(M1430-06-37) Toward Development of a New QCM-D Based Method for Rapid Detection of a Novel Biomarker of Morbidity and Mortality Risk in Sepsis Patients

Purpose: Millions of people around the world develop severe sepsis each year and nearly a third of them die from it. Multiple organ dysfunction syndrome (MODS) is a common and serious complication of sepsis that increases mortality by >3-fold. Clinical reports suggest that patient outcomes are greatly improved if patients at risk for MODS and/or mortality are identified in the first few hours after onset of sepsis. Such a fast response requires validated markers for prediction of the risk of MODS and an extremely rapid turnaround in analytics that measure the biomarker. In this regard, our research team has identified a novel bloodborne protein as a promising biomarker for MODS and mortality risk in sepsis. However, the current quantification method, a customized ELISA, requires approximately 36 hours to complete, which hinders its utility as a rapid test. Thus, the objective of this study is to assess the feasibility of a rapid and reproducible test for this biomarker in plasma using an immunosensor technology.

Methods: This study presents the use of Quartz Crystal Microbalance with Dissipation Probe (QCM-D) for real-time monitoring of the adsorption and binding processes of our biomarker (see Fig 1). The isoelectric point of the protein biomarker is between 5-6, and all experiments were conducted using PBS buffer at pH 7.4. The gold-coated quartz crystal was initially cleaned, and a stable baseline was achieved in standard buffer conditions. Next, the sensor surface was prepared by immobilizing our validated primary antibody for the protein marker to achieve complete saturation of the surface. Rinsing with buffer was performed to remove any loosely bound antibody. Subsequently, the sample containing biomarker was introduced, allowing for binding to antibodies on the surface of the sensor. Finally, a buffer rinse was employed to eliminate loosely bound molecules. The QCM-D system detected changes in mass and properties of the sensor surface. The initial adsorption rate and baseline frequencies at each step were calculated. To enhance adsorption conditions, various parameters were optimized, including the use of treated and untreated gold sensors, different concentrations of antibody (10 and 20 μg/ml), and different flow rates (10, 20, and 30 μl/min).

Results: Analysis revealed that the antibody treated gold sensor exhibited a higher frequency shift and initial adsorption rate of the biomarker. Regarding the immobilization of antibody from solution of different concentrations, a higher frequency shift and initial adsorption rate were observed with a 20 μg/ml antibody solution compared to 10 μg/ml. However, because of the marginal differences and cost considerations, the study primarily utilized the 10 μg/ml concentration. Investigating different flow rates showed that a flow rate of 10 μl/min enabled the detection of biomarker concentrations in the buffered solution within 2 hours, while a flow rate of 20 μl/min achieved detection in approximately 40 minutes (Fig 2). When the flow rate was increased to 30 μl/min, the biomarker was detected in approximately 20 minutes. However, our data revealed that the total amount of immobilized antibody and detected biomarker was not influenced by the flow rate (Fig 3). Taken together, these findings led us to select the flow rate of 20 μl/min as the optimal choice for developing a rapid test as it renders reliable data in under 1 hour and has the lowest requirements in terms of sample size and flow conditions.

Conclusion: These results demonstrate the feasibility of using QCM-D for rapid measurement (within an hour) of our biomarker. Optimizations, including the use of treated gold sensors, higher antibody concentration (20 μg/ml), and a flow rate of 20 μl/min, led to improved performance in terms of frequency shift and initial adsorption rate. These findings provide the basis for further development of a robust immunosensor that can detect the biomarker of interest in serum and/or plasma samples.

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Fig 1: Quartz Crystal Microbalance with Dissipation Probe (QCM-D) for real-time monitoring of the adsorption and binding processes of our biomarker.

Fig 2: A representative QCM-D experiment showing real-time shifts in resonance frequency at the flow rate of 20 µl/min. This analysis indicates that the concentration of biomarker in a sample can be detected within ~40 minutes.

Fig 3: The frequency shifts associated with irreversibly bound layer of anti-biomarker antibody and biomarker at different flow rates 10,20,30 µl/min.