Millifluidic System Research Articles

Biphasic gas–liquid millifluidic system for carbon dioxide plasma chemistry: Application to controlled selective epoxidation

The integration of nonthermal CO2 plasma for olefin epoxidation within a biphasic gas–liquid millifluidic system is presented. In this innovative approach, CO2 is efficiently reduced to CO and O atoms, facilitating the epoxidation of olefins. Analysis of the system’s effluent shows that epoxide production efficiency and yields, optimized to 90 %, significantly surpass those of traditional plasma methods, attributed to the enhanced gas–liquid mass transfer within the millifluidic environment. Moreover, O atoms generated from CO2 decomposition was observed to travel up to 30 mm in the post-plasma discharge area, enhancing the gas–liquid flow system’s efficiency. By capturing O atoms in the epoxidation reaction with olefins, the reverse reaction with CO is minimized, which in turn, markedly increases CO yield under the same conditions without olefins. These findings demonstrate that olefin epoxidation can be activated through CO2 plasma discharge in a controlled, selective, and catalyst-free manner under gentle conditions. This synergy between nonthermal plasma and millifluidic technologies paves the way for overcoming current limitations in plasma chemistry with industrial relevance.

A do-it-yourself benchtop device for highly scalable flow synthesis of protein-based nanoparticles

Synthesis of nanoparticles is typically carried out in batch procedures, which offer limited control of parameters, and a narrow range of possible batch volumes. In contrast, flow synthesis systems, usually having a microfluidic chip as a crucial part, are devoid of these drawbacks. However, large scale devices – millifluidic systems – may offer several advantages over microfluidic systems, such as easier and cheaper production, enhanced throughput, and reduced channel clogging. Here we report a millifluidic system for the generation of protein nanoparticles, using the flow format of the original swift thermal formation technology (STF), which can process batch volume ranging from 100 µl to any practically significant amount. Capabilities of the system are demonstrated with model synthesis of Epirubicin-encapsulated BSA nanoparticles. A better degree of scalability of the synthesis over batch procedure is shown: with a 10-fold working volume increase, hydrodynamic diameter and loading capacity changed by only 10 % and 1 % respectively, compared to 60 % and 30 % for the batch synthesis. Additionally, we provide all engineering drawings, electrical circuits, programming code and nuances of assembly and operation, so that our findings can be easily reproduced. The ease of construction of the device and the superior characteristics of the resulting nanoparticles compared to the batch method indicate application potential in both the biomedical research and industrial spheres.

Hydrogel-Integrated Millifluidic Systems: Advancing the Fabrication of Mucus-Producing Human Intestinal Models.

The luminal surface of the intestinal epithelium is protected by a vital mucus layer, which is essential for lubrication, hydration, and fostering symbiotic bacterial relationships. Replicating and studying this complex mucus structure in vitro presents considerable challenges. To address this, we developed a hydrogel-integrated millifluidic tissue chamber capable of applying precise apical shear stress to intestinal models cultured on flat or 3D structured hydrogel scaffolds with adjustable stiffness. The chamber is designed to accommodate nine hydrogel scaffolds, 3D-printed as flat disks with a storage modulus matching the physiological range of intestinal tissue stiffness (~3.7 kPa) from bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA). Computational fluid dynamics simulations were conducted to confirm a laminar flow profile for both flat and 3D villi-comprising scaffolds in the physiologically relevant regime. The system was initially validated with HT29-MTX seeded hydrogel scaffolds, demonstrating accelerated differentiation, increased mucus production, and enhanced 3D organization under shear stress. These characteristic intestinal tissue features are essential for advanced in vitro models as they critically contribute to a functional barrier. Subsequently, the chamber was challenged with human intestinal stem cells (ISCs) from the terminal ileum. Our findings indicate that biomimicking hydrogel scaffolds, in combination with physiological shear stress, promote multi-lineage differentiation, as evidenced by a gene and protein expression analysis of basic markers and the 3D structural organization of ISCs in the absence of chemical differentiation triggers. The quantitative analysis of the alkaline phosphatase (ALP) activity and secreted mucus demonstrates the functional differentiation of the cells into enterocyte and goblet cell lineages. The millifluidic system, which has been developed and optimized for performance and cost efficiency, enables the creation and modulation of advanced intestinal models under biomimicking conditions, including tunable matrix stiffness and varying fluid shear stresses. Moreover, the readily accessible and scalable mucus-producing cellular tissue models permit comprehensive mucus analysis and the investigation of pathogen interactions and penetration, thereby offering the potential to advance our understanding of intestinal mucus in health and disease.

