A nanoelectronics-blood-based diagnostic biomarker for ME/CFS (2019) Esfandyarpour, Davis et al

[Hutan]

Watching the recent video from Ron:

Ron Davis has summarized where they are

it sounds as though the cells stick to the sensors. "in this case, of cells that are attached to the electrodes". So, does the test measure the impedance of mostly just the cells immediately adjacent to the electrode or does the charge have to go through cells clustered on the electrode and then some plasma that is largely cell free? If it's the former, could it be that it is the 'stickiness' of the cells, that is the level of attraction the cells have for the electrodes that is different between the ME/CFS and healthy samples? Or have I completely misunderstood?

Even in this recent video, Ron seems satisfied that this is proof that ME/CFS cells are different to healthy cells. Like others here, I'm really concerned about the possibility that the sedentary nature of severe and moderate patients is causing the difference. Surely this is something the team has considered. I'd love to hear their response. Have they since tested the blood of any mildly affected ME/CFS people? I do hope we won't have to wait months to find out.

 

[Adrian]

Not a lot I am afraid. I was aware of it. It is just that as an immunologist familiar with suspensions of peripheral blood mononuclear cells I find it very hard to work out what was actually going on. Why didn't the cells walk off the electrodes from time to time?

I'm not sure what is happening in this case but in some cases of microfluidics you either move a droplet around (via activating electrodes) or you have a flow through something like a capillary action to move a small amount of fluid into a chamber for testing. Then you can measure things like impedance or heat the liquid, mix a sample with reagent and then test via things like light.

With the Nano engineering I think you may end up making the chamber to be the size of the cell so it doesn;t move (or maybe that is the needle bit to catch it). Others have gold fingers to hold a molecule and then use something like raman spectroscopy to examine the molecule.

I think the thing is to design the chip in a way which it correctly captures samples and I've no idea how that is done for nano tech but I did watch a video a while back around design tools for more conventional micro fluidics where the design process is quite similar to designing an silicon chip. Such techniques would tend to follow the flow of liquids and mixing etc.

 

[Barry]

So, does the test measure the impedance of mostly just the cells immediately adjacent to the electrode

"The impedance results are integrations of the impedance that are attributed to the media− sensor surface interactions (Zm-s), cell−cell interactions (Zc-c), cell−sensor surface adhesion (Zc-s), impedance of cells (membrane capacitance Cm, cytoplasm conductivity of the cells, σcp), resistance of the solution (Rs), and other components (e.g., proteins, exosomes, and lipids) in plasma. The assumption here is that the current flows radially into the space between the cell’s ventral surface and the substrate and then escapes between the cells. The current density is assumed to be consistent in the y direction, and the cells are disk-shaped objects with membrane surfaces and filled with a conducting electrolyte. Moreover, to identify the mechanisms and components involved at the cellular and molecular levels, we have started to investigate the plasma components (e.g., proteins, exosomes, and lipids) and individual cell types (e.g., T cells) separately. This investigation forms part of on-going studies that require further investigation before mechanisms may be suggested with a good degree of certainty."

I don't understand the biology, but it sounds like the anticipated mechanism could be much more than just interactions with the measurement probe.

 

[Jonathan Edwards]

"For each experiment, 50 μL of the prepared sample (SI Appendix) was injected into the microfluidic wells" ... I interpreted that to mean a total of 50μL into all the wells, not into each individual one.

But for 4000 wells that would be 0.0125 μL per well with an average of 2.5 cells. The smallest amount that a graduated pipette can discharge is about a micro litre. And that has to be discharged into a larger fluid volume otherwise it will just stick to one place by surface tension.

 

[arewenearlythereyet]

"The impedance results are integrations of the impedance that are attributed to the media− sensor surface interactions (Zm-s), cell−cell interactions (Zc-c), cell−sensor surface adhesion (Zc-s), impedance of cells (membrane capacitance Cm, cytoplasm conductivity of the cells, σcp), resistance of the solution (Rs), and other components (e.g., proteins, exosomes, and lipids) in plasma. The assumption here is that the current flows radially into the space between the cell’s ventral surface and the substrate and then escapes between the cells. The current density is assumed to be consistent in the y direction, and the cells are disk-shaped objects with membrane surfaces and filled with a conducting electrolyte. Moreover, to identify the mechanisms and components involved at the cellular and molecular levels, we have started to investigate the plasma components (e.g., proteins, exosomes, and lipids) and individual cell types (e.g., T cells) separately. This investigation forms part of on-going studies that require further investigation before mechanisms may be suggested with a good degree of certainty."

