title: Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis.
abstract: Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.
authors.affiliation: Proxomics, Institute of Chemical Biology, Imperial College London, London, SW7 2AZ, UK. ali.salehi-reyhani@imperial.ac.uk
authors.forename: Ali
authors.initials: A
authors.lastname: Salehi-Reyhani
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Sharma
authors.forename: Edward
authors.initials: E
authors.lastname: Burgin
authors.forename: Michael
authors.initials: M
authors.lastname: Barclay
authors.forename: Anthony
authors.initials: A
authors.lastname: Cass
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.forename: Oscar
authors.initials: O
authors.lastname: Ces
authors.forename: Keith R
authors.initials: KR
authors.lastname: Willison
authors.forename: David R
authors.initials: DR
authors.lastname: Klug
authors.forename: Aidan
authors.initials: A
authors.lastname: Brown
authors.forename: Miroslava
authors.initials: M
authors.lastname: Novakova
mesh_terms: D007118:Immunoassay; D046210:Microfluidic Analytical Techniques; D059010:Single-Cell Analysis; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*
chemical_list: D016159:Tumor Suppressor Protein p53