Fluorescence analysis offers speed and sensitivity, making it highly desirable for detecting environmental toxins. However, many toxins, including microcystins (a group of hepatotoxins, neurotoxins, and carcinogens produced by cyanobacteria), lack inherent fluorescence, posing a challenge for direct fluorescence detection. Current detection methods, such as ELISA, LC-MS, HPLC, electrochemical assays, and surface plasmon resonance bioassays, can be complex, expensive, or require specialized equipment. While fluorescent nanosensors using quantum dots, nanoclusters, nanodots, and polymer dots show promise, their reliance on direct analyte-fluorophore interactions limits their applicability to non-fluorescent substances and introduces issues such as environmental susceptibility and poor selectivity. Molecular imprinting technology offers a route to enhance selectivity and sensitivity by creating recognition sites within a polymer matrix. However, traditional MIP-based fluorescent sensors still often rely on direct interactions between the analyte and the fluorophore, again limiting their utility for non-fluorescent compounds. This study aims to overcome these limitations by developing a novel indirect fluorescence sensing mechanism using molecular imprinting coupled with a paper-based microfluidic chip for the sensitive and selective detection of microcystin-RR (MC-RR), a model non-fluorescent toxin. The development of a simple, cost-effective, and field-deployable method for detecting microcystins is crucial for environmental monitoring and public health.

The literature extensively documents the use of fluorescent nanosensors for various applications, highlighting the advantages of simplicity, high sensitivity, and high throughput. However, these sensors generally rely on direct interactions between the target analyte and the fluorescent nanoprobe, leading to limitations in applicability to non-fluorescent compounds and sensitivity to environmental conditions. Functionalization of fluorescent nanoprobes through chemical modification improves performance and selectivity by acting as recognition moieties. Molecular imprinting technology is well-established for creating selective recognition sites in polymers, and its incorporation into fluorescent sensors has demonstrated increased sensitivity and selectivity. However, most current MIP-based fluorescent sensors are limited to analytes exhibiting direct interactions (static or dynamic quenching, FRET, PET, IFE) with the fluorescent nanoprobe. Competitive binding assays, where the analyte competes with a fluorescent indicator for binding sites, offer an indirect sensing approach. Examples include the use of micro-array-imprinted membranes with quantum dots for triazophos detection. This study builds on these advancements by exploring an alternative sensing mechanism that is not reliant on direct analyte-fluorophore interactions, expanding the applicability of fluorescent sensors to non-fluorescent target molecules. Specifically, the study addresses the need for cost-effective, robust methods for microcystin detection, as existing methods using antibodies or aptamers are often expensive, sensitive to environmental conditions, and lack reproducibility.

This study employs a novel indirect fluorescence sensing mechanism (IFSM) using a dummy template approach in molecular imprinting. Microcystin-RR (MC-RR) is selected as the model analyte due to its lack of interaction with standard fluorescent probes. The method involves preparing zinc ferrite nanoparticles (ZnFe2O4 NPs) coated with a thin molecularly imprinted polymer (MIP) film. Arginine, a naturally occurring amino acid with structural similarity to a fragment of MC-RR, serves as the dummy template molecule during MIP synthesis. The choice of arginine is justified by its lower cost and reduced toxicity compared to MC-RR. The MIP-coated ZnFe2O4 NPs (ZnFe2O4@MIPs) are integrated onto a paper-based microfluidic chip. The chip’s surface is functionalized with amino-modified CdTe quantum dots (QDs). In the absence of MC-RR, the carboxyl groups in the MIP cavities interact with the amino groups of the QDs, leading to fluorescence quenching through mechanisms such as FRET and PET. When MC-RR is present, it binds to the imprinted cavities, blocking the interaction between ZnFe2O4@MIPs and QDs and leading to fluorescence recovery. This indirect fluorescence recovery is directly proportional to the concentration of MC-RR. The paper-based chip employs a slidable-clip design that allows for simultaneous analysis of dual samples, enhancing the efficiency of the method. Detailed characterization techniques including TEM, HRTEM, SEM, XRD, UV-Vis spectroscopy, FT-IR, and TGA are utilized to assess the morphology, structure, and composition of the materials. The indirect fluorescent sensing mechanism is meticulously investigated by examining the charge transfer processes, fluorescence lifetime, and the effects of different molecules. The performance of the device is thoroughly optimized by varying factors including synthesis conditions, buffer systems, concentration of sensing materials, and equilibration times. The selectivity and stability of the device are also evaluated. Finally, the developed method is applied to the analysis of real seawater and lake water samples, demonstrating its practical applicability for on-site detection.

The study successfully synthesized ZnFe2O4@MIPs with a thin, uniform MIP layer (2-3 nm thickness) as confirmed by TEM and HRTEM. XRD and UV-Vis spectroscopy confirmed the successful coating of the MIPs on the ZnFe2O4 NPs. The developed slidable-clip type paper-based microfluidic chip design facilitated simultaneous dual-sample analysis. Optimization studies established ideal reaction conditions (40°C, 700 rpm) for MIP synthesis and an optimal elution time of 24 hours. The HEPES buffer system showed superior performance in the sensing assay compared to PBS and Tris buffers. The IFSM demonstrated a linear response to MC-RR concentrations in the range of 0.5–50 µg/L (R²=0.955). The limit of detection (LOD) was determined to be 0.43 µg/L. The selectivity study showed excellent specificity for MC-RR with negligible response to other interfering compounds. The device demonstrated remarkable stability, with RSD values of 6.54% and 2.14% for fluorescence intensity and inter-device variability, respectively. Spiked recovery tests in real seawater and lake water samples yielded recoveries ranging from 92.1% to 109.8% with RSDs from 4.41% to 6.77%. Comparison with a standard LC-MS method in Taihu Lake water samples confirmed the consistency and rapid nature (under 20 min) of this novel detection strategy.

The results demonstrate the successful implementation of a novel indirect fluorescence sensing mechanism (IFSM) for the sensitive and selective detection of the non-fluorescent microcystin-RR. The use of arginine as a dummy template molecule significantly reduced the cost and risk associated with handling the highly toxic MC-RR. The paper-based microfluidic chip platform enhances the user-friendliness and portability of the assay, making it suitable for on-site detection. The IFSM overcomes limitations of existing direct fluorescence methods that rely on analyte-fluorophore interactions. This approach significantly broadens the potential applications of molecularly imprinted polymers coupled with fluorescent sensors for non-fluorescent targets. The high sensitivity and selectivity, coupled with the ease of use and cost-effectiveness of the paper-based device, mark a significant advancement in microcystin detection technology. The excellent performance in real water samples showcases the practical utility of the method for environmental monitoring.

This research successfully developed a universal IFSM for the rapid, selective, and sensitive detection of non-fluorescent microcystins. The method's implementation on a user-friendly paper-based chip greatly enhances its practicality for on-site analysis. This IFSM holds significant potential for the detection of a wide range of non-fluorescent target species, although further investigation is needed to optimize the synthesis of MIPs with higher affinity and improve anti-interference capability. Future work could focus on exploring alternative dummy templates and optimizing the MIP synthesis process for improved performance and exploring the application of this technology for other environmental toxins.

While this study demonstrated excellent performance, some limitations should be noted. The current LOD of 0.43 µg/L, while already a significant improvement, could be further improved by optimizing the MIP synthesis and enhancing the sensitivity of the fluorescence detection. The selectivity, although high, was tested against a specific set of interfering compounds, and further investigation with a wider range of potentially interfering substances would strengthen the claims. The applicability of the proposed method might be limited to water samples with specific characteristics. Extensive testing across various water matrices is warranted to determine the method's robustness and generalizability.