Electrochemical biosensor chip with structurally defined, electrochemically isolated detection modules for the multiparametric analysis of target molecules in biological fluids

Abstract

An electrochemical biosensor chip for the simultaneous quantitative detection of a plurality of different target molecules in a biological fluid, wherein the biosensor chip comprises: a) a substrate with a microfluidic structure designed to guide the biological fluid, b) a plurality of electrochemically isolated detection modules arranged along the microfluidic structure, c) wherein each detection module is structurally assigned to a specific target molecule and differs from the other detection modules by at least one irreversible structural feature selected from: -Electrode geometry, -Electrode surface, -Surface chemistry, - or a combination thereof, d) wherein each detection module comprises a working electrode, a counter electrode and a reference electrode, e) wherein each detection module is linked to a fixed electrochemical redox system which defines a module-specific electrochemical operating window, f) wherein the electrochemical operating windows of the detection modules do not overlap, (g) and wherein the biosensor chip comprises at least one non-functionalized internal reference electrode configured for hardware-based signal normalization, independent of the chemical identity of the target molecules.

Description

Summary

[0001] The invention relates to an electrochemical biosensor chip for the simultaneous, quantitative detection of multiple endothelial and angiogenesis-related biomarkers in human plasma. The chip has a microfluidic structure with several electrochemically isolated detection modules, each structurally uniquely assigned to a specific biomarker. Each detection module comprises a specially designed gold electrode with biomarker-specific geometry, defined surface chemistry, and a fixed electrochemical redox system. An integrated, non-functionalized reference electrode serves for hardware-based signal normalization. The biosensor is designed as a disposable chip and can be operated with an external, portable potentiostat. The system is particularly suitable for the highly sensitive, multiplex point-of-care analysis of vascular biomarkers.The biomarkers mentioned represent preferred embodiments; however, the invention is not limited to specific biomarkers, but is fundamentally designed for the detection of any target molecules in biological fluids. Technical field

[0002] The invention relates to the field of electrochemical biosensors, in particular microfluidic, multiplex-capable biosensor chips for the quantitative analysis of protein-based biomarkers in biological fluids. The invention is not limited to specific biomarkers, but generally relates to electrochemical biosensor chips for the structurally predetermined, multiplex detection of different target molecules in biological fluids. Background of the invention

[0003] Known electrochemical biosensors enable the detection of individual biomarkers or utilize non-specific electrode arrays in which different detection elements are immobilized on structurally identical electrodes. Such systems suffer from electrochemical crosstalk, signal drift, poor reproducibility, and limited comparability of parallel measurements.

[0004] In particular, for the simultaneous detection of endothelial and angiogenesis-related biomarkers such as NOSTRIN, eNOS, VEGF-A, sVEGFR1 and ET-1, there is a need for a platform that enables structurally defined, reproducible and low-interference multiplex analysis. Technical problem

[0005] The invention is based on the objective of providing an electrochemical biosensor chip that: • allows simultaneous quantitative detection of multiple biomarkers, • avoids electrochemical crosstalk, • ensures high signal stability and comparability, • and is suitable for portable, decentralized applications. Solution to the task

[0006] The task is solved by a biosensor chip that has several structurally predetermined, electrochemically isolated detection modules, each detection module: • is firmly assigned to a specific biomarker class, • possesses a biomarker-specific electrode geometry, • has a defined surface chemistry with a fixed linker length, • and is operated within its own electrochemical operating window. Designs

[0007] In a preferred embodiment, the electrochemical biosensor chip comprises several electrochemically isolated detection modules arranged along a microfluidic structure. Each detection module is structurally uniquely assigned to a specific biomarker and includes a working electrode with biomarker-specific geometry, a reference electrode, and a counter electrode.

[0008] In another embodiment, the detection modules also differ in the respective surface chemistry of the working electrodes, in particular in differently designed self-organized monolayers with defined linker length.

[0009] In another embodiment, the biosensor chip is designed as a disposable module and is intended for connection with an external, portable electrochemical evaluation unit. Supplementary disclosure regarding technical effect, structural design and electrochemical layout

[0010] "The numerical values ​​mentioned below represent preferred, exemplary embodiments and are not to be understood as mandatory limits. The numerical values ​​mentioned serve for technical illustration and do not exclude other, functionally equivalent embodiments." Technical effect and quantitative delimitation

[0011] The structural pre-assignment of the detection modules according to the invention leads to a measurable reduction of electrochemical crosstalk as well as to increased signal stability and comparability of parallel measurements.

[0012] In preferred embodiments, the electrochemical operating windows of the individual detection modules are separated from each other such that the respective redox potentials differ by at least 100 mV, preferably at least 150 mV, and particularly preferably at least 200 mV. This prevents the superposition of faradaic currents from adjacent detection modules.

[0013] Measurements with simultaneously activated detection modules show that the electrochemical crosstalk between adjacent modules is less than 5%, preferably less than 2%, relative to the respective biomarker-specific signal maximum. At the same time, signal reproducibility with a coefficient of variation (CV) of less than 10%, preferably less than 5%, is achieved.

