AVS2009 Session BI+AS+BM+MS-WeM: Array-Based Sensors and Diagnostics: Grand Challenges

Antibody-based microarray is a new proteomic methodology setting a novel standard for analysing complex, non-fractionated proteomes. The first generation of antibody micro- and nanoarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein maps, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody array set-ups have, however, proven to be challenging. In this presentation, the key technological challenges that must be resolved in a cross-disciplinary manner before true global proteome analysis can be performed using antibody array-based sensors will be presented and discussed.

In this context, we have successfully designed a set of state-of-the-art recombinant antibody array technology platforms for high-throughput proteomics. In more detail, we use human recombinant single-chain Fv (scFv) antibody fragments, microarray adapted by molecular design as probes, displaying an outstanding on-chip functionality and stability. Uniquely, the platforms allows us to target both water-soluble as well as membrane proteins in a highly multiplexed and sensitive (pM to fM range) manner in complete, i.e. non-fractionated, directly labeled complex proteomes. Platforms compatible with a wide range of proteomes, including serum, plasma, urine, cell lysates, tissue extracts, intact cells etc, have been successfully designed. In addition, the first steps towards implementing label-free sensing ( MS, MS -MS and SPRi) as well as designing self-addressable microarrays and miniaturized attovial-based nanoarrays as well as planar nanoarrays have been taken, clearly expanding the repertoire of technology platforms. The applicability of the platform(s) for differential high-content screening of clinical samples has been validated in a set of key applications within the field of oncoproteomics, autoimmunity, inflammatory diseases and allergy. The optimized antibody microarray technology platforms, as well as data from the screening analysis will be presented in context of the grand challenges the field experiences.

The Quansys Q-Plex (multiplex ELISA) Array is a fully quantitative ELISA-based test where up to 25 distinct capture antibodies have been absorbed to each well of a 96-well plate in a defined array. This array is composed of 20 nanoliter spots with 350µm diameters and a pitch of 650µm between spots. Each spot represents a different distinct capture antibody population.

This talk will highlight recent work from my laboratory that addresses new interfacial technologies to suppress noise (N) and amplify signal (S) leading to heterogeneous assays with extraordinarily high S/N. In the first demonstration, I will focus on the adventitious adsorption of proteins as the primary factor that controls the limit-of-detection (LOD) of protein microarrays and limits the measurement of analytes from complex mixtures such as serum or blood. I will show data on a new protein microarray assay where background adsorption is effectively eliminated through the use of a protein-resistant –nonfouling– polymer brush. These “zero background” protein microarrays were successfully used to quantify protein analytes in serum with femtomolar LOD and a dynamic range of six orders of magnitude of analyte concentration. These LODs are 100-fold lower when compared to the same protein microarrays spotted on a conventional polymer substrate that displays high binding capacity but significant adventitious protein adsorption. This study also provided the first demonstration of the interrogation of an analyte directly from undiluted, whole blood by a protein microarray with a LOD of ~15 fM. Next, I will summarize recent work in my laboratory on the development of a new isothermal fluorescence signal amplification and detection scheme that exploits the ability of terminal deoxynucleotidyl transferase (TdTase) to add up to 100 fluorescent nucleotides to the end of a short DNA tag with an exposed 3’-OH. I will show how DNA microarrays that are printed on the nonfouling polymer brush exhibit low background signal, yet allow on-chip fluorescence signal amplification, leading to DNA microarrays that exhibit a sub-picomolar LOD, which appears to be the lowest LOD reported for DNA microarrays, to date.

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation-switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody-fragments, which can be detected in quantities of less than 1 amol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response as well as by time-resolved fluorescence measurements. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.