Advanced microfluidic systems with temperature modulation for biological applications Free

Lab-on-a-chip devices, ranging from simple fluid-handling systems to sophisticated platforms with integrated optical, electrical, and thermal controls, have become indispensable in modern biological applications. These devices offer significant advantages, including drastically reduced sample volumes and analysis times.¹ Among their many functionalities, precise control of environmental conditions—especially temperature modulation—is a cornerstone for replicating physiological conditions in numerous biological applications,² rapid thermal cycling for DNA amplification in polymerase-chain-reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays,^3,4 localized hyperthermia for cancer treatment,^5–7 and maintaining optimal conditions for cell cultures, protein stability, and specific cellular activities.⁸ Such innovations have significantly expanded the utility of lab-on-a-chip systems, from point-of-care diagnostics to high-throughput drug screening, as exemplified by applications like SARS-CoV-2 detection.⁹ For example, by controlling protein digestion parameters—such as reaction temperature and analyte concentration—within these lab-on-a-chip devices, real-time mass spectrometric analysis of the resulting peptide fragments can be achieved.¹⁰ Despite these advances, traditional temperature control methods often fail to meet the unique scale and material constraints of microfluidic systems. Achieving precise and localized temperature control within these devices requires addressing challenges, such as non-uniform heat distribution,¹¹ thermal isolation,¹² and the seamless integration of microheaters with other microfluidic components.¹³ This review explores the specific temperature requirements for diverse biological applications and examines recent system developments designed to overcome these challenges, thereby enabling enhanced functionality and broader adoption of lab-on-a-chip technologies.

Precise and efficient thermal control is essential for numerous biomedical applications, particularly in epidemic prevention and personalized medicine. In particular, techniques like PCR and LAMP rely on rapid thermal cycling to amplify nucleic acids in a short time. Microfluidics-based miniaturized thermal cycling systems enhance these processes by offering reduced reaction volumes, minimized heat transfer times, lower power consumption, and improved sensitivity, resulting in faster and more efficient operations than conventional systems.^14,15 For instance, successful plasmid DNA amplification using lab-on-a-chip devices [Fig. 1(a)] demonstrates their effectiveness in accelerating thermal cycling workflows.^16,17

Recent advancements have enabled highly parallelized reverse transcription-PCR chips for single-cell gene expression analysis, capable of detecting RNA templates at extremely low copy numbers.¹⁸ Integrating PCR amplification with electrophoretic analysis further enhances the speed and accuracy of genetic testing, while improvements in thermal uniformity in microthermal cyclers significantly boost amplification reliability.^19,20 In contrast, LAMP amplifies DNA at a constant temperature [typically 60–70  $°$C, Fig. 1(b)], using specially designed primers and DNA polymerase with strand displacement activity.²¹ This allows for faster amplification compared to PCR, which requires precise temperature cycling through multiple thermal stages. However, despite its advantages, LAMP faces challenges, such as non-specific amplification, which can lead to false positives. Additionally, the detection methods in LAMP, such as fluorescence or turbidity measurement, are often less precise compared to the quantitative analysis capabilities of PCR-based devices. Advances in primer design, polymerase optimization, and enhanced detection techniques are crucial to overcoming these limitations.

When integrated into compact systems, these microfluidic chips hold great potential for portable and on-site diagnostics.²² For example, battery-powered mini thermal cyclers facilitate rapid DNA typing for forensic applications.²³ Moreover, solar-powered PCR systems eliminate the need for external power, enabling nucleic acid diagnostics in resource-limited settings.²⁴ These innovations allow for the rapid detection of infectious diseases and genetic disorders, thereby improving access to medical services for early disease diagnosis and monitoring.²⁵ 

Hyperthermia therapy, which raises tissue temperatures to therapeutic levels (40–43  $°$C), is a highly effective and selective strategy for targeting cancer cells,^26,27 as illustrated in Fig. 1(c). Unlike normal tissues, tumor tissues exhibit abnormal responses to elevated temperatures, making them particularly vulnerable to heat-induced damage. Integrating hyperthermia with microfluidic platforms enhances our understanding of cell death mechanisms and thermal resistance, enabling the optimization of treatment parameters for specific tumor types.²⁸ This approach is especially promising for personalized therapies, as microfluidic devices allow precise testing on patient-derived tumor samples, tailoring treatments to individual cases.²⁹ Beyond its direct effects, hyperthermia synergizes with other treatments, such as radiation and chemotherapy, by inducing heat shock proteins that stimulate immune responses for innovative combinations with immunotherapy.³⁰ 

