Project Summary Continuous molecular monitoring has advanced from sensors tested in benchtop beakers, into numerous demonstrations of sensors inserted into animal jugulars for blood monitoring, and is now emerging into monitoring in the skin’s interstitial fluid which is the clinically successful approach also used in wearable glucose monitors. However, major knowledge gap for molecular monitoring centers on a simple yet impactful question: while glucose monitors have found hypodermal sensor placement to be most accurate, for other analytes and diseases what tissue layer in the skin, if any, will provide clinically valuable continuous monitoring? This question must be addressed because the skin’s dermis and hypodermis are different in both diffusion resistance and cellular density, which for the examples of insulin or IL-6 could respectively confound measurement due to receptor uptake or generation. The objective of this R21 proposal is to fill this knowledge gap by creating and in- vivo testing the first ever multi-depth sensor array that enables simultaneous monitoring of analytes in the largely acellular dermis vs. the adipocyte-rich hypodermis. The central hypothesis is that a multi-depth aptamer bio- sensor array will reveal that most analytes <10 kDa in the dermis will have <20% mean absolute relative difference with capillary blood, while <20% mean absolute relative difference for analytes in the hypodermis will be limited to analytes <1 kDa. The rationale for an R21 is that it will provide basic proof-of-concept in rats before even more detailed R01 work can investigate in humans more advanced confounding factors of cellular analyte uptake (receptors) and local analyte generation. The PI Heikenfeld and co-I Kasting are exceptionally prepared given their deep expertise in biofluids for continuous monitoring, the PI’s recent demonstration of aptamer biosensors with >10-day longevity, the PI’s first in-human results with aptamer sensors, and active relationships with 2 of 3 glucose monitor companies. The team will achieve the R21’s overall objective and test the central hypothesis through the following aims. Aim 1 – create a linear array of 5 redundant sensor electrodes on glucose sensor needle dimensions and with electrical impedance validate simultaneous sensor insertion in the rat dermis and hypodermis. Aim 2 – with an aptamer-functionalized 5-sensor array simultaneously monitor in rat blood, dermis, and hypodermis infused concentrations of phenylalanine (165 Da), or vancomycin (1.5kDa), or NT- proBNP (8.5 kDa). The proposed work is innovative, because: (1) due to the challenging proximity of the dermis and hypodermis this will be a breakthrough tool to accurately enable their simultaneous measurement; (2) in clinical practice the 5-sensor array could also be the most reliable way to achieve and maintain dermal sensor placement. With respect to expected outcomes, R21 success will provide new fundamental insights for continuous monitoring and enable R01 pursuit of human testing for challenging but impactful analytes of chronic disease.

Project Narrative Continuous molecular monitoring has advanced from sensors tested in benchtop beakers, into numerous demonstrations of sensors inserted into animal jugulars for blood monitoring, and is now emerging into monitoring in the skin’s interstitial fluid which is the clinically successful approach used in wearable glucose monitors. However, a major knowledge gap for molecular monitoring centers on a simple yet impactful question: while glucose monitors have found hypodermal sensor placement to be most accurate, for other analytes and diseases what tissue layer in the skin, if any, will provide clinically accurate continuous monitoring? The objective of this R21 proposal is to fill this knowledge gap by creating and in-vivo testing the first ever multi-depth sensor array that enables simultaneous monitoring of analytes in the largely-acellular dermis vs. the adipocyte-rich hypodermis.