The nanoneedle salt stress test – too good a clue to leave abandoned on the lab bench?

[EndME]

I don't think it's necessarily true that it has failed. As I understand it's a mix of factors not to do with the results of tests. The engineer who designed it and made the prototype moving on to another job elsewhere, lack of funding from NIH, the pandemic putting a stop to all lab work for quite a long time, OMF not funding it because of the lack of a specialist engineer who could progress it in Ron's department, and Ron having other possible biomarker ideas to work on.

I agree that, that is the argument proposed by the OMF or by patients. However, if that were the case it would in my opinion be very unethical. If you have a solid biomarker, which is something you know for certain after 4 years, you put all your efforts into that. Engineers aren't irreplaceable. You'd hire a whole team of engineers if you believed in it. You'd put all your efforts into it. You'd pass it on to researchers working in the Long-Covid field and see if finding some funding is possible there, which it most likely would. You'd test it on Long-Covid patients. You wouldn't keep starting new projects that look at ideas for other possible biomarkers, that in the end lead nowhere and are a waste of time and resources if you already have a biomarker.

A biomarker would completely change everything, neglecting something like that would be extremely unethical.

As such it is for me far more logical that the nanoneedle didn't hold up as well as initially proposed.

 

[Trish]

I got the impression that the nanoneedle was originally being developed for a different purpose and testing it on ME cells was just an experiment, maybe suggested to the engineer by Ron. There may be something about who holds patents. That's a wild guess.
That's why I like the suggestion on this thread that testing the same idea with different equipment might be worth someone else pursuing.
If, as @Obermann says, measuring impedance on cell surfaces is a standard method, there must be other equipment to do that, and labs who could try out a salt experiment with a few healthy volunteers and a few patients blood.

 

[chillier]

Assuming this doesn't mean cells being accidentally incapacitated or killed through experimental error, wouldn't it still be pretty significant if ME cells were dying as a result of hyposmotic stress?

The researchers increased the plasma salt concentration by 65 mmol/ml to 200 mmol/ml, which they characterised as a middling level of hyperosmotic stress.

I know very little about this area, would you expect a relatively modest increase like this to kill or incapacitate cells?

Might depend on the cell type but I think cells have salt concentrations of about 150mM and so like to sit in an isotonic buffer of about 150mM NaCl. I've tried this before when you put suspended fibroblast cells in 300mM NaCl - a hypertonic solution - and they shrivel up dramatically as the water rushes out of the cells and into the media surrounding them. Similarly when using 0 or 50mM hypotonic solutions they look very firm like they're about to pop as the water rushes into the cells. They wouldn't last very long in either of these conditions.

Seems possible that 200mM NaCl would start to kill the cells after a while. This source seems to suggest a drop in viability in an immortalised T-cell line at these concentrations after 5 hours, another cancer cell line doesn't seem to mind

 

[Trish]

What happens when you put healthy cells in an only slightly hypertonic solution? I have assumed this is what Ron's team did, and the healthy cells were more able to use the sodium pump to keep the cytoplasm at optimum concentration, whereas the ME cells struggled to do so quickly enough, perhaps because they couldn't make ATP fast enough. Would that show up as difference in impedance on the cell surface?

 

[butter.]

FWIW, the nanoneedle could not differentiate between ME and MS.

 

[Hoopoe]

Would that show up as difference in impedance on the cell surface?

Various sources describe how salt affects cell membranes: it induces membrane depolarization, reduces stability, and other changes.

 

[Simon M]

As I replied earlier, it could be the difference is in how the electrodes react with the samples, with the cells themselves having no differences.

Missed that- what kind of difference though?

Were the samples from the patients and controls collected and processed in exactly the same way?
If they were collected at different times and places by different people and hence stored differently then that might explain the difference.

This is an important point. It looks like a mixed picture - but note that 5/5 seem to have been processed in sync, so I don't think that could explain the result (all the controls look much the same, however the samples were collected).

Sample collection

Ofthe 20 healthy controls studied, 5 were age- and gender-matched to 5 of our ME/CFS patients (with both sets of samples collected simultaneously), 10 were selected at random and recruited by us,and 5 were anonymous donors collected by the Stanford Blood Center. The recruited healthy controls (excluding the blood center samples) were not diagnosed with ME/CFS or related diseases and had no blood relatives diagnosed with ME/CFS.

Sample processing

Patient and control samples were handled exactly the same in all preprocessing steps. (See appendix S1 for more details.)

Assuming the methodology was consistent for all samples (healthy v. patients), then the critical fact here is that they did find a difference.
Why is another story.

Yes, And we don't need the nanoneedle to try to understand what lies behind that difference.

 

[Creekside]

Missed that- what kind of difference though?

Whatever the nanoneedles were made of, some molecules might bind to them, changing their electrical properties, changing the overall circuit impedance. I expect that reactivity of electrodes is pretty important for experiments. Try using potassium electrodes for an aqueous solution.l

 

[Jonathan Edwards]

That surprises me. It seems that impedance measurements of cells is a standard method for cell analysis.

