Lin Liu

Protein biomarkers play an indispensable role in the early diagnosis, prognosis assessment, therapeutic monitoring, and treatment evaluation of diseases. Optical biosensors have emerged as promising alternatives for protein detection due to their label-free, real-time, and highly sensitive characteristics. Molecularly imprinted polymers (MIPs), known as “plastic antibodies,” can provide chemically stable, low-cost, and tailorable recognition elements through the formation of target-complementary cavities in polymer matrices, making them highly attractive for integration with optical sensing platforms. In this work, we systematically summarize the advances in MIP-based optical biosensors for protein detection. The fundamental principles of biosensors and recognition elements are introduced, followed by a brief overview of the key components of imprinting technology, such as imprinting materials, functional monomers, cross-linking agents, polymerization mechanisms, template fixation strategies, and template removal conditions. Then, we comprehensively evaluate the integration of MIPs with various optical transduction techniques, including fluorescence, chemiluminescence, electrochemiluminescence, surface-enhanced Raman scattering, colorimetry, and surface plasmon resonance. Finally, the major challenges are discussed, and future development directions are proposed. This review aims to provide systematic theoretical references and useful insights for the rational design, performance improvement, and clinical translation of MIP-based optical biosensors for protein detection.

Early diagnosis of Alzheimer’s disease (AD) can facilitate the establishment and implementation of therapeutic interventions. The currently used diagnosis methods for AD mainly include cerebrospinal fluid analysis and positron emission tomography imaging. Due to their high invasiveness, high cost, and limited accessibility, these technologies are difficult to meet the needs of large-scale population screening, grading diagnosis, and treatment, thereby limiting the popularization of early diagnosis of AD. The detection of blood biomarkers has become an important breakthrough in early screening and diagnosis of different diseases due to its non-invasive, low-cost, and easy-to-operation advantages. Recently, blood proteins such as amyloid-beta (Aβ), total and phosphorylated Tau, light chain neurofilaments (NFL), and glial fibrillary acidic protein (GFAP) have been considered promising biomarkers for the diagnosis of AD. However, there is currently no effective, minimally invasive, and easily accessible detection method for clinical diagnosis and risk prediction of AD. Electrochemical and electrical biosensors are highly sensitive, simple, fast, and cost-effective analytical tools for disease monitoring, drug development, and target detection. In this work, we comprehensively and systematically overview the progress of various electrochemical and electrical techniques for determining AD-related blood protein biomarkers, mainly including electrochemistry, electrochemiluminescence, photoelectrochemistry, quartz crystal microbalance, field-effect transistor, and organic electrochemical transistor. This work can provide guidance for researchers to develop novel electrochemical and electrical biosensors for early and accurate diagnosis of AD.

Protein biomarkers can be used for monitoring the occurrence and development of diseases. Accurate, sensitive, and low-cost methods for protein detection can facilitate therapeutic intervention, improve clinical outcome, and reduce economic pressure for patients. Molecularly imprinted polymers (MIPs) have been considered as a type of biomimetic materials for developing biosensing technologies due to their advantages of high stability, low preparation cost, and good reusability over classical biometric recognition elements such as antibodies and aptamers. Electrochemical biosensors have become the most promising technology in sensing applications in view of their high sensitivity, fast response speed, cost-effectiveness, good stability, and ease of miniaturization. Efforts have been made to develop various electrochemical biosensors for protein detection with MIPs as recognition elements. This article provides an overview of the progress in molecular imprinting methods for the design and application of electrochemical protein biosensors. The strategies for imprinting and removing templates and preparing MIPs-modified sensing electrodes are comprehensively discussed. Finally, the challenges and future perspectives of protein-imprinted electrodes are addressed. This work will contribute to the development of innovative analytical devices based on MIPs for monitoring and managing various diseases by determining protein biomarkers.

Multiple-mode immunoassays have the advantages of self-correction, self-validation, and high accuracy and reliability. In this work, we developed a strategy for the design of triple-mode immunoassays with the self-assemblies of three-in-one small molecules as signal reporters. Pyrroloquinoline quinone (PQQ), with a well-defined redox peak and excellent spectroscopic and fluorescent signals, was chosen as the signaling molecule. PQQ was coordinated with Cu²⁺ to form metal–organic nanoparticle as the signal label. Hexahistidine (His₆)-tagged recognition element (recombinant streptavidin) was attached to the Cu-PQQ surface through metal coordination interaction between the His₆ tag and the unsaturated metal site. The captured Cu-PQQ nanoparticle released a large number of PQQ molecules under an acidic condition, which could be simultaneously monitoring by electrochemical, UV-vis, and fluorescent techniques, thereby allowing for the development of triple-model immunoassays. The three methods were used to determine the concentration of carcinoembryonic antigen (CEA) with the detection limits of 0.01, 0.1, and 0.1 ng/mL, respectively. This strategy opens up a universal route for the preparation of multiple-model signal labels and the oriented immobilization of bioreceptors for molecular recognition.

Reversible addition–fragmentation chain-transfer (RAFT) polymerization has become an efficient method in the field of polymer synthesis. Recently, the RAFT polymerization technique has been successfully used to prepare functional materials and develop various sensing methods used in different scenarios. The polymerization reaction can be initiated by thermal, electrochemical, photochemical, enzymatic, and mechanical stimulation. More interestingly, RAFT polymerization can be performed in situ by recruiting a large number of signal tags at the solid interface to amplify the signals. In this review, we addressed the latest achievements in the preparation of sensing materials and the design of different sensors based on the RAFT polymerization technique for sensing ions and small molecules and bioimaging of tumor cells and viruses. Then, electrochemical and optical biosensors through the signal amplification of the RAFT polymerization method were summarized. This work could provide inspiration for researchers to prepare fascinating sensing materials and develop novel detection technologies applied in various fields.

Nanoquenchers with a single quenching cofactor exhibit limited fluorescence quenching efficiency. In this work, a metal–organic hybrid with dual quenching cofactors (Cu²⁺ and pyrroloquinoline quinone or PQQ) was prepared by metal-coordinated assembly and used as a nanoquencher for a protease assay with enhanced quenching efficiency. The peptide substrate with an oligohistidine (His₆) tag was labeled with a fluorophore. Caspase-3 was determined as a protease example. The substrate was attached onto the surface of the Cu-PQQ nanoquencher by a metal coordination interaction between the unsaturated Cu²⁺ on the nanoparticle surface and the His₆ tag in the peptide. The cleavage of the peptide substrate by enzymatic hydrolysis led to the release of a fluorophore-conjugated segment from the nanoquencher surface, thus turning on the fluorescence. The nanoprobe was used to determine caspase-3 with a linear range of 0.01–5 ng/mL and a detection limit of 7 pg/mL. Furthermore, the method was used to evaluate inhibition efficiency and monitor drug-induced cell apoptosis. In contrast to other means of peptide immobilization, such as physical adsorption and covalent coupling, the strategy based on the metal coordination interaction is simple and powerful, thereby achieving assays of caspase-3 activity in lysates with a satisfactory result. The work should be valuable for the design of nanoquenchers with multiple quenching cofactors and the development of novel biosensors.

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal–organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu²⁺ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His₆) tag in CEA and the unsaturated Cu²⁺ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01–1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His₆ tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers.