IntroductionEnzymatic assays are a cornerstone of both clinical diagnostics and biomedical research, valued for their ability to detect and measure specific biological targets with high accuracy1. However, despite their widespread use, traditional assay methods are limited by lengthy run times, the need for large amounts of reagents, and a heavy reliance on fully equipped central labs. These drawbacks make it difficult to apply them in fast-paced or resource-limited environments2, especially in point-of-care testing (POCT), where quick and accessible results are essential3,4.Microfluidic technologies have emerged as a promising strategy to address these challenges by enabling precise manipulation of small fluid volumes, reducing reagent and sample requirements, and integrating multiple analytical steps within compact platforms4,5. Over the past two decades, a wide range of microfluidic systems, including digital, droplet-based, centrifugal, and chip-based architectures, have been explored for biochemical assays. These platforms offer advantages such as improved reaction efficiency, shortened assay time, and potential for automation and portability. However, the translation of enzymatic assays into microfluidic formats is not straightforward, as enzyme kinetics, stability and detection sensitivity can be significantly influenced by microscale conditions and device design6,7.Most previous reviews have focused on specific analytes, fabrication methods, individual microfluidic formats, or application domains, with comparatively limited attention to systematically comparing multiple microfluidic platforms from both design and assay performance perspectives. Consequently, the factors governing assay performance across different platform architectures and their implications for point-of-care (POC) systems remain insufficiently understood. In this review, we critically examine how enzymatic assays are implemented across different microfluidic platforms, with particular emphasis on the influence of device architecture and operating principles on analytical performance. Rather than simply summarizing previous studies, we provide a comparative analysis of different microfluidic strategies and their associated trade-offs for POCT applications. To maintain a focused scope, this review includes only studies in which complete enzymatic assays are performed within integrated microfluidic systems, while excluding non-enzymatic methods and devices lacking true microfluidic fluid control. Furthermore, the review highlights the major challenges and future directions for translating enzymatic microfluidic assays into practical clinical and POC applications. By identifying common design principles, current limitations, and future research priorities, this review provides a practical framework for the rational development of next-generation microfluidic enzymatic assays. Figure 1 provides an overview of the comparative framework used in this review, summarizing the four major microfluidic platforms, their predominant detection strategies, and the distribution of validation levels among the included studies, from proof-of-concept investigations to clinical validation.Fig. 1Full size imageConceptual overview of the four major microfluidic platforms for enzymatic assays, their predominant detection strategies, and the progression of included studies toward clinical translation. The percentages shown in the figure were calculated from the studies included in this review and represent the distribution of reported platform characteristics and validation levels within the reviewed literature.Microscale factors influencing Enzyme kinetics and assay performance in microfluidic platformsIn microfluidic enzymatic assays, precise temperature control is essential because enzyme activity and reaction kinetics are highly temperature-dependent. The small fluid volumes and high surface-area-to-volume ratio of microfluidic devices enable rapid thermal equilibration, shortening assay time and improving temperature uniformity. However, these characteristics also increase susceptibility to temperature fluctuations and thermal gradients, which may lead to non-uniform reaction conditions and reduced assay reproducibility8. Effective thermal management is therefore commonly achieved using integrated resistive microheaters, Peltier elements, or external thermostats in combination with real-time temperature monitoring and feedback control, ensuring consistent enzymatic performance9. The high surface-area-to-volume ratio also accelerates solvent evaporation, particularly in open or partially sealed systems. Even minor evaporation alters reagent concentrations, pH, and ionic strength, thereby affecting reaction kinetics and reproducibility. This effect is particularly important during prolonged or heated assays and is commonly minimized through device sealing or humidity control10. In addition to affecting heat and mass transfer, the high surface-area-to-volume ratio of microfluidic channels also increases interactions between biomolecules and channel walls, making nonspecific adsorption an important source of analytical error11. This effect is especially pronounced in hydrophobic polymers like polydimethylsiloxane (PDMS), where proteins and enzymes readily adsorb to channel walls, lowering their effective concentration. Glass and cyclic olefin copolymers (COCs) generally exhibit lower nonspecific adsorption than PDMS, whereas poly(methyl methacrylate) (PMMA) exhibits intermediate nonspecific adsorption characteristics. Surface modification strategies such as polyethylene glycol (PEG) grafting, bovine serum albumin (BSA) blocking, plasma treatment, and other antifouling coatings are therefore widely used to preserve enzyme activity and improve assay reproducibility12.Although nonspecific adsorption is generally undesirable, as it depletes active enzyme and perturbs reaction kinetics, intentional enzyme immobilization is widely employed in miniaturized systems to enhance enzyme stability, and improve long-term operational performance13,14. Despite these advantages, excessive surface binding or mass-transfer limitations may reduce catalytic efficiency15.Oxygen mass transfer is another important factor influencing enzymatic reactions in microfluidic devices, particularly for oxidase-based assays in which oxygen acts as a co-substrate. Although the high surface-area-to-volume ratio of miniaturized systems can facilitate oxygen transport, rapid oxygen consumption within confined reaction volumes may lead to local oxygen depletion, resulting in reduced reaction rates and compromised analytical accuracy. The extent of oxygen limitation depends on channel geometry, flow conditions, and device materials, all of which influence oxygen diffusion and availability16. In droplet-based systems, oxygen partitioning across the oil–water interface further influences oxygen availability17.Shear forces generated within microfluidic channels influence enzyme stability, mass transport, and mixing efficiency. Moderate shear enhances substrate transport, whereas excessive shear may induce conformational changes or partial inactivation of shear-sensitive enzymes. Therefore, optimizing flow conditions is important for maintaining stable enzyme activity and reproducible assay performance18.These microscale factors are closely interconnected and need to be considered during the design of microfluidic enzymatic assays, as they jointly determine enzyme kinetics, analytical sensitivity, and assay reproducibility.Biochemical assays using EnzymesGlucoseGlucose is one of the most extensively investigated biomarkers in clinical diagnostics because of its importance in diabetes management19. Microfluidic formats of enzymatic glucose assays have primarily relied on the hexokinase and glucose oxidase pathways, enabling reduced reagent consumption, shorter assay times, and integration into portable analytical platforms.Pressure-driven microfluidic systems, often integrated with electrochemical biosensors, have demonstrated reliable analytical performance in glucose detection. Several studies have reported high correlation coefficients (R² ≈ 0.95–0.99) and acceptable sensitivity values when compared with conventional laboratory analyzers20,21. For example, chip-based platforms used for monitoring glucose uptake and lactate production have enabled multiplexed analysis with reduced sample volumes while maintaining acceptable reproducibility22. These systems are particularly suitable for quantitative analysis; however, their performance can be influenced by channel design and sample distribution variability, which may affect reproducibility. In contrast, droplet-based platforms enable rapid reaction kinetics through enhanced mixing