Efficient numerical modelling of magnetophoresis in millifluidic systems.

Continuous flow magnetophoresis represents a common technique for actively separating particles within a fluid. For separation systems design, accurately predicting particle behaviour helps to characterise system performance, typically measured by the separation efficiency (SE). While finite element method (FEM) simulations offer high accuracy, they demand extensive computational resources. Alternatively, results can be achieved more quickly with simplified numerical models that use analytical descriptions of fluid flow, magnetic fields, and particle movement. In this research, we model a millifluidic system that separates magnetic particles using magnetophoresis. Therefore, we (1) develop a simple numerical model that can simulate continuous flow magnetophoresis for rectangular channels in two and three dimensions, (2) introduce a novel and simple approach to calculate the SE, and (3) quantify the effects of model assumptions in flow profile and dimensions on SE. Our method for estimating SE considers particle flux variation across the channel's cross-section due to the flow profile. The results are compared to an FEM model developed in COMSOL. The obtained three-dimensional simulation model computes results in seconds, around 180 times faster than the FEM approach, while deviating less than 2% from the FEM results. A comparison of the different two-dimensional and three-dimensional models underscores the significant influence of the flow profile and the SE calculation method on the result. The two dimensional models generally overestimate the SE of up to 15% due to their lower peak flow velocity. However, using a constant flow velocity leads to good agreement for high SE due to the overlap of differences in flow profile and SE calculation.

Cobalt/nickel purification by solvent extraction with ionic liquids in millifluidic reactors: From single-channel to numbered-up configuration with solvent recycle

This work describes the continuous separation of cobalt and nickel by solvent extraction in a numbered-up millifluidic system (1 mm ID) using Cyphos IL 101 (C101) as extractant, which is recycled. A dilution of 50 vol% C101 in p-cymene was chosen as the optimal trade-off between extraction performance and hydrodynamic properties of the solvent. The effects of channel length (20–100 cm), flow rate (1–2.5 mL min−1) and temperature (20–70 °C) were investigated on the extraction, scrubbing and stripping performance in a single-channel system. Subsequently, the developed manifold structure (five parallel reactors) was benchmarked against the single-channel system, which displayed a maldistribution of only 1% and performed equally well as the single-channel system. Then, a continuous separation process of one extraction and two countercurrent stripping stages was executed for 6 h with the proven manifold. A cobalt and nickel purity of, respectively, 99.7 and 97.7 % were achieved at a rate of 5 mL min−1. Stable slug flow was observed in the entire system throughout the operating period.

Non-equilibrium ordering of liquid crystalline (LC) films driven by external gradients in surfactant concentration

HypothesisGradients in the concentration of amphiphiles play an important role in many non-equilibrium processes involving complex fluids. Here we explore if non-equilibrium interfacial behaviors of thermotropic (oily) liquid crystals (LCs) can amplify microscopic gradients in surfactant concentration into macroscopic optical signals. ExperimentsWe use a milli-fluidic system to generate gradients in aqueous sodium dodecyl sulfate (SDS) concentration and optically quantify the dynamic ordering of micrometer-thick nematic LC films that contact the gradients. FindingsWe find that the reordering of the LCs is dominated by interfacial shearing by Marangoni flows, thus providing simple methods for rapid mapping of interfacial velocities from a single optical image and investigating the effects of confinement of surfactant-driven interfacial flows. Additionally, we establish that surface advection and surfactant desorption are the two key processes that regulate the interfacial flows, revealing that the dynamic response of the LC can provide rapid and potentially high throughput approaches to measurement of non-equilibrium interfacial properties of amphiphiles. We also observe flow-induced assemblies of microparticles to form at the LC interface, hinting at new non-equilibrium approaches to microparticle assembly. We conclude that dynamic states adopted by LCs in the presence of surfactant concentration gradients provide new opportunities for engineering complex fluids beyond equilibrium.

