Hydrophilic Polyelectrolyte Multilayers Improve the ELISA System: Antibody Enrichment and Blocking Free

In this study, polyelectrolyte multilayers were fabricated on a polystyrene (PS) plate using a Layer-by-Layer (LbL) self-assembly technique. The resulting functional platform showed improved performance compared with conventional enzyme-linked immunosorbent assay (ELISA) systems. Poly(diallyldimethylammonium chloride) (PDDA) and poly(acrylic acid) (PAA) were used as cationic and anionic polyelectrolytes. On the negatively-charged (PDDA/PAA)3 polyelectrolyte multilayers the hydrophilic PAA surface could efficiently decrease the magnitude of the noise signal, by inhibiting nonspecific adsorption even without blocking reagent adsorption. Moreover, the (PDDA/PAA)3 substrate covalently immobilized the primary antibody, greatly increasing the amount of primary antibody adsorption and enhancing the specific detection signal compared with a conventional PS plate. The calibration curve of the (PDDA/PAA)3 substrate showed a wide linear range, for concentrations from 0.033 to 33 nM, a large specific signal change, and a detection limit of 33 pM, even though the conventional blocking reagent adsorption step was omitted. The (PDDA/PAA)3 substrate provided a high-performance ELISA system with a simple fabrication process and high sensitivity; the system presented here shows potential for a variety of immunosensor applications.

[1]  Mitsuru Akashi,et al.  Polyelectrolyte multilayers-modified membrane filter for rapid immunoassay: protein condensation by centrifugal permeation , 2011 .

[2]  M. Akashi,et al.  Heterofunctional Interfaces Achieve Dual Protein Adsorption on Polyelectrolyte Multilayers , 2009 .

[3]  G. Sauerbrey.  Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung , 1959 .

[4]  M. Akashi,et al.  Layer-by-Layer Assembly on Hydrogel Surfaces and Control of Human Whole Blood Coagulation , 2003 .

[5]  Qiang He,et al.  Leucocyte Membrane-Coated Janus Microcapsules for Enhanced Photothermal Cancer Treatment. , 2016, Langmuir : the ACS journal of surfaces and colloids.

[6]  P. Schaaf,et al.  Polyelectrolyte multilayer coatings that resist protein adsorption at rest and under stretching , 2008 .

[7]  P. Pal,et al.  Interaction of ovalbumin with phospholipids Langmuir-Blodgett film. , 2007, The journal of physical chemistry. B.

[8]  Gero Decher,et al.  Buildup of ultrathin multilayer films by a self‐assembly process, 1 consecutive adsorption of anionic and cationic bipolar amphiphiles on charged surfaces , 1991 .

[9]  J. Schlenoff,et al.  Protein adsorption modalities on polyelectrolyte multilayers. , 2004, Biomacromolecules.

[10]  Zhihui Dai,et al.  An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites. , 2016, ACS applied materials & interfaces.

[11]  Young Keun Kim,et al.  A highly sensitive and selective diagnostic assay based on virus nanoparticles. , 2009, Nature nanotechnology.

[12]  Gregory L Baker,et al.  Use of porous membranes modified with polyelectrolyte multilayers as substrates for protein arrays with low nonspecific adsorption. , 2006, Analytical chemistry.

[13]  Hongwei Wang,et al.  Gold nanoparticle layer: a promising platform for ultra-sensitive cancer detection. , 2011, Langmuir : the ACS journal of surfaces and colloids.

[14]  Sung Jae Kim,et al.  Increasing the sensitivity of enzyme-linked immunosorbent assay using multiplexed electrokinetic concentrator. , 2010, Analytical chemistry.

[15]  Gero Decher,et al.  Toward Layered Polymeric Multicomposites , 1997 .

[16]  Zhimin Chen,et al.  Polyelectrolyte-free layer by layer self-assembled multilayer films of cationic phthalocyanine cobalt(II) and carbon nanotube for the efficient detection of 4-nitrophenol , 2016 .

[17]  J. Suchodolski,et al.  Development and analytical validation of an enzyme-linked immunosorbent assay for the measurement of feline tumor necrosis factor α in serum. , 2014, Veterinary clinical pathology.

[18]  Gero Decher,et al.  Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites , 1997 .

[19]  Y. Nagasaki,et al.  High-performance surface acoustic wave immunosensing system on a PEG/aptamer hybridized surface. , 2013, Langmuir : the ACS journal of surfaces and colloids.

[20]  Francesca Diana,et al.  Development and validation of a new qualitative ELISA screening for multiresidue detection of sulfonamides in food and feed , 2014 .

[21]  L. L. Del Mercato,et al.  Biological applications of LbL multilayer capsules: from drug delivery to sensing. , 2014, Advances in colloid and interface science.

[22]  Lin Yuan,et al.  Sensitive sandwich ELISA based on a gold nanoparticle layer for cancer detection. , 2012, The Analyst.

[23]  Y. Lvov,et al.  Sonication-Assisted Layer-by-Layer Assembly for Low Solubility Drug Nanoformulation. , 2015, ACS applied materials & interfaces.

[24]  H. Möhwald,et al.  Polyelectrolyte Multilayers: Towards Single Cell Studies , 2014 .

[25]  Olivier Lazcka,et al.  Pathogen detection: a perspective of traditional methods and biosensors. , 2007, Biosensors & bioelectronics.

[26]  H. Matsuno,et al.  A stereocomplex platform efficiently detecting antigen-antibody interactions. , 2007, Bioconjugate chemistry.

[27]  J. Schlenoff,et al.  Human mesenchymal stem cell osteoblast differentiation, ECM deposition, and biomineralization on PAH/PAA polyelectrolyte multilayers. , 2015, Journal of biomedical materials research. Part A.

[28]  J. Watanabe,et al.  Instantaneous determination via bimolecular recognition: usefulness of FRET in phosphorylcholine group enriched nanoparticles. , 2007, Bioconjugate chemistry.

[29]  Y. Nagasaki,et al.  Enhanced immunoresponse of antibody/mixed-PEG co-immobilized surface construction of high-performance immunomagnetic ELISA system. , 2007, Journal of colloid and interface science.

[30]  Jianlong Zhao,et al.  Nano-ELISA for highly sensitive protein detection. , 2009, Biosensors & bioelectronics.

[31]  Michiya Matsusaki,et al.  Layer‐by‐Layer Assembly Through Weak Interactions and Their Biomedical Applications , 2012, Advanced materials.

[32]  P. Fratzl,et al.  Light-Induced Water Splitting Causes High-Amplitude Oscillation of pH-Sensitive Layer-by-Layer Assemblies on TiO2. , 2016, Angewandte Chemie.

[33]  M. Fleming,et al.  A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis , 2015, Haematologica.

[34]  D. Davies,et al.  Three-dimensional structure of an intact human immunoglobulin. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Jianping Fu,et al.  Supporting information Ultrasensitive ELISA Using Enzyme-loaded Nanospherical Brushes as Labels , 2014 .