The monovalent binding affinity of high binding site density nanoparticle-antibody bioconjugates is shown to exceed the intrinsic affinity of the original, monoclonal antibody. The nanoparticle-antibody bioconjugates were prepared by covalent coupling of antibodies to long-lifetime fluorescent, europium(III) chelate nanoparticles, 107 nm in diameter. Experiments were carried out in standard microtitration wells to determine solid-phase association and dissociation rate constants, nonspecific binding, and affinity constants of the various binding site density nanoparticle-antibody bioconjugates and the conventionally labeled monoclonal antibody. The affinity constant for monovalent binding of a high binding site density bioconjugate (5.4 x 10(10) M(-1)) was 8-fold higher than the intrinsic affinity of the antibody (6.6 x 10(9) M(-1)). The separately measured association (2.5 x 10(6) M(-1) s(-1)) and dissociation (3.7 x 10(-5) s(-1)) rate constants of the bioconjugate were 2-fold higher and 4-fold lower, respectively, compared to the antibody. The dependence of the association rate constant of the density of the binding sites enhanced the kinetics and the affinity of the high binding site density bioconjugates. The nanoparticle labels with high specific activity, low nonspecific binding, and enhanced binding affinity of the nanoparticle-antibody bioconjugates contribute to the design of the next generation immunoassays with extreme sensitivity.

Electronically conducting polyanion and polycation based on poly(alkoxythiophene) derivatives, poly-3-(3'-thienyloxy)propanesulfonate (P3TOPS) and poly-3-(3'-thienyloxy)propyltriethylammonium (P3TOPA) have been synthesized. Both polymers are water-soluble and exhibit high conjugation length in solution and in the solid state. These polyelectrolytes were used to prepare conducting and electroactive polyelectrolyte multilayers by the sequential layer-by-layer adsorption technique. In aqueous solutions multilayers of P3TOPS with inactive polyelectrolytes (e.g., poly(diallyldimethylammonium chloride), PDADMA) displayed electrochemical and optical behavior similar to polythiophene films prepared in organic media. Their in-plane conductivity was low (ca. 1.6 x 10(-)(5) S cm(-)(1)). The conductivity could, however, be increased by a factor of ca. 40 in "all-thiophene" films, in which P3TOPA was substituted for the inactive polycation (PDADMA). The interpenetration of layers is of prime importance in films containing conducting components. The interpenetration of P3TOPS was studied by measuring the charge-transfer rate across an insulating polyelectrolyte multilayer between the substrate and the P3TOPS layer with modulated electroreflectance. The extent of interpenetration was 8-9 polyelectrolyte layers, the length scale (7-15 nm) depending on the nature of the insulating layer and, especially, on the ionic strength of the solution used for the adsorption of P3TOPS.

Page 2256, last paragraph, last sentence, should read, "The slope represented the dissociation rate constant of pure dissociation phase reaction, based on an integrated form of the rate 39 where R 0 was a response at t ) 0 and R n a response at t ) n."Page 2257, Table 1, headings of third and fourth columns should read "association rate const, k a (M -1 s -1 /1 × 10 5 ) a,c " and "dissociation rate const, k d (s -1 /1 × 10 -5 ) a ", respectively.

Background: The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay. Methods: We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 × 109 L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 × 1010 L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 × 1010 L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer. Results: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 × 105 molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2–10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%). Conclusions: The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.