Electrochemical Profiling of vWFA2 for Systemic Inflammatory State Detection

Introduction: The systemic inflammatory response (SIRS) is broadly defined as a set of non-specific symptoms presenting post-infection and/or inflammatory state. The overlap between conditions is complicated by the vagueness of the presenting symptoms, thus leading to inconclusive diagnostics and treatment delay. Von Willebrand Factor, domain 2A (vWF), a large multimeric glycoprotein circulating within blood as a series of heterogenous multimers, plays a critical role in physiologic hemostasis such as platelet adhesion to thrombogenic endothelial surfaces, platelet-to-platelet cohesion for thrombus plug formation, and factor VIII chaperoning, a cofactor for coagulation cascade acceleration. Altered functionality of vWFA2 leads to severe hemorrhagic consequences following defective formation of a platelet-rich thrombi and fibrin network. Currently, there is little research on the role of vWFA2 in inflammatory states, as the marker is traditionally studied in hemostatic disorders, thus its role in immuno-thrombosis is of peak interest. Since there is interest in exploring the tie between inflammation and coagulation, a sensing platform targeting vWFA2, a marker released in both systemic states, would aid clinicians in patient stratification. There are no point-of-care prognostic devices for vWFA2 detection in inflammatory states with very little research in electrochemical detection of this marker in broader terms. The first prognostic device that would be used for disease classification and symptom management should be highly sensitive and include a wide dynamic range for their target marker. Assay development of vWFA2 using electrochemical impedance spectroscopy techniques can provide clinicians with a tool to rapidly introduce treatment management for SIRS patients.

Materials and

Methods: The specific aim of this research is to develop a novel point-of-care (POC) diagnostic device for the rapid biomarker detection to aid in clinical management. The device, encompassed with a single-use disposable cartridge attached to a hand-held electronic reader, is functionalized to be highly sensitive for vWFA2 detection, as the electrode surface is prepared using specific capture probes, fabricated on an electrode sensor array of nanofilm semiconducting material to metal electrode interface. The response is measured using non-faradaic electrochemical impedance spectroscopy (EIS) techniques for vWFA2 detection in plasma samples. The EIS technique uses a potentiostat setup, which applies a controlled electrical signal to the system, and a frequency response analyzer, which measures the system’s response to the applied signal across a wide range of frequencies. The electrochemical performance of the biosensor was analyzed by measuring the resulting impedance response over a range of concentrations.
Specific recombinant human vWFA2 antibody and protein were purchased from R&D systems, USA. DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) and PBS (Phosphate Buffer Saline) were obtained from Thermo Fisher Scientific, USA. Healthy human lithium heparin (LiH) pooled plasma was obtained from Innovative Research, Inc, USA. All proteins, antibodies, reagents, and pooled human plasma samples were stored appropriately according to their respective storage instructions until use. All concentrations were individually aliquoted into smaller sample volumes to ensure the freeze-thaw cycles were limited to a maximum of 3 cycles. The antibody was diluted in PBS and the antigen concentrations were added to LiH pooled plasma analyze sensor property studies.

Results, Conclusions, and Discussions: *Plan to include Nyquist and Bode data once collected, CDR/SR doses tentative to change.

Sensor functionality was established in the range of 750 to 24,000 pg/mL, which encompasses both the physiological and pathological ranges of circulating vWFA2. An increase in the percent change in relation to the baseline zero dose was observed with the incremental increasing concentrations, with a r2 = 0.96. The S&R study showed a r2 = 0.99 by spiking a known concentration and evaluating percentage response to measure unknown concentrations. This robust response provides evidence for detection methods for vWFA2.

Cross-reactivity analyzes the response of an antibody to specific and non-specific molecules. The sensitive molecule, vWFA2, was cross-reacted with interferon gamma-induced protein 10 (IP-10), with bovine serum albumin. The response shows that the IP-10, at low and high concentrations, show a low response below the 20% threshold, and the specific molecule (vWFA2) shows a high response above the 80% threshold, indicating specificity of the functionalized sensor to the vWFA2 molecule in the presence of a non-specific molecule.

By functionalizing the Au-ZnO biosensor surface, detection of the vWFA2 antigen was successful. Precision, accuracy, and specificity were evaluated, confirming the biosensor’s efficacy in plasma. The findings confirm the importance of monitoring vWFA2 levels, a protein involved in the crosstalk between inflammation and hemostasis, to provide a comprehensive inflammatory status of patients. The POC biosensing platform aims to revolutionize detection mechanisms in inflammatory states. The novelty and importance of this assay lies in the ability to assess and confirm the importance of the role of vWFA2, a rather understudied inflammatory biomarker in terms of inflammatory states.

Acknowledgements (Optional): I would like to thank my supervising professor and mentor, Dr. Shalini Prasad, for her enthusiastic mentorship and guidance over this past year. Her teachings and support have ignited the passion within me to change the world of research through bioengineering. Her emphasis on always striving to learn more by never accepting complacency has shaped my vision as a researcher, and I am eternally thankful to her for her continued supervision.
I would also like to thank Dr. Sriram Muthukumar and Dr. Sasya Madhurantakam for their direction and mentorship over my research during the past year. They have patiently taught and trained me and continue to encourage my growth as an independent researcher. To my colleagues and undergraduate students, thank you for your continued support and guidance through this journey.