Recently, I worked on the lateral flow immunoassay using Hi-Flow Plus HF090 (Millipore) for the detection of protein and bacteria. Firstly, I checked five different kinds of buffer to find the optimized running buffer, including 20 mM, pH7.4 HEPES; 50 mM, pH 8.5 Borate; 10 mM, pH 7.4 Tris-HCl; 10 mM, pH 8.0 Tris-EDTA and 10 mM, pH 7.4 PBS. In this case, I found that Tris-EDTA showed the best result. The reviewer asked one question that why Tris-EDTA was best. I searched so many published papers, however, no paper mentioned the deteails of mechanism and reason.
Usually, buffers are selected empirically and not based on a specific mechanism. Therefore, the question of the reviewer seems to be strange. Perhaps you misunderstood this question and the reviewer wanted to know why (based on which criterion) you have chosen Tris-EDTA. Many researchers can not tell you what they mean with "best result".
Dear Prof. Weller,
Thanks very much for your valuable suggestion. I chose Tris-EDTA as the optimized running buffer because the OFF data was minimum when I used Tris-EDTA. The oher OFF results were relative higher when I used other running buffers. Please see the attached file. According to your experience, how to answer this question? If possible, could you please give me some professional suggestions at your earlist convinence? I will greatly appreciate your kind help.
Best regards,
Yours sincerely,
Rui Wang
I presume this is a SERS lateral flow assay? Gold or silver particles are sensitive to the type of ions in the running buffer, which may cause aggregation at the test line and your false positives: Tris HCl versus Tris EDTA; PBS has a high salt, NaCl content. Chlorine ions in particular may cause false positives? Examine the effect of your buffers on your SERS reporter particles aggregation state using UV-Visible absorption & DLS and / or NTA, with zeta potential analysis. The surface chemistry of your SERS reporters may be highly influenced by the running buffers - do they complex this surface and cause aggregation at the test line and a false positive?
You may wish to consider designing a non-fouling surface on your SERS reporter particles to prevent NSB? See links....
Detailed information on your assay design would determine if the above applies and is useful - I hope, however, you still find it helpful.
https://www.nature.com/articles/ncomms13437
Article Stealth Surface Modification of Surface-Enhanced Raman Scatt...
I think that your experimental results are quite convincing and do not need further explanations. Did you show this figure already in your manuscript?
Dear Dr. Clarkson,
Thanks very much for your valuable suggestion. It's SERS-based lateral flow. I prepared SERS probes as following procedure: MGITC as Raman reporter was immoblized on the surface of AuNPs, and then Borate buffer was added into the MGITC-labled AuNPs to adjust pH value. Then, the antibody was added to the above solution and shaked 2 h at room temperature. Finally, BSA was added to block the unbound surface of AuNPs. I will check the the effect of buffers on my SERS probes aggregation state using UV-Visible absorption & DLS.
According to my own understanding, different signals caused by running buffers may be also from the different binding performances between antibody and target because pH value and ionic strength can affect the binding affinity. I am not sure my understanding is correct or not.
Best regards
Dear Rui Wang,
sometimes reviewers do not read manuscripts carefully and hence asking for "improvements", which do not make sense. Unfortunately, it is often difficult to convince reviewers that they are wrong. To avoid that your manuscript is rejected based on this irrelevant point, I suggest that you answer the question pro forma. This means that you give a general explanation. John has already given some hints for a mechanistic discussion.
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