Nano-Calorimetry based point of care biosensor for metabolic disease management

Abstract

Point of care (POC) diagnostics represents one of the fastest growing health care technology segments. Developments in microfabrication have led to the development of highly-sensitive nanocalorimeters ideal for directly measuring heat generated in POC biosensors. Here we present a novel nano-calorimeter-based biosensor design with differential sensing to eliminate common mode noise and capillary microfluidic channels for sample delivery to the thermoelectric sensor. The calorimeter has a resolution of 1.4 ± 0.2 nJ/(Hz) utilizing a 27 junction bismuth/titanium thermopile, with a total Seebeck coefficient of 2160 μV/K. Sample is wicked to the calorimeter through a capillary channel making it suitable for monitoring blood obtained through a finger prick (<1 μL sample required). We demonstrate device performance in a model assay using catalase, achieving a threshold for hydrogen peroxide quantification of 50 μM. The potential for our device as a POC blood test for metabolic diseases is shown through the quantification of phenylalanine (Phe) in serum, an unmet necessary service in the management of Phenylketonuria (PKU). Pegylated phenylalanine ammonia-lyase (PEG-PAL) was utilized to react with Phe, but reliable detection was limited to <5 mM due to low enzymatic activity. The POC biosensor concept can be multiplexed and adapted to a large number of metabolic diseases utilizing different immobilized enzymes.

A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity

“…Both junction sets are located in the microfluidic channel, which allows for compensation for unwanted reaction enthalpies in differential calorimetry. We used this to subtract the heat of dissolution by exposing the reference junctions with denatured enzyme [19].…”

“…The microfluidic channel liquid and reaction zone were defined as water to properly simulate the diffusion of substrate and enzyme, as well as heat capacity and thermal conductivity to compute temperature profiles. The 3D model was then extended to include the calorimeter platform (Figure 2B) and assigned material-related thermal properties G i , taken from our previous modeling effort, to simulate the calorimeter platform's thermal response to the heat input from the enzymatic reaction [19]. The liquid channel was confined by the two thin Su-8 membranes and the two Su-8 channel walls with thermal properties G mem (thermal conductivity k = 0.2 W/(m*K); density ρ = 1123 kg/m 3 ; heat capacity C p = 1200 J/(kg*K)), and the sensing thermocouple junctions were embedded within the membrane.…”

“…We have successfully developed highly sensitive nanocalorimeter TELISA platforms and showed the detection of clinically relevant levels of herceptin and phenylalanine in serum [19,20]. The devices feature a small footprint, require nanoliter volumes of sample, and are mass produced by standard batch microfabrication techniques that can be commercially produced at a cost of less than $1 USD per device.…”

“…Previous work showed a calorimeter thermal time constant of 325 ms, and an energy sensitivity of 1.4 nJ/Hz 1/2 . For comparison purposes, this translates to an LOD of 25 femtomoles (fmol) for an acid-base neutralization reaction [19]. Nanocalorimeter fluid handling was driven by capillary forces, and assay readouts were obtained via direct voltages, showing promise for POC operation.…”

Nanoparticle-based Point of Care Immunoassays for in vitro Biomedical Diagnostics

“…metabolic disorders, different types of renal disorders depending upon the concentration of protein. [13][14][15][16] To address the diagnostics hurdles, and to enhance the sensitivity of the assay, nanoparticles-based immunoassays have been reported, which rely on the unique properties of nanoparticles and have improved detection limit up to 1000 times. [17][18][19][20]…”