I don't understand the biology, but it sounds like the anticipated mechanism could be much more than just interactions with the measurement probe.

I sort of understand the biology but I never thought of blood cells as having a ventral (and presumably dorsal) side ??? I always thought of them as being without a top and bottom....it was a long time since I studied cell biology though (1980’s) perhaps things have changed? I haven’t read the paper yet...is it erythrocytes specifically they have used?

ETA: discovered an answer to one of my questions ...they remove red blood cells since this interferes with the signal ..gives the opposite result. Maybe this is due to lack of mitochondria or other organelles? Still bemused by ventral comment though.

 

[mariovitali]

I was amazed to read that the test is capturing healthy controls near an experimentally set "gray region" because these controls have relatives with Fibromyalgia (..!)

Although the results of the nanoneedle are striking , the next hurdle is to identify whether the test differentiates ME/CFS from other conditions. I do not know if the following is relevant however searching about generating "hyperosmotic stress" on PBMC cells (as stated in the paper) :

a) It appears that Hyperosmotic stress on PBMCs induces inflammatory cytokines :

https://www.sciencedirect.com/science/article/pii/S0014482797934765?via=ihub

b) On a paper named "The role of hyperosmotic stress in inflammation and disease" we read :

Link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438915/

Hyperosmotic stress is linked to many maladies, including acute and chronic, as well as local and systemic, inflammatory disorders. Hyperosmolarity triggers cell shrinkage, oxidative stress, protein carbonylation, mitochondrial depolarization, DNA damage, and cell cycle arrest, thus rendering cells susceptible to apoptosis. However, many adaptive mechanisms exist to counter the deleterious effects of hyperosmotic stress, including cytoskeletal rearrangement and up-regulation of antioxidant enzymes, transporters, and heat shock proteins.

I think that this is interesting but also could be alarming in the sense that many types of disease could lead to the same result we have seen in the paper by Davis et. al ? In the paper linked above we find entries related to eye disease, diabetes, liver disease, inflammatory bowel disease and cardiovascular disease.

 

[Jonathan Edwards]

I don't understand the biology, but it sounds like the anticipated mechanism could be much more than just interactions with the measurement probe.

I certainly don't. Cells are not disk shaped.

The other thing I do not understand is why the electrodes are a few microns apart. Are they positive and negative, with current between, or is current transmitted to another electrode somewhere else? Cells are about 10 microns across so I would have thought current between two close electrodes would not pass through the cells much and certainly not between cells.

 

[Jonathan Edwards]

b) On a paper named "The role of hyperosmotic stress in inflammation and disease" we read :

Link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438915/

I think that paper just shows that you can find meaningless reviews on any idea you like these days. It seems to bear no relation to the reality of inflammatory disease whatever.

 

[Trish]

All the detail of the nanoneedle thing are beyond me, but there's a diagram on this video and Ron Davis talks a bit about it at about 18 minutes.

 

[Barry]

But for 4000 wells that would be 0.0125 μL per well with an average of 2.5 cells. The smallest amount that a graduated pipette can discharge is about a micro litre. And that has to be discharged into a larger fluid volume otherwise it will just stick to one place by surface tension.

In which case I'm out of my depth so don't know.

 

[Sly Saint]