Enabling continuous flow manufacturing of magnetic nanoparticles with a millifluidic system

From a translational perspective, magnetic nanoparticles (MNP) require manufacturing processes that can be performed reliably and at scale. Continuous manufacturing processes are particularly well-suited for this purpose compared to batch processes, which have high technical variability and low throughput production. In this study, a modularly built millifluidic system for the continuous flow synthesis of MNP by co-precipitation is presented. The system prevents fouling and clogging of passages and enables a reproducible and highly scalable MNP synthesis. The modular assembly allows an independent variation of the input parameters of each module. Here, several input parameters are investigated such as different ratios of iron ions (Fe3+/Fe2+) and base to iron ions (OH–/(Fe3++Fe2+)) as well as flow rates of the used reactant solutions. The basic MNP properties, e.g. core and hydrodynamic size, magnetization values, were determined. Further, magnetic hyperthermia, magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) studies were performed. MNP with core diameters of about 10 nm and low polydispersity index of up to 0.12 were successfully synthesized and show promising characteristics as MRI contrast agents (transverse relaxivity up to (465 ± 14) mM−1s−1), MPI tracers (amplitude ratio A5/A3 of the measured frequency components of up to 0.37) and heating agents (specific loss power of up to (236 ± 26)W/g).

High Temperature Continuous Flow Syntheses of Iron Oxide Nanoflowers Using the Polyol Route in a Multi-Parametric Millifluidic Device.

One of the most versatile routes for the elaboration of nanomaterials in materials science, including the synthesis of magnetic iron oxide nanoclusters, is the high-temperature polyol process. However, despite its versatility, this process still lacks reproducibility and scale-up, in addition to the low yield obtained in final materials. In this work, we demonstrate a home-made multiparametric continuous flow millifluidic system that can operate at high temperatures (up to 400 °C). After optimization, we validate its potential for the production of nanomaterials using the polyol route at 220 °C by elaborating ferrite iron oxide nanoclusters called nanoflowers (CoFe2O4, Fe3O4, MnFe2O4) with well-controlled nanostructure and composition, which are highly demanded due to their physical properties. Moreover, we demonstrate that by using such a continuous process, the chemical yield and reproducibility of the nanoflower synthesis are strongly improved as well as the possibility to produce these nanomaterials on a large scale with quantities up to 45 g per day.

A comprehensive parametric study for understanding the combined millifluidic and dripping encapsulation process and characterisation of oil-loaded capsules

Aim This study aimed to utilise and optimise the millifluidic and dripping encapsulation technique to develop and characterise the oil-core capsules. Methods Sodium alginate with Tween-20 (continuous phase) and sunflower oil (dispersed phase) were used in millifluidic system. After determining the surface and interfacial tensions and flow behaviour parameters, flow rates of phases and concentrations of alginate and Tween were optimised by the Taguchi method. The flow regime of droplets was also evaluated. Optimised millicapsules were characterised concerning morphology, dimension, encapsulation efficiency, SEM, FTIR and, DSC results. Results Dripping flow regime during droplet formation was observed. Reducing the interfacial tension between the continuous and dispersed phases resulted in about a 10.18% reduction in diameter. Optimised millicapsules depicted spherical shape (0.03 ± 0.01) with 3.95 ± 0.05 mm size and 97.5 ± 0.2% encapsulation efficiency. The FTIR and DSC results confirmed the entrapment of oil. Conclusion Millifluidic and dripping method effectively encapsulated sunflower oil in core-shell capsules.

Construction of enzymatic nanoreactors with high catalytic activity in millifluidic systems for cancer therapy

Enzymatic nanoreactors have shown great potential in cancer therapy due to their delighted anticancer results. Synthesizing biocompatible and highly active nanoreactors on a large scale is demanded in clinic translation. Herein, the therapeutic enzymatic nanoreactors with the structure of biocompatible micromaterials covered on enzymes were constructed in the millifluidic device. The results indicated that the nanoreactors were successfully synthesized in the droplet-flow millifluidic device for the first time and had high bio-safety even without post-processing. The nanoreactors could be internalized by cancer cells and possessed long blood circulation time in vivo (t1/2 = 25 h). Moreover, mild synthetic conditions and minimized synthesis pathway empowered nanoreactors with favorable catalytic activity about 96.5% ± 3.9% of native enzymes. Furthermore, enzymatic nanoreactors loaded with glucose oxidase presented a strong anticancer effect and low systemic toxicity. In summary, the assay provides a facile and promising strategy for synthesizing highly active nanoreactors on a large scale.

