Electrochemical evaluation of interstitial fluid cortisol through hydrogel microneedles

Introduction:: Over 25 million people in the US had a major depressive episode in 2020 [1]. Depression is associated with increases in cortisol concentrations throughout the body which return to normal levels following successful treatment. Monitoring cortisol concentrations over time will give insights into a patient’s status and inform if treatments are effective [2], however, current cortisol measurements are done with lab testing of biofluids such as blood and saliva, limiting the utility of these measurements for regular monitoring. This work shows advances toward a portable, rapid-response device for measuring cortisol directly from interstitial fluid (ISF) in patients with depression.

Using hydrogel microneedles, interstitial fluid (ISF) can be collected to measure cortisol concentrations without invasive techniques. Here, we demonstrate electrochemical measurement to quantify analyte concentrations directly through the hydrogel matrix using a surface-bound DNA aptamer targeting cortisol. This eliminates the need for sample harvesting from the microneedles and will enable portable cortisol measurement with a device as simple as current glucose meters (Figure 1a).


Materials and Methods:: Methacrylated hyaluronic acid (MeHA) hydrogel was synthesized with methacrylic anhydride and harvested via dialysis and lyophilization. Hydrogel precursor solution was made by dissolving MeHA at 50 mg/mL and a photoinitiator (Irgacure 2959) at 0.5 mg/mL in ultrapure water. Microneedle arrays were made by casting the hydrogel solution into a PDMS mold consisting of a 15x15 array of 600 µm long pyramidal microneedles. After drying, the hydrogel samples were removed from the molds and crosslinked via 365 nm UV for 10 minutes. To visualize fluid uptake, Rhodamine B was measured by inserting microneedles through a parafilm barrier into 1.4% agarose and removing needles at various timepoints for microscope imaging.

Commercial screen-printed gold electrodes were prepared through electrochemical cleaning with sulfuric acid and aptamer binding. A cortisol-specific aptamer sequence [3] containing a thiol modification was directly bound to the gold electrode surface followed by blocking with mercaptohexanol to prevent non-specific binding. MeHA samples were swelled with 100 µL PBS containing cortisol at concentrations from 5-20 ng/mL, which is within the range of cortisol found in human ISF [4]. Swelled hydrogel samples were then placed on screen-printed electrodes and electrochemically evaluated with potentiostatic electrochemical impedance spectroscopy (PEIS) for cortisol measurement using a frequency range of 100 mHz to 100 kHz, and an amplitude of 10 mV. EC-Lab Zfit software was then used to calculate the charge transfer resistance (R_CT) of the Nyquist plot by fitting the equivalent circuit (Figure 1d, inset) which was then compared across cortisol concentrations.


Results, Conclusions, and Discussions:: MeHA microneedles took up Rhodamine B dye from the base to the tip within 10 minutes (Figure 1b). Within 15 minutes, the microneedle patch was able to take up 131.7 ± 17.3 µL of fluid (Figure 1c), which is sufficient for measuring analyte concentration with electrochemistry. This fast swelling will aid in short sampling times and rapid results when analyzing cortisol concentrations from ISF.

Electrochemical evaluation showed successful measurement of analytes through the hydrogel matrix. A directly proportional relationship was found between the concentration of cortisol and the calculated R_CT through PEIS analysis (Figure 1d). The sensor responded linearly (R² = 0.991, slope = 380.11 ± 7.01 ohm/(ng/mL)) for concentrations from 5 to 20 ng/mL (Figure 1e). This demonstrates direct measurement of physiologically relevant cortisol concentrations from MeHA hydrogels without the need for additional sample harvesting or processing.

MeHA hydrogel microneedles are a good candidate for painless sampling of analytes from ISF. Combining these microneedles with direct electrochemical evaluation of analytes is a step toward portable sampling of important biomarkers such as cortisol. Future steps include testing signal resolution within the range of cortisol in ISF, and testing specificity of the sensor toward cortisol compared to other similar hormones. The sensing platform will be integrated with a miniaturized potentiostat and a smartphone app for sensing and readout using a handheld device. These studies lay the groundwork for portable, simple testing and tracking of cortisol concentrations over time which will lead to better management of depression and associated treatments.


Acknowledgements (Optional): :

References (Optional): : [1] “NIMH > > Major Depression.” https://www.nimh.nih.gov/health/statistics/major-depression
[2] Dziurkowska, E., (2021) J Clin Med, 10(21), 5204.
[3] Sanghavi, B. J. et al. (2016). Biosensors and Bioelectronics, 78, 244-252.
[4] Venugopal, M., et al. (2011). Sensors and Actuators A: Physical, 172(1), 154-160.