Claudio Parolo

Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes

The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the horseradish peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is presented here. Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an enhanced label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gaining sensitivity (up to 1 order of magnitude) without losing the simplicity of the LFIA format, opening the way to other LFIA applications including their on-demand performance tuning according to the analytical scenario.

Immunosensing using nanoparticles

Immunosensing technology is taking advantage of the lastest developments in materials science and inparticular from the nanomaterials field. Because of their unprecedented optical tunability as well as electrical and electrochemical qualities, we are seeing significant developments in the design of novel immunoassays; various conventional optical and electrical platforms which allow for future applications in several fields are being used. Properties of nanoparticles such as light absorption and dispersion are bringing interesting immunosensing alternatives. Nanoparticles are improving the sensitivity of existing techniques used for protein detection in immunoassays based on Surface Plasmon Resonance, Quartz Crystal Microbalance, Fluorescence spectroscopy etc. Electrochemical techniques are also taking advantage of electrical properties of nanoparticles. Redox properties of metal based nanoparticles, surface impedance change and conductance changes once nanoparticles are present as labelling tags or modifiers of transducer surfaces are also improving the technology. In most of the examples nanoparticle based biosensing systems are being offered as excellent screening and superior alternatives to existing conventional strategies/assays with interest for fields in clinical analysis, food quality, safety and security.

Design, preparation, and evaluation of a fixed-orientation antibody/gold-nanoparticle conjugate as an immunosensing label

Herein, we describe the development of a new, highly efficient label for immunosensing comprising an antibody/PEGylated gold-nanoparticle (AuNP) conjugate in which the antibody molecules are bound to the AuNP surface in a fixed orientation. Our method exploits the high density of positive charges on the major plane of antibodies that exists when the pH of the solution is lower than the isoelectric point of the antibody; the antibody molecules interact with the negatively charged AuNP surface through their major plane, enabling the antigen binding sites to move freely and therefore to reach maximum accessibility. This directed ionic interaction is reinforced by the formation of a peptide bond between the amino group of the Lys residues in the antibodies and the carboxylic groups of the PEGylated-AuNP surface via EDC chemistry. Electrochemical analyses revealed that the fixed-orientation conjugate offers a limit of detection that is 1 order of magnitude lower than that of a randomly oriented label. The performance of the new conjugate as an immunosensing label was assessed for the quantitative detection of IgG in human serum.

Gold nanoparticles decorated with a ferrocene derivative as a potential shift-based transducing system of interest for sensitive immunosensing

The condensation of 1,1-ferrocenedimethanol and lipoic acid yields a ferrocene derivative (FcD): 1,1-ferrocenyl bis(methylene lipoic acid ester), a bipodant linker which was used to aggregate gold nanoparticles (AuNPs). The FcD/AuNP aggregations displayed a very well-resolved signal related to the ferrocene oxidation to ferrocenium. The aggregates were found to form an efficient transducing system for a novel concept of immunosensing based on the shifting of the ferrocene oxidation potential. αHIgG/FcD/AuNP or chicken αGIgG/FcD/AuNP bioconjugates are used along with carbon SPEs as an immunosensing platform for the detection of HIgG or GIgG as model analytes. As shown by CV and DPV, the antigens addition triggers significant anodic potential shifts (up to 75 mV) of the ferrocene oxidation peak as an immunoresponse for the Ag-Ab recognition. The results from selectivity and specificity experiments using bovine serum albumin, ovalbumin and α-lactalbumin as potentially interfering proteins to HIgG Ag showed that these potential shifts would be solely due to the Ag-Ab recognition events with excellent selectivity and specificity in the presence of up to 1000-fold of the competitive proteins.