A millifluidic chip for cultivation of fish embryos and toxicity testing fabricated by 3D printing technology.

Zebrafish (Danio rerio) serves as a popular animal model for in vivo acute toxicity evaluation with the Fish embryo test (FET). Over the last few years there has been an effort to develop various systems for a high-throughput zebrafish embryo cultivation and FET. In this paper, we present a novel design of a millifluidic system fabricated by 3D printing technology and we evaluate its functional properties on Danio rerio embryos cultivation and toxicity testing. The development and the optimization of the millifluidic chip was performed by experimental measurements supported by numerical simulations of mass and momentum transport. The cultivation chip with two inlets and one outlet consisted of two individual channels placed on top of each other and separated by a partition with cultivation chambers. An individual embryo removal functionality, which can be used during the cultivation experiments for selective unloading of any of the cultivated embryos out of the chip, was added to the chip design. This unique property raises the possibility of detailed studies of the selected embryos by additional methods. Long-term (96 hours) perfusion cultivation experiments showed a normal development of zebrafish embryos in the chip. Model toxicity tests were further performed with diluted ethanol as a teratogen. Compared to the FET assays, an increased toxic effect of the ethanol on the embryos cultivated in the chip was observed when the median lethal dose and the percentage of the morphological end-points were evaluated. We conclude that the presented 3D printed chip is suitable for long-term zebrafish embryo cultivations and toxicity testing and can be further developed for the automated assays.

Coexistence of fluorescent Escherichia coli strains in millifluidic droplet reactors.

Understanding competition and cooperation within microbiota is of high fundamental and clinical importance, helping to comprehend species' evolution and biodiversity. We co-encapsulated and cultured two isogenic Escherichia coli strains expressing blue (BFP) and yellow (YFP) fluorescent proteins into numerous emulsion droplets and quantified their growth by employing fluorescence measurements. To characterize and compare the bacterial growth kinetics and behavior in mono and co-culture, we compared the experimental observations with predictions from a simple growth model. Varying the initial ratio (R0) of both cell types injected, we observed a broad landscape from competition to cooperation between both strains in their confined microenvironments depending on start frequency: from a nearly symmetric situation at R0 = 1, up to the domination of one subpopulation when R0 ≫ 1 (or R0 ≪ 1). Due to competition between the strains, their doubling times and final biomass ratios (R1) continuously deviate from the monoculture behavior. The correlation map of the two strains' doubling times reveals that the R0 is one of the critical parameters affecting the competitive interaction between isogenic bacterial strains. Thanks to this strategy, different species of bacteria can be monitored simultaneously in real-time. Further advantages include high statistical output, unaffected bacteria growth, and long-time measurements in a well-mixed environment. We expect that the millifluidic droplet-based reactor can be utilized for practical clinical applications, such as bacterial antibiotic resistance and enzyme reaction kinetics studies.

Stem-Like Cancer Cells in a Dynamic 3D Culture System: A Model to Study Metastatic Cell Adhesion and Anti-Cancer Drugs.

Metastatic spread is mainly sustained by cancer stem cells (CSC), a subpopulation of cancer cells that displays stemness features. CSC are thought to be derived from cancer cells that undergo epithelial to mesenchymal transition (EMT), thus acquiring resistance to anoikis and anti-cancer drugs. After detachment from the primary tumor mass, CSC reach the blood and lymphatic flow, and disseminate to the target tissue. This process is by nature dynamic and in vitro models are quite far from the in vivo situation. In this study, we have tried to reproduce the adhesion process of CSC to a target tissue by using a 3D dynamic cell culture system. We isolated two populations of 3D tumor spheroids displaying CSC-like features from breast carcinoma (MCF-7) and lung carcinoma (A549) cell lines. Human fibroblasts were layered on a polystyrene scaffold placed in a dynamically perfused millifluidic system and then the adhesion of tumor cell derived from spheroids to fibroblasts was investigated under continuous perfusion. After 24 h of perfusion, we found that spheroid cells tightly adhered to fibroblasts layered on the scaffold, as assessed by a scanning electron microscope (SEM). To further investigate mechanisms involved in spheroid cell adhesion to fibroblasts, we tested the effect of three RGD integrin antagonists with different molecular structures on cell adhesion; when injected into the circuit, only cilengitide was able to inhibit cell adhesion to fibroblasts. Although our model needs further refinements and improvements, we do believe this study could represent a promising approach in improving current models to study metastatic infiltration in vitro and a new tool to screen new potential anti-metastatic molecules.