18:19
whole new area for them so these aren't
18:21
bioengineers these are hardcore
18:23
electrical engineers and I like working
18:26
on them because when you've given them a
18:27
problem they say okay I'll go model that
18:29
and they go off and they they model it
18:35
to see Turman whether it will work or
18:37
not and they'll come back and say it
18:39
isn't gonna work and I you know of
18:41
course initially I was naive and said
18:43
nope wouldn't you shouldn't you try it
18:45
anyway and they said absolutely not it
18:46
doesn't work so it saves a lot of time
18:50
and money or they'll say yes it will
18:53
work and and they will get it to work so
18:58
this little device is nano fabricated
19:01
and has two electrodes of gold they're
19:04
very tiny 50 nanometers is very small
19:07
it's only it's it's large compared to
19:09
computer chips today but of course it's
19:12
so small you can't
19:13
so these guys make instruments that you
19:18
really can't see and it's a little scary
19:22
what they can actually make that you
19:23
can't see so all these kind of movies
19:26
you see sci-fi movies where they cut
19:28
something out of them come on that's
19:31
old-fashioned stuff what you would be
19:34
inserting next is something you couldn't
19:36
actually see it's too small so what you
19:42
can do with this is a measure the
19:44
electrical current and you can measure
19:47
different properties of the electrical
19:49
current and we determine what the right
19:55
cycling frequency cycling is and the
19:57
right voltage for it but in this we have
19:59
a trough with multiple electrodes and
20:01
there are 2500 electrodes in this device
20:05
so the reason you do that is so you can
20:07
get many many independent sampling and
20:09
then we sample it to make a collect a
20:15
measurement 200 times a second so it
20:17
creates an enormous enormous amount of
20:19
data and that gets rid of a lot of the
20:21
noise problems so what we what we put
20:25
into this then our in this case is blood
20:30
and what happens with blood is a healthy
20:38
person when you put in this device is
20:41
very very flat we figured that we had to
20:45
end the chronic fatigue syndrome it's
20:47
just the same so we decided we had to
20:49
stress the cell to make it respond
20:52
differently because there must be
20:54
something wrong with them and they can't
20:56
deal with energy very well so put in
20:58
sodium chloride they have to pump it out
21:01
so when we put in sodium chloride the
21:03
very first additions actually the very
21:05
first experiment you you see a small
21:08
effect of the adding the sodium and then
21:11
healthy's are flat mecfs patients
21:13
increase and this is the electrical
21:18
impedance which is a proper complex
21:20
property
21:24
no just a drop it's a drop of blood put
21:28
on the device and it's just measuring
21:32
the electrical impedance within the
21:34
blood sample we do get rid of the red
21:36
cells and the reason we get rid of the
21:38
red cells is they also have a signal but
21:41
it's in the opposite direction so when
21:43
you put the two together it kind of
21:45
doesn't show very much so we get a much
21:47
better response and we get rid of the
21:49
red cells now this is actually quite
21:54
cheap you get a real-time measurement it
21:59
reflects the different response to
22:00
sodium chloride we tried other salts
22:03
sodium fly it seems to work the best so
22:05
far it might be used as a diagnostic
22:08
don't know yet but we also think it
22:14
might be possible to use it as a drug
22:16
screen in other words if we put some
22:19
throw again that actually helps then the
22:21
signal might go away don't really like
22:24
using something where your signal goes
22:25
away because there might be a lot of
22:26
reasons why a signal would go away but
22:32
that often people say well you we've
22:36
done this on 20 patients that's not
22:38
nearly enough patience you need to do
22:39
hundreds and hundreds of patients well
22:41
that's the kind of studies where you
22:43
have a small signal when you have a
22:45
clean signal you don't need that many
22:46
and so we've done here's the twenty
22:50
patients they all go up they all go up
22:54
above this line here are the healthy
22:57
controls they're always below this line
22:59
so the probability that you could get
23:02
this from random chance is ten to the
23:06
minus nine so it's better than when the
23:09
lottery and so that should be sufficient
23:13
now we've increased that number now the
23:16
30 we still say the same response every
23:18
patient shows this response
23:21
no healthy controller shows it now that
23:25
doesn't mean that we aren't going to see
23:27
something when we look at other patients
23:28
and we're trying to do those experiments
23:30
now I wouldn't be surprised we see it
23:34
with with
23:35
patience and the reason I wouldn't be
23:37
surprised is that we know that chronic
23:39
fatigue syndrome is initiated by a
23:42
stressor other diseases are stressors
23:45
how do we know that they don't also have
23:48
chronic fatigue syndrome so like the
23:51
first person we started that the thought
23:53
about doing is MS so I talked to him a
23:56
specialist I said and I just went
23:59
through the list of the diagnostic
24:00
criteria you know what happens when if
24:03
they exercise oh they get worse are they
24:07
fatigued oh yeah they're always fatigued
24:10
that they fulfill all of the
24:12
requirements for chronic fatigue so now
24:14
I said well do they have chronic fatigue
24:15
syndrome
24:16
I said no no they have MS and and they
24:21
just they admitted to me that when you
24:23
have a clear diagnosis like MS all
24:26
symptoms that the patient has is caused
24:28
by the MS it wouldn't even consider it
24:31
that they have a second disease makes it
24:33
complicated for us
24:39
now we have tried this out on some doing
24:43
some drug screens and this is a very
24:48
plumeria result but just to just to
24:50
indicate that it might work it's only
24:53
been one patient it's been one patient
24:55
that we tried several times and there
24:58
are two drugs Copaxone which is an
25:02
immune modulator and a new drug called
25:05
SS 31 which is a drug that repairs
25:09
mitochondrial damage so those are tried
25:12
because of their involvement and what we
25:15
think might be involved in this disease
25:17
and they get rid of the signal if we
25:20
pretreat the cells we get no signal we
25:24
don't know if it's gonna work on a
25:25
patient or not at all
25:27
and that's this is something that we're
25:30
prepared now to work with some of the
25:32
local physicians that treat chronic
25:34
fatigue syndrome and have them try it
25:36
both of these drugs are probably pretty
25:39
safe this one is available this one is
25:42
not yet approved but will probably be
25:46
approved soon now we have done what's
25:51
called a plasma swap experiment and that
25:54
is where and I'm not gonna focus on this
25:56
heavily but that is for example where we
25:58
take plasma from a patient and mix it
26:01
with healthy cells and we get the signal
26:03
and if we do the reverse their signal is
26:05
gone so most of what we're seeing is
26:08
actually from the plasma but the cells
26:10
are required if you leave out the cells
26:12
no signal but so there's some
26:15
communication between the plasma and the
26:17
cell that gives us a signal now