and reduced diffusion distances. Studies such as that of Kim et al. reported low detection limits (0.03 mM) and short assay time of approximately 3 min, highlighting the potential of droplet systems for rapid biomarker detection23. These advantages notwithstanding, the dependence on optical detection methods, including polarized microscopy, may hinder their practical deployment in POC or portable diagnostic settings.Paper- and thread-based microfluidic devices offer an affordable option, making them well-suited for POC settings. They support straightforward colorimetric or electrochemical readouts and can be combined with portable detector tools such as smartphone-based analyzers and electrochemical biosensors, respectively24,25,26. Nevertheless, their performance tends to suffer from evaporation and poor flow control, which can compromise accuracy and repeatability under certain conditions27. Figure 2b illustrates a schematic design of a thread-based chip for the detection of glucose and urea. A thin layer of polyvinyl chloride (PVC) was applied to prevent evaporation of liquids, thereby reducing heating and improving system performance26. Centrifugal microfluidic systems, often referred to as lab-on-a-disc (LoaD) platforms, provide better automation and fluid management through controlled spin protocols. They allow precise timing and sample distribution, leading to dependable quantitative results with good reproducibility and correlation coefficients21,28. On the other hand, their dependence on external hardware may limit their use in fully resource-limited or decentralized settings. Recent studies have also introduced hybrid and alternative designs, including paper-disc combinations and hydrogel-supported open-channel systems and integrated paper-disc combinations, which have been introduced to improve operational flexibility while reducing overall device complexity29,30. Such designs reflect the continuous effort to strike a balance between analytical performance, ease of use, and manufacturing cost in microfluidic glucose assays.Fig. 2Full size imageRepresentative microfluidic platforms for enzymatic assays. a A diagram of the setup for lactate detection featuring peristaltic pumps and solenoid valves. Air gaps separate different sections, such as the calibration solution and sample solution in an automated pathway. Reproduced with permission from the original source22. b A thread-based chip coated with PVC for the enzymatic detection of glucose and urea, utilizing electrochemical biosensors. PVC improved the performance of the detection process. Reproduced with permission from the original source26. c A centrifugal microfluidic device for various integrated biochemical tests, including enzymatic assays for glucose, cholesterol, and triglycerides, as well as enzyme assays like AST and ALT, referenced in Table 1. Adapted with permission from the original source21.Comparison of various microfluidic platforms for glucose detection indicates that no single platform simultaneously satisfies the requirements for high analytical performance, low cost, ease of use, and clinical applicability. Instead, platform selection depends on the intended application, with electrochemical and droplet-based systems favoring analytical performance, and paper-based and smartphone-assisted platforms prioritizing accessibility and POC deployment.Table 1 summarizes the reported analytical performance of representative microfluidic platforms for glucose assay, highlighting variations in sensitivity, correlation, and detection limits across different system designs. For comparative purposes, the validation level of the reviewed studies was categorized into three groups: proof-of-concept (studies performed using standard or artificial samples), real sample validation (studies evaluated using biological specimens), and clinical validation (studies involving patient samples with comparison against established clinical reference methods). This classification is used consistently throughout the comparative tables.Table 1 Analytical performance of representative microfluidic platforms for glucose detection.Full size tableAmmoniaAmmonia, a product of amino acid metabolism, is an important biomarker for hepatic encephalopathy in patients with liver dysfunction34,35. Enzymatic determination is commonly based on glutamate dehydrogenase (GLDH), where ammonia consumption is coupled with Nicotinamide adenine dinucleotide phosphate (NADPH) oxidation and monitored spectrophotometrically at 340 nm1. Despite its clinical significance, the implementation of ammonia assays in microfluidic platforms remains relatively limited, mainly because of challenges related to enzyme stability, reaction specificity, and the low concentration of ammonia in certain biological samples.Recent studies have nevertheless demonstrated the feasibility of integrating enzymatic ammonia detection into microfluidic devices. For example, Kim et al. developed a wearable PDMS-based microfluidic platform for simultaneous detection of ammonia and ethanol in human sweat. Their multilayer design incorporates a superabsorbent polymer to collect sweat and trigger reagent release into reaction chambers, enabling controlled mixing without external pumping. Ammonia detection was based on an ammonia monooxygenase-mediated colorimetric assay, and the analytical performance was validated against liquid chromatography–mass spectrometry (LC–MS) measurements using digital image analysis35. These findings highlight the potential of wearable microfluidic platforms for enzymatic ammonia analysis in sweat; however, improvements in analytical sensitivity, reproducibility, and portable readout systems are still needed for broader clinical translation.LactateLactate is an important biomarker of tissue oxygenation and metabolic status, with clinical relevance in critical care, metabolic disorders, toxicology, and sports medicine1,36. Enzymatic determination is commonly based on lactate dehydrogenase (LDH) or lactate oxidase-mediated reactions coupled with spectrophotometric or electrochemical detection1,37. Integration of lactate assays into microfluidic systems has opened up a range of analytical possibilities, especially where real-time tracking and very small sample volumes are needed.Electrochemical and chip-based platforms have become widely adopted for continuous lactate monitoring in biological settings. One example is a hybrid microfluidic device with integrated electrochemical sensors that enables simultaneous measurement of glucose and lactate in liver cell cultures22. This system is especially useful for studying cell metabolism because it provides continuous readout while consuming minimal sample volumes. However, its performance may be affected by long-term sensor stability and biofouling, particularly when operating with complex biological fluids (Fig. 2a).Droplet-based microfluidics have shown significant potential for single-cell analysis and high-throughput applications. Several research groups have used droplet encapsulation to follow lactate release from individual cancer cells, shedding light on tumor metabolism and cell-to-cell variability38,39. These systems typically rely on fluorescence detection paired with LDH or lactate oxidase pathways, allowing time-resolved measurements. Monitoring fluorescence over time, rather than just a single endpoint measurement, has been shown to improve accuracy and reveal dynamic metabolic behavior38. For lactate oxidase-based assays, having multiple enzymes together in a confined space along with limited oxygen can trigger side reactions and dampen the signal. To get around this, researchers have proposed multi-step droplet strategies where enzymatic reactions are separated either in space or in time, which helps boost accuracy36. These strategies highlight the critical role of reaction engineering in microfluidic enzymatic systems.Wearable and flexible microfluidic platforms, especially those aimed at sweat analysis, have attracted increasing attention for non-invasive lactate monitoring. Flexible chip- and paper-based devices that incorporate lactate oxidase have demonstrated the feasibility of detecting lactate in human sweat quickly and with very small volumes28,40. These systems often pair with optical or electrochemical detection and can operate with sample volumes as low as 1 µL, delivering results in just seconds28. However, studies have pointed out that factors like how the enzyme is immobilized, the makeup of the sample matrix, and environmental conditions can all have a major impact on signal stability and reproducibility40.More recently, multilayer wearable microfluidic devices incorporating hydrogel-driven osmotic pumping and integrated electrochemical sensors have