Metrology, the science of measurement, plays a crucial role in ensuring the accuracy and reliability of flow measurements across various applications. Flow measurements are essential in numerous fields, including healthcare, pharmaceuticals, environmental monitoring, microfluidic technology and industrial processes. Accurate flow measurement is vital for maintaining product quality, ensuring safety, and optimizing performance but most of the instruments used to measure flow rate, especially in health applications and particularly those operating at the micro and nanoscale, have not been sufficiently studied regarding their flow accuracy and traceability. This lack of comprehensive research and validation raises concerns about the reliability and precision of these measurements, which are critical for ensuring patient safety and effective treatment outcomes. Furthermore, the absence of established protocols and standards for calibrating these instruments at such low flow ranges exacerbates the issue. Without standardized calibration procedures, it is challenging to verify the accuracy of flow measurements, leading to potential discrepancies and inconsistencies in the results. Addressing these gaps through rigorous studies and the development of robust calibration methods is essential for advancing the reliability and efficacy of flow rate measurements. In this work, new methodologies for calibration of syringe pumps, flow meters and microfluidic chips were developed and validated. The primary objective of this work is to enhance the gravimetric procedure described by Bissig et al. (Batista et al., 2020a) and to develop three innovative methods to ensure the traceability of micro and nanoflow measuring instruments. These instruments are increasingly being introduced to the market and are used in various applications, such as healthcare. In this work, various calibration methods were employed to measure flow, including gravimetry and newly developed techniques such as interferometry, pending drop, and the front track method. The front track method, in particular, was also described by Ogheard et al. (Bissig et al., 2015).

The gravimetric method, currently used at the IPQ-LVC laboratory, was improved and the lower limit 120 μL/h was extended to 10 μL/h. In the interferometry technique, an interferometer is used to measure the distance travelled by a pusher block of a syringe pump, over time, to determine the flow rate. The pending drop method uses high-resolution cameras to determine the growth of a drop over time. In the front track method, the cameras follow the meniscus of the liquid displacement in a close tube over time.

The methods developed in this work were validated internally or externally by the participation of IPQ-LVC in the EURAMET pilot project 1508 (Batista et al., 2020b).

The information described in this paper was the basis for the development of EURAMET guide cg 27 - Guidelines for the Calibration of Drug Delivery Devices and Infusion Device Analysers (EURAMET guide cg 27, 2024).

The development of the new methods has also allowed flow measurements (Batista et al., 2024) to be extended to another field–microfluidics and led to the publication of a new EURAMET Technical Guide 4 - Evaluation of flow related quantities in microfluidic devices (EURAMET, 2024).

The gravimetric method, interferometric method, front track and pending drop method were used to calibrate various microflow measuring instruments, namely, syringes pumps, flow meters and microfluidic chips in different flow ranges. The results were compared in terms of % of error and uncertainty mainly due to manufactures specification information. In general, 20 to 30 points were collected in each performed test.

The measurement error presented in this paper was determined according to the International Vocabulary of Metrology (VIM) (BIPM et al., 2012), as is defined as the measured quantity value minus a reference quantity value, Equation 1. Metrological error : A M e t r o = Q s e t − Q r e f Q r e f 100 % (1) where:

A _Metro is the relative flow measurement error or systematic error as defined by VIM (BIPM et al., 2012), Q _ref is the reference flow rate determined by the reference measurement method (e.g., gravimetric method), Q _set is the flow rate set or the indicated flow rate at the instrument under calibration (e.g., 1 mL/h).

A precision Nexus 3,000 pump with 1 mL glass syringe was calibrated using the methods described above at the following flow rates: 1,000 μL/h, 500 μL/h, 100 μL/h, 10 μL/h, 1 μL/h.

The results are presented in Figure 6 and Table 5.

From the figure above it can be seen that the results from all the methods are consistent on all points.

The method with the larger uncertainty in Figure 6 is the pending drop method, which is recommended to be used only above 100 mL/h with an expanded uncertainty from 5% to 10%.

In the gravimetric method, it was possible to measure down to 10 μL/h with an acceptable expanded uncertainty of 2.6%; this allowed a decrease of range in the volume and flow laboratory of IPQ that was previously of 120 mL/h with 2.5% uncertainty.

The front track method can go to 1 μL/h with an expanded uncertainty of 7%.

The method with the smaller uncertainty is the interferometric method, especially at low flow rates. However, the instruments need to have an external piston for this method to be employed.

Tests were also performed with a Cetoni pump (Figure 7) at 0.1 mL/h using a 100 μL glass syringe with the interferometric method. The results were really outstanding, with an error of 1.7% and an expanded uncertainty of 1.9%.

This interferometric method can calibrate flow meters and syringe pumps from 5,000 mL/h down to 1.6 nL/min (0.1 μL/h) with an expanded uncertainty range of (1.9–0.9) %. The calibration of a thermal Sensirion flow meter (Figure 8) was performed with the interferometer method and the front track method, tested at 1,500 nL/ min, 1,000 nL/min, 500 nL/min, 100 nL/min, 70 nL/min, 50 nL/min and 20 nL/min.

The results are presented Table 6 and Figure 8.

It can be verified from Figure 9 that the results are consistent in both methods used in the tests, and the uncertainty in general, smaller for the interferometer method.

In order to test the methods with an instrument used in real life application a perfusor space BBraun syringe pump, used in hospitals to administrate drug to patients (Figure 10), was calibrated with water at flow rates 5,000 μL/h, 1,000 μL/h, 500 μL/h, 100 μL/h, using the methods described in section 2.