 

[Barry]

The other thing I do not understand is why the electrodes are a few microns apart. Are they positive and negative, with current between, or is current transmitted to another electrode somewhere else? Cells are about 10 microns across so I would have thought current between two close electrodes would not pass through the cells much and certainly not between cells.

Maybe this is where the capacitance come in. If the cells have a capacitive characteristic to them, then that may be what they mean when they speak of "membrane capacitance Cm". Capacitors have (virtually) infinite resistance (the real component of impedance), but finite reactance (the imaginary component of impedance). For capacitors, the reactance reduces the higher the frequency and the larger the capacitance. i.e. The obstruction to current is inversely proportional to f and C. I think many natural structures have capacitance characteristics. Maybe the cell membrane is behaving as a dialectric.

 

[Jonathan Edwards]

The obstruction to current is inversely proportional to f and C. I think many natural structures have capacitance characteristics. Maybe the cell membrane is behaving as a dialectric.

Yes I still remember that stuff from school but they do not seem to tell us about this.

And I am still puzzled as to why the pairs of electrodes are so close together.

 

[Simon M]

I certainly don't. Cells are not disk shaped.

The other thing I do not understand is why the electrodes are a few microns apart. Are they positive and negative, with current between, or is current transmitted to another electrode somewhere else? Cells are about 10 microns across so I would have thought current between two close electrodes would not pass through the cells much and certainly not between cells.

According to figure 1 (A, B) The nano needle has two thin gold electrode layers sandwiched between three (Insulating?) oxide layers, presumably current flows between those two electrode layers via the fluid, but influenced by the close proximity of the cells, or even cell contact.

According to the scale on that figure 1, E), the nano needles themselves are spaced about 40 (Corrected from my earlier, higher figure) µm apart.

But perhaps I have misunderstood your point again

 

[Barry]

I am still puzzled as to why the pairs of electrodes are so close together.

As I write this I see @Simon M has also commented on this. I presume you mean, by electrodes, the two gold layers within the sensor, separated by an oxide layer. The superimposed circuit diagram shows that the two gold layers and sandwiched oxide layer, comprise a capacitor, as well as having resistance within the gold layers, and presumably any connective wiring. So there is a CR circuit as the diagram shows. So an a.c. current will be able to pass between the gold layers, thanks to the capacitance; it will also be affected by the resistances ... impedance overall.

Anything coming into contact with this sensor tip, if it has its own resistance and capacitance, would effectively then become part of the circuit, giving modified circuit properties; the impedance would therefore change. If the fluid and its content presents higher resistance and/or lower capacitance, then presumably the impedance reading would be higher than for fluid with lower resistance and/or higher capacitance. (Impedance would be highest of all when the sensor is not immersed at all).

Edited for clarity.

 

[anciendaze]

Just a comment about lack of a complete description of the test equipment. This is typical of initial disclosures of technical advances with significant economic value. I've seen this with patents, trade secrets and non-disclosure agreements in a completely different context. Simply delaying the time until competitors can bring a challenge to market can be worth fortunes. Even non-profit organizations are affected by potential profits down the line which might indirectly benefit them or otherwise impact donors. Ron Davis has already signaled that there may be more than the 2 million sufferers in the U.S. NIH assumes, so we are talking about substantial value for equipment that can distinguish them. This will also have a major effect on insurance, and even debates about national healthcare.

