this technique can be done in four hours if it is performed properly.While attempting this procedure it is important to remember that the type sink digestion time may differ from chip to chip. Following this procedure, other method like confocal laser-scanning microscopy, or even spectroscopy, electrochemical impedance spectroscopy can be performed in order to answer additional questions like assessment of the regeneration effect.

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