ABSTRACT
Miniaturized (Ø 10 µm), multiplexed (>5-plex), and high-density (>100 000 spots cm(-2)) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, Bioplume(TM)-consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels-to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced-based on miniaturized spot features (78.5 um(2), Ø 10 µm) at a 7-125-times increased spot density (250 000 spots cm(-2)), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.
Subject(s)
Antibodies/chemistry , Blood Proteins/analysis , Microchemistry/methods , Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Antibodies/immunology , Blood Proteins/immunology , Humans , Microchemistry/instrumentation , Microfluidics/instrumentation , Miniaturization , Pilot Projects , Recombinant Proteins/chemistry , Recombinant Proteins/immunologySubject(s)
Blood Cells/cytology , Blood Cells/metabolism , Blood Proteins/analysis , Cell Separation/instrumentation , Flow Cytometry/instrumentation , Microfluidic Analytical Techniques/instrumentation , Protein Array Analysis/instrumentation , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Humans , Nanotechnology/instrumentation , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A microspotting tool, consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels is introduced. This spotter, called Bioplume, is able to address on active surfaces and in a time-contact controlled manner picoliter of liquid solutions, leading to arrays of 5 to 20-microm diameter spots. In this paper, this device is used for the successive addressing of liquid solutions at the same location. Prior to exploit this principle in a biological context, it is demonstrated that: (1) a simple wash in water of the microcantilevers is enough to reduce by >96% the cross-contamination between the successive spotted solutions, and (2) the spatial resolution of the Bioplume spotter is high enough to deposit biomolecules at the same location. The methodology is validated through the immobilization of a 35mer oligonucleotide probe on an activated glass slide, showing specific hybridization only with the complementary strand spotted on top of the probe using the same microcantilevers. Similarly, this methodology is also used for the interaction of a protein with its antibody. Finally, a specifically developed external microfluidics cartridge is utilized to allow parallel deposition of three different biomolecules in a single run.