Measuring impedance across a whole cell makes sense although I never came across that in clinical or lab work. The odd thing about the nano needle is that the electrodes are so close together. I seem to remember that the assay was originally designed for some other purpose, like looking at impedance of protein solutions.

 

[chillier]

What happens when you put healthy cells in an only slightly hypertonic solution? I have assumed this is what Ron's team did, and the healthy cells were more able to use the sodium pump to keep the cytoplasm at optimum concentration, whereas the ME cells struggled to do so quickly enough, perhaps because they couldn't make ATP fast enough. Would that show up as difference in impedance on the cell surface?

They use 200mM NaCl in the nanoneedle paper which is why I mentioned that number above. You would think that would be enough to really hurt, as I say it seems to affect cell survival in one study for a T cell line but another cancer cell line HeLa cells don't mind - but then these are very hardy cells.

That surprises me. It seems that impedance measurements of cells is a standard method for cell analysis. Low-frequency (less than 1 kHz) and high-frequency (a few tens of kHz) impedance typically differ and give different information about the cell characteristics. Am I missing something here? https://www.axionbiosystems.com/technology/impedance-general

looks like in this case it's being used as a way to measure cell growth of adhesive cells in culture - which you can also do by counting the cells with a microscope but to be fair I suppose this would give you a way of monitoring it continuously. Your source also seems to suggest at these frequencies the cell membrane is mainly blocking current flow - so yeah could be the nanoneedle is not measuring anything inside the cell then.

The PNAS paper states the authors don't know what biological changes underpin the electrical differences they detect (though they speculate ). However, it does say that they used light microscopy and they observed no difference in cell sizes between patients and controls. Separately, they reported they didn't observe excessive cell death in ME cells. And they didn't report any other visual differences.

I couldn't see them mention cell death in the paper I guess the authors have mentioned this elsewhere? Still if there's some amount of cell death anywhere (PBMCs are a heterogeneous group of cell types anyway maybe some can handle the stress better than others) then the spilling out of highly charged molecules like DNA into the medium directly in contact with the sensors I would guess is an obvious candidate for signal changes. If not that maybe something is being secreted into the medium - cytokines or something.

 

[Trish]

Trying to make some sense of all this, rather than relying on my unreliable memory, I think it's worth going back to the paper they published which describes their theory of what happens:
https://www.pnas.org/doi/10.1073/pnas.1901274116
Quote (breaks added for easier reading):

Features of Assay and Theory of How Assay Operates.

Our assay is designed as an ultrasensitive assay capable of directly measuring biomolecular interactions in real time, at low cost, and in a multiplex format. The array processing and multiplexing is an important feature to enable high-throughput sensing and monitoring tasks on a large scale.

Fabrication of an array of thousands of sensors in parallel in a microfluidic channel integrated with their individual on-chip amplifiers and read-out systems can provide a single, portable platform that features a variety of clinical applications, including the detection of various biomarkers in parallel for improved early-stage disease diagnosis.

The way in which the assay operates is theorized as follows: The assay detects any impedance modulation due to the presence and/or interactions of biomolecules of interest at the active sensing region of the sensors.

Impedance (Z), briefly, is the ratio of applied voltage (V) to the induced current (A). It has two components: resistance, the in-phase component (Zre), and reactance, the out-of-phase component (Zim). With respect to an electrochemical system, the resistance component is commonly dominated by a change in local conductivity (e.g., change of ion concentration in osmolytes) (34), while the reactance component is commonly dominated by a change in local relative dielectric permittivity (34, 35) (e.g., local replacement of buffer molecules with biomolecules) (for further details, see SI Appendix).

Using our sensors, we recorded and calculated three parameters of in-phase impedance (Zre), out-of-phase impedance (Zim), and impedance magnitudes to further enhance the precision and accuracy of the sensors.

Utility of the assay for label-free detection of proteins and nucleic acids has previously been demonstrated (34–40). In this study, we applied the assay to objectively and continually quantify the effect of hyperosmotic stress on patients’ clinical samples. For each experiment, each of which lasted approximately 3 h, the sensors collected ∼40,000 data points with a sampling frequency of 5 Hz.

 

[Trish]

Whatever the nanoneedles were made of, some molecules might bind to them, changing their electrical properties, changing the overall circuit impedance. I expect that reactivity of electrodes is pretty important for experiments. Try using potassium electrodes for an aqueous solution.l

As far as I can see from the published paper I linked above, the only metal involved in the nanoneedle is gold.

 

[Barry]

Measuring impedance across a whole cell makes sense although I never came across that in clinical or lab work. The odd thing about the nano needle is that the electrodes are so close together. I seem to remember that the assay was originally designed for some other purpose, like looking at impedance of protein solutions.

