Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

Abstract

BackgroundRapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.Methods and findingsWe performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.ConclusionsOriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.

Improved monitoring dynamics through use of a tethered enzyme biosensor to detect and quantify neuron-specific enolase activity levels in biofluids

“…22 Briefly, His-Si4-PK and His-Si4-Luc were purified using Ni-NTA beads, and their enzymatic activity was tested in sodium phosphate buffer (50 mM) supplemented with MgCl 2 (5 mM) and KCl (20 mM). The forward reaction for PK is PEP + ADP → pyruvate + ATP.…”

“…22,31 NP-PK and NP-Luc were then supplemented with ADP, 2-PG, luciferin, Mg 2+ , and K + . A total of 10 µL samples of serum or plasma were added to individual wells in a 96-well plate, and the TET reaction mix (10 µL) was added just before luminescence measurements were initiated.…”

“…Here, we utilized our tethered enzyme biosensor technology (tethered enzyme technology [TET]), where pyruvate kinase (PK) and luciferase (Luc) are immobilized onto nanoparticles 22 to detect NSE via its enzyme activity and determine the rates at which NSE protein or its activity are changing in serum and plasma. We have previously shown, both as a broad observation for glycolytic enzymes, 23 and specifically for NSE detection, 22 that solid-state enzyme reactions using oriented protein immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes.…”

“…We have previously shown, both as a broad observation for glycolytic enzymes, 23 and specifically for NSE detection, 22 that solid-state enzyme reactions using oriented protein immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. This novel method enabled us to detect NSE in a rat model for brain injury and in human subjects with a rapid (<10 minutes), sensitive, and convenient assay.…”

“…This novel method enabled us to detect NSE in a rat model for brain injury and in human subjects with a rapid (<10 minutes), sensitive, and convenient assay. 22 Here, we hypothesize that through its advantages and improved temporal resolution, it will offer a better approach to utilizing the clinical value of using NSE A as a biomarker.…”