Thermal lysis, which employs localized heating (⁠ $≥$90  $°$C) to release intracellular contents such as DNA and proteins, is another critical application of temperature control in microfluidics³¹ as shown in Fig. 1(d). Microfluidic platforms offer significant advantages for cell lysis, including automation, integration, and miniaturization, which make them particularly valuable in resource-limited settings.³² Compared to other chemical or mechanical-based cell-lysis techniques, thermal lysis is a reagent-free and rapid approach that integrates seamlessly into lab-on-a-chip technologies. Using precise and localized heating mechanisms, such as resistive or microcantilever heaters, these platforms enable efficient intracellular content release.^33,34 Furthermore, combining thermal lysis with complementary methods like alkaline treatment enhances DNA recovery, yielding high-quality templates for advanced applications such as single-cell genomics.³⁵ 

Both hyperthermia and thermal lysis exemplify the potential of microfluidics in precision medicine and diagnostics. Optimizing these techniques, which includes ensuring the integrity of sub-cellular components, maximizing efficiency, and minimizing damage, remains critical for their continued advancement in clinical and research settings.^36,37

In cell culture applications, maintaining a physiologically stable temperature is essential to ensure uniform cell growth and viability, bio-molecule manipulation, or cell separation. For instance, research integrating a temperature control system into polymer-based cell culture chips has demonstrated long-term stable thermal conditions for drug testing and long-term cell observation.³⁸ Temperature gradient focusing, which balances electrophoretic mobility and bulk flow, is another example of effective temperature control that enables high-resolution separation of analytes like proteins and DNA.³⁹ Also, there is a study that uses negative dielectrophoresis barriers to prevent local overheating and cell damage near electrodes.⁴⁰ Innovative approaches, such as integrating conductive paths within microchannels, allow precise temperature control and gradient maintenance, enabling non-equilibrium studies and enhancing the versatility of microfluidic devices.⁴¹ These systems are particularly valuable for thermally activated reactions, such as enzymatic processes and protein structure analysis, highlighting the diverse applications of controlled-temperature systems in chemical and biological research.

Magnetic nanoparticles, particularly iron oxide, are widely used for hyperthermia therapies under alternating magnetic fields.^42–44 This localized heating approach raises tissue temperatures to therapeutic levels and can be precisely controlled for diverse biomedical applications [Fig. 2(a)]. As an experimental validation, magnetic nanoparticles achieved rapid, stable temperature reaching 93  $°$C after 22 min at a concentration of 10 mg/ml.⁴⁵ Earlier investigations also demonstrated that cuboidal superparamagnetic iron oxide nanoparticles can achieve high specific absorption rates,⁴⁶ while in vivo work on head and neck cancer models showed tumor center temperatures reaching approximately 40  $°$C within 5–10 min, resulting in significant tumor cell destruction.⁴⁷ 

Beyond oncology, nanoparticles have found utility in accelerating thermal cycling for PCR. A notable example is a nanodroplet PCR system employing infrared laser-assisted heating to complete 40 cycles in just 370 s, without affecting adjacent droplets.⁴⁸ However, challenges remain in achieving uniform nanoparticle distribution and consistent heating profiles in heterogeneous tissues. Real-time monitoring methods, such as luminescent nanoparticle thermosensors or embedded probes, can help overcome these issues by providing continuous feedback on thermal gradients and permitting dynamic adjustments in power or nanoparticle concentration. Additionally, careful consideration of particle size, shape, and surface functionalization is crucial for maintaining colloidal stability and minimizing toxicity, thereby ensuring repeatable heating outcomes.⁴⁶ Despite these challenges, nanoparticle-based induction heating presents substantial opportunities for enhanced temperature control, faster processing times, and increased efficacy in both clinical and research settings.

