A Variable Height Microfluidic Device for Multiplexed Immunoassay Analysis of Traumatic Brain Injury Biomarkers

Abstract

Traumatic brain injury (TBI) is a leading cause of global morbidity and mortality, partially due to the lack of sensitive diagnostic methods and efficacious therapies. Panels of protein biomarkers have been proposed as a way of diagnosing and monitoring TBI. To measure multiple TBI biomarkers simultaneously, we present a variable height microfluidic device consisting of a single channel that varies in height between the inlet and outlet and can passively multiplex bead-based immunoassays by trapping assay beads at the point where their diameter matches the channel height. We developed bead-based quantum dot-linked immunosorbent assays (QLISAs) for interleukin-6 (IL-6), glial fibrillary acidic protein (GFAP), and interleukin-8 (IL-8) using DynabeadsTM M-450, M-270, and MyOneTM, respectively. The IL-6 and GFAP QLISAs were successfully multiplexed using a variable height channel that ranged in height from ~7.6 µm at the inlet to ~2.1 µm at the outlet. The IL-6, GFAP, and IL-8 QLISAs were also multiplexed using a channel that ranged in height from ~6.3 µm at the inlet to ~0.9 µm at the outlet. Our system can keep pace with TBI biomarker discovery and validation, as additional protein biomarkers can be multiplexed simply by adding in antibody-conjugated beads of different diameters.

Poly(methyl methacrylate)-Based Nanofluidic Device for Rapid and Multiplexed Serological Antibody Detection of SARS-CoV-2

“…g-EBL enables a high degree of control over channel topography down to the sub-micron regime and allows for the facile integration of numerous 3D profiles with different height profiles. This is in contrast to nonlithographical techniques, such as HF-dipping, which also allow for the fabrication of wedge-shaped profiles in fluidic devices for similar applications. , Additionally, the referenced devices require active pumping, significantly larger sample volumes, do not achieve sub-micron channel heights, and have very limited design flexibility. Our g-EBL high-resolution structure is subsequently replica-molded to obtain numerous negative daughter stamps (Figure b).…”

The Current State of Traumatic Brain Injury Biomarker Measurement Methods

“…Clinically, protein biomarkers have been measured in CSF, serum, and plasma. Abbott is working on a test to measure biomarkers in whole blood, and Krausz et al demonstrated proof-of-concept GFAP measurements using spiked whole blood from a single donor [46,119]. Rickard et al demonstrated NAA measurements using fingerstick blood samples, but to the best of our knowledge, no one in industry has measured TBI protein biomarkers in a fingerstick blood sample, even though this sample is ideal for point-of-care biomarker measurements [48].…”

“…A summary of the assay characteristics for each early-stage sensor can be found in Table 2. [46] Notes: a LLODs were listed as ranges and were not separated by measurement technique, sample matrix, or biomarker. b LLODs and ranges were taken as the cutoff concentrations for each logic condition assignment.…”

“…Table 1 provides a detailed list of the TBI protein biomarkers measured by the devices discussed in this review. Severe TBI: 8-16 nM up to 100-800 nM in CSF [31] Gunawardhana and Lunte [33] Cleaved tau C-tau Neuronal damage (axons) [34] 2.48-66.54 pg/mL in serum [35] Severe TBI: 36.44-192.34 pg/mL in serum [35] Khetani et al [36] C-reactive protein CRP Prognosis [37,38] 0.642-2.785 mg/L in serum [39] mTBI: 2.110-30.932 mg/L in serum [39] Apori and Herr [40] Glial Fibrillary Acidic Protein GFAP Astrocyte damage [41] 7-20 pg/mL in plasma [17] mTBI: 69-1196 pg/mL in plasma [17] Agostini et al [ [51] 0.3-2 µmol/L in brain extracellular fluid [52] Severe TBI: >20 µmol/L in brain extracellular fluid [53] Halámek et al [54] Zhou et al [55] Interleukin-6 IL-6 Inflammation [56] ≤1.8 pg/mL in serum [57] Severe TBI: 0-1100 pg/mL in serum [57] Krausz et al [46] Interleukin-8 IL-8 Inflammation [56] ≤14.6 pg/mL in serum [58] Severe TBI: 0-2400 pg/mL in serum [57] Krausz et al [46] Lactate -Prognosis [59] 6.7-13.9 mg/dL in whole blood [60] Moderate to Severe TBI: 5. 11-17 pg/mL in serum [66] Severe TBI: 89-413 pg/mL in serum [66] Khetani et al [36] Neuron Specific Enolase NSE Neuronal damage [67] ≤0.15 µg/L in serum [67] >0.15 µg/L in serum [67] Cardinell et al [44] Gao et al [68] Li et al…”

“…Multiplexing in TBI assessment is important as the simultaneous assessment of two or more biomarkers increases the sensitivity and specificity of the assay. Krausz et al have recently developed a variable height device capable of passively multiplexing bead-based immunoassays for GFAP, interleukin-6 (IL-6), and interleukin-8 (IL-8) [46]. The variable height device gradually decreases in height between the channel inlet and outlet, causing assay beads to become physically trapped where their diameter matches the channel height.…”