The overall goal of this procedure is to measure the changes in the mechanical properties of underlying model bacterial cell targets caused by drug target binding interactions, and to quantify antibacterial resistance. This is accomplished by first depositing 20 nanometers of gold film onto one side of the pre-cleaned cantilever sensor array. The second step is to self-assemble appropriate surface targets, mimicking drug susceptible and drug-resistant bacterial phenotypes onto six of the cantilever sensors, while the remaining two cantilever sensors are assembled with irrelevant bacterial model targets to act as measurement controls.
Next, the cantilever sensor arrays are loaded into a liquid flow chamber. The final step is to prepare drug solutions at clinically relevant concentration ranges and sequentially inject each drug concentration over the cantilever sensor arrays. Ultimately, laser light focused onto the free ends of the cantilever sensor array is used to show cantilever deflections caused by the changes in surface stress that arise from drug target binding interactions.
The main advantage of this technique over existing methods like surface plus mon resonance and quotes crystal micro parlance, is that it detects the surface train generated by drug target binding interactions directly measuring specific ano mechanical forces caused by each drug binding to the surface targets. This method can help answer key questions in the pharmacology field, such as the speed, efficiency, and accuracy of dosing. Generally, individuals new to this matter will struggle because of tedious sample alignment.
However, we have recently innovated novel readout mechanism, which does not require initial sample alignment, and in future this method use will be much easier and more user-friendly to use. To begin, use a pair of Teflon tweezers to immerse a cantilever chip into a freshly prepared parama solution for 20 minutes. The cantilevers used in this experiment are 500 microns long, 100 microns wide and 0.9 microns thick.
After 20 minutes, remove the cantilever and rinse it thoroughly with deionized water. Inspect the cleaned chip using an optical microscope to look for impurities. Place chips with any remaining dust or dirt back into fresh piranha solution for an additional 20 minutes.
After a thorough cleaning in deionized water, rinse the chip in pure ethanol and dry it on a hot plate at 75 degrees Celsius for about 30 seconds to remove any traces of water. Then inspect it again using an optical microscope to confirm their cleanliness. Transfer the cleaned cantilever chip into an evaporation chamber loaded with titanium and gold sources.
Pump it down until a vacuum pressure of one times 10 to the negative seventh millibar is achieved. Once the required vacuum is attained, code one side of each cantilever array with a two nanometer thick layer of titanium. Then set up the system to deposit a 20 nanometer thick gold layer over the titanium, which acts as an adhesion layer.
Confirm the thickness of each layer with a quartz crystal monitor placed directly above the target source. Once the correct thickness is achieved, leave the gold coated cantilever chips in the chamber for one to two hours to cool under vacuum before opening the chamber. Next, transfer the cantilever sensor chip to a vacuum storage vessel filled with Argonne to prevent any form of contamination.
The cantilever chips can be stored here indefinitely. However, if no storage vessel is available, the chip must be used immediately just prior to the experiment. Arrange micro capillary tubes into channels on a functionalization stage according to the cantilever pitch size of 250 microns.
Then align them using a glass slide and clamp them into place to prevent them from moving during the experiment. Next, prepare two millimolar solutions of folated surface receptors made from drug sensitive and drug-resistant bacterial cell wall targets using pure ethanol. Next place a separate cantilever into each of the micro capillary tubes.
Ensure that the solutions of the surface capture molecules are confined to each individual cantilever sensor to avoid contamination. Then randomly assign a mixture of these surface capture molecules to the micro capillary tubes, and then inject eight microliters of the surface target solutions into their assigned tubes. Allow the cantilevers to incubate in the solutions for 20 minutes.
During this time, the freshly prepared gold coated cantilevers will become chemically active sensors through the self-assembly of bacterial muco peptide targets into monolayer on their gold coated surfaces. Begin by dissolving 0.1 molar mono and die basic sodium phosphate salts in ultrapure water and mix to yield a pH value of 7.4 and use 0.2 micron filters to filter the solution. Then add 0.002%of polysorbate 80 to the buffered solution.
This will minimize aggregation effects caused by non-specific interactions of drug molecules to the glassware. Next, dilute vancomycin and orin with freshly buffered solutions to the desired concentration of 100 pico molar to 250 micromolar, which will enable the calculation of KD vortex and sonicate the samples to ensure complete drug dissolution in buffer and serum to determine if the drug action is modified by serum proteins. Repeat this procedure with the same drug concentrations only this time, add them to whole serum.
Also vortex the serum samples for a total of 20 minutes during mixing to ensure complete solubility of the drugs. Lastly, purge the solutions with Argonne to begin the detection of surface stress across the cantilevers. First load the eight functionalized cantilever sensors on the chip into a liquid flow cell chamber.
Next, align the laser spot onto the free end of each sensor. Confirm the alignment by heating up the liquid chamber by one degree Celsius. All eight gold coated cantilever sensor arrays will undergo compressive downward bending due to the biome metallic effect caused by the differences in expansion rates of silicon and gold.
After heating for approximately 10 minutes, calculate the bending variation at the maximum bending signals between individual cantilever sensors. If the relative standard deviation of the signals is less than or equal to 5%then accept the alignment as desirable. Otherwise, repeat the process following alignment.
Allow the cantilevers to cool back to room temperature for 10 minutes to ensure equilibration. Next, measure the resonant frequencies of all eight cantilevers and calculate their spring constants. If the variation of the spring constant between each cantilever sensor is less than or equal to 1%then accept the calibration.
Otherwise, replace the cantilever chip sensor connective fluidic system like the syringe pump shown here to the accepted chip via a six-way valve. Set the flow rate of the system to run at a constant rate between 30 and 180 microliters per minute and set up the entire system in a temperature controlled cabinet at 25 degrees Celsius. Then begin to inject either buffer or serum alone with no drug to acquire a control baseline measurement lasting between five and 30 minutes.
Start automated lab view software to acquire the deflection data from each of the cantilevers simultaneously. Monitor the absolute bending signals of all eight cantilevers using a serial time multiplexed optical beam method with a single position sensitive detector. Once the baseline has been acquired, inject a drug solution for 30 to 60 minutes following the test sample.
10 millimolar HCL Wash is injected for 10 minutes to one hour in order to dissociate any bound drug complexes. Using this system nanoscale precision and unprecedented sensitivity to a single hydrogen bond deletion is possible here. The change on the end of a peptide chain from D alanine to D lactate results in a significant reduction of vancomycin binding rendering bacteria with the DLAC sequence resistant to this therapy when varying concentrations of the drug were tested on the D alanine terminating chain of peptides, vancomycin was shown to have a dose dependent effect with around 10 nanomolar being the lowest detectable concentration.
The ability of this system to detect antibiotics in a physiological environment was further investigated in serum at a clinically relevant concentration of seven micromolar. While the D alanine coated cantilevers still showed considerable bending with these conditions, the DLAC coated ones did not. Using data points from multiple drug concentrations, it's possible to create models which describe the drug target binding events.
The fit lines shown here, combine the langur absorption, isotherm, and a term to describe the large scale mechanical consequences of stressed network formation Once mastered, this technique can be done in minutes to confirm efficiency and accuracy of drug dosing, as well as any sign of and bacterial resistance if performed properly.