Could this be so the two electrodes, together with cell material in between acting as a dielectric, end up in combination as a capacitor. The dielectric layer within a capacitor is typically pretty thin. So by modelling it as a capacitor and testing to ascertain the dielectric properties of the capacitor, from that then identify characteristics of the cell wall itself? I've forgotten now, were they trying to better understand if the cell wall itself affected correct cell operation/interaction for pwME?

 

[Barry]

As far as I can see from the published paper I linked above, the only metal involved in the nanoneedle is gold.

My guess would be similar to other reasons gold is used in electronics, being a good non-corrosive conductor. From this it would likely not affect the measurements too much.

 

[Jonathan Edwards]

Trying to make some sense of all this, rather than relying on my unreliable memory, I think it's worth going back to the paper they published which describes their theory of what happens:
https://www.pnas.org/doi/10.1073/pnas.1901274116

Yes, two things strike me from that.

One is that this system does seem have been used or designed for looking at the impedance of solutions of molecules like proteins in fluid phase. Applying it to a cell by getting the cell to sit next to two electrodes seems to me very different and not really what you would expect for the set up.

The other thing is that they mention that there are masses of sensors for high throughput. Yet as far as I can see you only need one sensor per sample and there aren't that many patients. Maybe lots of sensors can give you an average but that is different from giving high throughput.

I am afraid I get the impression that this was a gadget someone had made that they thought might look good as a high tech assay system, without being very clear why they were using for ME cells.

 

[Creekside]

From this it would likely not affect the measurements too much.

Maybe not for large electrodes, but gold is bioreactive as nanoparticles, and maybe the electrode points qualify as nanoparticles (electron shells exposed). Gold also acts as a catalyst for biochemical reactions, so the electrodes, particularly when dimensions are nanometers or smaller, could be catalyzing reactions with some chemical that is common in people who are in less ideal health than the controls. All sorts of assumptions break down at some point when shrinking physical scales. Maybe they used .9999 pure gold, which is fine for macro-scale electrodes, but maybe parts per billion impurities makes an important difference at the nano scale.

"Our assay is designed as an ultrasensitive assay" To me that's a warning of new, unexpected problems. Whenever a new "ultrasensitive" technique is developed, someone is going to misapply it based on experience with less sensitive techniques, resulting in errors from incorrect assumptions. That will be followed by updated instructions on what to avoid when using the new technique. A lot of amazing, exciting claims of "free energy" and "inertialess drives" were based on pushing instruments beyond their reliable operating range.

Applying it to a cell by getting the cell to sit next to two electrodes seems to me very different and not really what you would expect for the set up.

Yes, a technique tested on molecules in a fluid may not provide reliable results with complex "lumps" in the fluid.

 

[Hubris]

FWIW, the nanoneedle could not differentiate between ME and MS.

That would be an even more compelling reason to keep working on it. A biomarker that could differentiate unspecified illness from controls would be a huge deal in medicine for a number of reasons. Even people with MS can get misdiagnosed with FND initially, just to make an example - so finding out when the impedance changes (at which stage of the illness) would be valuable. The thing about tenure and the engineer seem quite unlikely to me. If the nanoneedle really is a biomarker for unspecified neurological illness it would be quite easy to get an external group of researchers to replicate it. But even in the context of ME, as others have mentioned it shouldn't be hard to set up replication with long COVID research teams or hire another engineer.

The technical details here are over my head but FWIW maybe it might be useful for those who donated to put pressure on OMF to give us further information about what happened because the story doesn't really hold up.

What I'm saying is, the diagnostic methods in other neurological illness are quite flawed and everyone would absolutely love to have something like the nanoneedle. It almost seems like science fiction with how good the results were. So why is nobody pursuing this? It makes no sense.

 

[Kitty]

It almost seems like science fiction with how good the results were.

To me, the most plausible explanation for the lack follow-up is that the researchers discovered it might be.

 

[Simon M]

FWIW, the nanoneedle could not differentiate between ME and MS.

Interesting. Can you say any more - e.g. when is the info from and based on how many subjects?

One is that this system does seem have been used or designed for looking at the impedance of solutions of molecules like proteins in fluid phase.

Yes, the nanoneedle was developed to be used with a range of sensors that allowed them to detect very specific molecules. The use here seems to have been off-piste.

They use 200mM NaCl in the nanoneedle paper which is why I mentioned that number above. You would think that would be enough to really hurt, as I say it seems to affect cell survival in one study for a T cell line but another cancer cell line HeLa cells don't mind - but then these are very hardy cells.

So it's plausible that this level of hypertonic stress could kill PBMCs. However, live microscopy didn't see cells enlarging, which is what you would expect to see.

I couldn't see them mention cell death in the paper I guess the authors have mentioned this elsewhere?

It was in a blog, or on a video. I can't now remember if they said what they didn't see was cell death or cell lysis.

 

[butter.]

Interesting. Can you say any more - e.g. when is the info from and based on how many subjects?

I can't say more about it, only that it is true with a high degree of certainty.