Clogging of drippers caused by suspensions of kaolinite and montmorillonite clays

Clay particles under certain physico-chemical and hydrodynamic conditions can form agglomerates after passing through a filtering system, which favours clogging of the emitters. The main factors interfering with the aggregation potential of clay particles are the type of clay mineral, pH, and ionic strength of the irrigation water. This study analysed the influence of ionic strength and type of clay mineral on clogging, discharge variation, and particle deposition in turbulent-flow non-pressure compensating drippers. Two types of clay (kaolinite and montmorillonite) at a concentration of 500 mg L−1 and four values of ionic strength (0.31, 0.81, 0.02, and 0.01 mol L−1) promoted by the addition of different salts to the solution were used. Clogging tests were conducted with two commercial models of drippers (0.6 and 1.7 L h−1). The deposition zones along the labyrinth channel were analysed using a transparent milli-fluidic system coupled to an optical microscope. Deposition of particles inside dripper labyrinths was observed and this process was strongly influenced by the nature of the clay. The regions of highest particle deposition were vortices zones located in the first baffles of the labyrinths. Kaolinite particles had greater potential of accumulation in the labyrinths than montmorillonite particles. There were fluctuations in the drippers’ discharge during the clogging experiments, but the discharge variations observed were not sufficient to classify the emitters as clogged in any of the test conditions. Clay, as an isolated agent, did not cause full clogging in the emitters evaluated under any of the ionic strength conditions studied. Since particles did not accumulate in the region of the main flow, we suggest that clay particles alone have no potential to cause full clogging of drippers, but may contribute to clogging build-up.

Transport and Kinetic Effects on the Morphology of Silver Nanoparticles in a Millifluidic System

This work investigates the synthesis of silver nanoparticles of different shapes using silver nitrate as a precursor and ascorbic acid as a reducing agent in different reactors. The main focus of this work is to study the effect of capping agent concentrations and ratios of reducing agent to precursor concentrations (reactant ratios) on the shape and size of the nanoparticles synthesized. Three reactor configurations, namely, batch, straight millichannel, and spiral millichannel, are considered to study the transport effect on the morphology of silver nanoparticles synthesized. The kinetic effect on the particle morphology is studied by changing the capping agent concentrations and reactant ratios. It is shown that these variables play an important role in determining the nanoparticle shape and size. In this work, spherical nanoparticles (30–60 nm), triangular plates (80–200 nm), nanorods (130–150 nm), and bent wires (130–150 nm) morphologies are synthesized by controlling thermodynamic and kinetic parame...

Impact of hydrodynamics on clay particle deposition and biofilm development in a labyrinth-channel dripper

To better understand the physical and biological clogging in drip-irrigation, a study was conducted on the impacts of hydrodynamic conditions on clay particle deposition and biofilm development in drippers using an optical method. A transparent milli-fluidic system composed of labyrinth channels was used to identify areas most susceptible to particle clogging using two different types of clay suspensions: sodium bentonite and kaolin. The impact of salt addition [(NaCl) = 200 mg L− 1] on the clay deposition was also analyzed. Biofilm development was studied using the same methodology using a nutritive solution (chemical oxygen demand, COD = 200 mg L−1). In addition, fluid dynamics simulations were performed along the labyrinth channel to understand the effect of flow behaviour on the fouling. Computational fluid dynamics results show two types of flow topology: high velocity in the main flow (around 1 m s− 1) and low velocity in the vortex zones (less than 0.2 m s− 1) found in the channel corners. Using an optical method, kaolin deposition and biofilm growth in the dripper were observed to occur mainly in the inlet channel and initial vortex zones, which are characterized by lower mean velocity and turbulent kinetic energy values. This part of the dripper can be considered as a bottleneck that amplifies the fouling phenomena and which should be optimized. With the addition of NaCl, kaolin particles tend to form bigger flocs. Therefore, more significant particle deposition is observed, but this is not the case of bentonite for which no fouling is observed along the dripper.

