Using the tethered enzyme assay for rapid detection of NSE in human subjects.

<p><b>A)</b> Representative data as measured from a total of nine wells per subject (triplicate measurements of negative control with no 2PG; the test sample with 2PG; and positive control wells with 2PG and enolase (Eno); mean±STDEV) For calculation of NSE levels, slopes of averaged curves were calculated for the first 0.5–10 minutes of the enzymatic reactions (indicated by the red linear fit in between the dashed lines), then test wells were normalized to positive and negative wells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142326#sec007" target="_blank">methods</a> section). <b>B)</b> High correlation was found between NSE measurements by ELISA (ng/ml) vs. TET assay (AU represents the normalization of test reaction to the negative and positive reactions, as illustrated in A and described in the methods section) as of the human plasma samples. Line indicates best fit. Pearson’s r = 0.815, n = 20.</p

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

<div><p>Background</p><p>Rapid 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.</p><p>Methods and findings</p><p>We 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.</p><p>Conclusions</p><p>Oriented 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.</p></div

Using NP-PK and NP-Luc for rapid detection of NSE in a rat model for stroke.

<p><b>A)</b> Fluoro Jade-C staining for measurement of damaged brain tissue volume. The presence and absence of FJC-labeled degenerative neurons were imaged with epifluorescence under 20x magnification using filters for FITC. (a) Region with abundant FJC staining (bright cells) on lesioned side. (b) Region at the edge of FJC staining. (c) Region that is contralateral to (a) that did not show any FJC staining. Dotted line in B and low magnification inset indicates manually-mapped border between FJC positive and negative areas. <b>B)</b> FJC staining in control brain section. (a) and (b) are in area of craniectomy. <b>C-D)</b> Individual rat data (C-stroke and D-control) for NSE measurements at each time point (grayscale lines indicate data points for individual animals, line with symbol indicates the mean±standard deviation) as performed by tethered PK and Luc assay, and normalized to time point -1 hour. Plasma samples were collected from stroke induced or control rats pre (-1 hr), and post occlusion (0, 1, 3 and 6 hr). The mean slope of the trend (dashed line) was calculated averaging individual slopes in each group (stroke = 0.26 with 0.95 confidence interval, control = -0.02 with 0.38 confidence interval). <b>E</b>) Summary of rat stroke model experiments showing a statistically significant increase in NSE plasma levels in stroke (blue bars) vs. control (red bars) rats as soon as 1 hour post occlusion. <b>F)</b> Measurements of NSE in plasma from rats using ELISA showed elevated levels in stroke compared to control rats at the last time point (6 hr post-occlusion). P values– 0.05<*<0.1, 0.01<**<0.05. Data from 10 rats were included in our analysis, with the exclusion of 3 hour and 6 hour time points for one control animal that died (at 2 hour mark), and a 6 hour time point for a stroke animal which died and was found to have a large hemorrhage at the base of the brain.</p

Immobilization of PK and Luc on NPs improves coupled reaction efficiency.

<p><b>A)</b> Schematic illustration of the 6XHis-Si tag fusion constructs for PK or Luc. <b>B)</b> Schematic representation of the PK-Luc coupled reaction as used in the experiments described in C. <b>C)</b> PK and Luc coupled activity was assayed in 4 combinations as indicated by the color coded schematic illustrations: black- (NP-PK) + (NP-Luc), blue- (NP-PK) + (soluble Luc), green- (NP-Luc) + (soluble PK) or red- (soluble PK + soluble Luc). All combinations included equivalent amounts of PK and Luc. Maximal coupled reaction efficiency, as calculated from normalizing each time point data to t₀ (indicating the ratio of luminescence generated at each time point relative to the luminescence at the beginning of the reaction) and subtraction of the negative control well (no PEP, corresponding to the background luminescence signal), was observed when both PK and Luc were immobilized on NPs (each condition was tested in triplicates; data shown represents 3 individual experiments; AVG±STDEV).</p

Improved sensitivity for enolase detection via its enzymatic activity when using tethered PK and Luc.

<p><b>A)</b> Comparison of the detection sensitivity for enolase activity (Eno) as measured by NP-PK + NP-Luc (red) vs. PK + Luc in solution (blue). Increasing concentrations of Eno were added to immobilized or freely diffusing enzymes in reaction buffer. Luminescence was detected and integrated over 10 minutes at RT and plotted against Eno final unit amount (lines were added to guide the eye). The inset shows the ratio of the enzymes’ activities on NPs versus in solution as a function of units of ENO. Data presented as AVG±STDEV. <b>B)</b> NSE detection via PK and Luc coupled activity was performed as indicated in the schematic illustration (right). His-Si-PK and His-Si-Luc were immobilized on 500 nm NPs and mixed with reaction buffer supplemented with increasing concentrations of human NSE. Luminescence readout was normalized to t0 (first read) values and plotted as a function of time. Then, the luminescent signal was integrated for first 10 minutes, and a reading from the zero NSE well was subtracted from the collected signal. The sensitivity of the tethered enzyme assay was about 5 times higher than the reported average physiological NSE blood concentration (8.7 ± 3.9 ng/ml [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142326#pone.0142326.ref024" target="_blank">24</a>]). P values were calculated using student’s t-test for comparisons between NSE concentrations; values marked with ^<b>#</b>P were calculated for significance when compared against 0 ng/ml NSE.</p