Photothermal heating utilizes the conversion of absorbed light energy into heat, driven by localized surface plasmon resonance (LSPR) or the intrinsic absorption of nanomaterials. This mechanism enables precise temperature control at the microscale, enhancing applications in biosensing, cell lysis, and thermal property measurements.^49–51 Laser-assisted heating in gold-based materials and plasmonic nanoparticle droplets is reported to be capable of rapid, sub-second temperature modulation.⁵⁰ For instance, various gold nanostructures—nanoislands,⁵² nanoparticles (AuNPs),⁵³ and nanorods (AuNRs)⁵⁴—demonstrate photothermal efficacy for cell lysis, pathogen destruction, and biomarker detection, leveraging strong LSPR properties to generate localized heat. Additionally, nanodiamonds have been explored for dual photothermal therapy and in situ temperature sensing, underscoring the technique’s versatility and reliability in microfluidic environments.⁵¹ 

Future advancements in photothermal microfluidic systems should focus on addressing specific challenges to expand their applicability in biological research and medicine. Key priorities include improving biocompatibility by developing non-toxic photothermal materials and reducing power consumption to make systems more sustainable and portable. Integrating photothermal functionality with advanced sensing modalities—such as real-time fluorescence or Raman spectroscopy—could enable simultaneous analysis and treatment, broadening applications in personalized medicine. Moreover, scaling up photothermal microfluidics for high-throughput workflows, such as drug screening or clinical sample processing, remains an essential goal.

The integration of solid heaters into bulk microfluidic chips is the most convenient and easiest way for achieving precise temperature control in biological applications. This has been extensively studied, particularly regarding thermal uniformity, power consumption, and system complexity. External heaters, such as Peltier devices, have been widely used in applications requiring accurate temperature control over relatively large areas (e.g., cell culture and chemical synthesis).^2,8 A notable example involves a modular temperature control system employing individually addressable Peltier elements, which optimizes temperature gradients across the microfluidic chip.⁵⁵ 

For structuring microchannel, polydimethylsiloxane (PDMS) is a commonly used material for disposable PCR systems due to its low cost, easy fabrication, and biocompatibility [Fig. 2(b)].⁵⁶ For applications requiring transparent heating, indium tin oxide (ITO) films have also been successfully incorporated into PDMS microchips to provide efficient temperature control [Fig. 2(c)].⁵⁷ However, despite these advancements, fully integrating and miniaturizing temperature control in PDMS-based microfluidic devices remains challenging. One limitation is the relatively low thermal conductivity PDMS, often causing uneven temperature distribution. This has been addressed by developing ceramic-based PCR devices with closed-loop temperature monitoring, which improve temperature uniformity.⁵⁸ In addition, active compensation mechanisms in microheater arrays can significantly enhance thermal uniformity beyond traditional block-heater designs,¹⁹ and micro-immersion heaters that induce thermal convection within reaction chambers have been shown to improve temperature control performance.⁵⁹ 

Moving from PDMS-specific designs to more general bulk microfluidic platforms, the integration of solid heaters is one of the most straightforward ways to achieve precise temperature regulation. Design innovations further enhance heater performance in these bulk or printed circuit board (PCB)-based microfluidic systems. For instance, serpentine-shaped electrodes have been shown to improve temperature uniformity across PCR chips, while symmetric electrode arrangements exploit thermal crosstalk to maintain consistent heating profiles and boost PCR efficiency⁶⁰ [Fig. 2(d)].

While increasing complexity, heater-integrated microfluidic MEMS devices offer superior thermal management owing to their high precision and rapid thermal response.⁶¹ Silicon, known for its high thermal conductivity, facilitates fast temperature cycling and precise control.⁶² Accordingly, silicon-based micromachined PCR chips are introduced with platinum thin-film heaters and temperature sensors for efficient heat transfer.¹⁷ Some designs also utilize insulated membranes with embedded microheaters, achieving both low power consumption and fast thermal response to assist rapid nucleic acid amplification.²² These MEMS platforms are not limited to PCR; heater-integrated structures have been employed in membrane reactors for protein digestion and identification. Moreover, SiC electrodes have gained attention because of their excellent thermal/electrical properties, high thermoresistive sensitivity, and capacity to mitigate leakage current at elevated temperatures [Fig. 2(e)]. Researchers have demonstrated efficient and fully integrated thermal management solutions in SiC-based microheater-integrated system by addressing the current leakage issues at high temperature as they transfer of SiC onto a SiO $2$ substrate.⁶³ 