been developed for continuous lactate monitoring under low sweat conditions, improving operational stability and reducing user intervention41. These designs aim to reduce user intervention, minimize contamination, and maintain stable operation during prolonged monitoring. As illustrated in Fig. 4a, a recently reported microfluidic chip incorporating a backflow-prevention channel has also been reported for continuous lactate monitoring in cell culture. By separating the culture chamber from the detection zone, the device minimizes contamination while enabling fluorescent detection of lactate through a lactate oxidase-mediated assay42. In addition, the integration of controlled flow dynamics within the microchannel network supports stable signal acquisition over extended periods, making the platform suitable for dynamic metabolic analysis.Overall, current microfluidic platforms have substantially expanded the capabilities of enzymatic lactate analysis by enabling continuous monitoring, low-volume operation, and high spatiotemporal resolution. Nevertheless, improvements in enzyme stability, oxygen management, and signal reproducibility remain essential for the broader clinical translation of microfluidic lactate assays.CholesterolCholesterol is an essential lipid involved in membrane structure, steroid hormone synthesis, and bile acid metabolism, and is a key biomarker for cardiovascular risk assessment43,44. Enzymatic determination of total cholesterol typically involves sequential reactions catalyzed by cholesterol esterase (ChEt) and cholesterol oxidase (ChOx), with the generated hydrogen peroxide (H₂O₂) subsequently detected through peroxidase-mediated colorimetric reactions1. The implementation of cholesterol assays in microfluidic platforms is particularly challenging because of the multi-step enzymatic cascade required for accurate analysis, making reaction integration and assay stability critical design considerations.As summarized in Table 2, pressure-driven microfluidic platforms represent the most extensively investigated strategy for cholesterol detection. Most systems integrate electrochemical biosensors, in which nanomaterial-modified electrodes enhance electron-transfer efficiency and analytical performance45,46,47. In addition, precise fluid handling mechanisms, including syringe-controlled flow systems, contribute to enhanced assay reproducibility in chip-based assays48.Table 2 Microfluidic platforms for cholesterol detection: system types, detection strategies, validation level, and key features.Full size tableAlternative detection strategies, including fluorescence-based assays and thermal sensing approaches, have also been reported, demonstrating the adaptability of microfluidic platforms to different detection modalities while highlighting the trade-off between analytical performance and system complexity49,50. However, their practical implementation remains dependent on factors such as instrumentation requirements, integration complexity, and intended application.These disc-based systems enable automated fluid handling and parallel biochemical analysis, delivering results within relatively short timeframes (typically 20–30 min) and making them suitable for integrated lipid panel assessments21,51. However, like other LoaD systems, their dependence on external centrifugation equipment may limit portability and deployment in resource-limited settings.Recent developments have further explored portable and user-friendly detection approaches, including smartphone-based analysis and integrated sample preparation modules such as on-chip plasma separation20,33. These innovations aim to enhance the applicability of microfluidic cholesterol assays in POC settings by reducing reliance on complex laboratory infrastructure. An example of an integrated microfluidic platform for simultaneous diabetes and hyperlipidemia monitoring is presented in Fig. 3.Overall, the studies summarized in Table 2 indicate that electrochemical detection remains the predominant strategy due to its high sensitivity and compatibility with miniaturized systems. In contrast, emerging optical and integrated sample-preparation approaches continue to expand the capabilities of microfluidic cholesterol assays. Nevertheless, the integration of multi-step enzymatic reactions into reliable and reproducible microfluidic platforms remains a major challenge for decentralized testing.Fig. 3Full size imageSchematic illustration of a multi-analyzer chip. a Schematic of the fan-shaped chip, showing the components from the inlet port to the reaction chamber. The stop valves increase flow resistance, thereby directing fluid into previously unfilled chambers. b Photograph of the smartphone-assisted microfluidic chemistry analyzer (SMCA), a miniaturized device integrating an optical detection system, a motion control module, a signal processing unit, and a Bluetooth communication module, with an Android application for smartphone-based operation. c Schematic of the SMCA optical system: a white LED illuminates the reaction chamber from above. At the same time, transmitted light is collected by a macro lens through an aperture below the chip tray and detected by a CCD camera. Adapted with permission from the original source20.Triglyceride (TG)Triglycerides (TG) are the major form of lipid storage in the body and an important biomarker for assessing hyperlipidemia and cardiovascular risk19,51. Clinical enzymatic assays typically employ a sequential reaction involving lipase, glycerokinase, and glycerophosphate oxidase to generate H₂O₂, which is subsequently detected using established colorimetric methods1. The multi-enzyme cascade required for TG determination presents additional challenges for microfluidic integration compared with single-enzyme assays.The implementation of TG assays in microfluidic platforms is more challenging than that of single-enzyme assays because of the multi-step reaction cascade, which requires precise reagent handling and reaction control. Consequently, diverse microfluidic strategies have been developed to balance analytical sensitivity, operational simplicity, and assay stability. As illustrated in Fig. 4, pressure-driven microfluidic systems are frequently combined with different detection modalities, including electrochemical sensors and imaging-based approaches, enabling multi-analyte detection within a single platform.Electrochemical and nanomaterial-enhanced microfluidic systems have demonstrated promising analytical performance for TG detection. For instance, platforms incorporating magnetic nanoparticles as enzyme carriers have enabled efficient immobilization of TG-associated enzymes while maintaining long-term activity53. Nanostructured electrode interfaces improve electron transfer and analytical sensitivity, while replaceable enzyme-based carriers enhance operational flexibility and device lifetime.In parallel, optical and centrifugal microfluidic platforms have been explored for rapid and user-friendly TG analysis. Disc-based microfluidic systems have demonstrated the capability to perform enzymatic TG assays in human serum within short analysis times51. Although automated fluid handling and preloaded reagents improve ease of use and assay automation, dependence on external instrumentation may limit portability.Recent studies have increasingly focused on portable and low-cost microfluidic systems for decentralized TG analysis. Platforms incorporating dry-stabilized reagents, imaging-based analysis, Internet of Things (IoT)-enabled connectivity, and simple optical detection modules have demonstrated clinically relevant analytical performance while reducing reagent consumption and operational complexity54,55,56. The available evidence suggests that microfluidic TG assays reflect a broad spectrum of design strategies, ranging from high-performance nanomaterial-based systems to simplified portable devices. While significant progress has been made, challenges related to multi-step reaction integration, enzyme stability, and system standardization remain major challenges for translating these platforms into routine clinical use.Fig. 4Full size imageRepresentative microfluidic platforms for enzymatic analysis of biochemical parameters. a A pressure-driven chip platform for cell culture and lactate detection using fluorescence microscopy to detect resorufin, the fluorescent reaction product. Reproduced from the original source under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License42. b A microfluidic chip for simultaneous detection of chronic disease biomarkers in whole blood, including glucose, cholesterol, triglycerides, and uric acid, within 21 min. The device incorporates1 a plasma separation membrane within the plasma separation