How can one quantify capture efficiency of antigens in a microfluidic immunoassay chip ?
For example, if we have a anti-IgG coated microfluidic chip and we want to know the capture efficiency of the device. We are flowing say "x ng/ml" of IgGs for "t" seconds (till saturation happens) ?
Is this can be done using fluorescence measurements ? What exactly is the best manner to quantify "capture efficiency" ?
Hello, Ahmad.
Read about ELISA principle. You could replace the final detection system of an Enzyme linked sandwich component with FITC-linked molecules (either IgG or biotin-streptavidin system). Then it can be read by a spectrofluorometer in macroscale. There is a few systems that allow microfluorimetry or image presentation system using inverted fluorescence microscopy. Probably, your microdevice should first be adaptable to do the job. Under the microscope, the images of where assay takes place may be taken in a time-course manner. Due to the exosure to UV, you have to select spot that is not exposed for accurate measurement. Make a standard fluorescence intensity curve with known concentrations of IgG. You could tell in quantitative way. Not easy, but possible.
Reagards.
If you have known concentration of your antigen, you can check the concentration in remaining solution, deduct the value from flow through. It will give you approximately capture efficiency. Now you can check your chip binding by ELISA or fluorescence methods. You can calculate the binding efficiency from these two values.
Hope this is helpful.
I have the initial concentration of the antigen with me. How I am going to check the concentration in the solution obtained after flowing through the chip ?
Assuming I have 1ng/ml to 100 ng/ml of initial antigen concentration.
Bradford protein assay or you could do micro-dot blotting and image analysis for semi-quantitaion.
Try with the highest concentration, let's assume if you have an initial concentration 1ug/µl and you have 10 ml of the total solution.
Now if you are passing it with 100µl/second for 10, 20, 40, and 60 seconds. In 10 second you will pass 1 ml and in 20 seconds 2 ml and so on.
You need to collect the flow through to check the concentration.
Now you can check the concentration by BCA or nanodrop or running the page gel of flow through after each time interval. Now suppose you get the 10ng/µL in flow through you can calculate the total concentration in flow through. Now deduct that value from your initial loading concentration you will get the approximately bound protein.
For better understanding I recommend you to refer my paper Article Efficient intracellular delivery of biomacromolecules employ...
Where I have used the same method to check streptavidin binding and saturation on the nanowire. Figure S3.
If you have any fluorescence tagging, you can follow figure S2
Hope this can help you.
All the best for your experiments.
Hope you have found any solution by now, if not this paper can be helpful for your study they have also used IL-2 binding:
Article Injectable Polymeric Cytokine-Binding Nanowires Are Effectiv...
All the best
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