Molecularly Imprinted Polymer Research Articles

Recent Progress and Applications of Advanced Nanomaterials in Solid-Phase Extraction.

Sample preparation maintains a key bottleneck in the whole analytical procedure. Solid-phase sorbents (SPSs) have garnered increasing attention in sample preparation research due to their crucial roles in achieving high clean-up and enrichment efficiency in the analysis of trace targets present in complex matrices. Novel nanoscale materials with improved characteristics have garnered considerable interest across different scientific disciplines due to the limited capabilities of traditional bulk-scale materials. The purpose of this review is to offer a thorough summary of the latest developments and uses of SPSs in preparing samples for chromatographic analysis, focusing on the years 2020-2024. The techniques for preparing SPSs are examined, such as metal-organic frameworks (MOFs), covalent organic frameworks (COFs), carbon nanoparticles (CNPs), molecularly imprinted polymers (MIPs), and metallic nanomaterials (MNs). Examining the pros and cons of different extraction methods, including solid-phase extraction (SPE), magnetic SPE (MSPE), flow-based SPE (FBA-SPE), solid-phase microextraction (SPME), stir-bar sorptive extraction (SBSE), and dispersive SPE (DSPE), is the main focus. Furthermore, this article presents the utilization of SPE technology for isolating common contaminants in various environmental, biological, and food specimens. We highlight the persistent challenges in SPSs and anticipate future advancements and applications of novel SPSs.

Molecularly imprinted electrochemical sensor based on APTES-functionalized indium tin oxide electrode for the determination of sulfadiazine.

Anelectrochemical sensor was developedfor the sensitive and selective detection of sulfadiazine(SDZ), based on a molecularly imprinted polymer (MIP) film formed on an indium tin oxide (ITO) electrode through a self-assembly process. The SDZ-imprinted ITO electrode (SDZ-MIP/APTES-ITO) was prepared through in situ polymerization using sulfadiazine, methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), and 2,2'-azobisisobutyronitrile (AIBN) as the template, functional monomer, cross-linker, and initiator respectively. Before polymerization, the ITO electrode was functionalized with 3-aminopropyltriethoxysilane (APTES) to promote covalent attachment of the polymer to the electrode. After polymerization, the template molecule SDZ was removed to create selective recognition sites, forming the molecularly imprinted polymer electrode (MIP/APTES-ITO), which facilitates sulfadiazine detection. The sensor's performance was evaluated using cyclic and differential pulse voltammetry, demonstrating a linear response in the sulfadiazine concentration range 0.1 to 300μM, with a detection limit of 0.11μM. The MIP-based sensor exhibited goodreproducibility, repeatability, selectivity, and stability in sulfadiazine detection. Its practical applicability was confirmed by the successful quantification of sulfadiazine in spiked milk samples.

An MIP-Based PFAS Sensor Exploiting Nanolayers on Plastic Optical Fibers for Ultra-Wide and Ultra-Low Detection Ranges—A Case Study of PFAS Detection in River Water

In this work, a novel optical–chemical sensor for the detection of per- and polyfluorinated substances (PFASs) in a real scenario is presented. The proposed sensing approach exploits the multimode characteristics of plastic optical fibers (POFs) to achieve unconventional sensors via surface plasmon resonance (SPR) phenomena. The sensor is realized by the coupling of an SPR-POF platform with a novel chemical chip based on different polymeric nanolayers over the core of a D-shaped POF, one made up of an optical adhesive and one of a molecularly imprinted polymer (MIP) for PFAS. The chemical chip is used to launch the light into the SPR D-shaped POF platform, so the interaction between the analyte and the MIP’s sites can be used to modulate the propagated light in the POFs and the SPR phenomena. Selectivity tests and dose–response curves by standard PFOA water solutions were carried out to characterize the detection range sensor response, obtaining a wide PFAS response range, from 1 ppt to 1000 ppt. Then, tests performed on river water samples collected from the Bormida river paved the way for the applicability of the proposed approach to a real scenario.

