Quartz-Crystal Microbalance Biosensor-Based Biopanning to Study Protein-Drug Interactions

To prepare a QCM sensor-chip, attach a ceramic sensor-chip onto the oscillator of a 27-megahertz QCM apparatus, and record the intrinsic frequency in the air-phase before small-molecule immobilization.

After the recording, detach the chip, and carefully add a 20-microliter drop of a 1-millimolar small-molecule derivative solution in 70% ethanol to create a self-assembled monolayer on the gold electrode of the sensor-chip. Place the chip into a Petri dish lined with moistened tissue protected from light for 1 hour at room temperature, before gently washing the electrode surface with ultra-pure water.

Dry the chip with a gentle application of air, and load the chip onto the QCM apparatus. After 1 hour, record the reduction in frequency in the air-phase to measure the amount of the small-molecule that has been immobilized.

For T7 phage library bio-panning, place a cuvette with a dedicated magnetic stirrer onto the QCM biosensor set to 1,000 revolutions per minute, and add 8 milliliters of reaction buffer to the cuvette.

While the buffer is being stirred, attach the QCM sensor-chip to the oscillator. Pull down the arm of the oscillator to immerse the chip into the buffer and begin monitoring the QCM frequency. When the sensorgram equilibrates to around 3 Hertz per minute a frequency drift, inject 8 microliters of a T7 phage library into the cuvette, and mark the injection point on the sensor.

Monitor the frequency reduction caused by the T7 phages binding to the small-molecule immobilized on the gold electrode surface. After 10 minutes, stop the QCM frequency monitor and quickly lift the oscillator to remove the sensor-chip from the batch. Detach the sensor-chip from the oscillator and remove the buffer from the chip.

Place the dried sensor-chip into a humid Petri dish and add a 20-microliter drop of log-phase E. coli host cells onto the gold electrode. Incubate the dish onto a 96-well microplate mixer at 37 degrees Celsius and 1,000 to 1,500 revolutions per minute for 30 minutes protected from light to enhance the recovery of the bound T7 phages.

At the end of the incubation, transfer the 20 microliters of E. coli suspension into 200 microliters of LB medium. According to the general procedure, conduct the phage plaque isolation in the medium and DNA sequencing that encodes the drug-recognizing peptide sequences displayed on each phage capsid.