The detection and quantification of protein biomarkers in biological samples lies central to proteomics, drug design, disease prognosis and therapeutic development. The generation of viable protein microarrays is, though, challenging. Current antibody based optical microarrays are commonly based on sandwich assays in which antigen binding to the immobilised antibody is detected through the use of a secondary, labeled, antibody. Though sensitive, this method is laborious and requires a specifically-labeled secondary antibody for every antigen of interest. Labeling protocols are potentially perturbative, can also be time consuming and may lead to high background signals. Label free detection assays based on plasmon resonance or quartz crystal microbalance (QCM), for example, offer, typically, nM detection limits but require the use of sophisticated and very expensive pieces of equipment.
Other techniques using electrochemical principles have also been developed for assays. Assays are generated by controllably immobilizing receptive biomolecules (typically antibodies, nucleic acids or peptides) on electrodes and converting the target protein binding event into a measurable electrical signal. One of the most sensitive and powerful means of doing this is by impedance; that is through the application of a sinusoidal voltage to the supporting electrode and the measurement of the resulting current. Though it can be exceedingly sensitive, the measurement of impedance is that of only one interfacial electrical parameter and furthermore, is commonly interpreted only through the application of an “assumed equivalent circuit”. There are a variety of assumed equivalent circuits, and they are used to approximate the electrochemical interface (e.g. at the surface of a working electrode). Generally, the most appropriate assumed equivalent circuit must be chosen or devised to correctly interpret the data. For this to be successful, it requires a considerable degree of knowledge of the electrochemistry at the relevant electrochemical interface.
The present invention aims to provide an alternative to, ideally an improvement upon, the methods of the prior art. For example, it would be desirable to be able to calculate the concentration of biomolecules in solution at high accuracy using impedance data, but without having to interpret the data using an assumed equivalent circuit.