“To Catch or Not to Catch”: Microcapsule‐Based Sandwich Assay for Detection of Proteins and Nucleic Acids

Abstract

The development of new assays that specifically detect intermediate or final products of biotechnological processes or multiple analytes in biomedical research is important for diagnostics and biotechnology. This study presents a microcapsule‐based sandwich assay for detection of proteins and nucleic acids using flow cytometry as an optical readout. The main component of the assay are robust chemically cross‐linked microcapsules that are coated with adaptor proteins such as protein A or streptavidin. In the first approach, the ability of detecting the blood cancer biomarker beta‐2 microglobulin in the fM to pM concentration range with the help of protein A‐coated capsules is demonstrated. In the second approach, streptavidin‐coated capsules are used for detection of nucleic acids in the nM concentration range. The developed assay allows rapid quantitative analyte measurement, while providing high sensitivity and selectivity at very small sample quantities. In the future, protein A‐ and streptavidin‐coated microcapsules can be used as universal tools for detection of a broad range of analytes.

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids

“…We then tested both microcapsules and PS beads in a sandwich immunoassay for the protein analyte, human beta-2 microglobulin (hβ 2 m), which is measured in the plasma to detect cancer and in the urine to detect renal failure [ 26 ]. In this assay, the BBM.1 antibody on microcapsules or PS beads binds hβ 2 m, which is then detected with polyclonal rabbit anti-hβ 2 m antibody, followed by fluorescently labeled anti-rabbit secondary antibody ( Fig 2C ) [ 9 ]. A dose-response study comparing the microcapsules and the PS beads showed clearly that the microcapsules detected hβ 2 m with much higher sensitivity than the PS beads ( Fig 2D ).…”

“…Core-shell particles were prepared, crosslinked, and surface-modified with protein A as described in section 2.2 and 2.3 [ 9 ]. High-binding 96-well black well plates were coated with 100 μg mL -1 protein A in carbonate buffer (pH 9.6) overnight at 4°C and then blocked with 1% (w/v) BSA overnight.…”

“…In such particle-based assays, the use of flow cytometry has increased the sensitivity and largely removed the need for washing steps [ 8 ]. While most particle-based assays currently use solid beads made of polystyrene (PS) or other plastics, we have recently introduced the use of hollow polyelectrolyte microcapsules [ 9 ]. Their porous surface can be modified with large amounts of antibodies [ 10 – 12 ], and their great physico-chemical stability aids the development of assays that are fast and robust in a broad range of experimental conditions.…”

“…Their porous surface can be modified with large amounts of antibodies [ 10 – 12 ], and their great physico-chemical stability aids the development of assays that are fast and robust in a broad range of experimental conditions. One can also attach nucleic acids to the microcapsule surface for the sequence-specific detection of nucleic acids [ 9 ].…”

“…This study describes the validation and application of a highly sensitive and selective microcapsule-based immunoassay for the detection of proteins and nucleic acids [ 9 ]. We compare polyelectrolyte microcapsules with commercially available polystyrene (PS) beads in the detection of a protein (the disease marker human beta-2 microglobulin, hβ 2 m) and a nucleic acid.…”