The overall goal of the following experiment is to electrically deposit bio polymeric films and functionalize them with biological components such as proteins and cells. Electro deposition is achieved by taking advantage of the pH change that occurs locally at a biased electrode in combination with a pH responsive polysaccharide solution. Upon choosing a cathartic or anodic deposition strategy, the electrode is biased to trigger a soul gel transition, which generates a thin film having a pattern defined by the electro geometry.
Furthermore, biological components such as proteins and cells can be introduced throughout the electrode deposition process. In order to biologically functionalize the film results are obtained that show biological components are localized to the films based on fluorescence imaging. Additionally, the components are functional and able to interact with their environment demonstrated by electrochemical detection of enzymatic activity and the use of reporter cell response.
Biofabrication provides a novel toolbox for building the interface between biology and electronics. We believe its main advantages are its simplicity and its ability to assemble at near physiological conditions. Biology is expert in fabricating at the nanoscale through self-assembly and enzymatic means By enlisting these capabilities, we circumvent complex, hazardous, and time consuming synthesis methods that other assembly techniques utilize.
Our collaborative team got the idea of building the BioD device interface using biological materials and mechanisms when we observed their kites in could be electrode deposited. Kitan is a pH responsive film forming polysaccharide that can recognize device imposed electrical signals and deposit as a thin film. Once we saw that kitan could be electrode deposited, we realized we could access a broader suite of tools from biochemistry and molecular biology to build a BioD device interface.
The application of such technique extend towards labon chip settings where functional biological component are required as they provide a cheap and easy way to localize such components on an electrode where they can remain viable. Visual demonstration of this method is critical as a bio fabricate film can be dedicated and easily damaged. As such one must be careful not to be too harsh while washing or shooting the film.
As the film might be denominate easily disturbed. However, as an advantage, the electros can be reused because the film are easily removed. To begin this protocol, connect a power supply to the custom fabricated electrodes via patch cords using alligator clips.
An indium tin oxide or ITO covered glass slide will act as the anode or working electrode platinum foil will serve as the cathode or counter electrode position the electrode so that the IT o's surface to be functionalized opposes the counter electrode in a way that the IT O's surface is positioned either vertically to be dipped into the solution or horizontally, such that the solution can be contained on the surface. Next, prepare an alginate deposition solution by mixing 1%alginate and 0.5%calcium carbonate in distilled water and autoclaving the mixture. Then fluorescently label alginate with Fluor spheres by mixing and vortexing.
This will allow for fluorescence imaging of the resulting film After autoclaving, the solution submerge both electrodes in the prepared deposition solution apply a constant current density of three amps per meter squared for two minutes. Voltage will shift in the range of two to three volts After two minutes, disconnect the electrode and remove the non deposits solution. Gently rinse the film with sodium chloride to remove superfluous alginate.
Incubate the film briefly in 0.1 molar calcium chloride to strengthen the gel. The film is then incubated in the desired storage solution. Proceed to image the film using fluorescence microscopy prior to co deposition.
Grow a signal sender cell culture, and a signal receiver cell culture as directed in the written protocol accompanying this video. Next, prepare a deposition solution of alginate and calcium carbonate. Mix the solution with each cell culture in a one-to-one ratio to a final concentration of 1%alginate and 0.5%calcium carbonate with the cells diluted to about half the culturing density.
Use a glass slide patterned with two ITO electrodes contained by a poly dimethyl silane well and a platinum counter electrode. Connect one ITO electrode and the platinum electrode to a power supply. As before, immerse the electrodes in the deposition solution containing the receiver cells.
Set the power supply to a constant current at a density of three amps per meter squared, where surface area dimension is defined by the single electrode on which the deposition will occur. Apply the current for two minutes to allow for co deposition of the cells in the alginate matrix. Rinse the film with sodium chloride as before, switch the antic connection to the adjacent ITO electrode.
Repeat the deposition procedure but this time. Introducing the solution containing the sender cells. Incubate the two electrode chip containing the co deposited cells and calcium argenate overnight at 37 degrees Celsius in phosphate buffered saline.
Supplemented with 10%LB media and one millimolar calcium chloride. After incubation image the chip using a fluorescence microscope to begin chitosan electrode deposition. Connect the power supply to the electrodes.
Fire alligator clips. A gold coated silicon chip will act as the cathode and a platinum foil will serve as the anode. Position the gold electrode surface so that it deposes the counter electrode as before.
Prepare a Kato sand solution by mixing Kato sand flakes into water and slowly adding two molar hydrogen chloride to dissolve the polysaccharides. Following the procedure referenced in the text, the Kato sand solution can be fluorescently labeled in order to image the electrode deposited film by fluorescence microscopy Next place. The electrodes into the kaizan solution completely submerging the desired area for deposition.
For two minutes apply a constant current calculated at density of four amps per meter squared based on the working electrodes surface area. In this case, a current of 16 microamps is applied to a four millimeter squared gold electrode voltage will shift within the range of two to three volts after rinse in the electrode with deionized water to remove superfluous chitosan. Proceed to image the film using a fluorescence microscope, copos chitosan, and glucose oxidase from a solution at a current density of four ramps per meter squared onto a patterned electrode.