module and2 smartphone-based analysis of the chromogenic reaction. Adapted with permission from the original source33.Urea-BUN and creatinineUrea and creatinine are the primary biomarkers for renal function assessment. Urea is commonly determined through urease-catalyzed hydrolysis, followed by quantification of the generated ammonia using enzymatic or colorimetric reactions1,32. In contrast, creatinine determination typically requires multi-step enzymatic cascades involving creatininase, creatinase, or creatinine deaminase19. The implementation of these assays in microfluidic platforms requires careful consideration of the need for multi-step reaction integration, sample preparation, and reliable enzyme immobilization. As summarized in Table 3, a broad range of microfluidic approaches has been developed to address these challenges. Pressure-driven microfluidic platforms represent the most extensively investigated approach, particularly when integrated with electrochemical biosensors. These platforms offer rapid response times and high sensitivity, making them suitable for real-time monitoring applications57. Improved assay reproducibility has been achieved through the integration of microvalves, pneumatic micropumps, dialysis membranes, and advanced electrochemical sensor designs, including 3D-printed arrays and ratiometric sensing strategies58,59,60.Table 3 Articles investigating on urea/creatinine assay on various types of microfluidic platforms utilizing different detection methods.Full size tableEnzyme immobilization remains a major challenge in urea and creatinine microfluidic assays because it directly influences long-term analytical performance. Various materials have been explored for this purpose, including alginate microbeads, silk fibroin membranes, and hydrogel-based supports, each offering different advantages in terms of biocompatibility and enzyme retention14,32. These approaches aim to extend operational lifetime while maintaining catalytic activity under continuous-flow conditions. Beyond conventional chip-based systems, alternative microfluidic architectures have also been studied. Thread-based and paper-based microfluidic devices provide low-cost and flexible platforms, although they may offer less precise control over fluid dynamics62. Similarly, low-temperature co-fired ceramics (LTCC)-based microfluidic systems have demonstrated the ability to perform assays using very low sample volumes (as low as 1 µL) while maintaining acceptable stability over extended periods64.Centrifugal microfluidic platforms have also been applied to multiplexed renal function biomarker analysis, enabling automated fluid handling and simultaneous determination of urea and creatinine within approximately 15 min using integrated optical detection65. However, their reliance on external spinning instrumentation may limit portability for decentralized testing. Recent advances have introduced hybrid and advanced detection strategies, including the integration of CMOS imaging systems, Raman spectroscopy, and open-channel hydrogel-based designs29,68. These approaches aim to improve sensitivity, reduce system complexity, and enable real-time or minimally invasive monitoring.The studies summarized in Table 3 indicate that electrochemical detection remains the predominant strategy for pressure-driven microfluidic platforms because of its compatibility with miniaturization and rapid response. Continued advances in enzyme stabilization, integrated sample preparation, and multiplexed analysis will be essential for the broader clinical translation of microfluidic renal function assays.Uric acidUric acid is the end product of purine metabolism and an important biomarker for disorders such as gout and renal dysfunction19. Clinical determination is primarily based on uricase-mediated enzymatic reactions that generate H₂O₂, which is subsequently detected using colorimetric, fluorometric, or chemiluminescent methods1,19,69. The integration of these assays into microfluidic platforms has enabled diverse detection strategies with reduced sample consumption and improved suitability for POCT systems.Chemiluminescence-based microfluidic systems have demonstrated high analytical sensitivity owing to their low background noise and efficient optical signal generation. One example is a paper-based microfluidic device integrated with a chemiluminescent biosensor. In such systems, urate oxidase is immobilized inside the microchannels, and the H₂O₂ generated during the reaction subsequently participates in luminescent reactions, producing a signal that directly correlates with uric acid concentration70. Such systems are particularly attractive for low-cost and portable applications, although signal stability and reagent handling may require careful optimization.In addition, fluorescence-based microfluidic platforms have been explored to enhance detection sensitivity through signal amplification strategies. Thin-film microfluidic devices incorporating enzyme immobilization strategies within polymer matrices, such as polyvinyl alcohol (PVA), have been reported to enhance optical signal intensity and enable the analysis of multiple biological fluids, including whole blood and saliva71. The use of resorufin-based detection in such systems provides a sensitive and quantitative readout, although performance may depend on enzyme stability and matrix effects.Multiplexed and integrated microfluidic systems represent another important direction for uric acid detection. Pressure-driven microfluidic platforms have been developed for the simultaneous analysis of multiple chronic disease biomarkers, including uric acid, using uricase-based pathway coupled with colorimetric detection33. The incorporation of smartphone-based readout systems enables quantitative analysis through image processing, while integrated modules such as on-chip plasma separation improve sample handling and reduce sample preparation steps. As illustrated in Fig. 4b, these integrated designs support multi-analyte detection within a single platform, enhancing their potential for POC applications.Collectively, the reviewed studies highlight the adaptability of uric acid assays to a wide range of microfluidic platforms, with pressure-driven and paper-based platforms emerging as particularly suitable for optical detection. Recent advances in multiplexed assay integration and smartphone-assisted analysis further strengthen the potential of these platforms for decentralized monitoring of metabolic disorders.Enzymes assayTissue injury releases intracellular enzymes into the bloodstream, where they serve as important biomarkers of tissue damage. Clinical laboratories routinely measure changes in the activity of these enzymes in serum or plasma, as they are normally present only at low concentrations under physiological conditions. The characteristic pattern of enzyme activity changes provides valuable information for identifying the affected tissue and underlying pathology. The following sections review clinically relevant enzyme assays implemented on microfluidic platforms.Alkaline phosphatase (ALP)Alkaline phosphatase (ALP) is an enzyme that hydrolyzes monophosphate esters under alkaline pH conditions. In clinical diagnostics, serum ALP activity is primarily an indicator of hepatic function; however, its levels can also be altered by non-hepatic factors such as age, neoplasia, endocrine diseases, gestation, and certain drugs19,72. In enzymatic assays, ALP activity is commonly measured through the hydrolysis of p-nitrophenyl phosphate (pNPP), generating yellow p-nitrophenol (pNP), which can be quantified by colorimetric detection at 410 nm19.The integration of ALP assays into microfluidic platforms has enabled a wide range of applications, particularly in immunoassays, liver function testing, and single-cell analysis. Multiplexed biochemical assays and immunoassays employing ALP as a detection enzyme have been extensively implemented on LoaD platforms. These platforms enable automated fluid handling through centrifugo-pneumatic mechanisms and dissolvable film valves, allowing precise control of sequential assay steps73,74. For example, disc-based systems have been developed for liver panel analysis, enabling simultaneous measurement of multiple biomarkers, including ALP, from small sample volumes (~ 50 µL) within approximately 20 min. Reported coefficients of variation (CV) was 7.9%, indicating acceptable reproducibility for clinical applications74. In addition, similar platforms have been adapted for ELISA-based detection, where ALP serves as a reporter enzyme and signal readout is obtained through optical intensity measurements. The incorporation of pneumatic energy storage further enhances fluid control and assay automation, as