A note on HNB dye

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Over the past few weeks we’ve heard of great success stories in implementing LAMP as well as some roadblocks and strange problems people have been facing. A particularly interesting one has popped up with HNB dye. We hear reports of one group of users buying and using a hydroxynaphthol blue dye that simply did not dye the reaction mix upon the addition of dye to a total concentration of 120 µM as is stated in the protocol. We got a sample of their HNB dye and tried it out in our lab. It turns out that there are two kinds of HNB being used for metal pH titrations, the disodium salt of hydroxynaphthol blue and the trisodium salt of hydroxynaphthol blue. What’s the difference? It is very visible, see for youself.

The top Falcon tube contains 3 mM trisodium salt of hydroxynaphthol blue solution. The bottom Falcon tube contains 3 mM disodium salt of hydroxynaphthol blue solution. The difference in colour and its intensity is striking at first sight. Whereas the trisodium salt solution is very dark, non-transparent, the disodium salt solution is see-through and light. When added to an RT-LAMP mastermix at a 120 µM final concentration, the disodium salt does not dye the reaction mix at all. It stays colourless and no colour change is visible before or after incubation. Therefore, HNB disodium salt is entirely useless for the purposes of colorimetric RT-LAMP detection.

Both of these products come as a dried powder but differ in their price points substantially, the disodium salt being significantly cheaper than its trisodium counterpart.

We can strongly discourage the purchase or use of HNB disodium salt, even though it may be cheaper. We have successfully used and tested HNB trisodium salt from the suppliers Sigma-Aldich (formerly Merck) and Hach. It is important to measure out HNB precisely on analytical scales, and to dissolve the powder in nuclease-free water and nothing else. We are certain that if used properly, HNB is a robust way to signal amplification in HNB reactions.