Computational and experimental investigation on functional monomer selection for a novel molecularly imprinting polymer of remdesivir

Molecularly-imprinted polymers (MIPs) are a sample preparation technique with a functional cavity that can rebind the same or similar analyte to the template. It also can increase the selectivity of the analytical method. Computational approaches are capable of making predictions about the most optimal synthesis conditions, including the most appropriate monomers and solvents. This study used the DFT method, GFN2-xTB, and molecular dynamics simulation to predict MIP synthesis’s best functional monomer, solvent, and stoichiometric ratio. Remdesivir (RMD), used as a template, has also never been developed to be made as MIP. The monomers were acrylic acid (AA), 2-hydroxyethyl methacrylate (HEMA), and acrylamide (AM). Laboratory testing, association constant, and Job plot analysis are carried out to confirm the results of computational tests. The polymer was synthesized using precipitation and characterized using SEM, FTIR, and TGA. The adsorption capacity was evaluated in two different solvents, i.e. acetonitrile (ACN) and water: ACN=9: 1 (v/v). Computational study results identified acrylamide as the most interactive ligand with RMD as a template. The results of molecular dynamics simulations identify that the RMD: AM=1: 1 complex is the most stable in terms of the radial distribution function, hydrogen bond occupancy, and Gibbs bond free energy. The adsorption ability test results show that MIP 1 and MIP 2 have imprinting factor values of 1.36 and 1.15, respectively.

Flexible Au@Ag/PDMS SERS imprinted membrane combined with molecular imprinting technology for selective detection of MC-LR

In this study, a core–shell structured bimetallic nano-cube, Au@Ag NCs, was prepared by seed-mediated growth procedure. The array structure of Au@Ag NCs was achieved at the interface through the autonomous assembly technique at the three-phase boundary. Employing polydimethylsiloxane (PDMS) as a flexible carrier, the array structure was effortlessly transferred to the PDMS membrane, bypassing the need for rigid substrates through a simple “pasting” method. This yielded a highly flexible and transparent SERS substrate with an array structure (Au@Ag NCs/PDMS membrane, AAP). In order to promote the selective detection property to the practical samples, molecularly imprinted polymers (MIPs) were coated on the surface of membrane to prepare the imprinted membrane (Au@Ag NCs/PDMS-MIMs, AAP-MIMs). It was demonstrated from the results that the AAP-MIMs exhibited high SERS sensitivity, stability, and uniformity. Furthermore, the flexible substrate possessed commendable mechanical strength, and facilitated the detection of analytes on irregular surfaces. In summary, this substrate held promising potential for practical on-site detection and analysis of specific target substances.

Anchor carbon dots inside NH2-MIL-88B via ship-in-a-bottle strategy for dual signal enhancement in colorimetric-fluorescent sensors

The increase in oxytetracycline (OTC) pollution has become a significant risk to ecological stability and human health because of excessive use. Therefore, developing a precise and reliable sensor for trace OTC detection is critical. In this work, a dual-mode sensor of colorimetric-fluorescent (CL-FL) with dual signal-on was developed for OTC detection. Firstly, a novel carbon dots encapsulating in cavities of MOFs composite (CDs@NH2-MIL-88B) was constructed by the ship-in-a-bottle approach, which enhanced peroxidase-like activity and fluorescent intensity. Further, combining with surface molecularly imprinted polymer (MIP), the dual-mode sensor was constructed to develop selectivity (CDs@NH2-MIL-88B@rMIP). Finally, the dual signal enhancement mechanism of the sensor comes from the interaction between OTC and identifying binding site involving strong hydrogen bonding and metal complexation from imprinted cavity and CDs@NH2-MIL-88B, respectively. The prepared sensor has a linear detection range of 1.00 × 10-9 - 1.00 × 10-5M with the limit of detection (LOD) of 1.47 × 10-10M for the CL mode and a detection range of 1.00 × 10-11 -1.00 × 10-7M with the LOD of 1.84 × 10-12M for the FL mode. Meanwhile, the sensor shows high sensitivity, accuracy and reliability in OTC detection. Overall, this work provides a promising CL-FL dual-mode sensing systems with signal-on in the detection of antibiotics and other hazardous pollutants.

Enzyme Activity Inhibition of α-Amylase Using Molecularly Imprinted Polymer (MIP) Hydrogel Microparticles.