[0014] These effects are not solely due to a spatial separation of the electrodes, but result from the combined and fixed assignment of electrode geometry, surface chemistry and electrochemical working window to a detection module. Structural predetermination of the detection modules

[0015] A “structurally unambiguous assignment” within the meaning of the invention is understood to mean that each detection module is irreversibly distinguishable from the other modules at the manufacturing level by at least one of the following features: • a biomarker-specific defined electrode geometry, selected from different surfaces, outline shapes, edge lengths or aspect ratios, • a fixed positioning within the microfluidic structure, • an irreversible surface functionalization with biomarker-specific self-organized monolayer, • or a combination of these characteristics.

[0016] The structural assignment is therefore independent of software-based addressing or subsequent user assignment and is an integral part of the chip.

[0017] In preferred embodiments, the working electrodes of the detection modules differ in their effective electrode area by at least 20%, preferably at least 40%, resulting in different electrochemical characteristics that provide an additional functional separation of the modules. Electrochemical redox systems and operating windows

[0018] Each detection module is assigned a fixed electrochemical redox system whose formal redox potential lies within a biomarker-specific operating window. Suitable redox systems include, but are not limited to: • Ferrocene and ferrocene derivatives, • Quinone / hydroquinone systems, • Methylene blue or toluidine blue derivatives, • Ruthenium or osmium complexes.

[0019] In preferred embodiments, the redox systems are selected such that their respective redox potentials lie within a voltage range of -0.4 V to +0.6 V (against Ag / AgCl), with the operating windows of the individual detection modules not overlapping.

[0020] The fixed assignment of the redox system to a detection module ensures that each module is read exclusively within its defined electrochemical window, thus enabling simultaneous, interference-free multiplex measurement. Surface chemistry and linker lengths

[0021] The self-assembled monolayers of the working electrodes exhibit biomarker-specific defined linker lengths, preferably in the range of 0.5 nm to 5 nm. Different linker lengths allow for targeted manipulation of the electron transfer rate and the accessibility of the redox system, contributing to further electrochemical decoupling of the detection modules.

[0022] The combination of linker length, electrode geometry and redox system is fixed for each detection module and is not interchangeable. Internal reference electrode and hardware-based normalization

[0023] The biosensor chip has at least one non-functionalized internal reference electrode that is electrically connected to all detection modules and serves as a hardware-based reference for signal normalization.

[0024] In preferred embodiments, the reference electrode is made of gold, platinum, or carbon and is spatially arranged within the microfluidic structure such that it is exposed to the same electrochemical environmental conditions as the detection modules. Signal normalization is performed at the hardware level of the connected potentiostat before or during data acquisition and is independent of software-based post-processing. This reduces drift, matrix effects, and potentiostat-induced variations. Production and irreversibility

[0025] The detection modules are preferably manufactured on a common substrate using lithographic or microstructuring methods.

[0026] The geometric design, surface functionalization and redox system assignment take place during manufacturing and are not reversible after completion of production.

[0027] This ensures that the structural and electrochemical pre-assignment of the detection modules cannot be changed by the user. Summary of technical delimitation

[0028] The combination according to the invention consists of: • structurally predetermined electrode geometries, • biomarker-specific surface chemistry with defined linker length, • fixed electrochemical redox systems with separate operating windows, • as well as hardware-based internal reference normalization represents a complete technical solution that goes beyond a mere aggregation of known features and enables reproducible, low-noise and highly sensitive multiplex analysis. Advantages

[0029] The biosensor chip according to the invention enables a parallel electrochemical analysis of several biomarkers within an integrated microfluidic structure.

[0030] The structural pre-assignment of the detection modules ensures high signal stability and comparability.

[0031] The modular design allows for a compact form and easy integration into portable analysis systems. Industrial applicability

[0032] The electrochemical biosensor chip can be manufactured industrially and is suitable for use in analytical measurement systems for the quantitative determination of protein-based biomarkers in biological samples.

[0033] It is particularly suitable for portable analysis systems as well as for applications in medical research and development. Basic technical concept

[0034] The basic technical concept of the invention is based on the structural separation and electrochemical decoupling of several detection modules on a common support substrate.

[0035] By assigning electrode geometry, surface chemistry and electrochemical operating window to a specific detection module, a reproducible and low-interference multiplex analysis is enabled. System architecture

[0036] The biosensor chip is designed as a standalone sensor module and is intended for electrical connection with an external evaluation unit.

[0037] The evaluation unit can be designed as a portable potentiostat and powered by a low-voltage DC power source.

[0038] The biosensor chip is preferably designed as a disposable module, while the evaluation unit is reusable. Reference of the illustrations to the claims:

[0039] The elements shown in the figures correspond to the components of the biosensor chip described in claims 1-6. Reference numbers 1 to 10 show the working electrode (WE), counter electrode (CE), reference electrode (RE), microfluidic channel, self-assembled monolayer (SAM), and the five biomarker-specific detection modules with their associated electrochemical operating windows. The figures serve solely for illustration and are not intended to limit the claims or alter the scope of protection of the invention. Legend of the drawings: 1 working electrode (WE) 2 Counter electrode (CE) 3 Reference electrode (RE) 4 Microfluidic channel / Plasma flow 5 Self-organizing monolayer (SAM) 6 NOSTRIN detection module / redox window 7 eNOS detection module / redox window 8 VEGF-A detection module / redox window 9 sVEGFR1 detection module / redox window 10 ET-1 detection module / redox window Disclaimer

[0040] The illustrations are for illustrative purposes only and are not intended to limit the scope of the invention or to alter the invention set forth in the description and claims.

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