illustrated in Fig. 5a75.Droplet-based and single-cell microfluidic platforms have also expanded the application of ALP detection to high-resolution biological studies. In cancer research, droplet-based microfluidic systems have been used to track ALP activity down to the single-cell level. This approach enables characterization of cellular heterogeneity and assessment of treatment-induced responses in individual cells76. In such systems, advanced detection techniques, including Surface-Enhanced Resonance Raman Scattering (SERRS), have been employed to improve sensitivity by leveraging nanoparticle-enhanced signal amplification. As illustrated in Fig. 5b, these approaches enable precise quantification of enzymatic activity within individual microdroplets.Similarly, trap-based microfluidic devices have been developed for single-cell analysis in non-mammalian organisms, such as microalgae. These platforms use fluorescence to detect ALP activity and can be integrated with computational tools, including machine learning, to classify cell subpopulations based on differences in enzymatic activity (Fig. 6b)77. Overall, the reviewed studies demonstrate that microfluidic ALP assays span applications ranging from routine clinical diagnostics to single-cell analysis. Centrifugal platforms are particularly well-suited for automated and multiplexed biochemical testing. In contrast, droplet-based and trap-based systems provide powerful tools for investigating enzymatic activity at the single-cell level.Fig. 5Full size imagea Schematic representation of a centrifugal microfluidic platform1. Fluidic layout for immunoassay analysis2. Schematic illustration of antibody capture within individual array chambers75. b Schematic overview of the droplet-based microfluidic SERRS workflow for single-cell encapsulation and detection of secreted ALP. Following cell encapsulation and incubation, ALP activity is quantified at the single-cell level using SERRS76. Both images have been reproduced with permission from the original sources.Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are clinically important biomarkers of liver injury. Liver diseases are the primary cause of elevated serum aminotransferase activities, with ALT generally showing greater increases than AST. Persistent ALT elevation for more than six months is suggestive of chronic hepatitis19. Clinical measurement of AST and ALT is based on coupled enzymatic reactions in which the target aminotransferase is linked to a specific dehydrogenase, and enzyme activity is determined by monitoring NADH absorbance at 340 nm19,21.Microfluidic implementation of ALT and AST assays has largely focused on integrated biochemical panels rather than standalone assays. A centrifugal disc-based microfluidic platform has been developed for the simultaneous determination of ALT and AST activities in human serum using kinetic assays at 340 nm. In addition to these liver function markers, the platform performs multiple biochemical assays within approximately 22 min using optical detection21. Beyond conventional biochemical testing, microfluidic systems have also been applied to model complex physiological processes involving aminotransferases. For example, An and coworkers developed a laminated organ-on-chip device to recapitulate key processes of drug absorption, distribution, metabolism, and excretion (ADME). The multilayer PDMS platform incorporated interconnected microchannels and porous membranes to support the co-culture of hepatocytes with other tissue types, enabling physiologically relevant drug transport and metabolism. Within this system, ALT activity was measured in the culture medium as an indicator of hepatotoxicity and quantified using fluorescence microscopy. As illustrated in Fig. 6a, this organ-on-chip platform provides a physiologically relevant model for investigating hepatic metabolism, drug distribution, and hepatotoxicity while reducing reliance on animal models78. In another study, a modular open-platform microfluidic system was developed for liver function testing, supporting immunoassays, molecular assays, and biochemical analyses, including ALT determination. Individual assays were performed on dedicated microfluidic cartridges incorporating standardized functional modules for fluid storage, valving, mixing, heating, and detection. Depending on the assay type, detection was achieved by optical absorbance for immunoassays and biochemical analyses or fluorescence-based detection for molecular assays. The cartridges were fabricated by injection molding using polymeric materials, facilitating scalable manufacturing. Analytical results obtained with the microfluidic platform showed good agreement with those of conventional laboratory methods79.The reviewed studies indicate that AST and ALT assays have primarily been incorporated into integrated microfluidic platforms rather than developed as standalone diagnostic systems. Their principal value lies in multiplexed liver function testing and organ-on-chip models, where aminotransferase measurements complement the investigation of hepatic physiology, drug metabolism, and hepatotoxicity.Gamma-glutamyl transferase (GGT)Gamma-glutamyl transferase (GGT) is a peptidase involved in γ-glutamyl group transfer and is widely used as a biomarker of hepatobiliary disorders. Although GGT is distributed in several tissues, circulating GGT activity originates primarily from the hepatobiliary system. Because of its limited specificity, GGT is typically interpreted alongside other liver biomarkers rather than as an independent diagnostic marker1,19. Clinical assays are based on the hydrolysis of L-γ-glutamyl-p-nitroanilide in the presence of glycylglycine, producing p-nitroaniline, which is measured spectrophotometrically at 405 nm1,19,74.Similar to AST and ALT assays, microfluidic implementations of GGT assays are relatively limited and are primarily incorporated into multi-analyte liver function platforms rather than developed as standalone systems. For example, centrifugal LoaD platforms enable simultaneous measurement of multiple liver biomarkers, including GGT, from small sample volumes through automated fluid handling. These systems typically employ centrifugo-pneumatic valving combined with dissolvable film valves to achieve automated plasma separation and precise reagent metering, thereby facilitating integrated biochemical analysis with minimal user intervention74. In addition to these integrated diagnostic platforms, microfluidic systems have also been explored in physiologically relevant models. Organ-on-a-chip approaches have incorporated GGT measurement for real-time monitoring of tissue function and toxicity. In such systems, enzymatic activity is assessed using substrate analogs such as γ-glutamyl-p-nitroanilide, while detection can be coupled with optical or fluorescence-based methods to enable continuous analysis without interrupting system operation80. The reviewed studies indicate that GGT assays have primarily been incorporated into integrated microfluidic platforms, particularly liver function panels and organ-on-chip models, rather than developed as standalone assays. This trend reflects both the complementary clinical role of GGT and the inherent advantages of microfluidic technologies for multiplexed biochemical analysis.Fig. 6Full size imageMicroscopic images of cultured and trapped cells on microfluidic platforms. a1 Fluorescence microscopy image of Caco-2 cells stained with Cell Tracker™ Red and Hoechst 33,342;2 HUVEC cell layer stained with Cell Tracker™ Green and Hoechst 33,342; and3 brightfield image of hepatocyte cells cultured on porous membranes for cytotoxicity analysis. Red bars indicate the normalized fluorescence intensity corresponding to ALP activity from the cells. Adapted from the original source under the terms of the Creative Commons Attribution (CC BY) license78. b Fluorescence microscopy images (brightfield and FITC using AP live stain) of trapped C. reinhardtii cells for ALP activity analysis:1 diminished-growth cells and2 activated-growth cells. Adapted with permission from the original source77. c Microfluidic sorting system based on AMY activity of A. oryzae cells within microdroplets. Adapted from the original source under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) license81.Amylase (AMY)Amylase (AMY), together with lipase, is one of the principal enzymes evaluated in pancreatic function assessment. Clinical AMY assays are commonly based on the hydrolysis of synthetic oligosaccharide substrates conjugated to 4-nitrophenyl (4-NP), with the released 4-NP quantified spectrophotometrically at 405 nm19. Fluorogenic substrates have also been employed, particularly in single-cell and high-throughput microfluidic