Doomed to repeat history

I still "owe" the world and the EIC of Applied Materials and Interfaces a whistleblower-style disclosure of all the correspondence related to the Fe³⁺ sensor paper discussed in my previous posts (1, 2, 3). The problem is that I need to carve a few days out of my schedule to document everything properly. In the interim, I came across a couple of related papers recently that bear examination in more than 140 character tweets.

In reverse chronological order, today I saw the ASAP notification of this paper by Magri in RSC Advances. RSC Advances has been a significant contributor to potentially bogus Fe³⁺ sensor papers in the past, but seems to be aware of the problem now as evidence by this paper that has been in ASAP limbo for >7 months (tangent - there are ASAP papers there from 2013). Magri's paper follows a similar path to my groups study on another popular Fe³⁺ sensor. Magri's results demonstrate that the earlier study (cited 59 times according to Google Scholar) makes incorrect conclusions about the mechanism of fluorescence signal transduction. In short, the fluorescence quenching is not due to metal ion binding to the sensor's receptor, but rather inner filter effects stemming from Fe³⁺ inherent incompatibility with water at pH >4. I'd encourage people to read both papers because it will give great insight to the types of problems plaguing the fluorescent sensor field. The original Tetrahedron Letters paper lacks any absorption spectra, which would provide direct evidence of the expected inner filter effects however. Magri does the fluorescent sensor community a great service in publishing a thorough investigation like this.

This leads me to an ASAP Applied Materials and Interfaces paper that ruffles my feathers. The paper claims a signal transduction mechanism that involves forming a 5-coordinate Fe³⁺ complex with monodentate phenolic ligands in water (unbuffered, deionized). There is a component of the project that involves nanoparticle upconversion of light to excite the Nile Red fluorophore; however, at its core this system is essentially on a small molecule sensor. The fluorphore-ligand dyad (Nile Red-phenol) is reported to bind Fe³⁺, which quenches the fluorophore emission. Whether the excitation of the fluorophore is direct or from upconversion is not particularly relevant to the sensing mechanism. There are several red flags that suggest this cannot be the actual mechanism:

1. In addition to Fe³⁺ being incompatible with non-acidic water, monodentate phenols are unlikely to have strong interactions with Fe³⁺ in the presence of excess water ligands (unlike catechols, which take advantage of the chelate effect). It's even less likely that FeL₂, FeL₃ and higher order species will form in high concentrations. You will not find many (any) inorganic chemists who will buy into the author's coordination chemistry. Furthermore, evidence for these complexes being reasonable is demonstrated in non-aqueous solution (in DCM, SI Figure S8). Changing the solvent completely changes the  coordination chemistry and hydrolytic stability of the Fe³⁺ ion.
2. The absorption spectrum (Figure 3) has the hallmarks of particulate formation/light scattering. The individual spectra increasingly deviate from the baseline as Fe³⁺ is added
3. The procedures for working with Fe³⁺ in cells do not match the recommended protocols. Adding Fe³⁺ to the extracellular fluid does not result in an increase in free intracellular Fe³⁺. I do not believe anyone has studied this in enough detail to know what happens when you do this. I suspect a broad stress response (which could lead to all kinds of intracellular changes) and/or uptake of iron nanoparticles by endocytosis; however, this is speculation.

Clearly, the authors see a change when adding Fe³⁺, but their mechanism is wrong. There are many possibilities - inner filter effects, formation of iron nanoparticles, pH-induced changes from Fe³⁺ hydrolyisis. Every published study that fails to account for inorganic and photochemisty just perpetuates the myth that these protocols for handling Fe³⁺ in aqueous solution and cells provide valid results. This facilitates the publication of the next dubious study. I would alert the journal to this problem before publishing this blog; however, they've made it abundantly clear through our previous correspondence that they do not care whether or not papers published in the journal contain conclusions that are consistent with well-establish chemical concepts.