A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

The overall goal of the following experiment is to observe the effect of DNA hybridization events on impedance spectra using a microfluidic electrochemical device. To do this, an electrochemical chip is fabricated to enable an array of electrode sensors and a microfluidic PDMS chip is fabricated. To facilitate the introduction of micro volume samples, the two chips are bonded together and functionalized with single stranded DNA probes.

Target single stranded DNA is then added upon DNA hybridization, the stronger repulsion force between the negatively charged double stranded DNA and the negatively charged cyanide Ferris cyanide molecules results in higher charge transfer resistance. Ultimately, DNA hybridization events can be detected by measuring the changes in electrochemical impedance spectra. The main advantage of this technique over existing methods, like using a plate reader or J shift assay, is that the devices can perform the same technique with low sample volume and at a fraction of the cost with without sacrificing sensitivity or specificity.

This method can help answer key questions in cancer research, influenza detection, and genetic engineering by enabling quick specific DNA sequence screening and investigation of the binary kinetics of single stranded DNA sequences. Because this rapid high throughput DNA hybridization analysis method can perform with low sample volume and without complicated sample preparation steps, the implications of this technique extend to our diagnosis of genetic disorders, various forms of cancer or illnesses with specific DNA markers such as influenza. Generally individuals new to the method will struggle because the development of micro devices requires control processing parameters and an iterative approach to solving problems such as solution leakage.

Following assembly of the device To pattern gold electrodes onto the electrochemical chip, hold a blank prime grade four inch silicon wafer with tweezers and use a wash bottle to rinse the surface of both sides of the wafer with acetone, methanol, and then isopropanol. This process is referred to hereafter as an A MI clean under a stream of deionized water. Rinse the isopropanol from the wafer, then use a nitrogen gun to dry the wafer.

Load the wafer into plasma enhanced chemical vapor deposition or P-E-C-V-D equipment. Enter the parameters to yield a one micrometer thick silicon dioxide ation layer and start the automated process next, using a DC sputtering tool deposit a layer of chromium 200 angstroms thick onto the wafer, then deposit a layer of gold 2000 stroms thick. Load the wafer on a spinner, dust the surface with a nitrogen gun and using a paster pipette apply spin PR one positive photo resist spin to create a uniform film about 1.6 micrometers thick.

Then pre-bake the wafer for one minute at 100 degrees celsius on a hot plate. Load the wafer with the photo resist into the mask aligner equipment. Then initiate an exposure of 190 millijoules per centimeter squared at 405 nanometers in which the mask aligner will open the shutter and expose the photo resist to light through a mask with the designed features.

After the exposure, submerge the wafer in a glass dish containing 3 5 2 developer and incubate for 30 seconds with gentle shaking. Then rinse the wafer under a stream of di water and dry with the nitrogen gun as before. Next, submerge the wafer in a glass dish containing gold etching solution and incubate for 1.5 minutes with gentle shaking.

Then transfer the wafer to a glass dish containing chromium mentioned and incubate with gentle shaking until the chromium marks on the wafer disappear. This should take about 30 seconds. Rinse the wafer with di water and dry with the nitrogen gun holding the wafer with tweezers.

Strip the photo resist using the a MI clean procedure. Then rinse with DI water and dry with the nitrogen gun wearing two layers of gloves. Submerge the wafer in a Teflon dish with piranha clean solution for one minute.

Then rinse the wafer with di water and dry with a nitrogen gun. Next to pattern the platinum electrodes, load the wafer on a spinner. Dust the surface using a nitrogen gun and using a paster pipette dispense PR two positive photo resist on the surface spin to create a uniform film about 1.4 micrometers thick.

Place the wafer on a hot plate at 100 degrees Celsius and pre-bake for one minute. After the pre-bake, expose the wafer at 30 millijoules per centimeter squared 405 nanometers through a photo mask. Then place the wafer on a hot plate at 125 degrees Celsius and bake for 45 seconds.

Remove the mask from the mask, aligner and flood. Expose the wafer with 1000 millijoules per centimeter squared at 405 nanometer wavelength without a photo mask. After the exposure, develop the wafer by submerging it in a glass dish containing six to one a Z 400 K developer solution.

Incubate for two minutes with gentle shaking. Then rinse the wafer with di water and dry with a nitrogen gun. Using an E-beam evaporation system deposit a 400 angstroms thick layer of titanium followed by a 1600 angstroms thick layer of platinum on the wafer.

Place the wafer in a glass dish filled with acetone and place the dish in an ultrasonic bath for five minutes to lift off the photo. Resist then holding the wafer with tweezers. Use a wash bottle to rinse both surfaces of the wafer with acetone and then isopropanol.

Rinse the wafer with di water and dry with the nitrogen gun. To prepare the mold for the assay channels, rinse a blank four inch test grade quality silicon wafer with the a MI clean procedure. As before, rinse the isopropanol from the wafer with di water, followed by drying with a nitrogen gun.

Load the wafer on a spinner, dust the surface of the wafer with a nitrogen gun and using a pasti pipette dispense PR three negative photo resist on the surface to create a uniform film about 100 micrometers thick. Place the wafer on a hot plate to pre-bake it using a two-step baking procedure. First, ramp up the temperature from room temperature to 65 degrees Celsius at 300 degrees Celsius per hour.

