Multi-Biomarker Thread-Based Biosensors for Monitoring Acute Compartment Syndrome

Acute compartment syndrome (ACS) is a serious condition arising from pressure buildup due to swelling or internal bleeding of tissue, mostly as a result of trauma [1]. ACS is prevalent in combat settings in military personnel as it could result from polytraumatic, blunt, or crushing accidents from explosions and the application of tourniquets [2]. Extreme trauma frequently results in ischemia, which can have long-term consequences like limb loss and death. Our overarching hypothesis is that early diagnosis of ACS may be possible through continuous and real-time assessment of physiological indicators, such as intramuscular oxygen, lactate, pH, and glucose [1,3]. Towards that goal, we propose the design of label-free thread-based electrochemical sensors for in-vivo measurement of these biomarkers inside the muscle tissue to capture physiological indicators for compartment syndrome detection. Compared to other flexible platforms, threads provide the most flexibility for tissue-embedded sensors, microfluidics, and bioelectronics [4-5]. For the enzymatic sensor (glucose and lactate), an amperometric method uses a two-electrode electrochemical set-up. The working electrode (WE) is composed of a composite Glutaraldehyde- Bovine Serum Albumin- Glucose Oxidase- Prussian Blue- carbon coated-suture (GA/BSA/GOx/PB/C) for glucose and GA/BSA/LOx/PB/C-coated suture for lactate sensing. An Ag/AgCl-coated suture as reference (RE) and counter electrodes (CE) is used. To measure pH, a composite silver/silver chloride (Ag/AgCl)-coated suture was utilized as the RE to determine the open-circuit potential of a polyaniline-carbon (PANI-C) coated suture. Fabrication on sensors starts with the cleaning of the suture (5/0 Polysorb^TMby COVIDIEN) in isopropyl alcohol (sonication 15 min), followed by plasma treatment (5 min) and carbon-ink deposition [4-5]. To create enzymatic (glucose and lactate) sensors, PB was electrochemically deposited on a carbon-coated suture in an electrolyte solution (0.10 M HCl and 0.10 M KCl) containing potassium ferricyanide and iron chloride for 90 seconds, followed by a cyclic voltammetric scan from -0.20 to 1.0 V at 0.10 V/sec vs Ag/AgCl electrode and washed with distilled water. To restrict the sensing region, a polyurethane (PU 5% w/v) layer is coated on the sensor leaving both ends of the sensor exposed. One end (carbon coated) is for the potentiostat connections and the other end is for further deposition of the enzyme matrix, which makes up the sensing region. PU/PB/C-suture was incubated in an enzyme solution containing BSA, and GA in 0.10 M phosphate-buffered saline (PBS, pH 7.4) then washed with PBS and dried. To fabricate the pH sensor, pH-sensitive dye polyaniline was electrochemically deposited at 0.80 V for 900 sec and washed with distilled water. The RE was fabricated by dip-coating a cleaned suture in conductive Ag/AgCl paste [5] and then baking at 60°C for 30 min. An amperometric response was noted on an Ag suture (as a cathode) for the oxygen sensor. To protect the enzyme layer from biofouling, an alginate-calcium hydrogel was drop cast on the sensing region, dried, then washed with PBS. For a demonstration of sensor performance, the electrochemical measurements were performed with cannulated sensors submerged in a phantom skin-gel environment (2.0 % agarose solution in 0.10 M PBS, pH 7.4). Under optimal experimental conditions, the devised pH sensor showed an excellent sensitivity of ~28 ± 3 mV/pH. Dissolved oxygen exhibited a linear relationship with change in current and a sensitivity of 0.66 µA/mgL^-1, whereas the glucose and lactate sensor sensitivity was considered adequate at 18.53 µA/mM and 30 µA/mM, respectively. Our results demonstrate that the thread-based flexible platform is capable of detecting physiological biomarkers in skin-mimicking models and could be particularly useful for continuous and real-time monitoring of several key metabolites such as glucose, lactate, pH, and oxygen in tissue.