Measurement of adsorption constants of laccase on gold nanoparticles to evaluate the enhancement in enzyme activity of adsorbed laccase

Abstract

Adsorption of enzymes to nanoparticles and the mechanisms responsible for enzyme activity modulation of adsorbed enzymes are not well understood. In this work, gold nanoparticles were used for electrostatic adsorption of a plant-derived laccase. Adsorption constants were determined by four independent techniques: dynamic light scattering, electrophoretic light scattering, agarose gel electrophoresis and fluorescence quenching. Stable bionanoconjugates were formed with log K in the range 6.8-8.9. An increase in enzyme activity was detected, in particular at acidic and close to neutral pH values, a feature that expands the useful pH range of the enzyme. A model for the adsorption was developed, based on geometrical considerations and volume increase data from dynamic light scattering. This indicates that enzymes adsorbed to gold nanoparticles are ca. 9 times more active than the free enzyme.

Development of a Gold Nanoparticle-Based Lateral-Flow Immunoassay for Pneumocystis Pneumonia Serological Diagnosis at Point-of-Care

“…These AuNP-RSA conjugates were characterized and optimized through AGE assays which allow the separation of the conjugates according to their differences in size and surface charge (Guirgis et al, 2012;Franco and Pereira, 2013;Cavadas et al, 2016;Kim et al, 2016;Almeida et al, 2018). Analysis of AuNP-RSA conjugates with increasing RSA:AuNP molar ratios after electrophoresis (Figures 4A,C) showed that as more RSA is adsorbed at the AuNPs surface, the formed conjugate migrates less in the agarose gel.…”

“…The antigen concentration to use in the conjugation process to guarantee the maximum coverage of the AuNPs surface but also the colloidal stability of the AuNP-RSA conjugates was optimized. For that purpose, AuNP-Msg and AuNP-Kex1 conjugates were formed through electrostatic interactions established between increased molar ratios of each RSA and the AuNPs in solution, as described previously (Guirgis et al, 2012;Cavadas et al, 2016;Almeida et al, 2018). A solution of 0.06nM AuNPs was incubated overnight (≈15 h) at 4 • C with molar ratios ranging from 0 to 5000 of Msg and Kex1 stock solutions of 0.16 and 0.11 mg.mL −1 , respectively.…”

“…The gels were run at constant voltage of 180 V with a 21 cm electrode spacing for 20 min in TAE 0.125 × using the E865 CONSORT power supply. Digital pictures were acquired (WAS-LX1A Huawei P10 Lite camera) and processed through eReuss software (a gel analysis application freely available at https://github.com/ lkrippahl/eReuss), providing an accurate measurement of the red bands migration in agarose, which allows the calculation of their electrophoretic mobility (Ferard, 1994;Almeida et al, 2018). Electrophoretic mobility [µ (µm.cm/V.s)] is defined as the observed rate of migration of a component [ν (µm/s)] divided by the electric field strength [E (V/cm)] in a given medium.…”

“…The functionalized AuNPs produced were conjugated to Msg (AuNP-Msg) or Kex1 (AuNP-Kex1) RSA of P. jirovecii, in order to synthesize probes for the detection of IgM anti-P. jirovecii antibodies in sera of patients with PcP. Agarose gel electrophoresis assays were used to characterize the AuNPs before and after conjugation with the RSA (Guirgis et al, 2012;Cavadas et al, 2016;Kim et al, 2016;Almeida et al, 2018). As shown in Figures 4A,C, the migration distance of the RSA-conjugated particles (AuNP-Msg and AuNP-Kex1), compared to the non-conjugated AuNPs, decreases as the RSA:AuNP ratio increases.…”

Design and Simple Assembly of Gold Nanostar Bioconjugates for Surface-Enhanced Raman Spectroscopy Immunoassays

“…Conformational changes of the proteins resulting from interactions with metal NPs can affect physicochemical properties of each counterpart, which leads to a loss of reactivity and impaired applicability of the bioconjugates in detection methods. A broad variety of techniques such as ultraviolet-visible – near infrared (UV-Vis-NIR) absorption [13], fluorescence [14], differential centrifugal sedimentation (DCS) [15], dynamic light scattering (DLS) [16], and circular dichroism spectroscopies [17,18,19] or even SERS [20] can elucidate the mechanism for these interactions. Several studies have demonstrated enhanced enzymatic activities for enzymes conjugated with NPs [12,17,21].…”