A GMR enzymatic assay for quantifying nuclease and peptidase activity

Abstract

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Keywords: DNA substrate; GMR; disease monitoring; enzymatic activity; giant magnetoresistive sensor; peptide substrate; point-of-care testing.

FIGURE 1

Graphical illustration of a hydrolase assay that uses magnetoresistance to quantify substrate cleavage. (A) A biotinylated substrate is covalently attached to the sensor surface through amine coupling. The addition of streptavidin-coated magnetic nanoparticles increases the magnetoresistance signal as the nanoparticles are tethered to the biotin substrate close to the sensor surface. The substrate is cleaved by a hydrolase enzyme and releases the magnetic nanoparticle. (B) Illustration of how the magnetoresistance signal changes with time. In the absence of magnetic nanoparticles, a low signal is detected. Upon the addition of streptavidin-coated magnetic nanoparticles, the signal increases with time as the particles bind to the substrate and are orientated close to the GMR sensor surface. The signal then decreases in proportion to the concentration of active hydrolase enzyme added.

FIGURE 2

Sensor Functionalization and Stability of the Substrate. (A) The sensor surface is cleaned and activated by ultraviolet-ozone treatment before poly (allylamine) (PAAM) is added. The hydrolyzed poly (ethylene-alt-maleic anhydride) (PEMA) is added to create a layer of maleic groups. When the amine-containing substrate is spotted on the sensor surface, the maleic groups form a covalent bond with the amines on the substrate. (B) Time-dependent loading of streptavidin-MNP onto a sensor surface containing PEG₁₁-biotin. (C) Change in magnetoresistance for a fully assembled PEG₁₁-MNP complex that was stored for 21 days at 4°C and then sequentially incubated at various conditions. The change is recorded from one condition to the next. (D) Time-dependent decrease in signal in the presence of an extremely basic reagent (pH 13.5).

FIGURE 3

Nuclease Assay. (A) Graphical illustration of the nuclease assay. The signal decreases when the nuclease cleaves the substrate. (B) Hydrolysis of the ACTAGT (Bcu I substrate) and TACATG (Bcu I scrambled) sequences by DNase I results in a time-dependent decrease in the MR signal. (C) Hydrolysis of ACTAGT and not TACATG by the restriction enzyme, Bcu I. (D) Sequential addition of Bcu I and DNase I shows that Bcu I specifically cleaves the ACTAGT substrate, while DNase I cleaves the TACATG sequence.

FIGURE 4

Human Neutrophil Elastase Assay. (A) Illustration of the neutrophil elastase assay. The peptide substrate is covalently attached to the sensor surface and then bound to streptavidin-MNPs via a biotin group on the peptide. This results in an increase in magnetoresistance. The addition of human neutrophil elastase cleaves the substrate, decreasing the signal. (B) An example of how the signal is loaded and then reduced by adding human neutrophil elastase. The signal is displayed as a percentage of the loading signal after the magnetic nanoparticle binding has saturated. (C) Human neutrophil elastase assays with inhibition by sivelestat sodium. All three assays have 20 μg/mL of neutrophil elastase, but the inhibitor concentration increases from 0 to 20 μg/mL. (D) Human neutrophil elastase titration serial diluting from 125 to 3.9 nM by a factor of 2 in PBS containing 0.01% Tween-20. The magnetic assay results are shown as the maximum velocity.

FIGURE 5

Validation of Magnetic Neutrophil Elastase Assay in Buffer and Sputum. (A) Validation of the magnetic human neutrophil elastase assay by comparison to a traditional AMC assay readout with a spectrometer. Each point represents a different concentration of human neutrophil elastase in buffer readout by the GMR readout station (shown in the y-axis) and the spectrometer (shown in the x-axis). The concentration ranges from 3.9 to 125 nM of human neutrophil elastase. (B) Validation of the magnetic human neutrophil elastase assay compared to a traditional amc assay readout with a spectrometer. Each point represents a different human sputum sample of patients with cystic fibrosis measured by the GMR station (shown in the y-axis) and the spectrometer (shown in the x-axis).