(M1230-01-03) Optimized PEG-Biotin Functionalized QCM-D Sensor for Rapid Detection of a Sepsis-Associated Biomarker

Purpose: Multiple organ dysfunction syndrome (MODS) is a frequent and life-threatening complication of sepsis, increasing mortality by >3-fold. There are currently no specific therapies for MODS, and early identification of high-risk patients remains a substantial unmet need. Critical care specialists have clearly indicated that a validated biomarker which could be rapidly measured (i.e. within minutes) at time of hospital admission would be invaluable for identifying patients at risk for MODS, thereby enabling aggressive monitoring and intervention. However, no such biomarker or rapid diagnostic test currently exists. Our team has identified a bloodborne protein that dramatically increases in concentration during the early stages of sepsis. Plasma concentrations of this protein biomarker, measured 12 hours after sepsis induction via cecal ligation-puncture surgery, accurately predicted cardiac dysfunction and early mortality in a rat model. Building on this discovery, we developed a prototype piezoelectric immunosensor based on quartz crystal microbalance with dissipation monitoring (QCM-D), capable of detecting this biomarker in plasma samples in < 30 minutes. The objective of this work is to further improve the sensitivity and robustness of our immunosensor and rigorously test its performance in experimental sepsis models. To achieve this, we implemented a surface modification strategy for the gold sensor, employing a mixed self-assembled monolayer of PEG-thiol and biotinylated PEG-thiol to minimize non-specific protein adsorption and provide functional groups for antibody immobilization. Sequential addition of streptavidin and biotinylated protein A enabled antibody orientation and stability upon attachment, ensuring accessibility of biomarker binding sites and enhancing assay specificity and reliability. Our optimized immunosensor demonstrated successful detection of the biomarker in plasma samples, highlighting its potential for rapid, point-of-care use in the clinic.

Methods: Gold-coated QCM-D sensors were cleaned and incubated for over 12 hours in an ethanolic solution containing 1 mM mixtures of thiol-PEG hydroxyl and thiol-PEG biotin at varying ratios (10%, 50%, and 100% biotinylation) to optimize surface functionalization. To further refine the sensor coating, we evaluated these biotinylation ratios using three different molecular weights of thiol-PEG: 1000 Da, 2000 Da, and 3400 Da. After thorough ethanol rinsing to remove unbound thiol-PEG, the QCM-D sensor was mounted in the system flow cell and sequential steps were performed as outlined in Figure 1 and as follows: Sensor was sequentially exposed to streptavidin (75 µg/mL in PBS, 50 µL/min flow) until saturation, followed by biotinylated protein A (35 µg/mL in PBS, 50 µL/min flow). Anti-biomarker antibody (10 µg/mL, 20 µL/min flow) was then introduced, and the performance of the sensor construct was evaluated using rat plasma samples with increasing concentrations of recombinant biomarker. All QCM-D measurements were conducted at 22 °C, and frequency shifts normalized by overtone number were monitored. Data were collected at the fundamental and multiple overtones, but only the 3rd overtone was reported due to minimal variation among overtones.

Results: To determine impact of biotin sensor coating, two key parameters were assessed: (i) the amount of streptavidin binding to the biotinylated surfaces, and (ii) the subsequent immobilization of biotinylated protein A onto the streptavidin-modified surface. As shown in Figure 2, increasing the fraction of thiol-PEG biotin resulted in enhanced binding of both streptavidin and protein A, with the highest immobilization observed at 100% biotin content. Based on these findings, we selected the 100% thiol-PEG biotin with a molecular weight of 1000 Da for further sensor development. Using the optimized sensor surface, we successfully detected the target biomarker in rat plasma samples spiked with known concentrations of the recombinant protein (Figure 3). These results demonstrate the effectiveness of our surface modification strategy and confirm the suitability of the QCM-D-based immunosensor for rapid and sensitive detection of the novel sepsis biomarker in complex biological matrices.

Conclusion: We successfully developed a robust QCM-D immunosensor with optimized PEG-biotin surface chemistry that enables rapid detection of a novel protein biomarker in plasma. This platform shows strong potential for point-of-care diagnostics to identify patients at high risk for MODS early on during the course of sepsis, supporting timely clinical intervention to reduce morbidity and mortality.

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Figure 1. Schematic of novel biomarker capture surface generated through a biotin-streptavidin layer formation.

Figure 2. Frequency shifts for streptavidin and protein A binding as a part of optimization of the second-generation robust sensor.

Figure 3. Frequency shifts recorded for rat plasma samples spiked with known concentrations of the biomarker.