A novel point-of-care diagnostic prototype system for the simultaneous electrochemiluminescent sensing of multiple traumatic brain injury biomarkers

Abstract

Traumatic brain injuries (TBI) are typically acquired when a sudden violent event causes damage to the brain tissue. A high percentage (70-85%) of all TBI patients are suffering from mild TBI (mTBI), which is often difficult to detect and diagnose with standard imaging tools (MRI, CT scan) due to the absence of significant lesions and specific symptoms. Recent studies suggest that a screening test based on the measurement of a protein biomarker panel directly from a patient's blood can facilitate mTBI diagnosis. Herein, we report a novel prototype system designed as a precursor of a future hand-held point-of-care (POC) diagnostic device for the simultaneous multi-biomarker sensing, employing a microarray-type spatially resolved electrochemiluminescence immunoassay (SR-ECLIA). The small tabletop prototype consists of a screen-printed electrode compartment to conduct multi-analyte ECL sandwich assays, a potentiostat module and a light collection module, all integrated into a compact 3D-printed housing (18.2 × 16.5 × 5.0 cm), as well as an sCMOS detector. Based on this design concept, further miniaturization, system integration, performance optimization and clinical evaluation shall pave the way towards the development of a portable instrument for use at the site of accident and healthcare. To demonstrate the system's feasibility, current performance and efficiency, the simultaneous detection of three mTBI biomarkers (GFAP, h-FABP, S100β) in 50% serum was achieved in the upper pg mL^-1 range. The proposed device is amenable to the detection of other biomarker panels and thus could open new medical diagnostic avenues for sensitive multi-analyte measurements with low-volume biological sample requirements.

Fig. 1. Prototype build-up: a) a layer rendering image of the prototype main components; b) photo of the prototype device; c) schematic representation of the light collection module; d) schematic rendering of the electrode placed inside the electrode compartment: (i) depicts the system when the electrode and potentiostat compartments are disconnected (some parts are made transparent here), and ECL read buffer is manually added; (ii) depicts the system during the measurement step when the compartments are engaged and properly positioned beneath the light collection module (transparent blue); e) software architecture diagram. The graphical interface is shown in the SI-3.

Fig. 2. Detection of a BSA@SULFO-TAG microarray on SPCE: a) ECL images obtained from SPCE spotted with 5 × 5 spot patterns with: 5 drops (1.75 pg, ∼250 μm spot diameter), 10 drops (3.50 pg, ∼300 μm spot diameter), and 20 drops (7.00 pg, ∼400 μm spot diameter) of 1 μg mL⁻¹ of the BSA@SULFO-TAG protein. Images were treated with ImageJ by 8 × 8 binning (sum) and dark subtraction; b) conditions of the ECL microarray established on the SPCE by spotting various quantities of BSA@SULFO-TAG solutions (from 0.0625 μg mL⁻¹ to 1.0000 μg mL⁻¹); c) correlation between spotted quantities of BSA@SULFO-TAG on SPCE from b) and obtained ECL signals intensities. The top inset image shows zoomed-in single lines of microarrays from images presented in a) obtained from spots with different number of 5, 10, and 20 drops. Second inset shows zoomed-in part of curve in the range of 0.1–1 pg of BSA@SULFO-TAG. The error bars represent the standard deviation for different spots; bars are in some cases smaller than the data symbol employed. Images treated with ImageJ color palette “red-hot”, 8 × 8 binning (sum) and dark subtraction. MSD Read Buffer T 2× (75 μL) was used for ECL read-out.

Fig. 3. Microarray-type spatially resolved (SR-ECLIA) CRP singleplex assay performance in “clean target” (buffer solution) using POC prototype detection system: a) capture antibody spotting pattern (letter “C” – BSA@SULFO-TAG control spots; letter “I” – inflammatory (CRP) biomarker spot; and corresponding ECL raw data images generated at various CRP concentrations (0, 25, 50, 100, and 250 pg mL⁻¹) (8 × 8 binning and dark subtraction, but no other postprocessing); b) obtained calibration curve. The error bars represent the standard deviations from two replicates (n = 2) and the fitting was performed with a 4-PL equation; dashed horizontal curve represent the evolution of the background measured on the electrode surface, in the surrounding area of the spots. The signals were not normalized. Dotted vertical line represent the LOD (calculated at 34 pg mL⁻¹). Assay conditions are listed in SI-5.

Fig. 4. Microarray-type SR-ECLIA 3-plex protocol: a) schematic representation of the assay established for detection of three mTBI biomarkers (GFAP, h-FABP, S100β) on a single SPCE. STEP 1 includes the spotting of small quantities of capture antibodies of each biomarker on the predefined spot positions on the SPCE using a spotter device (BSA labelled with SULFO-TAG was used for control/alignment purposes); STEP 2 includes the typical sandwich assay protocol composed of a blocking step, addition of three antigens, and addition of detection antibodies modified with the SULFO-TAG label carrying Ru(bpy)₃²⁺; STEP 3 is the detection step that includes addition of the tripropylamine (TPA) co-reactant and applying a potential on the SPCE to trigger the ECL signal generation from each biomarker spot; STEP 4 comprises the raw data processing; b) optimization of capture antibody amount per spot (cAb amount for all biomarkers: 7.00 ng per spot, 2.10 ng per spot, 0.70 ng per spot, 0.18 ng per spot). The antigen concentrations were 0, 1, 10 and 50 ng mL⁻¹. The other assay conditions are listed in SI-5.

Fig. 5. Microarray-type SR-ECLIA 3-plex assay performance in 50% serum (v/v) using POC prototype detection system: a) capture antibody spotting pattern (letter “C” – BSA@SULFO-TAG control spots; letter “G” – GFAP biomarker spots; letter “H” – h-FABP biomarker spots; letter “S” – S100β biomarker spots); and corresponding ECL raw data images generated at various biomarker concentrations (0, 250, 500, 750, and 1000 pg mL⁻¹) (8 × 8 binning and dark subtraction, but no other postprocessing), and b) obtained 3-plex calibration curves. The error bars represent the standard deviations from two replicates (n = 2) and the fitting was performed with a 4-PL equation; c) assay specificity heatmap at single antigen concentrations of 2.5 ng mL⁻¹.

Fig. 6. Model of the future device: The envisioned “next generation” fully integrated POC diagnostic system for the simultaneous mTBI multi-biomarker detection with key components is shown. The five bars (i.e., biomarker concentrations above or below a pathological threshold) visible on the device's display represent results from a putative 5-plex SR-ECLIA. Designed by MADI and Marc E. Pfeifer. Copyright HES-SO Valais-Wallis.