The results presented in Figure 11 are consistent for all methods and in all flow rate points. The uncertainty values are very similar for all methods regarding each calibration point. More results on this pump can be found in (Batista, 2022).

Finally, a microfluidic Lab-On-a-Chip device (Figure 12) for passive mixing and magnetic separation of bioanalytes with square channel cross-section and obstacles that promotes a mixture of components for sample preparation was also characterized using the front track method and the gravimetric method.

The chips were tested at 600 μL/h because this is the flow rate at which this chip is used. Water was used as a calibration liquid. The total acquisition time was 15 min, with one data point obtained at every 30 s. Tests were performed with and without the chip and three replicates were performed for each method. All the results, including the average of the replicates, are presented in Figure 13.

The results in Figure 13 were consistent for the two methods used. The uncertainties values were very similar for the two methods, being higher for the front track method, probably due to the short acquisition time arising from the limitations of the capillary used.

To validate the methods developed, especially the interferometric method that gives a smaller uncertainty and can go down to 0.1 μL/h, IPQ participated in EURAMET project 1508. The results for the calibration of a Cetoni precision pump are presented in Figure 14 (EURAMET, 2022). The results of IPQ are consistent with the reference value, which was estimated based on the weighting mean of all participants at all points.

In Figure 15 is possible to see summarizes the range of application and uncertainty of the methodologies developed in the scope of this work for flow measurements.

The developed interferometric method demonstrated exceptional performance, enabling flow measurements as low as 0.1 μL/h with uncertainty values below 2%. It is compatible with any flow generator equipped with an external motor, where the pusher block and the interferometer reflector cube are added (e.g., Nexus syringe pump). However, despite its precision, the method can be costly to implement in a laboratory setting due to the high price of the required instrumentation. Additionally, successful deployment necessitates specialized technical knowledge in interferometry.

The front track method offers a viable option for measuring flow rates as low as 0.1 μL/h across various flow generators, flow meters, insulin pumps and microfluidic chips. However, the method currently exhibits high uncertainty levels. To improve accuracy, testing with smaller capillaries is recommended, as this could reduce the measurement range and extend testing time—potentially achieving acceptable uncertainty levels between 2% and 3%. Despite its limitations, the method is characterized by its ease of implementation, straightforward handling procedures, and low cost.

In this work, the gravimetric method was successfully extended to measure flow rates down to 10 μL/h, compared to the previous limit of 100 μL/h. However, there remains potential for further improvement in both measurement range and uncertainty. This method is compatible with any flow generator, flow meter, or microfluidic device that work down to 10 μL/h, though it generally exhibits higher uncertainty than the interferometric method. Similar to the front track method, it is easy to implement and operate, and it involves relatively low costs.

The pending drop method also presents significant potential for improvement, particularly in controlling evaporation, which currently contributes to higher uncertainty values compared to the interferometric, gravimetric, and front track methods. Additionally, its operational range is more limited. Nevertheless, the method is highly versatile, suitable for use with many microfluidic or flow measurement device that work up to 100 μL/h. It is also easy to use and cost-effective to implement in a laboratory setting.

The implementation of the methodologies explored in this work was designed to enable the calibration of various types of instruments with different characteristics, such as precision syringes, perfusion syringes, microchips, and flowmeters, which are commonly used in the health and pharmaceutical industries as well as in microfluidic technologies. This calibration is crucial for ensuring the accuracy and reliability of these instruments.

It is essential that this information is disseminated not only to the scientific community but also to the medical and biomedical communities. Sharing these findings with healthcare professionals and researchers can foster collaboration and innovation, leading to further advancements in medical technology and practices. Additionally, manufacturers of medical instruments and other flow devices should be informed of these methodologies to ensure that their products meet the highest standards of accuracy and reliability.

In this work, the gravimetric method was improved for microflow measurements but there is still room for improvement. Three new methods were developed, and microfluidic chips were manipulated and tested for the first time at IPQ-LVC.

The interferometric method had the best performance regarding all methods tested but has some limitation in the type of instrument used (it must have an external motor) and cost of installation. The front track method is a good option that can be used for any type of flow measuring instrument or microfluidic device but more work is needed in order to decrease the uncertainty. The pending drop method has the worst performance of the four methods but is easy to use and low-cost i2mplementation.

All methods were internally validated by comparison with each other in the calibration of several flow measuring instruments.

This work served as the basis for the development of the EURAMET Guide CG-27, “Guidelines for the Calibration of Drug Delivery Devices and Infusion Device Analysers” (EURAMET guide cg 27, 2024). This document provides standardized procedures for testing microflow and nanoflow instruments aiming to improve the accuracy and comparability of measurement results Additionally, it contributed to the publication of the new EURAMET Technical Guide 4, “Evaluation of Flow-Related Quantities in Microfluidic Devices (EURAMET, 2024), but despite advancements in flow measurement techniques, challenges remain, particularly in microfluidics applications such has organ-on-chip. Ensuring traceability and accuracy at these scales requires ongoing research and development.

The development of standardized calibration methods and advanced measurement techniques for validating manufacturing, performance, and safety are essential in shaping the future of healthcare and microfluidic technology. The new EPM MFMET II project aims to fill these gaps by developing protocols and guidelines to support standardization efforts (Mfmet.eu, 2025).

Metrology is a key enabler of innovation and patient safety in healthcare. By ensuring the accuracy and reliability of drug delivery systems and diagnostic devices, like organ-on-chips, metrology helps reduce errors, enhance treatment efficacy, and save lives.

Going forward, standardized calibration methods, advanced measurement techniques, and collaborative research will be essential in shaping the future of healthcare technology.