The demand for highly sensitive, fast and low-cost biosensors for reliable quantification of small biomolecules or cancer biomarkers is leading to the development of a new class of devices able to change the techniques currently used for diagnosis in oncology. Lab‐on‐fiber (LoF) optrodes offer several advantages over conventional techniques for point‐of‐care platforms aimed at real‐time and label‐free detection of clinically relevant biomarkers. Moreover, the easy integration of LoF platforms in medical needles, catheters and nano-endoscopes offers unique potentials for in vivo biopsies and tumor microenvironment assessment. Here, we demonstrate the capability to improve the immobilization strategies through the use of hinge carbohydrates by involving homemade antibodies that demonstrated a significantly improved recognition of the antigen with ultra‐low detection limits. In order to create an effective pipeline for the improvement of biofunctionalization protocols to be used in connection with the LoF platform, here we first, optimized the protocol using a microfluidic Surface Plasmon Resonance device. Then we transferred the optimized strategy on LoF platform, based on Optical Fiber Meta‐tip (OFMT), for the final validation. As a clinically relevant scenario, we focused on a serological biomarker, Cripto‐1, for its ability to promote tumorigenesis in breast and liver cancer. Reported results demonstrate that the proposed approach based on oriented antibody immobilization is able to significantly improve Cripto‐1 detection with a ten‐fold enhancement versus the random approach. Therefore, our work opens new avenues in the development of high‐sensitivity LoF biosensors for the detection of clinically relevant biomarkers in the sub‐ng/mL range.
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