According to the Chitosan electrode deposition procedure, a kaizan film entrapped with glucose oxidase will be generated. Attach the treated electrode to a three electrode system as the working electrode, a platinum wire as the counter electrode and silver, silver chloride as the reference electrode, immerse the electrodes into a phosphate buffer solution containing sodium chloride. Electrochemically conjugate the protein to the Kazam film by applying a constant voltage for 60 seconds using chrono pyrometry following conjugation.
Place the chip in phosphate buffer and wash for 10 minutes on an orbital shaker to remove any unreactive sodium chloride and unconjugated glucose oxidase. Reattach the three electrode system and immerse a solution of five millimolar glucose. Then set the desire parameters for cyclic vol telemetry to sweep the potential in a positive direction to 0.7 volts.
Remove the electrodes from the glucose solution and rinse with phosphate buffer. Then place the electrodes inter 10 milliliter beaker containing eight milliliters of phosphate buffer on a magnetic stir plate bias. The GOX functionalized chip to 0.6 volts to serve as the working electrode.
After setting the desire parameters, collect a baseline recording. Add aliquots of glucose to the buffer to increase the glucose concentration by four millimolar for each aliquot In this experiment, 400 microliter aliquots of 80 millimolar glucose are added to the buffer. Correlate the current output to each glucose concentration To generate a standard curve to perform protein functionalization use a glass slide patterned with an adjacent gold and ITO electrode contained within A-P-D-M-S.
Well bias the gold electrode with a cathartic potential to electrode deposit tizen as shown previously. After rinsing the film, add a solution of blue fluorescently labeled auto inducer two synthase supplemented with tyrosinase. Following a one hour incubation at room temperature.
Rinse the film with PBS as before, apply an anodic potential to the ITO electrode immersed in an alginate deposition solution containing receiver cells. Continue with the same steps outlined in the co deposition of cell populations in alginate to generate the transmitted signal enzymatically. After rinsing the films add a solution of five micromolar, SAH and PBS supplemented with 10%LB media and one millimolar.
Calcium chloride cover the electrodes to prevent evaporation of the solution and incubate overnight at 37 degrees Celsius. This will allow for a receiver cell response by generating a red fluorescent protein DS red adjacent electrodes may be imaged with fluorescence microscopy by justing. The filters to capture the blue fluorescence of the also inducer two synthase and the red fluorescence expressed by the co-opted receiver cells imposed electrical signals can create localized microenvironments near an electrode surface and these stimuli can trigger the self-assembly of polysaccharides such as alginate and razan to deposit as a hydrogel film on the electrode surface.
Because this soul gel transition occurs at the electrode surface, the resulting film is electro addressed with its geometry matching the electrode pattern. Using alginate unique cell populations have been co deposited at separate addresses. Evidence of their electrode addresss is observed upon interaction between the sender and receiver cell population.
The molecule auto inducer two diffuses from the sender cells and is taken up by the receiver cells resulting in expression of the DS red fluorescent protein.Here. Red fluorescence is observed only at the electrode where receivers are addressed. Conversely, the biosensing enzyme glucose oxidase provides the ability for detection of glucose through an enzymatic reaction by producing hydrogen peroxide, which can then be electrochemically oxidized to produce an output current.
In this way, a chemical signal can be transduced to electrical. This plot shows that films in which Geo X was chemically conjugated produce a strong antic signal in the presence of glucose as opposed to those films containing no Geo X.These results indicate Geo X can be assembled onto a deposited zam film and or retain catalytic activity. Furthermore, a step increase in an otic current is produced in response to increasing glucose concentrations.
The standard curve also present shows that step increases proceeded in a near linear fashion dependent on the amount of glucose added. These results show that the enzyme also retains its sensitivity upon conjugation to the Razza film as depicted here, the I two synthase includes a Penta tyrosine tag. Tyrosinase acts on the tyrosine tag oxidizing the residues phenol groups to o quinones, which then covalently bind to kazan's amine evidence of Kazam film functionalization within the a i two synthase by Tyrosinase assembly is observed in this fluorescence image showing a kaza film on gold where the functionalized I two synthase has been fluorescently labeled blue because a I two synthase generates a I two from the substrate SAH in the same way as the sender cells, its proximity to co-opted receiver cells in the presence of SAH also causes the receiver cells to fluorescently respond by expressing ds.
Red red fluorescence of the receiver cells again demonstrates interaction between addresses due to the diffusion of ai, two from one to the other, and further indicates that 10 enzymes immobilized to tizen retain activity once covalently bound. After watching this video, you should have a good understanding of how to electro deposit polysaccharide films and how to functionalize them using a variety of techniques. We have demonstrated the versatility in these functionalization steps allowing us to work with a multitude of biological components in further testing.
Once mastered, this technique can be performed in as little as a few minutes in order to generate well-defined film patterns onto which biological components can be precisely grafted. And so in this way, we are able to very easily generate biologically patterned and active surfaces on designated electrodes, all done in an organized fashion and yet able to achieve complex biological interactions. Following this procedure, creativity can be employed both in the design of the electro array and in the choice of biological components in order to achieve the design interaction.
Our hope is that the biofabrication tools will bridge two disparate fields, biotechnology and microelectronics, each of which has individually revolutionized our lives. By interfacing these two fields, we believe that we will create new solutions to some of society's most pressing problems. For example, we envision that Biofabrication might provide simple and versatile methods for new ways of sensing, for point of care, diagnosis of disease and sensing biological and chemical threats.
One example far reaching might be that the next next generation arterial stent would also be able to monitor your metabolic profile as well as keep your artery open.
