High-Performance Immunolatex Possessing a Mixed-PEG/Antibody Coimmobilized Surface: Highly Sensitive Ferritin Immunodiagnostics

Abstract

To create a high-performance immunoassay system based on a nanosphere/antibody complex, pentaethylenehexamine-ended poly(ethylene glycol), N6-PEG comprising N6-PEG-5k (M(n) = 6000 g/mol) and N6-PEG-2k (M(n) = 2000 g/mol) was employed as a novel blocking agent to modify the surface of nanospheres. Both the antibody (antiferritin) and the N6-PEG were covalently bonded onto the nanospheres by the linkage of their amino groups with the activated carboxyl groups of those particles. The quantification of antiferritin and tethered N6-PEG polymer was carried out using the copper reduction/bicinchoninic acid reaction (the Micro BCA method). Dynamic-light-scattering (DLS) and electrophoretic mobility (mu(e)) measurements were performed to characterize the nanosphere/antiferritin/N6-PEG complex, which was prepared under various conditions. Simultaneously, the immune response of the complex obtained in this manner was measured by the turbidimetric monitoring method in phosphate buffer (10 mM, pH = 7.4). On the basis of all the results, the optimum conditions for preparing an acceptable nanosphere/antiferritin/N6-PEG complex were determined. Interestingly, compared to the blocking treatment with bovine serum albumin (BSA), which is a well-known blocking agent, surface modification with N6-PEG, especially that using a mixture of N6-PEG-5k and N6-PEG-2k, improved the performance (increased immune response yield and decreased detection limit) of the nanosphere/antiferritin complex to a remarkable degree in both phosphate buffer and 100% fetal bovine serum (FBS), thus significantly demonstrating the potential of the nanosphere/antibody/mixed-PEG complex as central to a high-performance immunoassay system.

Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

“…In addition, the presence of PEG layer facilitates the efficient loading and conjugation of antibodies to the surface of QDs without significant reduction in the optical properties of the fluorescent core usually described by our peers. [46][47][48] This can also be attributed to the enhanced hydrophilicity and reduced steric hindrance caused by PEG, which prevent the nanoparticles aggregation in the antibody conjugation process and increase the stability of final conjugates. 46,47 Moreover, the flexible PEG motif on the surface of nanoparticles could maintain the natural conformation of the antibody, 47,48 and thus improve the affinity of the QD-antibody conjugates.…”

In vitro and in vivo characteristics of core–shell type nanogel particles: Optimization of core cross-linking density and surface poly(ethylene glycol) density in PEGylated nanogels

“…4. We have previously reported the construction of a mixed-PEG brush surface with co-immobilization of long and short PEG chains with different molecular weights to increase the PEG density and improve the non-fouling character of the substrate surface [31,32]. In order to improve the surface character, the effect of the addition of a short PEG brush on the PEGylated nanogels was examined.…”

Improvement of the thermal stability of streptavidin immobilized on magnetic beads by the construction of a mixed poly(ethylene glycol) tethered-chain layer

“…Streptavidin, ethanolamine and N6-PEGs were attached to the surface of carboxylated magnetic beads through the active ester reaction method in the same manner as previously reported. 9,11 Magnetic bead suspension (10 ml) was poured into a 1.5-ml plastic tube, and the tube was placed on a magnetic separator to remove the supernatant. The remaining beads were dispersed into 1 ml of NaH 2 PO 4 buffer solution (10 mM, pH 4.7).…”

“…The reaction time for streptavidin immobilization was 15 min at room temperature. To construct a densely packed PEGylated surface, we used our original blocking procedure [9][10][11][14][15][16] in this study, in which a mixed PEG tethered-chain layer was constructed on the SA-MB surface by the use of a mixture of long PEG (5k) and short PEG (2k), as follows: immediately after the immobilization of streptavidin, the beads were dispersed into 1 ml of N6-PEG solution and incubated overnight. The N6-PEG solution was a mixture of 600 ml of N6-PEG (5k) (1.5 wt%, pH 7.4) and 400 ml of N6-PEG (2k) (1.5 wt%, pH 7.4) aqueous solutions.…”

“…Poly(ethylene glycol) (PEG) is known as an excellent blocking agent. Because of the nonionic properties, hydrophilicity and large steric-exclusion effect of PEG, 5 PEGylated surfaces and nano-and microscale particles show excellent non-fouling properties with various molecules 6 and high-dispersion stabilities, [7][8][9] respectively. Furthermore, in our recent studies, we discovered that PEGylation improves the functioning of immobilized proteins on solid surfaces and particles.…”