Hi, hope all is well. I am now working on lateral flow assay development. I am using a FITC-ssDNA-Biotin probe to conjugate with streptavidin coated AuNP (40nm). The test lines are biotin-BSA conjugates (1ng/ml) and control lines are anti-FITC (1ng/mL). A crispr/cas assay (cas12a) is used as pre-step to lateral flow, so if the target is present, the ssDNA probe will be cleaved and then captured by test lines.
The assay works perfectly fine with 1) only AuNP 2) DNA probe + AuNP 3) crispr/cas assay negative controls, however, when it comes to the positive controls, there is only faint line shown on the control and on the test lines, and sometimes even no line is shown. I am pretty sure the lateral flow itself is working as all experiments are done on the same batch. Could anyone please suggest what is going on? Photos are attached.
The running buffer is triton x-100 (0.1%), 0.01M PBS and reaction volume is 100 uL.
Many thanks in advance!
Hi there. Whats the concentration of Cas12 and DNA target that you are trying to detect? Are you amplifying the target DNA before the Cas12 reaction? My first guess would be that your system does not have the required sensitivity. Hope this helps.
Hi Diogo Diogo Figueiredo , thanks for your reply. The conc. of cas12a is 250 nM (final conc.) and the whole CRISPR/Cas system is validated using fluorescent measurement (probe change from LFA to fluorescent). The products are amplified before CRISPR/Cas as well.
I think the reaction does happen as there is a clear change between NC and PC in control line, just I don't know for some reason the test line are not capturing the cleaved probe. Could you please help me?
Best, Wenliang
Given that, I would say the kinetics of the trans cleavage of your reporter are not fast enough to be detected by your current protocol. Do you incubate the Cas12a reaction (after amplification) with your reporter? try to incubate this mixture at 37 degrees for 30 minutes and then add this mixture to the lateral flow strip.
Best,
Diogo
Hi Diogo Figueiredo thank you for your reply! I repeated my experiments today and pre incubated my CRISPR/Cas mixture for 30 min but that does not change the result...
These were just general guidelines, did you incubate the CRISPR reaction with the ssDNA reporter? what is the sequence of the reporter? did you test different incubation times? Whats the initial sample you are using for the amplification assay? have you confirmed primer design and sensitivity/specificity?
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