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


RESULTS
We 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.


CONCLUSIONS
The 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.
Evans
David
D
School of Electronic and Electrical Engineering, University of Leeds, Leeds LS2 9JT, UK. een1dae@leeds.ac.uk
Johnson
Steven
S
Laurenson
Sophie
S
Davies
A Giles
AG
Ko Ferrigno
Paul
P
Wälti
Christoph
C
D052158:Aptamers, Peptide / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D006046:Gold; D046228:Microarray Analysis / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics*; D013057:Spectrum Analysis
D052158:Aptamers, Peptide; D020033:Protein Isoforms; D006046:Gold