The overall goal of the following experiment is to demonstrate a rapid and generalizable strategy to reversibly, immobilize small molecules and functionalized nanoparticles on the surface of sensor chips. This is achieved by first immobilizing anti GST antibodies on a sensor surface. As a second step capture of GST antigen.
Functionalized with trans cyclo, octine, or tetra zine groups provides a reactive surface for subsequent cyclo edition reactions with small molecule tet sine derivatives or nanoparticle trend cyclo octane conjugates, which are subsequently functionalized with small molecule tet sine derivatives. Next, the functionalized surface may be used for interaction analysis with a desired solubilized target. In the last step, the surface is regenerated, enabling subsequent capture cyclo edition cycles binding results show on ship bio orthogonal reactions preserve the ability of immobilized small molecules or situ two functionalized nanoparticles to interact with the FK BP 12 target and illustrate how this approach can extend the range of applications for SPR.
The main advantage of this on-chip capture cycle edition technique is that it enables rapid reversible and mobilization of small molecules with control over orientation and I mobilization density. Typically, the capture cycle edition process can be performed within minutes, significantly decreasing surface preparation times, demonstrating the procedure will be Jason Sandler, a student from our laboratory To begin this procedure at eight microliters of a 50 millimolar T-C-O-N-H-S solution in DMSO to 100 microliters of a one milligram per milliliter GST solution in PBS. After shaking the mixture at room temperature for one hour, remove the excess reagent using a Zeba spin desalting column.
Next, add six microliters of a 25 millimolar tetra NHS Ester solution in DMF to 75 microliters of a one milligram per milliliter solution of GST in PBS and shake the mixture at room temperature after one hour. Dilute the reaction mixture with 25 microliters of PBS and purify using a spin desalting column. Then add 100 microliters of the 50 millimolar T-C-O-N-H-S solution to 150 microliters of an emanated nanoparticle solution and shake the mixture in the same manner as before.
After mixing, remove the excess reagent by gel filtration using an NAP 10 column. Alluded with PBS buffer. Collect the colored band containing the N-P-T-C-O product.
Transfer the purified sample to a centrifuge tube containing a centrifugal filter device with a molecular weight cutoff of 100, 000. Then concentrate the sample to a final volume of approximately 150 microliters following centrifugation. Add 50 microliters of 0.1 molar cynic anhydride solution in DMSO to the N-P-T-C-O solution.
After shaking the mixture at room temperature for one hour, purify the N-P-T-C-O product using an NAP 10 column eluding with PBS using a surface plasma in resonance instrument, select immobilization on flow cells one and two in the AM immobilization wizard template. Modify the Amin method to activate surface carboxylic groups by injection of a one-to-one solution of 0.4 molar EDC and 0.1 molar NHS for 480 seconds. At a flow rate of 10 microliters per minute, set the injection of ethanolamine to quench the remaining activated esters to a 422nd contact time at a flow rate of 10 microliters per minute.
Input the ligand name anti GST and set to inject for a contact time of 420 seconds at a flow rate of 10 microliters per minute. Next place the previously prepared solution vials in reagent rack two. Making sure to match the positions with the content list after assigning the file name and destination where data will be saved.
Start the immobilization. Edit the immobilization wizard to only inject 20 micrograms per milliliter of the GST ligand solution for 420 seconds at five microliters per minute over reference flow cell one. Place the vials containing G-S-T-T-C-O-A previously prepared 10 micromolar TERAZ olaine solution and the regeneration solution in reagent rack two.
Select the manual run method, set the flow rate to five microliters per minute and the flow path to flow cell two. Inject the G-S-T-T-C-O solution for 420 seconds followed by the Teraz Benz Lamine solution for 600 seconds. Following this, regenerate the surface with two 32nd injections of regeneration solution.
After placing the GST TERAZ zine, N-P-T-C-O, teraz Belamine and regeneration solution vials in reagent rack two, select the instrument manual run method, then set the flow rate to five microliters per minute and the flow path to flow cell two. Inject a solution of GST Tetra zine for 60 seconds, followed by a solution of N-P-T-C-O for 60 seconds. Finally, inject a solution of tetras zine belamine for 60 seconds.
When finished, regenerate the surface in the same manner as before. Next, select a new method and input the general parameters, which include data collection, rate detection, sample compartment, temperature, concentration units, and buffer settings. In the assay steps panel, create a new step and name it sample.
Then set the purpose and connect based settings to sample in the cycle types panel, create a new cycle step and insert the following commands. Capture sample regeneration one and regeneration two. Select the capture command and input G-S-T-T-C-O as the ligand.