Molecularly imprinted polymers (MIPs) are a class of synthetic recognition materials that offer a cost-effective and robust alternative to antibodies. While MIPs have found predominant use in biosensing and diagnostic applications, their potential for alternative uses, such as enzyme inhibition, remains unexplored. In this work, we synthesized a range of acrylamide-based hydrogel MIP microparticles (35 μm) specific for the recognition of α-amylase. These MIPs also showed good selectivity toward the target protein with over 96% binding of the target protein, compared with the control nonimprinted polymer (NIP) counterparts. Specificity of the MIPs was determined with the binding of nontarget proteins, trypsin, human serum albumin (HSA), and bovine serum albumin (BSA). The MIPs were further evaluated for their ability to inhibit α-amylase enzymatic activity, showing a significant decrease in activity. These findings highlight the potential of MIPs as enzyme inhibitors, suggesting an innovative application beyond their conventional use.

A novel molecularly imprinted electrochemical sensor based on quasi-three-dimensional nanomaterials Nb2CTx/AgNWs for specific detection of sulfadiazine.

A novel molecularly imprinted electrochemical sensor (MIECS) was constructed for the specific detection of sulfadiazine (SDZ) in food. Niobium carbide (Nb2CTx) as a typical two-dimensional lamellar nanomaterial has good electrical conductivity and unique structure, which was assembled with one-dimensional silver nanowires (AgNWs) to form quasi-three-dimensional composite nanomaterials (Nb2CTx/AgNWs). As spacer material, AgNWs prevented the aggregation of Nb2CTx and the collapse of Nb2CTx layers. At the same time, a fast electron transport channel was constructed through the synergistic effect between nanomaterialsthe two. The Nb2CTx/AgNWs realized the enhancement of electrical signals. Molecularly imprinted polymers (MIPs) endowed the sensor with selectivity, achieving the specific detection of sulfadiazine. Under the optimal experimental conditions, the method has a wide linear range (1 × 10-8-1 × 10-4 mol L-1) and a low limit of detection (1.30 × 10-9 mol L-1). The sensor was used to detect sulfadiazine in pork, chicken, and feed samples, and the recovery was 82.61-94.87%. The results were in good agreement with the HPLC results, which proved the accuracy and practicability of the method.

Lossy Mode Resonance Optical Fiber Enhanced by Electrochemical-Molecularly Imprinted Polymers for Glucose Detection.

Noninvasive glucose sensors are emergent intelligent sensors for analyzing glucose concentration in body fluids within invasion-free conditions. Conventional glucose sensors are often limited by a number of issues such as invasive and real-time detection, creating challenges in continuously characterizing biomarkers or subtle binding dynamics. In this study, we introduce an efficient lossy mode resonance (LMR) optical fiber sensor incorporating the molecularly imprinted polymers (MIPs) to amplify glucose molecules. A molecularly imprinted recognition platform is created on an LMR sensor surface through a convenient one-step electrochemical (EC) polymerization method, in which 3-Aminophenylboric acid and glucose serve as the functional monomer and template molecule, respectively. LMR resonance wavelength shift induced by the coupling of the optical lossy mode and the fiber core mode is employed as the parameter to characterize biomolecules. Due to its high sensitivity to surrounding environment changes, a limit of detection (LOD) of 4.62 × 10-2 μmol/L for glucose can be achieved by this optical fiber sensor. Additionally, the prepared EC-MIPs LMR sensor is capable of detecting glucose molecules in human saliva samples with high accuracy, endowing its potential for practical applications.

A novel method for quality evaluation of Radix Angelica sinensis based on molecularly imprinted electrochemical sensor

Rapid and sensitive detection of n-butylidenephthalide (NBP) is crucial for quality control of Radix Angelica Sinensis (RAS) and its related pharmaceuticals due to their shared pharmacological effects, such as immune enhancement and anti-tumor properties. Current detection methods struggle to quantify NBP quickly and accurately. A molecularly imprinted polymer (MIP)-based electrochemical sensor has been developed, forming a film on PCN-222(Fe) via electropolymerization for the rapid and selective detection of NBP. o-Phenylenediamine (o-PD) was polymerized onto PCN-222(Fe), utilizing its high surface area and porous structure to create a high-performance MIP (MIP/PCN-222(Fe)) sensor. This sensor detects NBP binding at the molecularly imprinted sites through a redox probe, with current changes reflecting the NBP content in the sample. This sensor exhibits a strong affinity for NBP, with a linear detection range from 200 nM to 1 mM, a detection limit of 76 nM, and high specificity towards similar phthalide compounds. Experimental results show that the MIP/PCN-222(Fe) sensor can accurately quantify NBP in real samples, offering a simplified method with promising applications for RAS quality evaluation.

