A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

The overall goal of this new fluoro metric drug screening method is to determine the binding affinity of small drug molecules to a target protein by forming fluorescent gold nano clusters within the drug loaded protein template. This method can help answer key questions in the field of protein targeting drug discoveries such as how the vidding strength of drug molecules can affect the stability of proteins from unfolding leading to def formation of fluorescent gold nano clusters of different intensities. Main advantage of this tenix is that we can turn a protein target into a fluorescent biosensor to enable fast screening of small molecular drugs without the needs of sophisticated instrument and tedious labeling steps.

The combination of my own knowledge as well as co-author, she's discovery li loaded the protein, has a distinct stabilization effect on plasmonic. Nanoparticle inspired the idea for this method. Begin this procedure with preparation of the chemical reagents for drug screening as detailed in the text protocol to prepare the human serum albumin or HSA templated gold nano clusters, placed two glass vials each containing a micro magnetic stir bar on two separate temperature controllable magnetic stirs.

Set the temperature of one magnetic stir to 60 degrees Celsius. Next, add 200 microliters of human serum albumin solution, 200 microliters of ultrapure water and 200 microliters of gold chloride solution to each vial under constant stirring to allow the encapsulation of gold ions inside the protein template, two minutes later, add 20 microliters of sodium hydroxide solution to each vial so as to activate the reducing capability of human serum albumin informing gold nano clusters. Start to record the reaction time at zero minutes every 20 minutes.

Draw 50 microliters of each sample and transfer to a 384 well black plate. Measure the emission spectrum with a microplate reader. Stop the magnetic stir after 100 minutes.

Cool down the glass vials under running water in a sink. Plot the photo emission spectra at all times for each sample to acquire the formation kinetics of gold nano clusters at different temperature conditions. To prepare the bovine serum albumin or BSA templated gold nano clusters, place a glass vial containing a micro magnetic stir bar on the top of a magnetic stir.

Leave the temperature as room temperature. Mix 200 microliters of BSA solution, 200 microliters of urea and 200 microliters of gold chloride solution in the glass vial under constant stirring. Two minutes later at 20 microliters of sodium hydroxide solution.

To activate the reducing capability of BSA to form gold nano clusters and start to record the reaction time as zero minutes. Measure the photo emission spectrum of the reaction mixture hourly. Place five glass vials each containing a magnetic stir bar and pre-labeled with a drug or pure HSA on top of a temperature controllable multi-point magnetic stir.

Add 200 microliters of HSA to each vial. Then add one microliter of each previously prepared drug solution to the four corresponding files. Also, add one microliter of DMSO to the control switch on the stir r.

Set the spin speed to 360 RPM and incubate for one hour to allow the drug binding to HSA one hour later at 200 microliters of milli Q water and 200 microliters of gold chloride solution to each vial under constant stirring. Set the temperature to 60 degrees Celsius under constant stirring for 10 minutes. After 10 minutes at 20 microliters of 1.5 molar sodium hydroxide to each file and start to record the reaction time at zero minutes.

Joint reaction solutions for measurement should follow the same sequence of adding drugs and should be practiced as quickly as possible to minimize the time lag effect Quickly draw 50 microliters of solution from each vial. Transfer to a 384 well black plate and measure the emission spectrum. Record the emission spectra of each sample every 10 minutes, collecting at least four spectra at four different times.

Repeat these steps several times to obtain results with a consistent trend. Plot the time resolved photo emission spectra for each sample. Identify the peak intensity of all samples and plot against the time to compare the formation kinetics of gold nano clusters in different drug loaded protein templates to measure the binding constant between each drug and the HSA.

Replace the drug solutions with ibuprofen solutions at four different concentrations. 10 minutes later, cool down the glass vials under running water in a sink. Draw 50 microliters of the above solutions from each vial to a 384 well black plate and measure the emission spectrum.

Repeat these steps twice more to obtain another two sets of results at different drug concentrations before stopping the magnetic stir. Then plot the photo emission spectra and analyze the results for the raw obtained in each individual batch. Plot the photo emission spectra of each sample in software such as Origin Pro and determine the peak intensity.

Calculate and plot the relative fluorescence intensity against the drug concentration. Then calculate the binding constant by fitting the data to a single site binding model. Using the Mics cementin equation to do this in software such as Origin Pro.

Select the menu analysis fitting non-linear curve fit, then select hail function from the growth sigmoidal category on the settings function selection page. Click fit to display. The fitted results shown here are representative results of the time resolved photo emission spectra of gold nano clusters produced at different protein denaturing conditions.

It can be observed at gold nano clusters form faster at higher temperature. Urea denaturing condition can also facilitate the direct synthesis of gold nano clusters using BSA protein as template. Typical results of fast fluoro metric drug screening based on the fluorescence intensity of gold nano clusters using drug loaded HSA as template are shown here.

The higher the fluorescent intensity, the weaker the binding strength of a drug molecule to HSA. These spectra show the effect of drug loading concentration on the formation rate of fluorescent gold nano clusters inside the ibuprofen loaded HSA. The higher the drug concentration, the lower the fluorescent signal of the resultant gold nano clusters shown here is a representative result of protein drug binding constant determination.

It is calculated by fitting the relative fluorescence intensity of gold nano clusters synthesized in the HSA template preloaded with ibuprofen drug of varied concentrations using the MICUs cementin equation Once mastered, this technique can be done in 30 minutes for multiple drug screening in parallel if it is performed properly. After watching this video, you should now have a good understanding of how to synthesize rein, go nano cluster in the drug loaded protein, and to determine the binding affinity of drugs to a target protein as detectable by the fluorescent signal emitted from the resulting gold nano clusters.