Then hold at 65 degrees Celsius for 10 minutes. Ramp up to 95 degrees Celsius at 300 degrees Celsius per hour and then hold for 30 minutes. Expose the wafer with 2, 500 millijoules per centimeter squared at 405 nanometer wavelength through a photo mask.

Then place the wafer on a hot plate and post bake the wafer by ramping up from room temperature to 95 degrees Celsius at 300 degrees Celsius per hour. Hold at 95 degrees Celsius for 10 minutes. Submerge the wafer in a glass dish containing propylene glycol, monomethyl ether acetate or P-G-M-E-A developer and allow it to develop for 10 minutes.

Rinse the wafer with isopropanol and dry with a nitrogen gun to fabricate assay channels to find an enclosure around the mold wafer with aluminum foil and slowly pour the uncured poly dimethyl suboxane or PDMS over the mold. Bake the mold with the PDMS in a box furnace using a two-step baking procedure in which the temperature ramps up from room temperature to 80 degrees Celsius over five minutes and remains at 80 degrees Celsius for 17 minutes. After the mold has cooled, peel away the PDMS from the mold and place on aluminum foil.

Cut the assay chips with a surgical knife and define holes with a biopsy punching tool as shown here. Manually align the PDMS assay channels with the working electrodes on the electrochemical chip. PDMS sticks well to the electrochemical chip, sealing the channels and preventing leakage.

Connect flexible tubing to a plastic elbow or straight connector using a short flexible tube as an adapter to functionalize the vertical micro channels. Connect a syringe containing the single stranded DNA probe to the tubing and place it in a syringe pump. Attach the connectors to each of the whole punched inlets in the device.

The single most difficult aspect of this procedure is flowing the solution in the microchannels without generating bubbles. To ensure success, flow the solution very slowly To achieve homogenous filling of the channel, Initiate the pump at a flow rate of 200 microliters per hour to slowly fill each of the three separate microchannels with a different single stranded DNA probe incubation solution. After incubating for one hour, exchange the syringe tubing and connector to rinse the microchannels with PBS Next exchange the syringe tubing and connectors to fill the microchannel with a solution consisting of one millimolar six mer CAPTO one H ethanol MCH in 10 millimolar PBS 100 millimolar sodium chloride, and 1.395 millimolar TCEP incubate for one hour.

Then rinse with PBS as before using tweezers. Lift off the PDMS rotated 90 degrees to a horizontal orientation and placed down. This will expose separate rows of reaction chambers with each row containing a unique counter and reference electrode for the DNA hybridization analysis.

Exchange, the syringe tubing and connectors to fill the micro channels with a control measurement solution of Forex concentrated saline sodium citrate SSC buffer incubate for 20 minutes after the incubation exchange the syringe to fill the micro channels with five millimolar Ferris cyanide five millimolar Pharaoh cyanide in PBS solution. Then to record the background impedance values of the electrochemical system for different frequencies by electrochemical impedance spectroscopy or EIS connect a potential stat to the working counter and reference electrodes using probe tips. Input the parameters of the EIS measurement into the computer software and then initiate the measurement.

Repeat this measurement for each working electrode in the micro channels to measure DNA hybridization for each non complimentary and complimentary single stranded DNA target exchange. The syringe tubing and connector. Fill the micro channels with a four x concentrated SSC buffer containing one micromolar of the target single stranded DNA and incubate for 20 minutes.

Take measurements by initiating the EIS measurement with a software on the computer for each target single stranded DNA solution to validate the electrochemical activity of the bio chipp cyclic vol. Telemetry measurements were taken in the micro channels filled with an electroactive redox Couple cyanide cyanide shown here are cyclic vol telegrams of nine working electrodes. The similar shape, peak heights, and peak separation for all electrodes demonstrate high reproducibility to demonstrate the ability of the biochips to detect DNA hybridization events.

Electrochemical impedance variations were measured for hybridization events between three single stranded DNA probes and their complimentary and non complimentary single stranded DNA targets higher charge transfer resistance changes were observed when complimentary single stranded DNA target was introduced and hybridized with the probe compared to the introduction of non complimentary single-stranded DNA targets to demonstrate the sensitivity of the biosensor. Different concentrations of complimentary single-stranded DNA target were introduced to the sensing probe shown here is a nyquist plot of electrochemical impedance spectroscopy measurements following the introduction of increasing target single stranded DNA concentrations indicated by the arrow on the graph. Increased impedance values were seen at low frequencies about 15 hertz for higher target, single stranded DNA concentrations due to the stronger repulsion forces between the double stranded DNA and the ferro cyanide Ferris cyanide molecules.

Overall, the BIOCHIPS shows the ability to rapidly sense the presence of DNA hybridization events. After watching this video, you should have a good understanding of how to develop and fabricate a microfluidic electrochemical device and use it to detect DNA hybridization events. While attempting this procedure, it's important to remember to optimize various bios parameters such as signal to nurse ratio, stability and sensitivity Following this procedure.

Other methods like introduction of single stranded DNA targets with slightly different sequences of nucleic acids can be performed in order to answer additional questions like DNA mutation combination and competition. Don't forget that working with micro fabrication process solutions can be extremely hazardous, and body eye and hand protection should always warn while performing this procedure.