A fluidic device for continuous on-line inductive sensing of proteolytic cleavages

Proteases, an important class of enzymes that cleave proteins and peptides, carry a wealth of potentially useful information. Devices to enable routine and cost effective measurement of their activity could find frequent use in clinical settings for medical diagnostics, as well as some industrial contexts such as detecting on-line biological contamination. In particular, devices that make use of readouts involving magnetic particles may offer distinct advantages for continuous sensing because material they release can be magnetically captured downstream and their readout is insensitive to optical properties of the sample. Bioassays based on giant magnetoresistance sensors that detect the binding or release of magnetic materials have been widely explored for these reasons, but they typically require expensive consumables. Here, we develop a simpler protease sensor based on inductive detection of particle release with pulsed magnetic fields, leveraging a design that incorporates both the pulse coil and gradiometer coils into a printed circuit board. Our fluidic chips are formed from casts of 3D printed molds, such that both the sensor and the consumable components could be relatively easy to mass produce. Using pulses ranging up to 10 s of mT, we show that our device has a limit of detection below 1 μg of iron and that its duty cycle can be varied to control temperature through Joule heating. By chemically functionalizing the glass surface of our fluidic chips with zwitterionic polymer and incorporating a PEG block co-polymer into the PDMS component, we are able to suppress the nonspecific binding of albumin by 7.8 times inside the chips. We demonstrate a layer-by-layer approach for covalently linking magnetic nanoparticles to the chips <i>via</i> cleavable peptide substrates. Finally, we observe the release of the magnetic particles from the chips under conditions of proteolytic cleavage and measure resulting changes in inductive signals, demonstrating a detection sensitivity for chymotrypsin in the hundreds of nM. The methods we establish here have the potential to aid progress toward sensors comprised of disposable fluidic chips measured by inexpensive detection devices that may one day facilitate ubiquitous protease activity monitoring.