applications82,83.Microfluidic implementations of amylase assays can be broadly categorized into clinical diagnostic platforms and research-oriented high-throughput systems. Microfluidic implementations of amylase assays in clinically relevant settings remain relatively limited but demonstrate a clear trend toward miniaturization and rapid analysis. Several studies have translated conventional spectrophotometric assays into microfluidic formats using small sample volumes and integrated detection systems, achieving results comparable to standard laboratory analyzers. These platforms often incorporate on-chip sample handling components, such as plasma separation and localized reaction zones. They are particularly suited for POC applications due to their reduced assay time and minimal reagent consumption84,85. More recently, droplet-based microfluidic systems have extended these capabilities toward rapid and sensitive measurements. By integrating fluorescence-based detection with enzymatic reactions, these platforms enable fast analysis with high sensitivity while maintaining agreement with conventional methods. The use of fluorogenic substrates and integrated optical detectors, such as photomultiplier tubes, allows continuous signal monitoring and significantly shortens assay duration, in some cases to only a few minutes86. Figure 7b represents a schematic view of this detection system.Beyond clinical diagnostics, droplet microfluidics has emerged as a widely adopted platform for high-throughput enzyme screening and microbial strain engineering. In these systems, individual cells or enzyme variants are encapsulated within microdroplets containing fluorogenic substrates, enabling enzymatic activity to be monitored at the single-cell level through fluorescence signals. Integration of droplet generation, incubation, and fluorescence-activated droplet sorting (FADS) allows rapid screening of large mutant libraries, identification of high-producing microbial strains, and optimization of recombinant enzyme production82,83,87. These platforms have been successfully applied to bacterial, yeast, and fungal systems, highlighting their versatility for industrial biotechnology and enzyme engineering applications.More advanced droplet-based platforms combine droplet microfluidics with downstream analytical technologies such as flow cytometry and omics-based analyses. These integrated workflows enable high-throughput strain selection, subsequent genetic engineering, and detailed phenotypic characterization, thereby extending the role of microfluidics beyond enzyme screening toward industrial biotechnology applications81,88. Representative integrated workflows are illustrated in Figs. 5c and 6a.The reviewed studies indicate two major directions for microfluidic amylase assays. Pressure-driven platforms are primarily aimed at rapid clinical diagnostics and POC testing. In contrast, droplet microfluidics has emerged as a leading approach for high-throughput enzyme screening and microbial strain engineering. These complementary application areas highlight the versatility of microfluidic technologies in addressing both clinical diagnostic needs and biotechnology-oriented research.Fig. 7Full size imageRepresentative microfluidic platforms employing different detection strategies for enzymatic assays. a A droplet microfluidic system for screening yeast cells with high α-AMY production levels from a cell library. Fluorescence microscopy was used to detect AMY secretion by yeast cells, enabling fluorescence-based sorting. Adapted from the original source under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) license88. b A droplet microfluidic setup for real-time detection of pancreatic α-AMY in postoperative patients. A photometry system with a PMT detector analyzed the colorimetric reaction in droplets. Adapted with permission from the original source86. c Chip-based microfluidic platform integrated with an electrochemical biosensor for monitoring multiple analytes, including LDH, urea, uric acid, and methotrexate. Reproduced with permission from the original source60.LipaseLipase (LPS) is a pancreatic enzyme that hydrolyzes lipids at the hydrophobic/hydrophilic interfaces. Although it is primarily produced by the pancreas, small amounts are also derived from the stomach and intestinal mucosa. Clinically, serum lipase activity serves as an important biomarker for pancreatitis19. Clinical lipase assays are based on the hydrolysis of synthetic triglyceride analogs, generating chromogenic or fluorogenic products that are quantified spectrophotometrically or fluorometrically19,89. Unlike several other enzymatic biomarkers discussed in this review, lipase detection in microfluidic systems is predominantly investigated using droplet-based devices, particularly for research-oriented applications such as enzymatic screening and kinetic analysis. As summarized in Table 4, most studies employ fluorescence-based detection strategies, reflecting the compatibility of droplet microfluidics with high-sensitivity optical readouts.Table 4 Summarized articles focusing on the lipase detection on various types of microfluidics.Full size tableDroplet microfluidic platforms have been extensively applied to investigate lipase activity under well-controlled microenvironmental conditions. These systems support kinetic analysis of lipid digestion, enabling evaluation of enzyme activity and reaction dynamics through fluorescence microscopy and related optical techniques90,91. Beyond mechanistic studies, droplet microfluidic technology has become a powerful platform for high-throughput screening of lipolytic enzyme libraries, where individual enzyme variants are compartmentalized into isolated microreactors. Integration of FADS, laser-based detection, and dielectrophoretic sorting enables rapid identification of high-activity enzymes while maintaining high analytical sensitivity92,93,94.In addition to fluorescence-based approaches, alternative detection strategies have also been explored. Photometric detection has been applied in both droplet and pressure-driven microfluidic systems to determine kinetic parameters such as Vmax and Km, demonstrating the feasibility of translating conventional enzymatic analysis into microscale platforms89,95. Moreover, confocal fluorescence microscopy has been used to monitor the release of fluorescent lipid digestion products, such as beta-carotene derivatives, providing spatial and temporal resolution of enzymatic activity within microdroplets96.Collectively, the reviewed studies indicate that microfluidic lipase assays are primarily directed toward mechanistic enzymology, lipid digestion studies, and high-throughput enzyme screening rather than routine clinical diagnostics. The predominance of droplet-based platforms reflects their unique capability to compartmentalize reactions and perform kinetic measurements with high temporal resolution, making them particularly valuable for enzyme engineering and biotechnology applications.Creatine kinase (CK)Creatine kinase (CK) is a clinically important biomarker of skeletal and cardiac muscle injury. Conventional CK assays are based on coupled enzymatic reactions that generate NADPH, which is quantified spectrophotometrically at 340 nm19.Despite the widespread clinical use of enzymatic CK assays, no studies were identified that implemented enzymatic CK detection on microfluidic platforms. This represents a notable gap in the current literature and may provide an opportunity for future development of microfluidic enzymatic assays. Instead, reported microfluidic approaches have primarily relied on immunoassay-based detection using pressure-driven platforms with optical readout98,99,100,101,102. In addition, electrochemical pressure-driven microfluidic systems employing nucleic acid aptamers have been developed for CK detection, providing an alternative affinity-based sensing strategy103,104.Lactate dehydrogenase (LDH)Lactate dehydrogenase (LDH) is a key enzyme in cellular energy metabolism and an important clinical biomarker of tissue injury. Because LDH is widely distributed throughout the body, elevated serum activity is observed in various pathological conditions, particularly hematological malignancies and cancer19. Clinical measurement of LDH activity is based on kinetic spectrophotometric monitoring of NAD⁺/NADH interconversion at 340 nm, where the rate of absorbance change is proportional to enzyme activity1,19.Microfluidic LDH assays have been implemented using antibody-based, droplet-based, and electrochemical detection strategies, reflecting the diverse analytical applications of this enzyme. Antibody-based microfluidic platforms enable the selective capture of LDH from complex biological samples using