The overall goal of this procedure is to introduce a versatile electrochemical microfluidic biosensor platform based on low-cost, dry film photoresist technology which is capable of gaging various types of analites depending on the subsequently used immobilized enzyme linked assay. This method can help answer key questions in the field of point of care testing such as the quantification of different drugs, like antibiotics, or diagnosing various diseases such as cancer. The main advantage of this technique is that the here in presented biosensor is mainly based on low-cost materials, easy to use, and highly versatile in terms of its application.
Visual demonstration of this method is critical, as the steps related with dry film photo resists are difficult to master because the handling requires a lot of training and practice. Demonstrating the on-chip assay immobilization and measurement will be Eva Grether, a student assistant from my group. To begin, cut a polyimide, or PI substrate into six inch round wafers.
Then, put the PI wafer in an oven at 120 degrees Celsius, for roughly one hour for a dehydration bake. To perform the first photolithography step for the liftoff process, program the spin coder to a 30 second spinning time at 3000 rpm with an acceleration of 2000 rpm per seconds. Place the PI wafer on the spin coder and fix it applying a vacuum.
Then start the spin coder program and dispense two milliliters of a resist, enabling the liftoff process. Remove the wafer from the spin coder and soft bake the PI wafer on a hot plate for two minutes at 100 degrees Celsius. Adjust the desired mask for patterning the electrodes on the PI wafer with the naked eye and expose it to 400 millijoules per square centimenter of UV lights.
Then place the wafer in a bowl filled with a resist matching developer on an orbital shaker and let it shake slightly for one minute. After the access resist is removed, use a shower to rinse the PI wafer with deionized water on a wet bench, before drying it using compressed air. Next, deposit platinum for the formation of the electrodes by using a standard physical vapor deposition process to deposit 200 nanometers of platinum onto the wafer.
After placing the wafer in a bowl, add the matching remover to the bowl and remove the liftoff resist, while slightly shaking the wafer on an orbital shaker until all access platinum is removed. Following rinsing and drying the PI wafer as before, perform the second photolithography step and clean the electrodes as described in the text protocol. Next, passivate the platinum contact pads of the wafer with a UV sensitive adhesive tape.
To do so, cut the foil into five millimeter wide and 11 centimeter long strips and attach them to the wafer, protecting the parts not to be deposited with silver. For the silver deposition, put a sonic bath into a fume cupboard and insert a container of silver electrolyte solution into the bath. Set the sonic bath to room temperature and depower to 10%Connect the bulk contact of the reference electrodes to a constant current source.
Then, connect the counter electrode, a silver wire that is immersed in the silver electrolyte solution, to the current source, using standard connecting cables. Set the current source to DC and to a current density of about 4.5 milliampere per square centimeter, resulting in a silver deposition rate of approximately 0.3 microns per minute. Start the sonic bath and let the current source run for 10 minutes.
After 10 minutes, rinse the wafer with deionized water. To perform the third photolithography step, first cut the dry film photoresist layers to a similar size as that of the wafer. Fix the desired mask onto the exposure unit and align the DFR layer onto the mask using the naked eye.
Then, illuminate the resist with 250 millijoules per square centimeter of UV light. Remove the protective foil of the photoresist and develop the resist for roughly two minutes in a 1%sodium carbonate solution, using a standard sonic bath, preheated to 42 degrees Celsius and to sonic power of 100%To stop the reaction immediately, shake the resist for one minute in a 1%hydrochloric acid bath on an orbital shaker. Rinse and dry each DFR layer as before.
To laminate the DFR layers onto the PI substrate, place the PI wafer onto a transparency overhead foil and fixate using standard adhesive tape. Adjust the channel DFR layer on the PI wafer under a microscope using the respective alignment structures. For the lamination, use a standard hot roll laminator and preheat the top roll to 100 degrees Celsius and the bottom roll to 60 degrees Celsius.
Set the pressure to three bar with a forward speed of 0.3 meters per minute. Place the wafer with the fixed DFR layer in the middle of the laminator and start it so that the DFR is pushed through the laminator. Repeat the step after rotating the wafer by 180 degrees.
