I have to develop a paper strip for detection of a small molecule, a nucleoside. I need some suggestions on the arrangement of different pads as I am doing the paper based sensor for the first time. Though worked with conventional ELISA, paper seems to be little tricky. We have cellulose pads for using as sample and absorbent pads, nitrocellulose membrane (0.2um), glass fiber to employ as conjugate pad. Some of you might be knowing what are the several problems one faces in dealing with paper sensor to translate the work in eppendorf tubes to a paper. What would be several things to follow and not to follow ?
I am assembling these four pads on a microscopic slide using a glue. I Treated conjugate pad with gold nanoparticle-antibody conjugate. Spotted corresponding secondary antibody on the nitrocellulose membrane using a pippete. Dried all the pads at 40 degrees Celsius before assembling them. I am Adding water on to the sample pad and trying to see the elution of nanoparticle conjugate and its binding to secondary antibody. Things seem to not work. Can anyone suggest what could be the possible flaws to avoid.
Your experience and suggestions and advice would help me a lot.
Thanking you in advance.
If you have not referred this paper till yet, maybe this can help. got a lot of useful suggestions from this.
Thank you very much Aditya for the paper. But, I could not find the information about the assembly of different pads and the presence of blocking agents. May be I have to look into the references of the article.
Thank you again
Dear Gangula
Can you please tell me which type of nitrocellulose membrane , Conjugate release pad , and sample pad you are using for your assay? LFA usually depends upon the individuals optimization and it varies from person to person. The flow of solvents/ analytes completely depend upon the assembly of LFA materials and its suitable treatments with the respective buffer. The assembly of LFA papers upon glass slides might not work due to its diameter. I can suggest you one thing buy one LFA strips from market and design accordingly.
Best
Properly gluing the nitrocellulose to the slide is difficult and, depending on the glue, you may be clogging the nitrocellulose pores. I suggest using commercially available. plastic-backed nitrocellulose to avoid this problem.
Thank you Prabir. Will try to optimize.
Thank you Ridchard, Will try with plastic backed membrane.
Can anyone help with some of these queries ?
How do we know how many microlitres of sample to add to the sample pad ?
What is the rate at which we add the sample ? Do we add all at once or drop by drop ?
How do we know that the assay on paper strip is done ? will the colour developed be a indicator that test is done ? How do we maintain that running time is same for all the strips to ensure reproducibility.
If I am dispensing the test lines and control lines manually with a pipette, what are the things to be careful about ? and how much should be the approximate volume to be dispensed if I am using 1mg/ml solution.
Thank you so much for your time
Abilash
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