Colloidal gold clusters formation and chemometrics for direct SERS determination of bioanalytes in complex media.

In this work, we report the sensitive and selective sensing of the purine bases adenine and guanine in urine matrix by using surface-enhanced Raman spectroscopy (SERS) and a colloidal SERS substrate. To identify suitable conditions for quantitative analysis, the pH dependence of spectra of adenine, guanine, urine simulant and their mixtures was studied on gold nanoparticles suspension. Interestingly, although the urine matrix promotes the analytes signal suppression and overlapping bands, it can also cause an improvement in repeatability of the SERS measurements. This effect was associated to the relatively controlled formation of small-sized gold clusters and it was investigated both experimentally and theoretically. Furthermore, a correlation constrained multivariate curve resolution-alternating least squares (MCR-ALS) method was developed to resolve overlapping SERS bands and to quantify physiologically relevant (micromolar) concentrations of the bioanalytes. The performance of the proposed MCR-ALS approach (assessed in terms of figures of merit) was similar to that obtained by using partial least squares regression, but with the additional advantage of retrieving valuable spectral information. Therefore, this method can be used for improving selectivity of colloidal clusters in qualitative and quantitative SERS analysis of complex media, avoiding the need for tedious nanoparticle-surface modification or preliminary chromatographic separation.

A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles.

Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag NPs) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. To illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-STV) pair interaction was studied. Controlled aggregates (dimers) of STV functionalized Ag NPs were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag NPs, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.

Enzyme-Free Immunoassay Using Silver Nanoparticles for Detection of Gliadin at Ultralow Concentrations.

Determination of biomarkers in clinical or food samples is of crucial importance for monitoring, prevention, and care of public health. The standard procedure used for this purpose is the enzyme-linked immunosorbent assay (ELISA), which makes use of the specific antibody-antigen biorecognition and the catalytic effect of the enzymes. One of the main shortcomings of this technique is the use of enzymes that often present low chemical and thermal stabilities compared to other chemicals. Other drawbacks include the nonspecific binding process that could lead to false-positive results, the use of relatively large amounts of the sample, and the number of time-consuming steps involved. Recently, an enzyme-free and ultrasensitive analytical method for antigen detection denoted as intensity depletion immunolinked assay (IDILA) has been proposed by our laboratory. The assay is based on the inhibition to form Ag nanosphere dimers linked by a specific antibody in the presence of the corresponding antigen. In this work, we go a step further demonstrating how the performance of this method could be improved by using silver nanoparticles (Ag NPs) of different diameters (58 and 78 nm). The experiments are performed for detecting gliadin, an antigen of utmost importance in celiac disease, and the results are compared with ELISA, the standard technique homologated by the Food Codex Alimentarius. It is found that the IDILA assay could be around 1000 or 10 000 times more sensitive than ELISA, also having lower limits of detection, depending on the conditions explored (fraction of dimers and Ag NP diameter). Using the appropriate conditions, the IDILA assay is shown to be able to detect femtomolar concentrations of the antigen, besides being robust, reliable, cheap, rapid (around 2 h), and of easy implementation using the standard equipment and biomolecular reagents used for the ELISA assay.