Comparative Analysis of Pharmaceutical Persistence in Canadian Wastewater Treatment Plants Utilizing Molecularly Imprinted Polymers and Commercial Solid Phase Extraction Sorbents

In this study, the performances of a synthesized Molecularly Imprinted Polymer (MIP) and a commercial Solid Phase Extraction (SPE) cartridge were compared in terms of the removal efficiency and quantification of pharmaceutical compounds in wastewater samples. Concentration analysis of Emtricitabine, Tenofovir, Naproxen, Diclofenac, Ibuprofen, and Efavirenz was conducted in influent, primary effluent, secondary effluent, and post-chlorination water samples using liquid chromatography-UV detection. Regressions analysis revealed high linearity (R2 values from 0.9980 to 0.9999) for the compounds, alongside recoveries ranging from 90.9% to 100%. The method detection limits (MDLs) were also determined. Removal efficiencies were computed, with Naproxen and Ibuprofen demonstrating complete removal (100%), while other compounds displayed different of removal efficiency levels. The MIP was found to perform slightly better than traditional SPE cartridges in terms of selectivity, efficiency, and overall performance. This study provides valuable insights into the concentrations of target compounds at various treatment stages, emphasizing the importance of choosing extraction cartridges in wastewater treatment processes.

Molecularly imprinted electrochemiluminescence sensor based on luminol functionalized Co-MOF for rifampicin detection.

A highly sensitive molecularly imprinted electrochemiluminescence (MIECL) sensor was developed for detecting rifampicin (RIF) based on luminol@Co-MOF. Co-MOF had a significant enhancement of ECL signaling in the luminol-O2 system. Molecular imprinted polymers (MIPs) with the introduction of RIF provide new properties for the specific recognition of RIF. A noteworthy decrease in ECL intensity was observed with higher concentrations of RIF. Consequently, the ECL signal was controlled by RIF elution from and adsorption by the MIP, thus establishing a new method for RIF detection. Under optimal conditions, this sensor exhibited linear detection ranges of RIF between 1.0 × 10-11mol L-1 and 1.0 × 10-6mol L-1, with a detection limit of 3.3 × 10-12mol L-1 (S/N = 3). The recoveries ranged between 98.1 and 106.0% in fish samples. This method can be used as a sensitive and rapid method to detect RIF in real samples.

Enhancing mycotoxins detection through quantum dots-based optical biosensors

Abstract Quantum dot-based optical biosensors represent a significant advancement for detection of mycotoxins that are toxic secondary metabolites produced by fungi and pose serious health risk effects. This review highlights the importance of detection of filamentous fungi such as Aspergillus, Penicillium, Fusarium, Claviceps, and Alternaria in mycotoxin production, leading to contamination of agricultural products and subsequent health issues. Conventional detection methods such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC), and enzyme-linked immunosorbent assay (ELISA) are discussed with their respective advantages and limitations. Then the innovative use of quantum dots (QDs) in fabrication of biosensors is discussed in the present review, emphasizing their unique optical properties, such as size-tunable fluorescence and high photostability. These properties enable the development of highly sensitive and specific biosensors for mycotoxin detection. The application of QD-based biosensors, based on their applied bioreceptors including antibodies, molecularly imprinted polymers (MIPs) and aptamer, is explored through various detection strategies and recent advancements. The review concludes by underscoring the potential of QD-based biosensors in providing portable, cost-effective, and efficient solutions for real-time monitoring of mycotoxin for enhancing food safety and protecting public health.

Dual Recognition Strategy‐Based Transistor Sensor Array for Ultrasensitive and Multi‐Target Detection of Antibiotics

AbstractMulti‐target detection is a notable development direction in the field of analytical methods, boasting significant potential for breakthroughs in complex sample detection. Herein, an extended gate field‐effect transistor (EGFET) sensor array for ultrasensitive and multi‐target detection of antibiotics is developed. To enhance the recognition ability for diverse antibiotics, a dual‐recognition strategy based on tailor‐made molecularly imprinted polymer (MIP) and aptamer is adopted to fabricate the extended gate electrode (sensing electrode). The dual‐recognition strategy‐based EGFET exhibits superior sensitivity, selectivity, and stability compared to single recognition probes due to the synergistic effect of the affinities of MIP and specific aptamer. The developed sensor array can detect three types of antibiotics, including ampicillin, amoxicillin, and kanamycin, at the same time with record‐low detection limit (fM level) and a detection range spanning six orders of magnitude. The practical detection of antibiotics in various samples (tap water, river water, milk, honey, and artificial urine) further validates the feasibility of the sensor array in practical applications. The key advantages of flexibility, high sensitivity and selectivity, and multifunctionality demonstrate the high potential of EGFET sensor array for simultaneous detection of multiple target molecules.

