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Long Considered Inaccessible for Dx Use, Interstitial Fluid Collection May be Enabled by Pin-and-Paper Method


NEW YORK (360Dx) – The fluid that passively bathes cells in the skin may be a snapshot of the body's systemic state, but so-called interstitial fluid (ISF) has heretofore been too inaccessible to be used in diagnostics.

Now, a paper-based technology developed by researchers at the Georgia Institute of Technology and Washington University in St. Louis  may enable this sample type to be collected and used in diagnostics, although more research needs to be done.

As described in ACS Sensors earlier this month, the technology is the marriage of a patch made of tiny pins with plasmonic paper — a type of cellulose filter paper that is painted with nanoparticles, in this case polystyrene sulfonate coated gold nanorods.

First author on the study, Chandana Kolluru, helped develop the technology while completing her doctoral work in the lab of Mark Prausnitz, a renowned researcher pioneering painless, self-administered patches of dissolving microneedles for vaccine delivery.

Kolluru and her colleagues had initially developed a microneedle patch to deliver polio vaccine, but she then became interested in whether the patch could also be used to collect interstitial fluid. For diagnostics applications, "The cool thing about this fluid is, it is clear and it does not clot," Kolluru said in an interview.

The ISF is generated by the filtration of blood, she said, so it potentially provides information about both local tissue as well as systemic states. But applications of ISF are generally not well explored, Kolluru said, because it has previously been too difficult to collect.

Yet, although there are no ISF-based tests cleared by the US Food and Drug Administration to date, "interstitial fluid is a hot topic right now," Kolluru said. For diagnostic use, it might be useful for things like detection of cancer biomarkers, exposure to chemical toxins, or rapid infectious disease exposure testing at airports, she added.

The fluid is also a focus for noninvasive glucose monitoring technologies, also called continuous glucose monitors (CGMs), and this application has been in development in a number of labs and startup companies with varying degrees of success to date. For example, the Glucowatch from Cygnus Inc. was cleared in 2002 and used microneedles to monitor ISF, but it was discontinued in 2008 due to unreliable readings.

By contrast, Abbott makes an implantable metal wire that monitors glucose in interstitial fluid. Called FreeStyle Libre, the device was cleared by the US Food and Drug Administration in 2016, with a 2018 expansion for a 14-day system. Roche, meanwhile, has collaborated with Senseonics to distribute an FDA-cleared 90-day CGM called Eversense that is essentially an ISF sensor injected beneath the skin. And a company called Nemaura Medical is also developing a wearable patch technology called SugarBEAT that purports to use electric pulses to attract glucose from ISF for measurement.

All of these devices, however, are more invasive than microneedles and involve larger and more complex wearable devices.

The patch Kolloru helped develop is easy to use, at least in animals, and requires no expensive devices, she said. It is also easy and inexpensive to fabricate. She added that there are now hopes in the field of incorporating ISF monitoring into wearable devices for other types of systemic biomarkers besides glucose.

The microneedle patch does not have hollow needles, but rather it is cut from a flat sheet of medical grade stainless steel, Kolluru said, perhaps more like pins than needles. By placing the patch on the skin multiple times and inserting the tiny needles, "the pumping action from the insertion causes the fluid to come from the skin onto the surface," she said. Then, the fluid flows into filter paper.

"The insertion is easy – you just hold the skin and do multiple insertions of the microneedles until you see the fluid come up" and the paper becomes saturated with ISF, she said. She has also tested the microneedle device on herself.

"The needles are really small," she noted, so "You can feel that the needles went in and came out, but they are so tiny that it doesn't hurt," she said. Nor does the insertion bleed or leave a mark on the skin.

Initial work published in PNAS characterized the needles in terms of thickness and length and narrowed down an ideal filter paper. The reseachers  also measured fluid collection in pig skin to quantify how much could be captured, and did preliminary human studies, but with a patch attached to a suction device to draw up the fluid.

Subsequently, as described in Advanced Healthcare Materials, the team showed it was possible to collect about 2 microliters of ISF from rat skin within one minute.

And, as published in BioMedical Microdevices, the group then managed to use non-functionalized filter paper to collect ISF and detect polio-specific antibodies in rats after vaccinating them. The study also described an assessment of vancomycin — a drug that has a narrow therapeutic window delineating ineffective and toxic doses and that must be continuously monitored — and showed that ISF and blood have similar pharmacokinetic profiles.

The problem was that the ISF was collected onto plain, non-functionalized filter paper and dried, so it would then have to be rehydrated for testing. This required a centrifuge for ideal extraction efficiency, which limited the method to use in a lab, and the researchers could not be certain if some of the biomarker of interest was actually still trapped in the paper.

That's when Kolluru came across a report about plasmonic paper from Srikanth Singamaneni's lab at WUSTL. The method involves putting functionalized nanoparticles onto paper and using them to grab biomarkers.

It was a "wow" moment for Kolluru and seemed like a great solution to the problem. "We have a microneedle patch and they have the paper," she said, so the two groups ultimately initiated a collaboration to merge the technologies.

Singamaneni's team functionalized gold nanorods and put them into a solution that was then filled into a pen-like instrument, Kolluru said, and this was used to write onto standard cellulose filter paper, with miniscule pieces being used to absorb ISF after the microneedles were applied to skin.

Specifically, the piece of functionalized paper is 1 millimeter by 7 millimeters, so the gold nanorods needed to cover it should not be extremely expensive, and they could also potentially be extracted from the paper and reused if needed, Kolluru noted.

As a proof-of-concept, the group showed in ACS Sensors that functionalizing the gold nanorods with a negatively charged polystyrene compound could capture positively charged rhodamine in ISF from rats that had been injected intravenously with the dye.

"Just from electrostatic interaction, we could have dye particle attach onto the paper," Kolluru said. This enabled on-patch detection with no additional sample processing.

The dose of dye was high, admittedly, but that was mainly because rhodamine tends to be sequestered by proteins in the blood, she said, but collecting ISF onto non-functionalized paper that was subsequently extracted also showed similar results.

So far, the method of combining microneedles and plasmonic paper has only been tested in animal models, but Kolluru said human tests are in the works with the ultimate goal of integrating the patch into a wearable device.

Others homing in on ISF collection patches include the lab of Ronen Polsky at Sandia National Labs. That group has used hollow microneedles to reveal that ISF had similar transcriptome and proteome signatures as serum and plasma samples, and that exosomes can be found in the fluid, too.  Alternatively, the lab of Ryan Bellamy at Queen’s University Belfast uses hydrogel microneedles that swell upon insertion as they imbibe interstitial fluid, Kolluru said.

The Prausnitz lab has filed a patent for the microneedles patch and collection method.