Hollow Microneedle Patch integrated with paper-based analytical device for Multiplexed Quantification of Biomarkers in the Interstitial Fluid

Real-time monitoring of relevant biomarkers in biological fluids is essential for management of stress and inflammatory disorders. Interstitial fluid (ISF) offers a relatively painless alternative to blood as a reliable source of biomarkers for routine monitoring. In this study, we introduce the combination of a microfluidic paper-based analytical device (µPAD) integrated within a 3D printed hollow microneedle platform with a vacuum-based suction system for reliable sampling and multiplexed quantification of inflammatory and stress biomarkers in the ISF, namely interleukin 6 (IL-6), glucose, and lactate. The microneedles are 3D printed offering high resolution structures for penetration and sampling. In our µPAD, a dry-binding protein assay is employed for colorimetric IL-6 determination, reducing the use of expensive reagents like antibodies and simplifying the analysis procedure. Correspondingly, glucose and lactate monitoring are based on the oxidation reaction with their enzymes and potassium iodide (KI). The variation of the colour changes in each detection zone is proportional to these biomolecule levels. With these characteristics, the linear range of our µPAD sensor is from 10.0 to 30.0 pg mL-1 for IL-6 and from 1.25 to 15.0 mmol L-1 for both glucose and lactate. Additionally, detection limits (LODs) of 2.0 pg mL-1, 0.25 mmol L-1, and 0.32 mmol L-1 for IL-6, glucose, and lactate, respectively, were obtained. By integrating µPAD into the hollow microneedle platform, the workflow begins with positioning the device into the skin. The interstitial fluid is sampled reliably through the hollow microneedles using a handheld vacuum pump causing it to flow into the µPAD, where the colorimetric reactions are initiated. Studies were performed in tissue models of agarose gel and ex vivo pig skin samples revealed the recovery range of 96.0-108.0% with the highest RSD of 4.72%. This demonstrates the remarkable reliability and precision of the proposed technique. Consequently, the proposed device offers a first of its kind inexpensive wearable platform that integrates a complete workflow from sample acquisition to multiplexed sensing and visual readout without the need for trained personnel or expensive instrumentation. The device also holds significant potential as a wearable device for monitoring and management of chronic conditions such as post-traumatic stress disorder or PTSD.