Microfluidic impedimetric biosensor for blood congulation

In clinical coagulation diagnostics, there is a great need for bed-side testing devices to determine the coagulation status. Discontinuous or inaccurate measurements can lead to bleeding or life- threatening thrombosis. The conventional coagulation tests are either associated with long turnaround times or require sample preparation, costly equipment, and skilled staff. So, they are mainly not appropriate for on-site use. Furthermore, in many cases, clinical findings, based on a single biomarker, are not enough for an accurate diagnosis. Thus, it is highly desirable to measure various biomarkers simultaneously. This project aims to implement a microfluidic impedimetric sensor using aptamers for simultaneous and label-free on-site detection of blood coagulation status. Aptamers can recognize their target molecules due to their 3D structure with a high specificity and affinity. Impedimetric biosensors enable label-free detection of biomolecular interactions. Their low system complexity and high miniaturization capability favor them for the development of portable systems. However, current impedimetric sensors suffer mainly from the influence of the ion strength of the sample solution. With our nanogap IDA sensor the influence of the sample solution can be minimized. The successful proof-of-concept of such a microfluidic impedimetric biosensor using aptamers would be a game- changer in the blood coagulation testing and beyond as it could also be applied in many other application fields and pave the way for the real-time and online monitoring of various biomarkers in future.

The aim of this project was the development of a microfluidic impedance biosensor system for label-free, point-of-care diagnostics (POCT), which allows various blood coagulation markers to be determined in the blood. This should enable a more precise determination of blood coagulation during or after treatment (e.g. cardiac surgery or dialysis) in order to prevent life-threatening thromboses. The microfluidic biosensor chip developed is intended to replace the current lengthy and time-consuming analysis. Sample preparation (preparation of whole blood into blood and plasma) is completely replaced by our microfluidic chip. Blood plasma is the key information source of our organism and provides important information about the hemostatic balance of the body and contains a vast amount of essential biomarkers such as proteins, enzymes, small molecules and nucleic acids. Ultra-pure plasma is necessary for reproducible measurement of the biomarkers, as otherwise the blood cells would interfere with the highly sensitive electrochemical measurements. Our microfluidic chip successfully extracts ultra-pure plasma continuously without filters or centrifugation by exploiting two hydrodynamic effects-the Fahraeus-Lindqvist effect and the double Fung effect-in a passive separation approach. The chip's microfluidic geometry was optimized through COMSOL Multiphysics simulations and fabricated using microfabrication techniques and conventional 3D printing. The resulting chips achieve excellent plasma separation efficiency (99.9%) across a wide flow rate range (0.05-0.2 ml/min), with hemolysis levels outperforming those from centrifugation. Subsequently, the PDMS-based plasma separation chip was integrated with silicon-based electrode structures for biomarker detection, located immediately downstream of the separation zone. For specific detection of thrombin, fibrinogen, plasmin, and beta-thromboglobulin, individual aptamers were selected via the SELEX method (systematic evolution of ligands by exponential enrichment). Binding assays using quantitative RT-qPCR identified the highest-affinity aptamers, with increased selection pressure favoring those with strong and specific protein binding. Next-generation sequencing (NGS) confirmed that all selected aptamers bind specifically to their targets, while negative controls (bovine and human serum albumin) showed no binding. Currently, we are developing an innovative assay for reliable detection of blood coagulation markers using these aptamers. In parallel, we are testing various interdigital electrode designs, including nano- and micro-gap structures, to further enhance system sensitivity and specificity. The goal is to establish a robust, versatile platform meeting the highest technological and analytical standards.