functionalized membranes, followed by fluorescence-based detection via NADH-mediated resazurin conversion. These systems provide high analytical sensitivity while preserving enzyme activity, making them well-suited for multi-enzyme analysis in microscale formats105. LDH assays have also been incorporated into automated droplet-based microfluidic platforms for multiplexed cytotoxicity assessment, demonstrating the versatility of LDH as a functional biomarker in integrated biochemical analyses106. In parallel, electrochemical microfluidic systems have quantified LDH activity through enzymatically generated NADH using nanostructured electrode interfaces. Integration of these sensing strategies with 3D-printed microfluidic arrays enables simultaneous analysis of multiple biomarkers and supports applications in cancer-related diagnostics60. A representative schematic of this electrochemical platform is shown in Fig. 7c.The reviewed studies indicate that LDH assays have been successfully integrated into diverse microfluidic formats, ranging from antibody-based analytical platforms to multiplexed electrochemical systems. Beyond conventional enzyme quantification, these approaches demonstrate the potential of microfluidics for integrated cytotoxicity assessment, multi-enzyme analysis, and cancer-related biomarker profiling.Newborn screening enzymesNewborn screening enzyme assays are among the most important tests, especially during the first weeks of life, for the early detection of congenital diseases. These tests are performed using dried blood spot (DBS) samples collected from the bottom of the heel. The most commonly studied enzymes for this purpose are lysosomal enzymes, in which dysfunction can cause severe diseases that profoundly affect people’s quality of life. Pompe, Gaucher, Fabry, Hunter, and Hurler diseases, as well as biotinidase deficiency, are well-known diseases in this area1.Like other enzyme tests mentioned above, microfluidic platforms have also been introduced to improve newborn screening tests using DBS samples, and the most common type of microfluidic platform in this field is digital microfluidics (DMF)107. Instead of pumping fluids through continuous channels the way conventional droplet microfluidics does, DMF handles individual droplets sitting on a grid of electrodes, most often relying on electrowetting-on-dielectric, or EWOD. Because droplets can be moved, combined, divided, and stirred on demand, the platform lends itself well to running enzymatic assays automatically and in parallel108.GAA (acid α-glucosidase) for Pompe disease, GLA (α-galactosidase A) for Fabry disease, GBA (β-glucosidase) for Gaucher disease, IDUA (α-L-iduronidase) for Hurler disease, IDS (iduronate-2-sulfatase) for Hunter syndrome, and biotinidase for biotinidase deficiency are six important enzymes that have been tested using DMF, and their commercial platforms have been introduced worldwide107,108,109 (Fig. 8). These DMF platforms have been evaluated at various stages of validation, and some of them have been used to analyze approximately 900 DBS samples110. However, in our review, most of the enzymes investigated using DMF were related to newborn screening, with lysosomal enzymes representing the majority. In all of the studies mentioned above, enzyme activity was measured using optical fluorimetric detection. Collectively, these studies indicated that DMF differentiated normal from affected samples with good accuracy and, in several cases, corresponded closely with conventional fluorometric reference methods. Nevertheless, most failed to report formal estimates of sensitivity and specificity.Fig. 8Full size imageDigital microfluidic platform for multiplex newborn screening of Pompe, Fabry, Hunter, Gaucher, and Hurler diseases. Reproduced with permission from the original source107.DiscussionEnzymatic assays remain essential in biomedical research and clinical diagnostics, yet conventional methods are often limited by long processing times, high reagent consumption, and reliance on centralized laboratory infrastructure111,112. Microfluidic technologies address these challenges by enabling miniaturized, automated, and integrated platforms that reduce sample and reagent consumption, shorten assay times, and support advanced capabilities such as real-time monitoring and multiplexed detection. Despite significant technological advances, however, the clinical adoption of microfluidic enzymatic assays remains limited due to challenges in standardization, long-term enzyme stability, reproducibility, scalable manufacturing, and regulatory validation113.Analysis of the studies included in this review published since 2010, summarized in Fig. 9, reveals distinct application trends among the four major microfluidic platforms. Pressure-driven microfluidics was the most extensively investigated platform for enzymatic assays, whereas droplet-based microfluidics was more frequently employed for enzyme activity studies, particularly microbial screening, enzyme library screening, and single-cell analysis.The observed distribution of studies reflects the distinct strengths and applications of the reviewed microfluidic platforms. Pressure-driven microfluidics predominated in enzymatic assays, likely because of its compatibility with diverse biosensing and detection strategies, although its reliance on external pumping and relatively complex fabrication may limit scalability. In contrast, centrifugal microfluidics offers automated liquid handling, straightforward multiplexing, and simple operation, making it well-suited for POC diagnostics. Droplet-based microfluidics, while providing exceptional throughput, low reagent consumption, and single-cell resolution, is primarily employed for enzyme discovery and microbial screening, with broader clinical translation constrained by its dependence on fluorescence-based detection and sophisticated optical instrumentation. DMF platforms, in contrast, were predominantly applied to newborn screening assays, particularly for lysosomal enzyme activity, with optical fluorimetric detection used across the studies reviewed. Overall, the reviewed studies suggest that platform selection is influenced not only by analytical performance but also by manufacturability and clinical translation requirements.Despite the encouraging analytical performance reported across all four platforms, relatively few studies have progressed beyond proof-of-concept demonstrations to comprehensive clinical validation. Based on our analysis of the reviewed literature, 44 studies were limited to proof-of-concept investigations, 30 included validation using real biological samples, and only 14 involved clinical validation using patient samples together with comparisons against established clinical reference methods. Even among platforms with relatively advanced clinical applications, such as DMF systems for newborn screening, several studies relied primarily on analytical validation or limited comparisons with reference methods, whereas comprehensive diagnostic performance metrics, including formal sensitivity and specificity against established clinical reference standards, were frequently unavailable. Five of these clinically validated studies employed pressure-driven microfluidic platforms40,53,68,71,84, whereas four used centrifugal microfluidic platforms20,21,51,74, four used DMF platforms107,108,109,110 and one utilized droplet microfluidics86. Although pressure-driven platforms accounted for the largest number of clinically validated studies, the limited number of reports precludes definitive conclusions regarding platform superiority for clinical translation. Nevertheless, systematic evaluation of inter-run reproducibility, device-to-device variability, long-term enzyme stability, interference resistance, and large-scale clinical validation remains limited. Consequently, future progress will depend not only on improving analytical sensitivity but also on enhancing reproducibility, standardization, scalable manufacturing, and comprehensive clinical validation.Another important observation is that microscale physicochemical factors should be considered alongside analytical performance in the design of microfluidic enzymatic assays. As discussed earlier, these factors can substantially influence enzyme kinetics, catalytic efficiency, and assay reproducibility. Their integration into device design and optimization will therefore be important for developing robust and clinically translatable microfluidic platforms.Overall, the reviewed studies suggest that pressure-driven and centrifugal platforms are currently better positioned for clinical translation, whereas droplet-based systems remain predominantly research-oriented. Platform selection is therefore