Validation of a smartphone-compatible MIP-based sensor for bisphenol A determination in wastewater samples.

A handheld smartphone-compatible molecularly imprinted polymer (MIP)-based sensor was developed for the analysis of bisphenol A (BPA) in wastewater samples. Sensing elements based on ethylene glycol methacrylate phosphate (EGMP)-containing MIP films were designed and optimized using molecular dynamics simulations. The highly porous MIP films were synthesized via in situ polymerization, employing a fragment-based approach. The colorimetric response was based on the 4-aminoantipyrine method, while the MIP films were further utilized to detect BPA with a smartphone. The proposed sensor exhibited a wide linear range from 5 to 250μM, with a limit of detection (LOD) of 5μM (S/N = 3). Furthermore, the designed analytical system demonstrated excellent analytical performance in terms of selectivity, stability, and reproducibility. During sensor validation, real wastewater samples were successfully tested for BPA, showcasing the feasibility of the smartphone-compatible MIP-based sensor. Recovery values of 87.1-114.6% underscored the efficacy and reliability of the developed sensor system.

Molecularly Imprinted Electrochemical Sensor Constructed by Ferrocene‐Functionalized Carbon Nanotubes for Rapid Recognition and Determination of Tryptophan

AbstractMolecular imprinting is an advanced technology in the fabrication of novel chemical sensors recently applied, enabling rapid responses and highly selective binding to specific molecules, thereby enabling efficient detection of material constituents. In this study, a new molecularly imprinted electrochemical sensor (MIES) utilizing ferrocene‐functionalized carbon‐nanotubes (Fc‐CNTs) was developed for the determination of tryptophan (Trp). The formation of molecularly imprinted polymer (MIP) involves polymerization in dimethyl sulfoxide (DMSO) using Trp, methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) as the template molecules, functional monomers, and cross‐linking agents, respectively. Due to the modification of MIPs, the sensor achieves high performance. The sensor demonstrates a linear range from 1 to 45 μM and from 45 to 330 μM, with the limit of detection of 0.44 μM, exceeding that of the majority of sensors reported in the literature. Furthermore, the sensor can effectively detect Trp in real samples, including human serum and amino acid oral liquid. The recovery rate of Trp in human serum samples is 94.2 % to 105.30 %, and in amino acid oral liquid is 94.3 % to 101.68 %. This study provides a promising approach for the effective detection and analysis of tryptophan constituents.

Kinetics and Mechanism Study of Different Types of Surface‐Imprinted Polymers for CO2 Adsorption

ABSTRACTThe increased level of CO2 in the atmosphere has led to global warming and climate change. To mitigate these problems, solid adsorbents have become attractive materials for capturing excess CO2 in the post‐combustion method. Molecularly imprinted polymer (MIP) can be applied to prepare highly selective adsorbents that could capture CO2 molecules. The MIP was prepared using the surface imprinted polymer technique in this preliminary work. Only two types of support materials were used (graphite and silica gel) to screen the best support material that could enhance the CO2 adsorption capacity of the resulting adsorbent, which was analyzed using a fixed‐bed column reactor. Graphite‐imprinted polymer (GMIP) was found to be a good potential for CO2 adsorption. The Avrami model best described the adsorption system, while the fixed‐bed curve data fit the Yoon–Nelson model well. The semi‐empirical method was used to assess the interaction mechanism of the molecularly imprinted polymer with CO2 during adsorption. This investigation involved testing four different ratios of the template complex to functional monomers. The ratio of 1:4 (CO2:Allylthiourea) demonstrated the highest binding energy, with a higher formation of hydrogen bonds. Film diffusion and intraparticle diffusion were the main rate‐limiting steps that played vital roles at different stages of CO2 adsorption. This preliminary work has enhanced the development of MIP for CO2 adsorption and showcased the integration of computational approaches in tailoring specific MIPs.