Next, remove the protective layer from the front side of the DFR channel layer by placing standard adhesive tape at one end of the DFR and pulling it upwards. Use a hand dispenser with 0.004 inch tubes to dispense small droplets of dissolved polytetrafluoroethylene into the wells of the insulation layer. To seal the microfluidic chip, laminate the cover DFR layer onto the channel layer as before.
Then, hardbake the microfluidic biosensor by first removing all protective foils on the cover and backside DFR layers. Cut the biosensors into strips using an ordinary pair of scissors. Finally, cure the chips in an oven at 160 degrees Celsius for three hours.
To absorb avidin into the immobilization area of the channel dispense two microliters of the avidin solution into the inlet of the biosensor. To ensure that the fluidic keeps stopping at the barrier during the whole incubation time, dispense two microliters of deionized water on the outlet of the chip. Incubate the chip at 25 degrees Celsius for one hour in a closed container.
Remove excess reagents through the inlet of the chip by applying a vacuum. Then, wash the channel with 50 microliters of wash buffer dispensed on the outlet while the vacuum is being applied. Dry the channel for 30 seconds with vacuum.
Block the channel surface to inhibit unspecific binding by pipe heading two microliters of 1%BSA solution on the inlet and two microliters of deionized water on the outlet of the channel. Incubate the chip at 25 degrees Celsius for one hour in a closed container before removing access reagents and drying the channel as before. Then, incubate DNA oligos labeled with biotin and six FAM by dispensing two microliters of one concentration of the DNA solution on the inlet and two microliters of deionized water on the outlet.
Incubate the chip at 25 degrees Celsius for 15 minutes in a closed container before removing access reagents and drying the channel. To immobilize the glucose oxidase labeled six FAM antibodies, introduce two microliters of the antibody solution to the inlet and two microliters of deionized water to the outlet of the channel. Again, incubate the labeled antibodies at 25 degrees Celsius for 15 minutes in a closed container before removing the excess reagents.
Apply the fluidic PMMA spacer onto the biosensor and electrically connect the biosensor to the potentiostat. Realize the fluidic connection of the syringe pump with the biosensor by using the fluidic adapter. Start the syringe pump manually with a 0.1 molar PPS at a flow rate of 20 microliters per minute.
At the working electrode versus the on-chip reference electrode apply 30 cycles of an alternating voltage of 0.8 and 0.05 volts for five seconds each. Following this, oxidize the working electrode by applying a voltage of 0.8 volts for 60 seconds. To start the assay signal readout, wait until the measured current signal stabilizes.
Stop the syringe pump and switch the reagent from 0.1 molar PPS to a 40 millimolar glucose solution before starting the software at the syringe pump controller. After automatically stopping the flow for one, two, or five minutes, the syringe pump will then restart the flow again. Observe the resulting currents peak.
The electrochemical biosensor contains one single microfluidic channel with two distinct areas separated by the hydrophobic stopping barrier. The immobilization area, where the assay is immobilized by simple absorption of the biomolecules and the electrochemical cell where the amperometric readout of the assay is performed. For the amperometric readout of the biosensor, the so-called stop-flow technique is used.
First, a constant flow of the enzyme substrate glucose is applied. After the signal stabilizes, the flow is stopped. During the stopping phase, the glucose is converted into hydrogen peroxide and is accumulated in the channel.
By restarting the flow, the hydrogen peroxide is flushed through the electrochemical cell where it is detected, resulting in a current peak. Depending on the amount of bound analyte, and therefore the amount of bound glucose oxidase, the signal height varies, which results in an example calibration curve, as seen here. Once mastered, the fabrication of the biosensor can be done in roughly 10 hours if it's performed properly.
While the assay incubation and readout time depends on the employed assay, but usually can be realized within three hours. After watching this video, you should have a good understanding about the fabrication on chip assay preparation and operation of the electrochemical dry film photoresist based biosensors. While attempting this procedure, it is important to ensure that all protocol steps are strictly followed and performed with great caution.
Since the sensor performance strongly depends on the fabrication process. The implications of this technique extend towards a personalized medicine covering the onsite diagnosis of different types of diseases and drugs, and thus, facilitating in individualized therapy. Don't forget that working with silver electrolyte solutions can be extremely hazardous and precautions such as wearing safety goggles and appropriate gloves should always be taken while performing this procedure.
