The overall goal of this protocol is to verify bioprobe immobilization and subsequent DNA biosensing on polysilicon nanowire field-effect transistors. This method can help answer important questions in the bioelectric sensing field, such as bioprobe immobilization and nucleic acid detection. Our biosensing device is a promising transistor for real-time, labor-free, and also high-sensitivity biosensing application.
The implications of these biosensors extend toward clinical diagnosis of emerging infectious disease, because it can readily, sensitively, and accurately detect the specific bio-target. Though this method can provide insight into nucleic acid detection, it can also be applied to detection of other molecules, such as cytokines, hormones, proteins, and viruses. Generally, individuals new to this method will struggle because it requires interdisciplinary collaboration between biology and the electrical engineering fields.
The preparation of the polycrystalline silicon nanowire field-effect transistor is covered by the text protocol. The device is formed from a six-inch wafer. The silicon nanowires on the device are about 100 nanometers wide and 1.6 microns long.
To pre-treat the device, first remove it from storage in an electronically dry cabinet and open the sealed vacuum bag that contains it. Clean the biosensor in a Sonicator loaded with pure acetone for 10 minutes. Then, change the bath to pure ethanol and repeat the sonication for another five minutes.
After this second bath, use a stream of nitrogen gas to dry the device. Next, treat the device with oxygen plasma at 18 watts for 30 seconds. Now take AC conductance measurements and measure the drain current electric properties on the device, as explained in the next section.
Then, proceed with immobilizing DNA on the device surface while taking additional measurements, both AC conductance and drain current, after each modification. First, prepare for the pH profiling. Make 10 millimolar sodium phosphate tribasic dodecahydrate in deionized water, which has a pH of 11.6.
Then, make a 10 millimolar phosphoric acid solution in deionized water, which has a pH of 2.35. Now make seven 10 millimolar buffer solutions with pH values ranging from three to nine by mixing the two stock solutions. With the test solutions and microfluidic channel prepared, measure real-time conductance using a lock-in technique.
Place two needle probes on each of the source and drain pads. Convert the AC current signal into an AC voltage signal using a low-noise current preamplifier. Then set the optimal liquid gate voltage and proceed with delivering each buffer solution while measuring conductance at a drain voltage of 10 millivolts.
Next, measure the drain current with neutral buffer. Flow pH 7 buffer onto the device from a syringe pump at five milliliters per hour. Then, using commercial semiconductor analysis software, measure the drain current in N MOSFET mode.
Set the bias voltage to 0.5 volts and sweep the gate potential from negative one to three volts at 0.2-volt intervals. Now run the test and obtain the data. Begin with immersing the biosensor in 2%APTES in ethanol for 30 minutes to covalently link the nanowire surface.
Then wash the biosensor three times for 30 seconds with ethanol, and then clean it in a Sonicator loaded with ethanol. Now, to create amine groups on the silicon nanowires, place the biosensor on a 120-degree Celsius surface for 10 minutes. Following the heating step, take measurements on the device as described in the previous section.
Next, to create aldehyde groups on the silicon nanowires, immerse the device in 12.5%glutaraldehyde in 10 millimolar sodium phosphate pH 7 for one hour at room temperature. Limit any light exposure during this step. After the glutaraldehyde treatment, wash the device with 10 millimolar sodium phosphate buffer three times for 30 seconds per wash.
Then blowdry the biosensor under pure nitrogen gas and take measurements to confirm the surface modification. The next step is to incubate the biosensor in one micromolar of probe solution overnight to thus apply the DNA probe. The following day, block the unreacted aldehyde groups by immersing the device in 10 millimolar Tris with four millimolar sodium cyanoborohydride for half an hour.
Then wash the device off with 10 millimolar sodium phosphate buffer at pH 7 three times, followed by blowdrying the device under nitrogen gas, as before. Then take a final set of measurements to confirm the surface modification before proceeding to use it as a biosensor. Begin with taking a baseline measurement.
Flow pH neutral sodium phosphate buffer into the device for 10 minutes. During this procedure, the solution flow rate is always five milliliters per hour. Then wait 30 minutes, flow pH neutral buffer over the biosensor for 10 minutes again, and measure the current of the device.
Next, load 10 picomoles of DNA, complementary to the probe, onto the silicon nanowire surface by flowing the solution over the device for 10 minutes. Then incubate the biosensor for 30 minutes. As a negative control, apply 100 picomoles of non-complementary DNA to the device.
After the incubation, flow pH neutral buffer over the biosensor for 10 minutes to wash off unbound target DNA. Following this step, measure the current of the device as before. Next, load one nanomolar of recovery DNA onto the DNA probe immobilized silicon nanowire surface for 10 minutes.
Then incubate the biosensor for 30 minutes, as before. Finally, flow pH neutral buffer over the device for another 10 minutes, and then take current measurements once again. A DNA probe was immobilized on the silicon oxide surface and each modification step was confirmed using X-ray photoelectron spectroscopy.
For the unmodified pSNWFET containing the native oxide layer on the SNW surface, the hydroxyl groups were ionized to form charged groups with increasing pH values. Conductance likely varied at different pHs. The shift in the electric properties of the device after different modifications confirms the change in the wire surface.
In such experiments, the current voltage curve of the unmodified device was used as the baseline. After the device was immersed in APTES, the current voltage curve of the device shifted to the left because of the positive charge on the wire surface. After conjugating glutaraldehyde to the APTES modified device, the current voltage curve shifted back to the right because of neutrally-charged MI bond formation.
Finally, a five prime amine-modified DNA probe was introduced to bind to the APTES glutaraldehyde-modified device. The backbone of the DNA caused the current voltage curve to shift to the far right. After watching this video, you should have a good understanding of how to confirm the bioprobe immobilization and DNA sensing application on polysilicon nanowire field-effect transistors.
Once mastered, this technique of the preparation for nucleic acid detection can be done within one hour, if it is performed properly, which can be performed in advance. The final biosensing takes less than 10 minutes. While attempting this procedure, it's important to remember to analyze the electric properties of the device at each step of the bioprobe modification.
This technique paved the way for researchers in the field of semiconductor-based sensors to its broader application of biosensing. Following these procedures, other methods like immobilizing cancer biomarker probe, can be performed in order to answer additional questions in fields such as clinical cancer diagnosis.
