Improving ELISA Technology using Ultra-efficient Peroxidase Mimics - Project Summary/Abstract Enzyme-linked immunosorbent assay (ELISA) technology has been widely used in clinical diagnostics for decades. Nevertheless, its limit of detection (in the range of picogram/milliliter to nanogram/milliliter) has not been substantially improved in recent years, which inhibits many critical applications such as early detection of cancers. In conventional ELISAs, enzyme-catalyzed reactions are responsible for the generation of detectable color signal and thus largely determine the detection sensitivity. Therefore, the detection limit of conventional ELISAs is inherently limited by the performance of enzymes. In this research, we propose to develop a novel signal generation paradigm which breaks the intrinsic limitations of enzymes for ELISA technology. Specifically, in this technique, M-Ptn core-shell nanoparticles (M = Ni, Co, or Fe; n: atomic ratio of Pt to M) (referred to as M-Ptn NPs ) are employed as alternatives to enzymes for ELISA. Herein, the M-Ptn NPs act as highly effective peroxidase mimics, catalyzing colorimetric reaction at a rate much higher than that horseradish peroxidase (HRP, a typical enzyme for ELISA) can achieve. Note, materials cost of the M-Ptn NPs used in ELISA is low because of the minimal usage amount (nanogram level per test). We hypothesize that the magnitude of signal amplification of such M-Ptn NP system could be maximized by carefully controlling the structure and elemental composition of M-Ptn NPs. In our preliminary study, using carcinoembryonic antigen (CEA) as a model cancer biomarker, we have demonstrated that the detection limit of conventional ELISA could be lowered ~340 times (approaching the regime of femtogram/milliliter) by using Ni-Pt NPs as alternatives to HRP. Our research program is novel and innovative in its approach because such signal generation system for ELISA has several distinctive advantages over existing designs: i) the use of M-Ptn NPs as peroxidase mimics with record-high catalytic efficiency, which could generate color signal that is orders of magnitude stronger than HRP does; ii) the M-Ptn NPs-based ELISA retains the simplicity of conventional HRP-based ELISA, without involving additional assay procedures and/or devices; iii) the sizes of M-Ptn NPs are designed to be relatively small (comparable to the dimension of HRP molecule), eliminating the steric hindrance of biomarker capture during assay; and iv) the M-Ptn NPs are much more stable than natural enzymes, making the new ELISA more reliable. This research program involves three specific aims: 1) to build the M-Ptn NP system that possess maximized catalytic efficiencies, 2) to establish the M-Ptn NPs-based ELISA, of which detection limit is ≥1,000- fold lower than conventional HRP-based ELISA, and 3) to demonstrate clinical use of M-Ptn NPs-based ELISA by testing different disease biomarkers in human blood samples. The success of this research will i) break through the existing detection limit barrier of ELISA technology; ii) have a clinical and translational impact on disease biomarker detection; and iii) impact the general field of in vitro diagnostics by offering a type of ultra- efficient peroxidase mimics that are suitable for many diagnostic technologies beyond ELISA.