In addition to thermal cycling, heater-integrated microfluidics also have a great potential in calorimetry, which requires precise thermal measurements and sample amount control. Thus, highly sensitive microfluidic calorimeters have been developed using heater and thermometer-integrated MEMS to measure nanoscale thermal responses. For instance, parylene-based microfluidic calorimeters combine vacuum encapsulation for thermal isolation and gold resistive heaters to achieve exceptional sensitivity and control (4.2 nW resolution), allowing precise measurements of biochemical reactions at a single-nanoliter scale.¹² Another example is a microfluidic calorimeter for single-cell studies, such as in situ lysis and protein interaction analysis, where heater integration ensures a stable and localized thermal environment during protein–protein interaction assays.⁶⁴ These examples demonstrate the importance of integrating heaters within microfluidic calorimeters to enhance the resolution and reliability of thermal measurements, particularly for applications that require sensitivity to minute thermal variations.

Real-time and localized temperature monitoring is essential for controlling temperature-sensitive biomedical processes, such as enzymatic reactions, PCR, and cell culture. Moreover, maintaining a good repeatability at optimal temperature conditions is necessary to prevent sample degradation and ensure experimental reproducibility. Various approaches have been developed to address the challenges of in situ temperature measurement in microfluidic systems. One common strategy is to integrate a temperature sensor alongside a heater and regulate the temperature via feedback control. For example, multiscale integrated temperature/flow velocity sensor patches have been embedded into PDMS microfluidic chips, measuring temperatures up to 100  $°$C with a resolution of 1.25  $°$C and capturing real-time temperature changes from 23 to 50  $°$C in microchannels.⁶⁵ Another approach involves thermoelectric microfluidic sensors leveraging a Bi/Sb thin-film thermopile, which can detect extremely small temperature variations down to $10 − 4$ over a $1.2×6$ cm $2$ junction [Fig. 3(a)].⁶⁶ Using this thermopile, the heat released during DNA hybridization reaction was successfully measured.⁶⁷ While such large thermopiles excel at measuring subtle temperature fluctuations, they primarily measure the average temperature across the entire device rather than providing localized temperature data. In many biomedical applications, however, small temperature gradients at the microscale can critically affect reaction kinetics and cell responses. Consequently, localized sensing solutions are needed.

In this context, Geitenbeek et al.⁶⁸ introduced a locally integrated temperature-sensing strategy [Fig. 3(b)] to address the limitations of large thermopiles. Their droplet-flow silicon/glass microfluidic chip incorporates built-in platinum heaters and two platinum temperature sensors (TS1 and TS2), allowing on-chip heating and real-time local temperature monitoring within 150  $μ$m-high, 200  $μ$m-wide channels. They further explored PDMS/glass and glass/glass chips using platinum wires or electrodes for integrated heating and sensing, achieving accuracies of about 0.34 up to 120  $°$C. In a separate study, a 500  $μ$m-sized pyroelectric temperature sensor using polyvinylidene fluoride demonstrated rapid and sensitive monitoring at 0.3  $°$C accuracy,⁶⁹ underscoring the ongoing drive for miniaturized, localized thermal detection in microfluidics.

While significant progress has been made, fully integrating temperature sensors into microfluidic platforms for specific applications remains challenging. Factors such as sensor size, sensitivity, and compatibility with diverse microenvironments must all be carefully balanced. Future sensing technologies should aim for high spatial resolution, minimal noise, and robust integration to ensure reliable and reproducible thermal measurements essential for advanced biological processes.

Localized temperature measurements using fluorescent or luminescent dyes have been extensively explored in biomicrofluidics due to their in situ monitoring capability, high spatial resolution, and relatively straightforward implementation.⁷⁰ As depicted in Fig. 3(c),⁶⁸ placed luminescent particles between two resistance temperature detectors maintained at 40 and 60  $°$C, respectively, and employed confocal luminescence spectroscopy to validate the localized temperature changes between them. Their approach uses ratiometric band shape luminescence thermometry based on thermally coupled energy levels of Er³⁺ in NaYF₄ nanoparticles, enabling accurate in situ temperature mapping up to at least 120  $°$C with a precision of 0.34  $°$C. The spatial resolution is further improved down to approximately 9  $μ$m, making it highly suitable for applications that demand high-resolution thermal measurements.

