Abstract
Introduction
The progress in multi-marker biosensors is expected to induce changes in medicine beyond the mere innovation of diagnostic screening tests. In the near future, decentralized point-of-care testing and therapy is expected to foster the birth of personalized medicine, revolutionizing the classical hospital-centered analysis laboratory. Low-cost, automatic, fool-proof diagnostic systems and parallelized analyses will represent key technologies for the implementation of such epochal change. Most multi-marker assays are nowadays performed with surface-bound strategies. These derive directly from the parallelization of classical techniques that involve complex multi-step procedures. In a more modern, but still largely experimental approach, most of the assay specificity relies on the specific binding of the analyte to its probing molecule that has been covalently immobilized on a designed surface. The detection of such binding can then be measured with label-free techniques that are sensitive to a change of a physical property related to the binding of the analyte material. This can be a change in dielectric constant, mass, or ion conductivity. In order for such a strategy to be successful, it is at least required that i) the probe molecules are highly specific, exposed efficiently to the surface and stably bound, ii) the solid-liquid interface is stable over time and is not subject to non-specific interactions with molecules in the analyte, iii) the measuring technique is sensitive enough. As an additional set of desired features, the entire process should be amenable to parallelization, easy to run, inexpensive, and possibly provide real-time data.
Results
The instrumentation we developed responds to variations of electrical properties as a consequence of the affinity binding of biological material on an derivatized electrode. The biosensing principle relies on a custom application of 2-electrode chronoamperometry. Such instrumentation can work in parallel on a large number of measuring microelec-trodes embedded in a microfluidic system. Time-drift is a common instability problem of electrochemical measurements for biosensing, as the measurement can modify the interface. Our instrumentation applies such a low excitation signal that it does not perturb the sensitive interface, hence minimizing time-drift to a value negligible in the timeframe of the analysis (about 0.1 %/min). A simple variation of the standard electronics used for chronoamperometry makes the measurement independent of the specific composition of the measuring buffer, and makes it possible to perform tests in moderately complex buffers. Along with the low time-drift, this is an essential feature for real-time analysis, which can be performed with this setup.
The biosensor was employed to study the dynamic assembly and substitution of self-assembled monolayers on a clean gold surface, and it proved sensitive enough to follow such processes in real time. Likewise, the dynamics of the non-specific adsorption of proteins on gold electrodes has been observed. Such experiments can be performed with many techniques, but our simple, parallel and compact electronic approach is a valuable practical improvement.
For the purpose of biological assays, the microelectrodes have been functionalized with a monolayer based on mixed oli-goethyleneglycol alkanethiols, used to immobilize and expose biological probe molecules to the analyte. Such monolayer reduces unwanted non-specific adsorption. The advances presented herein, in particular the high stability, parallelism and the possibility to measure in moderately complex buffers, could open new perspectives in the development of electrochemical techniques for diagnostic multi-marker applications.