Continuous synthesis of gold nanoparticles in micro- and millifluidic systems

Abstract Gold nanomaterials have diverse applications ranging from healthcare and nanomedicine to analytical sciences and catalysis. Microfluidic and millifluidic reactors offer multiple advantages for their synthesis and manufacturing, including controlled or fast mixing, accurate reaction time control and excellent heat transfer. These advantages are demonstrated by reviewing gold nanoparticle synthesis strategies in flow devices. However, there are still challenges to be resolved, such as reactor fouling, particularly if robust manufacturing processes are to be developed to achieve the desired targets in terms of nanoparticle size, size distribution, surface properties, process throughput and robustness. Solutions to these challenges are more effective through a coordinated approach from chemists, engineers and physicists, which has at its core a qualitative and quantitative understanding of the synthesis processes and reactor operation. This is important as nanoparticle synthesis is complex, encompassing multiple phenomena interacting with each other, often taking place at short timescales. The proposed methodology for the development of reactors and processes is generic and contains various interconnected considerations. It aims to be a starting point towards rigorous design procedures for the robust and reproducible continuous flow synthesis of gold nanoparticles. Graphical Abstract:

A Millifluidic System for Analysis of Daphnia magna Locomotory Responses to Water-born Toxicants

Aquatic toxicity testing in environmental monitoring and chemical risk assessment is critical to assess water quality for human use as well as predict impact of pollutants on ecosystems. In recent years, studies have increasingly focused on the relevance of sub-lethal effects of environmental contaminants. Sub-lethal toxicity endpoints such as behavioural responses are highly integrative and have distinct benefits for assessing water quality because they occur rapidly and thus can be used to sense the presence of toxicants. Our work describes a Lab-on-a-Chip system for the automated analysis of freshwater cladoceran Daphnia magna locomotory responses to water-born toxicants. The design combines a Lab-on-a-Chip system for Daphnia sp. culture under perfusion with time-resolved videomicroscopy and software tracking locomotory activity of multiple specimens. The application of the system to analyse the swimming behaviour of water fleas exposed to different concentrations of water-born toxicants demonstrated that Lab-on-a-Chip devices can become important research tools for behavioural ecotoxicology and water quality biomonitoring.

Measurement of reaction kinetics of [177Lu]Lu-DOTA-TATE using a microfluidic system.

Microfluidic synthesis techniques can offer improvement over batch syntheses which are currently used for radiopharmaceutical production. These improvements are, for example, better mixing of reactants, more efficient energy transfer, less radiolysis, faster reaction optimization, and overall improved reaction control. However, scale-up challenges hinder the routine clinical use, so the main advantage is currently the ability to optimize reactions rapidly and with low reactant consumption. Translating those results to clinical systems could be done based on calculations, if kinetic constants and diffusion coefficients were known. This study describes a microfluidic system with which it was possible to determine the kinetic association rate constants for the formation of [177Lu]Lu-DOTA-TATE under conditions currently used for clinical production. The kinetic rate constants showed a temperature dependence that followed the Arrhenius equation, allowing the determination of Arrhenius parameters for a Lu-DOTA conjugate (A = 1.24 ± 0.05 × 1019 M-1 s-1, EA = 109.5 ± 0.1 × 103 J mol-1) for the first time. The required reaction time for the formation of [177Lu]Lu-DOTA-TATE (99% yield) at 80 °C was 44 s in a microfluidic channel (100 μm). Simulations done with COMSOL Multiphysics® indicated that processing clinical amounts (3 mL reaction solution) in less than 12 min is possible in a micro- or milli-fluidic system, if the diameter of the reaction channel is increased to over 500 μm. These results show that a continuous, microfluidic system can become a viable alternative to the conventional, batch-wise radiolabelling technique.

Droplet millifluidics for kinetic study of transketolase.

We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme. The oxaloacetate formed is reduced to malate by NADH in the reaction catalysed by malate dehydrogenase as the second auxiliary enzyme. The extent of oxidation of NADH was measured by spectrophotometry at 340 nm. This system gives a direct, quantitative, generic method to evaluate the TK activity versus different substrates. We demonstrate the accuracy of this strategy to determine the enzymatic kinetic parameters and to study the substrate specificity of a thermostable TK from thermophilic microorganism Geobacillus stearothermophilus, offering promising prospects in biocatalysis. Millifluidic systems are useful in this regard as they can be used to rapidly evaluate the TK activity towards various substrates, and also different sets of conditions, identifying the optimal operating environment while minimizing resource consumption and ensuring high control over the operating conditions.