Biosensors and lateral flow immunoassays: Current state and future prospects

The advent of paper-based biosensors represents a novel paradigm in point-of-care (POC) diagnostics, emerging as versatile tools. However, the broad term "biosensors" can be misleading, encompassing a range of techniques such as dipstick assays, electrochemical, microfluidics and immunoassay-based biosensors (including lateral flow (LFA), vertical flow and nucleic acid-based immunoassays). This narrative review aims to consolidate the vast and dispersed information on biosensors into a systematically organized resource addressing both practical and theoretical aspects for researchers developing paper-based biosensors. It offers a comprehensive classification of biorecognition elements and labels, insights into various conjugation techniques, and characterization methods for both labels and conjugates. Following the development and optimization of biological reactions, this review emphasizes the careful selection of membranes and reagents to effectively reproduce molecular reactions on paper. Membranes are critical to biosensor efficacy, with fluid dynamics influenced by factors such as pore size, protein holding capacity and wicking rate. While POC diagnostics have traditionally provided binary (yes/no) results, advancements now allow for semi-quantitative and quantitative results. Technologies such as in-text, printers, various software's and smartphone can be used as colour analysis utilizing colour models beyond RGB like XYZ, grey intensity, CMY, CMYK, HSV and HSL that can analyse and process the colour intensity. AI integration further simplifies result analysis through image analysis, interpretation, predictive modelling, clinical decision support, enhancing detection, data integration and management. This review also emphasizes validation and stability studies in accordance with regulatory guidelines, ensuring the reliability of biosensors. The review ultimately covers: (i) A foundational understanding of various biosensor techniques, focusing on the self-sufficient LFA technique. (ii) Strategies to enhance sensitivity through pre- and post-assay modifications. (iii) A comprehensive troubleshooting section addressing common challenges in bioassay and fabrication. (iv) Multiplexing approaches enabling the simultaneous detection of multiple analytes for enhanced biomarker confirmation. By amalgamating knowledge from these approaches, this review offers the potential to elevate a basic traditional LFA strip into a highly sensitive diagnostic tool. It serves not only as a repository of knowledge but also as a roadmap for researchers and practitioners navigating the burgeoning field of paper-based biosensors.