Greenness assessment of a molecularly imprinted polymeric sensor based on a bio-inspired polymer

Methyldopa, a synthesized dopamine substitute with phenolic, amine, and carboxylic groups, was used to create a selective molecular imprinted polymer (MIP) for detecting formoterol fumarate dihydrate (FFD), a long-acting beta2-agonist for asthma and COPD. The bio-inspired polymer (MD) was electro-grafted onto a pencil graphite electrode (PGE) using cyclic voltammetry in a phosphate buffer (pH 6.5). An indirect method involving a redox probe (ferrocyanide/ferricyanide) and differential pulse voltammetry measured FFD binding to the MIP’s 3D cavities. The sensor showed a linear response range from 1 × 10⁻⁹ M to 2 × 10⁻¹⁰ M, with a detection limit of 1.7 × 10⁻¹¹ M. The polymethyldopa (PMD) and FFD interaction was assessed by UV spectroscopy, and the method was validated per ICH guidelines. Green analytical approaches, including RGB and GAPI, were also implemented. The goal was to use advances in molecularly imprinted polymers to develop a more precise and selective electrochemical sensor for FFD quantification.

Comparative study of bisphenols in e-cigarette liquids: Evaluating fabric phase sorptive extraction, ultrasound-assisted membrane extraction, and solid phase extraction techniques

To address the under-researched risk of bisphenols (BPs) in e-cigarette liquids, comprehensive studies have been conducted to propose optimum sample preparation and analysis methods. To determine twelve BPs in refill liquids for e-cigarettes, three sample preparation methods based on distinct operational and working principles were employed. These included fabric phase sorptive extraction (FPSE), ultrasound-assisted solvent extraction of porous membrane-packed samples (UASE-PMS) and solid phase extraction (SPE) utilizing molecularly imprinted polymers (MIPs) technology. Each extraction method was combined with ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Key parameters of FPSE and UASE-PMS procedures were optimized. This optimization included selection of the FPSE membrane types, durations of extraction and the choice solvents. Comprehensive validation was conducted, demonstrating linearity across a range from 2 to 60 ng/mL for all BPs (BPS, BPA, BPF, BPE, BPB, BPC, BPZ, BPFL, BPBP, BPP, BPG and BPM). Determination coefficients were above 0.9913, signifying linear relationship. The limits of detection (LODs) were established below 0.90 ng/mL, while the limits of quantification (LOQs) were lower than 2.5 ng/mL. Notably, the method based on UASE-PMS was successfully applied to the analysis of refill liquids for e-cigarettes samples. A comparative analysis of the methods highlighted variances in precision, accuracy, and applicable aspects, such as adjustment of parameters, sample preparation time, cost, handling, availability and possible limitations. Three methods have been identified as suitable for analysing BPs in e-cigarette refill liquids, highlighting the necessity to examine their presence in these products.

Hydrogel-based fluorescence assay kit for simultaneous determination of ceftazidime and avibactam

Monitoring the concentration of antibiotics rapidly and cost-effectively is crucial for accurate clinical medication and timely identification of drug-induced illnesses. Here, we constructed a novel fluorescent assay kit to monitor Zavicefta, an effective antibiotic composed of avibactam (AVI) and ceftazidime (CFZ) to treat carbapenem-resistant gram-negative bacteria infections. AVI can emit fluorescence, but CFZ cannot. To enable simultaneous measurement of both in one kit, we designed molecularly imprinted polymer (MIP) modified quantum dots (QDs) for CFZ determination. MIPs have received significant attention as an artificial antibody due to their exceptional specificity for various targets, particularly drugs with small molecular weight. Under the excitation wavelength of 350 nm, the detection process involves a decrease in QDs’ fluorescence signal at 600 nm owing to the “gate effect” between MIP and CFZ and the internal filtration effect between CFZ and QDs. Simultaneously, a fluorescence emission characteristic peak at 420 nm for AVI emerges. In addition, to simplify the operation procedure and improve determination throughput, the detection agents were incorporated into a hydrogel and placed in a 96-well plate, enabling concurrent quantification of AVI and CFZ within the respective range of 80–1000 μM and 1–1000 μM. The developed assay kit successfully determined AVI and CFZ in human serums and therapeutic drug monitoring in a live rabbit model. Recoveries of AVI and CFZ were 92.7–114%, with relative standard deviations below 6.0%. Moreover, a smartphone was employed to read the fluorescence signals, which was beneficial for cost reduction and out-of-lab analysis. This study will deliver a pragmatic resolution to developing high-throughput assay kits for drug determination.Graphical