Polyethylene imine-based receptor immobilization for label free bioassays

Abstract

Polyethylene imine (PEI) based immobilization of antibodies is described and the concept is proved on the label free assay of C-Reactive Protein (CRP). This novel immobilization method is composed of a hyperbranched PEI layer which was deposited at a high pH (9.5) on the sensor surface. The free amino groups of PEI were derivatized with neutravidin by Biotin N-hydroxysuccinimide ester and the biotinylated anti-CRP antibody immobilized on this layer. Direct binding assay of recombinant CRP was successfully performed in the low µg/ml concentrations using a label free optical waveguide biosensor.

Microfluidic channels laser-cut in thin double-sided tapes: Cost-effective biocompatible fluidics in minutes from design to final integration with optical biochips

“…For interrogating the waveguide sensors and recording the effective refractive index changes of the waveguide modes a benchtop GCI setup [11] and a BIOS OWLS system (Microvacuum Ltd., Hungary) [14] were used. Before integration, the sensor chips were cleaned with chromsulfuric acid according to published protocols [15,16] and dried with a stream of clean nitrogen gas.…”

Bulk and surface sensitivity of a resonant waveguide grating imager

“…Using these results and Eq. (3), we estimate that the imager has a limit of detection of adsorbed mass down to 0.078 ng/cm 2 , which is approximately one order of magnitude better than OWLS ($1 ng/cm 2 ), 16 but similar to a PC biosensor fabricated on a glass substrate (0:042 ng=cm À2 ), 27 and SPR ($0:1 ng=cm À2 ). 13 Assuming that the average size of a protein is $50 nm 2 and the surface coverage of immobilized and functional proteins is 50%, the imager is estimated to be capable of detecting a small molecule of 100 Da binding to $8% immobilized proteins under optimal conditions, suggesting that, similarly to SPR and PC biosensors, the imager can be applied for fragment screening under optimal conditions.…”

“…With the bulk refractive index sensitivity of 113.85 nm/RIU and the resolvability of 0.25 pm wavelength shift of the imager, we estimated its lower limit of detection in Dn (Dn min ) to be 2.2 Â 10 À6 RIU. This is approximately one order of magnitude better than the bulk refractive index resolution of OWLS [14][15][16] or a PC biosensor using plastic substrates (3.4 Â 10 À5 RIU), but is largely within the range for SPR (10 À5 -10 À8 RIU). 13 However, the practical limit of detection for Dn min observed was $10 À4 under the non-optimal experimental conditions used (i.e., manual washing, uncontrolled temperature, and the open environment).…”

“…The imager only measures the resonant wavelength of the zeroth order TM mode (but not that of the transverse electric (TE) mode) of the biosensor, preventing direct determination of the mass surface coverage of adsorbed species. Therefore, we used dual mode (TE and TM modes) OWLS as a complementary technique [14][15][16] to determine both the thickness d A and effective refractive index n A of the adsorbed layer. The adsorbed mass density M can then be obtained using the de Feijter formula 19,20 …”

Sample handling in surface sensitive chemical and biological sensing: A practical review of basic fluidics and analyte transport

“…label-free detection with a typical temporal resolution of 10-20 s, which makes it an ideal research tool in several fields, including the online monitoring of protein adsorption [19,20,21,22], bacterial [23,24] and mammalian cell adhesion [25,26,27,28,29,30], supported lipid-bilayer deposition [31,32], polyelectrolyte multilayer build-up [33,34,35], nanoparticle surface adhesion and assembly [36,37], and receptor-ligand interactions [38,39].…”