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.
Plasmonic nanoparticle aggregates are one of the most widely employed nanostructures as colloidal surface enhanced Raman spectroscopy (SERS) substrates mainly due to their ability to generate huge near-field enhancements. However, the available definitions to determine the enhancement factor, a key parameter to quantitatively describe the quality of SERS substrates, exhibit important limitations to adequately describe the performance of colloidal plasmonic nanoparticle aggregates as SERS platforms. Herein, we introduce a new figure of merit named active concentration enhancement factor (ACEF) to assess the SERS enhancement factor of colloidal gold nanoparticle aggregates, which shows significant improvements to characterize the SERS performance with respect to formerly reported parameters. The determination of the analyte active concentration, that is, the concentration of analyte molecules that effectively contributes to the SERS signal, was achieved according to a strategy that involves a rigorous modeling of the extinction spectra of the colloidal gold nanoparticle aggregates. Furthermore, the experimentally obtained ACEF values can be directly compared with theoretically calculated electromagnetic field enhancement factors. We believe that the figure of merit introduced might help to strengthen the development and design of SERS substrates.
Surface-enhanced Raman spectroscopy (SERS) has demonstrated to be a powerful technique for the ultrasensitive detection of different types of analytes and particularly biomolecules, with the rational design of SERS substrates being one of the most relevant issues for the development of effective detection protocols. In this work, a colloidal SERS substrate consisting of a pair of Au nanorods linked by the molecular bridge biotin/streptavidin/biotin has been obtained and employed for the detection of picomolar quantities of the biotinylated antibodies gliadin IgG and Ara h1 IgG, which is of great interest in food science. As a consequence of the bioconjugation strategy implemented, the SERS substrate, that is, the Au nanorod dimer, presents the advantage of combining in a single nanostructure the capabilities for both direct and indirect detection of the biotinylated antibodies. Furthermore, the experimental results are supported by detailed electrodynamics simulations which takes into account not only the gradient of the near-field enhancements within the hot spot but also the volume occupied by the respective biomolecules. The SERS substrate presented here could be straightforwardly employed for the ultrasensitive detection of other biotinylated biomolecules.
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