influenced not only by analytical performance but also by manufacturability and clinical implementation requirements.Fig. 9Full size imageComparison of the four types of microfluidics reviewed in this article for (a): enzymatic assays and (b): enzyme assays, along with the ratios of the four types of detection methods.Future perspectives and emerging research frontiersThis review suggests that Future progress in microfluidic enzymatic assays will depend not only on continued improvements in analytical performance but also on the integration of emerging technologies that address current barriers to clinical translation.Nanozymes have emerged as a promising avenue for improving the performance and stability of enzymatic assays conducted within microfluidic systems. Since the intrinsic peroxidase-like activity of Fe₃O₄ nanoparticles was first reported, nanozymes have attracted growing research interest as substitutes for, or complements to, natural enzymes, particularly within low-cost paper-based POC testing. Their catalytic mechanisms can differ substantially, with some systems exhibiting self-consuming behavior, whereas others can sustain catalytic activity through regeneration mechanisms. This distinction may influence their suitability for single-use assays versus continuous monitoring applications114. Recent investigations have reported the incorporation of nanozymes into paper-based microfluidic platforms for colorimetric assays, including glucose detection via nanozyme–glucose oxidase systems that serve to amplify the signal115,116,117. Nanozyme-mediated detection has likewise been integrated into centrifugal microfluidic architectures featuring automated sequential reagent delivery and assay processing118. Although applications to date have predominantly addressed biosensing and immunoassays, these developments underscore the feasibility of embedding nanozymes within diverse microfluidic configurations. Further advances in nanozyme-assisted droplet and digital microfluidic systems may enable the convergence of catalytic signal amplification with automated fluid handling and miniaturized detection.Beyond nanozyme integration, artificial intelligence (AI) and machine learning have profoundly contributed to the design and performance improvement of microfluidic platforms in recent years119,120. Advances in ultrasensitive detection have also played an important role in microfluidic-based detection platforms, particularly for single-molecule detection and the analysis of generally low-abundance targets. In addition, multiplexed and multimodal detection methods using complementary signal detectors can improve the simultaneous analysis of multiple targets121. Ultimately, the successful clinical translation of microfluidic technologies depends not only on analytical validation but also on standardized production, regulatory validation, reproducibility, and further development toward commercialization122,123. Collectively, these advances can accelerate the transition of microfluidic enzymatic assays from proof-of-concept studies toward clinically applicable diagnostic tools.Data AvailabilityData from published articles were categorized and summarized graphically.ReferencesHenry’s clinical diagnosis and management by laboratory methods. 24th ed. McPherson RA, Pincus MR, editors. St. Louis, MO: Elsevier; 2021.Google Scholar Siavashy S, Soltani M, Rahimi S, Hosseinali M, Guilandokht Z, Raahemifar K. Recent advancements in microfluidic-based biosensors for detection of genes and proteins: Applications and techniques. Biosensors and Bioelectronics: X. 2024;19:100489.Article CAS Google Scholar Jung W, Han J, Choi J-W, Ahn CH. 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Lab on a Chip. 2024;24(7):1867–74.Article CAS PubMed PubMed Central Google Scholar Download referencesAcknowledgementsThis work was supported by Shahid Beheshti University of Medical Sciences (SBMU) under the Pajoohan Grant 43008197 and by the Iran National Science Foundation under Grant No. 4024817. The authors thank the anonymous reviewers for their constructive comments, which significantly improved the clarity and structure of this manuscript.Author informationAuthors and AffiliationsDepartment of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranBahareh Mohammadi Jobani, Siamak Salami & Zahra ShahsavariBio-microfluidics Laboratory, Department of Electrical Engineering and Information Technology, Iranian Research Organization for Science and Technology, Tehran, IranEsmail PishbinSchool of Mechanical Engineering, Iran University of Science and Technology, Tehran, IranAmin DehghanPathology and Stem cell Research Center, Kerman University of Medical Sciences, Kerman, IranSiamak Salami & Amid RahiAuthorsBahareh Mohammadi JobaniView author publicationsSearch author on:PubMed Google ScholarEsmail PishbinView author publicationsSearch author on:PubMed Google ScholarAmin DehghanView author publicationsSearch author on:PubMed Google ScholarSiamak SalamiView author publicationsSearch author on:PubMed Google ScholarAmid RahiView author publicationsSearch author on:PubMed Google ScholarZahra ShahsavariView author publicationsSearch author on:PubMed Google ScholarContributionsLead Autor: Siamak Salami Conceptualization: Bahareh Mohammadi Jobani. Literature review: Bahareh Mohammadi Jobani, Amin Dehghan. Writing – original draft: Bahareh Mohammadi Jobani, Amin Dehghan. Writing – review & editing: All authors. Visualization: Bahareh Mohammadi Jobani, Amin Dehghan. Supervision: Siamak Salami, Esmail Pishbin, Amid Rahi, Zahra Shahsavari. Validation: Esmail Pishbin, Amid Rahi, Zahra Shahsavari, Siamak Salami. Funding acquisition: Esmail Pishbin, Zahra Shahsavari.Author BiographiesBahareh Mohammadi Jobani is a Ph.D. student in Clinical Biochemistry at Shahid Beheshti University of Medical Sciences, Tehran, Iran. Her research focuses on microfluidics for clinical and biomedical applications, tissue engineering, and cancer cell studies. She has experience in the design and fabrication of microfluidic platforms for biomedical research and diagnostic applications. She developed the original concept and framework of the manuscript.Esmail Pishbin received his Ph.D. in Mechanical Engineering from Iran University of Science and Technology, Iran. He is currently an Assistant Professor and Project Manager at the Iranian Research Organization for Science and Technology (IROST), Tehran, Iran. His research focuses on bioengineering and experimental microfluidic phenomena, with particular interest in biomedical and microfluidic applications.Amin Dehghan received his Master’s degree in mechanical Engineering from Iran University of Science and Technology, Iran. His work focuses on microfluidics and microfabrication, with a strong emphasis on applying engineering solutions to the medical industry. He has expertise in designing and fabricating microfluidic systems, centrifugal microfluidics, droplet manipulation, and lab-on-a-chip technologies, complemented by proficiency in COMSOL Multiphysics and fluid mechanics.Siamak Salami received his Ph.D. from Tarbiat Modares University of Medical Sciences, Tehran, Iran. He is currently a Full Professor and Project Manager in the Department of Clinical Biochemistry at Shahid Beheshti University of Medical Sciences, Tehran, Iran. His research interests include microfluidics for clinical applications, cancer cell studies, and molecular biology. He is the Lead Author of this review, providing senior scientific oversight and conceptual guidance for the manuscript.Amid Rahi received his Ph.D. in Medical Nanotechnology from Shahid Beheshti University of Medical Sciences, Tehran, Iran. He is currently an Assistant Professor at the Pathology and Stem Cell Research Center, Kerman University of Medical Sciences, Kerman, Iran. His research interests include nanoscience, biosensors, and microfluidics for clinical applications.Zahra Shahsavari received her Ph.D. in Clinical Biochemistry from Tarbiat Modares University of Medical Sciences, Tehran, Iran. She is currently an Assistant Professor in the Department of Clinical Biochemistry at Shahid Beheshti University of Medical Sciences, Tehran, Iran. Her research interests include cell-death signaling pathways, cancer cell studies, molecular biology, and microfluidics for clinical applications.Corresponding authorsCorrespondence to Esmail Pishbin, Amid Rahi or Zahra Shahsavari.Ethics declarationsCompeting interestsThe authors declare no competing interests.Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary informationBelow is the link to the electronic supplementary material.Supplementary Material 1 (download JPG )Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. 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