Additional techniques include fluorescence lifetime imaging microscopy combined with optical tweezers, which enable non-invasive temperature measurements using temperature-sensitive fluorescent microprobes.⁷¹ By leveraging fluorescent dyes in PDMS microchannels, temperatures up to 100  $°$C can be measured with a resolution of 1.25  $°$C, facilitating real-time observation of thermal gradients between 23 and 50  $°$C.⁶⁵ Moreover, CdSe/ZnS quantum dots can serve as temperature indicators for droplet-based heating, synchronized with AuNPs or AuNRs under photothermal laser irradiation.⁵⁰ 

Recently, nanodiamonds with nitrogen-vacancy (NV) centers have gained prominence for their excellent biocompatibility and nanoscale temperature-sensing capabilities, enabling investigations of intricate cellular processes.^72,73 NV centers can operate at room temperature and detect nanoscale fields, making them suitable for applications ranging from cancer biomarker detection to neural activity monitoring.⁷⁴ The optically detected magnetic resonance (ODMR) spectra of these NV centers are used in temperature sensing through the linear response of zero-field splitting to thermal variations.^75,76 Integrating NV-based nanodiamonds into biomicrofluidic platforms opens up advanced applications, including nanoscale thermometry inside a cell for localized heating and selective cell-cycle acceleration with focused laser beams,⁷⁷ as well as photothermal therapy for cancer treatments.⁵¹ By providing precise, real-time temperature monitoring, these biocompatible localized thermometry methods not only enhance our understanding of cell death mechanisms but also optimize parameters for cancer therapies and other temperature-sensitive biomedical processes.

Building on their capability as high-resolution temperature sensors, nanodiamonds containing NV centers also show significant promise for magnetic field detection.⁷⁸ Although the ODMR spectra of NV centers are widely used for accurate temperature sensing (via the linear response of zero-field splitting to temperature changes), these same quantum properties allow NV centers to function as nanoscale magnetometers.⁷⁹ As illustrated in Fig. 4(a), a 532 nm laser optically pumps and reads out the spin states (⁠ $m s=0,±1$⁠) through fluorescence, while the Zeeman splitting (⁠ $2 γ N VB$⁠) induced by the external magnetic field enables precise measurement of the field strength.

Recent studies have also investigated the integration of NV centers with complementary metal–oxide–semiconductor technology to create compact, portable quantum sensors for biomedical applications.⁸⁰ Beyond NV centers, other quantum-based approaches such as superconducting quantum interference devices and spin-exchange relaxation-free atomic magnetometers have proven valuable for magnetocardiography and the magnetic imaging of living cells.⁸¹ Remote chip-scale quantum sensors now enable magnetic field measurements in unshielded environments, offering high sensitivity and spatial resolution for medical diagnostics.⁸² Potential future uses include probing neural activity, monitoring magnetic nanoparticles for targeted drug delivery, and studying intracellular biochemical processes in real time.⁸³ The biocompatibility and stability characteristics of nanodiamonds are particularly useful for in vivo applications such as mapping magnetic fields within tissues or detecting paramagnetic ions in physiological media.

The suspended microchannel resonator (SMR) is a MEMS device that uses a resonance frequency to measure the mass of particles or cells flowing through an embedded microchannel^84,85 [Fig. 4(b)]. SMRs have revolutionized biological and biomedical fields by enabling real-time mass measurements of individual cells,⁸⁶ particles,⁸⁷ and biomolecules with attogram-level precision.⁸⁸ This capability enables researchers to monitor critical processes such as cell growth, division, and drug responses.⁸⁹ In biomedical applications, the SMR has facilitated the detection of tumor cells,⁹⁰ characterization of extracellular vesicles, and precise quantification of single-cell biophysical properties,⁹¹ providing critical insights into disease mechanisms and potential therapeutic targets.⁹² The integration of heaters into SMRs has enhanced their functionality, enabling simultaneous thermal sensing and mechanical measurements.⁹³ For more effective and precise temperature control, heater-integrated microchannel resonators have been developed,⁹⁴ though not yet widely applied in biological systems. With the aid of simultaneous thermal and mechanical sensing, precise thermophysical property measurements of liquids filled in the microchannel are implemented. Unlike conventional biomedical devices with flat, non-resonant surfaces, the resonating structure of the SMR is more susceptible to external environmental factors, such as temperature variations caused by forced convection. Convective heat loss is minimized by maintaining atmospheric pressure within the channel while placing the surrounding structures under vacuum.⁹⁵ Future directions also include combining SMRs with other spectroscopic methods such as micro-Raman spectroscopy for noncontact temperature measurements or additional feature extraction like chemical fingerprints.^96,97