Name 120 seconds as the contact time, five microliters per minute as the flow rate. And set the flow path to second. Then select the sample command and input a 182nd contact time, a 62nd dissociation time, a 30 microliter per minute flow rate and set the flow path to both.
After this, select the regeneration one command and input the regeneration solution. Name a 32nd contact time at 30 microliters per minute and set flow path to second. Repeat the same procedure for the regeneration.
Two command select set, run, and set the flow path to two dash one. Select next and fill the sample list with the tetra Belamine analyte solution and a concentration series of 1.5 to 20 micromolar at a one to two dilution. Then select next to rack position panel.
After setting the solution vials in reagent rack two, place the dilution series. In a 96 well plate. Making sure to match the positions with the content list.
Save the method template and start the binding assay. Say once a new method and the general parameters have been selected, create and name the capture sample and regeneration steps in the assay steps panel. Following this, select the corresponding purpose and connect base settings.
Then select the cycle types panel and create three cycle steps. Capture, sample and regeneration. Insert the capture command twice under the capture cycle.
Step in the capture one panel. Select the capture solution as variable. Then set the contact time to 300 seconds at a flow rate of five microliters per minute and set the flow path to second in the capture two panel.
Select the capture solution as a variable. Set the contact time to 250 seconds at a flow rate of five microliters per minute and set the flow path to second. Next, insert the sample command under the sample cycle step.
Select the sample panel and set the contact time to 60 seconds. The dissociation time to 200 seconds at a flow rate of 30 microliters per minute and set the flow path to both. Insert the regeneration command twice under the regeneration cycle step, select the regeneration one panel, input the regeneration solution.
Name a contact time of 30 seconds at 30 microliters per minute, and set the flow path to second. Repeat the same procedure for the regeneration. Two panel select set up, run and set the flow path to two dash one select next and input G-S-T-T-C-O and teraz AP 1, 497 conjugate as the capture solution names FKBP 12 as the sample name and 1.5 to 96 nano molar at a one to two dilution as the concentration series.
After placing the solution vials in reagent rack two and the dilution series in a 96 well plate save the method template and start the binding assay. Efficient reversible immobilization of bioactive small molecules with control over orientation and density plays a key role in the development of new biosensor applications. Shown here is the real-time monitoring of teraz zine Benzoin immobilization.
A solution of G-S-T-T-C-O is injected over a pre immobilized anti GST surface resulting in approximately 400 R RU rise. In response, a second injection with Teraz zine Benzo Lamine shows a fast 15 RU rise in response. No dissociation of the dert antigen was observed after switching to running buffer, providing evidence for structure stability.
The surface was regenerated in the last step of the cycle to enable multiple capture cyclo edition cycles. Using this procedure and replacing the tetra zine, benzyl Lamine moiety with the teraz zine AP 1, 497 conjugate generates a bioactive surface, which is used for interaction studies with its target. FK BP 12 disruption of the antibody antigen interaction regenerates the anti GST surface, allowing a new molecular assembly to be built.
In this case, A GST TERAZ zine injection is followed with an injection of N-P-T-C-O, effectively immobilizing the nanoparticle to the sensor surface. Unre reacted TCO groups on the nanoparticles are available for cyclo edition with injected teraz zine benzo lamine. Alternatively, TET INE AP 1, 497 conjugate is used to monitor functionalized nanoparticle FK BP 12 interactions.
No dissociation of the multi-component structures were observed providing evidence for structure stability and bioactivity with capabilities for real-time immobilization reaction monitoring and surface regeneration. The straightforward characterization of the association rate KA is accomplished as shown here. Knowledge of reaction kinetics provides guidelines for controlling immobilization density and important parameter for biosensor assays.
Injecting increasing concentrations of teraz BELAMINE or N-P-T-C-O in duplicate over G-S-T-T-C-O or GST teraz zine surfaces respectively generates the binding data. The binding data for two sequential analyte samples of the same concentration over the same surface are nearly super imposable reflecting minimal loss of antibody binding capacity. Due to multiple cycles of capture cyclo edition and regeneration, the evaluation software provides the best fit kinetic analysis for the cyclo edition reaction rates.
We have developed an approach that utilizes antibody antigen capture, coupled to fast, highly specific cyclo edition reactions between teraz zine and trans cycline that enables rapid reversible immobilization of small molecules. For SPR chip based studies, the capture cycle edition method requires short contact times with less than 10 micromolar concentrations of small molecule ligands. After watching this video, you should have a good understanding of how to immobilize small molecules or multi-component structures on a sensor surface using the capture cyclo edition method.
In order to achieve this, it is important to use reagents of good quality for conjugation reactions and properly set up assay parameters on the SPR instrument.
