Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays

Abstract

BackgroundThe dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection.ResultsWe present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background.ConclusionThe use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

Label-free affinity biosensor arrays: novel technology for molecular diagnostics

“…For example, electrochemical cleavage of the gold-thiol bond has been used to direct the immobilization of different proteins onto 20-µm diameter metallic electrodes separated by 15 µm [11]. The electrode microarray was subsequently used to demonstrate label-free, electronic detection of cyclin-dependent protein kinases in whole-cell lysates, with no measurable cross talk between functionalized electrodes.…”

Trends and Perspectives in Immunosensors

“…Additionally, electrode geometry has proven important with interdigitated electrode arrays providing improved sensitivity [39,48]. Impedance-based sensors have been used to monitor protein-carbohydrate [49] and proteinprotein [50,51] interactions. For example, Yu et al [52] reported multiplexed protein monitoring with an array of gold electrodes.…”

Target Profiling of Small Molecules

“…Additionally, the method was applied to probing protein-protein interactions in an array format by Evans and co-workers [32]. In this study, a selective chemical masking process was used specifically to derivatize 20-μm-wide gold electrodes.…”