The overall goal of this technique is to detect and quantify low abundant biomolecules in highly complex samples with high sensitivity, simplicity, low cost, speed, and without the use of any labeling. This method can help answer key questions in the biotechnology, biochemistry, and biomedicine fields, such as environmental monitoring, food safety, drinking water quality, and medical diagnosis. The main advantages of this technique are its inherent rapidity, simplicity, high sensitivity, low cost, easy manipulation, and real time detection without using any labeling reagents.
The implications of this technique extend toward diagnosis of diseases due to its ability to quantitate low abundant biomolecules. For example, different biomarkers in real time. To clean the glass coverslips, immerse them in 10 milliliters of 1.0 molar HCl for 10 minutes in an ultrasonic cleaner at room temperature.
Then, immerse them in deionized water and 1.0 molar Sodium Hydroxide under the same conditions. Dry the glass coverslips with nitrogen gas. Then, immerse the clean and dried coverslips in 10 milliliters of an APTES solution for one hour to introduce amino groups on the coverglass surface at room temperature.
Rinse the coverslips with deionized water before drying them again with nitrogen gas. Immerse the APTES modified coverslips in a 10 milliliter solution of glutaraldehyde for two hours to activate the amino groups on the surface at room temperature. Following incubation, rinse the coverslips with 10 millimolar phosphate buffer to remove excess glutaraldehyde from the surface.
Dry the coverslips with nitrogen gas. Next, prepare 1.0 milliliter of template solution in 10 millimolar phosphate buffer in a 0.1 milligram per milliliter concentration. Drop 200 microliters of this template solution onto the modified coverslips and incubate at four degrees Celsius overnight.
To clean the electrodes, first immerse the electrodes in a small beaker containing five milliliters of 70%ethanol for 10 minutes in an ultrasonic cleaner at room temperature. Then, sequentially immerse the electrodes in deionized water, acetone, deionized water, Acidic Piranha Solution, and deionized water under the same conditions. Dry the electrodes with nitrogen gas.
To perform the electropolymerization of Tyramine, prepare eight milliliters of 10 millimolar Tyramine solution in 10 millimolar phosphate buffer containing two milliliters of ethanol. Perform 15 cycles of cyclic voltammetric scans in this solution using a potentiostat covering a potential range of zero to 1.5 volts and a scan rate of 50 millivolts per second. Then, rinse the electrodes with deionized water before drying the electrodes with nitrogen gas.
Immerse the electrodes in a solution containing 30 millimolar acryloyl chloride and 30 millimolar trimethylamine in toluene at room tempereature over night before rinsing and drying the electrodes again. Prior to polymerization, prepare a monimer solution containing monomers and cross-linker in 820 microliters of ultra-pure water. Then, prepare 5%volume to volume TEMED in ultra-pure water.
Add 20 microliters of the TEMED solution into the monomer solution, and purge with nitrogen gas for five minutes. Then, add 20 microliters of freshly prepared APS into the monomer solution. Pipette 1.5 microliters of the monomer solution onto the modified gold electrode surface.
Now, start the polymerization by bringing the template stamp into contact with the monomer treated surface, and leave it for five hours in room temperature. Following polymerization, remove the template stamp from the surface carefully, by using forceps. Next, rinse the electrode with deionized water and dry with nitrogen gas.
Immerse the electrodes in one milliliter of ten millomer of one dodecanetheol prepared in ethanol for twenty minutes in order to cover pinholes on the electrode's surface. Finally, rinse the electrodes with deionized water and dry the electrodes with nitrogen gas before characterizing their surfaces with scanning electron microscopy. Insert the imprinted capacitive gold electrodes into the electrochemical flow cell integrated to a capacitive biosensor.
Prepare 100 milliliters of regeneration buffer and one liter of running buffer. Start the analysis with the injection of regeneration buffer to regenerate the system and running buffer to re-equilibrate the system for 25 minutes. Prepare standard template solutions in the desired concentration range in running buffer.
Then, inject 250 microliters of these standard solutions sequentially onto the system in optimum conditions. The surface characterization of the gold electrodes is performed by scanning electron microscopy. The bare gold surface is shown.
Also shown, are bacteriophages adherent to the electrode's surface in different magnifications. Shown here is real-time bacteriophage bonding to the imprinted gold electrode through a sensogram. A stable base line is established after regeneration and re-equilibration of the system before the injection of sample.
There are phage-specific cavities on the electrode. Injection of the sample-inducing bacteriophages results in a reduced capacitance, due to the binding of target bacteriophages to the imprinted cavities on the gold electrodes'surface. Calibration curves show the changing capacitance versus protein injection in bacteriophage injection.
From the equations in these graphs, it is possible to calculate the concentration of bacteriophage or protein in a sample. After watching this video, you should have a good understanding of how to develop an ultra-sensitive, fast, and real-time detection system. Once mastered, this technique can be done in five hours and thirty minutes, excluding the waiting times in between.
While attempting this procedure, it's important to prepare all the agents fresh, in the same day. Following this procedure, other metrics like atomic force microscopy, scanning electronic microscopy, ellipsometer, and quantum angle measurements can be performed to determine if the polymerization or imprinting was successful, and to detect any significant difference between the surfaces of bare and imprinted gold electrodes. After its development, this technique paved the way for researchers in the field of biochemistry, biotechnology, and biomedicine to explore an ultra-sensitive detection of a biomolecule in food safety and medical diagnosis.
Don't forget that working with functional monomers, cross-linkers, initiators, and other agents can be extremely hazardous. And, precautions such as wearing gloves, laboratory coats, and performing the polymerization experiments inside the fume hood, should always be taken, while performing these experiments.
