Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

Abstract

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100

Microarray methods for protein biomarker detection

“…An excellent and efficient alternative is microarray-based technologies. 14,19,[24][25][26][27][28][29][30] Although microarray methods are wellestablished for high-throughput nucleic acid studies, their application for protein detection has been limited by issues such as the surface immobilization of protein capture probes without loss in bioactivity as well as the availability of highly specific probes suitable for use in complex biological samples where there is a high risk of assay cross-reactivity. Herein, we discuss some of the latest developments aimed at expanding the applicability of microarray biosensors for the detection of biomarkers for disease analysis.…”

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

“…We demonstrated the feasibility of the printer for producing miniaturized, high-density planar recombinant antibody arrays for protein expression profiling. Hence, the early design of this printing device should be added to the short list of currently available instruments capable of generating such miniaturized arrays [25,30,32,33,34,48].…”

“…A set of technologies are available for producing such nanoarrays, e.g. atomic force microscopy (AFM) [25], dip-pen nanolithography (DPN) [26], nanofountain probe [27,28], nanoimprint lithography [16], and various nanodispensers [20,22,29]. Albeit successful, these nanoarray layouts have been found to be associated with a set of key methodological shortcomings.…”

“…Using a DPN-based desktop printer (NLP3000 TM ), we have produced the first 12-and 48-plex planar recombinant antibody arrays, based on 78.5 um 2 (Ø 10 μm)-sized spots at a density of 38 000 spots cm −2 , interfaced with a fluorescent-based readout [33,34]. To the best of our knowledge, only one other complete microprinter system is currently at hand, the Nano eNabler TM , relying on a microcantilever-based surface patterning tool for printing [25,32]. Noteworthy, a novel microspotting tool, Bioplume TM -consisting of an array of micromachined silicon cantilevers with integrated microfluidics channels-was recently introduced [34][35][36][37].…”

“…Recently, it was highlighted that these three technological bottlenecks might be bypassed by aiming for miniaturized antibody arrays based on submicron-sized (Ø 10 μm) rather than nanosized (Ø < 1 μm) spot features [25,[30][31][32][33][34]. Using a DPN-based desktop printer (NLP3000 TM ), we have produced the first 12-and 48-plex planar recombinant antibody arrays, based on 78.5 um 2 (Ø 10 μm)-sized spots at a density of 38 000 spots cm −2 , interfaced with a fluorescent-based readout [33,34].…”