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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.

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