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Immunoassays are commonly used methods for detection of small molecules that typically require numerous steps of the labeling between immune-recognition reagents and tracers, immobilization and recurrent washing, making them time consuming and difficult to adapt into point of care formats. Here we describe a "ready-to-use" homogeneous competitive immunosensor with an assay time of 10 min that is based exclusively on recombinant reagents. The signal is produced when the split fragments of the nano luciferase (Nluc) are brought together by the interaction of a heavy chain only variable domain (VHH) with a peptidomimetic of the target small molecule. A VHH to 2,4-dichlorophenoxyacetic acid (2,4-D) was used to isolated the peptidomimetic (NGFFEPWQVVYV) from phage display libraries using six panning conditions. Then the peptidomimetic and VHH were fused with the larger (LgN) and smaller piece (SmN) of split fragments of Nluc, respectively. In order to optimize the signal and sensitivity of the immunosensor, we explored the effects of the spacer between the peptidomimetic and LgN, the copy number of peptidomimetics, and the spacer between SmN and VHH, generating 24 combinations that allowed to conclude on their respective roles. Eventually, the developed "ready-to-use" immunosensor performed excellent signal-to-noise ratio and sensitivity, and could be applied to the detection of 2,4-D in real samples. Meanwhile, the immunosensor totally realizes labeling-free, immobilization-free and washing-free, also can be produced in a highly cost effective way., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC(50) of 0.15 ngmL(-1) (linear range from 4.4 × 10(-3) to 10 ngmL(-1)). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9 × 1000 nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors., (Copyright © 2011 Elsevier B.V. All rights reserved.)