Influence of Pluronic F127 on the distribution and functionality of inkjet-printed biomolecules in porous nitrocellulose substrates

Autor: Liyakat Hamid Mujawar, Aart van Amerongen, Willem Norde
Přispěvatelé: Polymers at Surfaces and Interfaces
Jazyk: angličtina
Rok vydání: 2015
Předmět:
Mycoplasma bovis
Immunoconjugates
Laboratorium voor Fysische chemie en Kolloïdkunde
protein microarrays
FLOW
hydrophobicities
Analytical Chemistry
chemistry.chemical_compound
block-copolymer
Nitrocellulose
antibody
BLOCK-COPOLYMER
membrane
Physical Chemistry and Colloid Science
Microscopy
Confocal

biology
Collodion
Pluronic
Fluorescence
Primary and secondary antibodies
Antibodies
Bacterial

PROTEIN MICROARRAYS
flow
Protein microarray
Printing
additives
Biological Assay
Porosity
Streptavidin
DNA
Bacterial

ADDITIVES
Fluorophore
Surface Properties
SPOT MORPHOLOGY
Protein Array Analysis
Poloxamer
surfaces
Buffers
Corynebacterium
orientation
Immunoglobulin Fab Fragments
Surface-Active Agents
Orientation
Fluorescent Dyes
Chromatography
SURFACES
Substrate (chemistry)
HYDROPHOBICITIES
BBP Bioconversion
Inkjet printing
chemistry
ANTIBODY
spot morphology
biology.protein
MEMBRANE
Conjugate
Zdroj: Talanta, 131, 541-547. ELSEVIER SCIENCE BV
Talanta, 131, 541-547
Talanta 131 (2015)
ISSN: 0039-9140
Popis: The distribution of inkjet-printed biomolecules in porous nitrocellulose substrates often results in a non-homogeneous spot morphology commonly referred to as 'doughnut-shaped' spots. We have studied the influence of Pluronic F127 (an amphiphilic surfactant) on the functionality of inkjet-printed primary antibody molecules and on the final assay result by performing a one-step antibody binding assay in the nitrocellulose substrate. The primary antibody was printed with and without Pluronic, followed by the addition of double-labelled amplicons as antigen molecules and a fluorophore-labelled streptavidin as detection conjugate. The distribution of the fluorescence intensity down into the nitrocellulose substrate was investigated by confocal laser scanning microscopy in 'Z' stacking mode. Each horizontal slice was further analysed by applying a concentric ring format and the fluorescence intensity in each slice was represented in a colour-coded way. The mean and total fluorescence intensity of the antibody binding assay (fluorescent streptavidin) showed a peak at 0.2% (w/v) Pluronic F127. In addition, an improved spot morphology was observed also peaking at the same Pluronic concentration. Subsequently, we investigated the direct influence of Pluronic F127 on the location of the primary antibody molecules by labelling these molecules with the fluorophore Alexa-488. Our results show that upon increasing the concentration of Pluronic F127 in the printing buffer, the spot diameter increased and the number of primary antibody molecules bound in the spot area gradually decreased. This was confirmed by analysing the distribution of fluorescently labelled primary antibody molecules down into the membrane layers.We conclude that a particular ratio between primary antibody and Pluronic F127 molecules in combination with available substrate binding capacity results in an optimal orientation, that is Fab-UP, of the primary antibody molecules. Consequently, an increased number of antigen molecules (in our case the labelled amplicons) and of the fluorescent detection conjugate (streptavidin) will give an optimal signal. Moreover, distribution of the primary antibody molecules was more homogeneous at the optimal Pluronic F127 concentration, contributing to the better spot morphology observed. (C) 2014 Elsevier B.V. All rights reserved.
Databáze: OpenAIRE