When anything involves really large sums of money I warn people to "stand-by for maximum weirdness." When the numbers involve millions of people who are either fully or partially disabled the economic cost is staggering. We are getting into the range where it is impossible to distinguish rational economic self interest from psychosis, based on behavior alone.

(I've personally seen some extremes that would cross the line into criminal behavior, but have good reasons not to make them public due to laws about libel and slander. This is not simple cowardice, because I don't have important information and would be likely to make incorrect charges. Some of the information I do have may have been false and planted. Even good people will withhold information when faced with serious consequences, and there is no question bad people willing to distort perceptions do exist. )

I wish we could have rational discussions without the distortions introduced by narrow financial interests, some of them always hidden, but my view of current politics and economics suggests this is impossible. We will need patience and cunning to filter the information available until matters are firmly settled. We are piecing together a puzzle based on just a few pieces available to us. This is not the scientific ideal, but it is typical of reality.

 

[lansbergen]

We are piecing together a puzzle based on just a few pieces available to us. This is not the scientific ideal, but it is typical of reality.

Yep

 

[Jonathan Edwards]

We will need patience and cunning to filter the information available until matters are firmly settled. We are piecing together a puzzle based on just a few pieces available to us. This is not the scientific ideal, but it is typical of reality.

It may be fairly typical of biomedical commercial development but it is not good science. And from my point of view if people are not prepared to publish clear good quality science I am inclined to ignore the stuff completely. I never concealed anything from anyone during my scientific career and I don't regret it.

 

[wigglethemouse]

And I am still puzzled as to why the pairs of electrodes are so close together.

This to me is one of the most interesting questions. I don't understand the ratio of plasma + salt soln to PBMC volume. Perhaps this is why the electrodes are close? Another reason could be that the closer together the electrodes, the higher the current, so background noise is much less. These are very tricky measurements and high impedance's are very difficult to measure in labs full of 60Hz background noise (paper describes needing faradic cage to minimise electrical lab noise).

Many people have done cellular impedance measurements on small circuit boards and add drugs to see the impedance change. These of course have larger geometries, but you can buy interdigitated circuit boards with 50um channels between the electrodes for ~$100 in one off quantities.

If one of the available types of commercial equipment could be adapted to incorporate the nanoneedle circuit, or the test adapted to use existing commercial equipment, then this test could be scaled up and replicated much quicker than just continuing with the nanoneedle alone. Indeed that is my hope. Just need $$$, ideas, and researcher availability + funding.

One experiment that would be really interesting would be to have Cornell measure cellular impedance of activated T-cells using commercial equipment (I read a paper on this) as they presented data at the NIH conference on activated T-Cells using Seahorse and also special staining. So you could tie the impedance change to two other measurables. The reason you activate T-Cells is to "stress" their energy complex. I guess it's probably not as simple as I write.........

But I guess the first job is to replicate the nanoneedle experiments in a larger group more diverse group using the existing setup before moving on to new experiments. Walk before you can run.

 

[anciendaze]

It may be fairly typical of biomedical commercial development but it is not good science. And from my point of view if people are not prepared to publish clear good quality science I am inclined to ignore the stuff completely. I never concealed anything from anyone during my scientific career and I don't regret it.

I don't expect Ron Davis to conceal matters for long, but he has already indicated there are aspects they don't understand. I'm sure you have been careful about making claims you were not ready to back up.

I'm also assuming we will see patent applications derived from this work before long. Once you start talking to university attorneys about intellectual property, things become more complicated, and they will forbid discussion of matters a scientist sees no harm in mentioning.

My background is not in commercial biomedical research, but I'm well aware of moral quandaries. I can understand all too well how Boeing got into the habit of downplaying the role of the MCAS features in the 737 Max 8, and how marketing types thought that making a few dollars by selling aircraft without the optional warning indicators about discrepancies from angle of attack sensors was good business. I'm sure they are reassessing that decision at present.

I left one job over disagreements concerning the Space Shuttle's thermal protection system, and regret that I could not do more to prevent the loss of Columbia years later. Perhaps, if I had not driven myself to the point of collapse, I might have made a difference.