Despite the promise of SMRs for high-throughput and highly accurate measurements, challenges remain in fabrication and scalability. Recent efforts have shifted from optical measurements to integrated electrical readouts and parallelized sensor arrays, capable of screening up to 40 000 particles per hour.⁸⁷ However, to translate this level of performance into practical use, it is crucial to incorporate not only sensor parallelization but also careful microfluidic design. Very small channel diameters can impose additional challenges when analyzing large sample volumes, because variable particle sizes lead to significant size-distribution effects. A potential solution may involve using a preprocessing chip for size-based separation, routing particles into separate SMRs optimized for specific size ranges. Another key limitation is the high cost and complexity of sensor fabrication. The mainstream approach to SMR fabrication typically requires a multi-mask photolithography process (often exceeding 20 masks) and two wafer-bonding steps to embed silicon channels—an inherently difficult and expensive procedure. Alternatives such as the silicon-on-nothing approach^98,99 aim to streamline channel embedding in resonator fabrication, but yet achieved in optically measured silicon dioxide microtubes. Thus, achieving practical designs for high-throughput with integrated electrodes remains an ongoing challenge.

Incorporating advanced sensor modalities—such as quantum magnetometers based on NV centers and SMRs (Fig. 5)—represents a major step toward building more comprehensive biomedical platforms. NV centers enable highly sensitive, nanoscale temperature and magnetic field detection for applications like neural mapping and cancer biomarker analysis, while SMRs permit real-time mass measurements at the single-cell level. Recent innovations in SMR technology, including on-chip heaters and resistive thermometry,⁹⁴ now enable direct measurement of thermal properties within microchannels. Even before the integration of the system, facilitating the thermal sensing with SMR for the analysis of processes like DNA denaturation⁹³ is studied. Additionally, both silicon-¹⁰⁰ and glass-based¹⁰¹ SMRs, combined with optical spectrum and reflectivity measurements, have been used to monitor bacterial or tumor cell viability. Looking ahead, incorporating nanodiamond structures with NV centers inside microfluidic channels could provide quantum sensing capabilities—ranging from pH and temperature to magnetic and electric fields—while simultaneously performing mass measurements. These multimodal platforms also open possibilities for advanced microfluidic control, where integrated electrodes can direct nanomaterial delivery to precise locations in wet samples, expanding potential applications in nanoscale manipulation¹⁰² and drug delivery.

Achieving these multi-modal functionalities in a single, miniaturized device will require careful attention to fabrication, microfluidic design, and sensor parallelization. Automated and modular systems could expedite clinical and industrial translation by enabling high-throughput applications in drug screening and personalized medicine. For instance, coupling SMR-based mass measurements with microcalorimetry may elucidate how metabolic pathways influence both heat output and cell mass, while fluorescence-based temperature sensors combined with NV-center quantum sensors promise finer resolution of intracellular thermal gradients. Even in-cell nuclear magnetic resonance (NMR) could be integrated to provide chemical/biological structural and dynamic data in real time. Ultimately, with streamlined fabrication and cost-effective solutions, these next-generation platforms could make multi-modal sensing accessible to a wider community of researchers, driving further innovation in both fundamental biological research and clinical diagnostics.

This research was conducted with the support of the Technology Innovation Program (No. 00144157, Development of Heterogeneous Multi-Sensor MicroSystem Platform) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea), the Korea Evaluation Institute of Industrial Technology (KEIT) funded by the 2024 Materials and Components Technology Development Program (Ministry of Trade, Industry and Energy, Republic of Korea) (No. RS-2024-00443505), and the National Research Foundation of Korea (NRF) grants funded by the Korean government (Ministry of Science and ICT) (No. RS-2024-00416835).

The authors have no conflicts to disclose.

J. Ko: Conceptualization (lead); Investigation (lead); Writing – original draft (lead); Writing – review & editing (lead). J. Lee: Funding acquisition (lead); Supervision (lead); Writing – original draft (supporting); Writing – review & editing (supporting).

The data that support the findings of this study are available within the article.