Droplet digital PCR (dPCR)
The science of microfluidic liquid handling for droplet digital PCR (ddPCR)
- Higher accuracy
- Higher sensitivity
- Absolute quantitation
Main applications using droplet digital PCR technology
Liquid biopsy
Liquid biopsies are non-invasive tests performed on blood samples to detect cancer cells circulating in the blood (circulating tumor cells, CTCs) or pieces of DNA from tumor cells in the blood. This technique is increasingly used for cancer detection and monitoring, as it is low risk for the patient and helps doctors understand what kind of molecular changes are taking place in the tumor. Droplet Digital PCR (ddPC) provides the level of sensitivity required for liquid biopsy.
Copy number variation
A key measurement challenge in diagnostic research involves identifying small changes in nucleic acid sequence that are commonly associated with genetic diseases. Changes in the genomic DNA leading to an abnormal copy of a DNA sequence are called copy number variations (CNVs). They are present in complex diseases such as Down’s Syndrome and many cancers.
Pathogen Detection and Microbiome Analysis
Droplet digital PCR is extensively used in microbiology.. Digital PCR’s ability to amplify low concentration targets in complex backgrounds and show higher sensitivity than standard PCR makes it the technology choice for microbiome analysis.
Resources
Importance of fluid handling for droplet digital PCR applications
Droplet digital PCR relies on random distribution of dPCR mix containing target molecules on the partition of equivalent volumes (here, the droplets). In this way, some partitions contain no target molecules, while the remaining partitions contain at least one molecule. Partitions are next categorized and counted as positive or negative depending on their fluorescence intensity. The calculations are derived from a Poisson model, which can be impacted by partition volume since the model assumes the partition to be monodisperse. As a consequence, a heterogeneous droplet population can affect the droplet digital PCR process. In fact, several groups demonstrated through different studies that partition droplet volume variability can cause a bias on the accuracy of the measurements.
More information can be found in the paper written by Emslie et al.: Droplet Volume Variability and Impact on Digital PCR Copy Number Concentration Measurements The impact of flow rate on droplet size using a microfluidic system is today well described in the literature. Thus, to avoid heterogeneous droplet populations that can affect the droplet digital PCR process, one should consider using a precise flow controller.
Flow control systems for digital PCR
Flow rate stability is thus critical for having repeatable reactor volumes and reproducible results in droplet digital PCR experiments. Syringe pumps are commonly used for generating droplets. Depending on the model in use, syringe pumps show limited flow control. As a consequence, the droplet size, proportional to the flow rate, is affected. In addition, the actual flow rate cannot be monitored with such devices. The flow rate value is displayed on the device, but no information on the time required for reaching a set flow rate is given (the time for flow equilibrium may vary depending on the microfluidic setup, and flow can oscillate depending on the instrument). An alternative to syringe pumps is pressure-based flow controllers. These show that high-precision flow control, fast reaction time, and flow monitoring are possible.
We compared the production of water-in-oil emulsions using microfluidic syringe pumps and pressure-based flow controllers. Using pressure control, the desired droplet size is quickly obtained (< 6 s), and monodisperse droplet generation is ensured over time. Thus pressure controllers are the instruments of choice for droplet digital PCR.
The benefits of choosing Fluigent for your droplet digital PCR system
- Best in class stability: < 0.5% due to our field-proven, patented FASTAB™ technology allowing optimal flow control with the robustness required in demanding industrial environments.
- Straightforward workflow automation included in Fluigent’s software
- An expert engineering time specializing in microfluidic design and mechanical and software integration
References
1. Whale, A. S. et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Research40, (2012).
2. Emslie, K. R. et al. Droplet volume variability and impact on digital pcr copy number concentration measurements. Analytical Chemistry91, 4124–4131 (2019).
3. Emslie, K. R. et al. Supporting information Droplet volume variability and impact on digital PCR copy number concentration measurements Author names and affiliations.
Introduction to digital PCR
What is digital PCR?
Digital droplet-based assays offer promising opportunities for the absolute quantitation of low concentration analytic species. During the last decade digital-PCR assay (dPCR) became one of the most prominent assays for this class of analytical methods.
For performing the assay, the sample volume is split into multiple droplets in such a way that each droplet contains either one or none of the target DNA molecules.
Due to the small droplet volume, the PCR reaction runs very efficiently even from a single molecule.
How does digital PCR work?
During amplification, a fluorescent dye is formed or activated. The positive droplets become fluorescent. Absolute quantitation of the number of target molecules is simplified to the count of fluorescence active droplets in the generated droplet collection. Not regarding the simplicity of the approach, its technical implementation is challenged by stabilizing the droplets collected over the complete assay avoiding unwanted droplet coalescence or crosstalk between the droplet ingredients. This has been solved by utilizing perfluorinated mineral oils as the carrier oil in combination with advanced perfluorinated surfactants, which stabilize the emulsion and avoid crosstalk and DNA exchange between the individual droplets.
In this application note we are investigating the usability of the commercially available surfactant dSurf for an exemplary digital PCR assay.
What are the materials and equipment for digital PCR experimentation?
The Fluigent droplet kit was employed for the experiments, utilizing the Fluigent EZ Drop chip with three microfluidic droplet generators per chip. Interconnection between the chip and fluid reservoirs was achieved using 2m PEEK 1/32″ tubing with an outer diameter of .010″ and two sleeves with 1/16″ outer diameter, .033″ inner diameter, and 1.6″ length. More details and dimensions of the droplet generation chip can be found on the Fluigent website.
Pressure-driven flow control was managed through the „Fluigent-MFCSTM-EZ“ pressure control system. DNA amplification took place using the Eppendorf Mastercycler Gradient thermocycler. For optical readout, droplets were loaded into a disposable 10 µl cell counting chamber called „Countess™“ without a grid, manufactured by EVETM NanoEnTek.
Image acquisition involved a standard fluorescence microscope (Axiovert-MAT-M, Carl Zeiss AG, Germany) equipped with a Zeiss Fluar 10x magnification NA 0.5 objective, HBO 100 light source, FITC-filter set, and an Andor-Neo sCMOS camera (Oxford Instruments, Abingdon, UK) with a 5-second exposure time for fluorescence images.
What is the method of dpcr?
Droplets were generated at a working pressure of 240 mbar for the dSurf and 140 mbar for the PCR-Mix. The chip was connected with PEEK 1/32” tubing OD x .010” and 2x sleeves 1/16” OD x.033” ID x 1.6”, tubing length: 200 mm. Generated droplets were collected into a 0.2 ml PCR vial. Amplification was performed in a conventional thermocycler with the following settings:
To acquire images, the amplified droplets were transferred to a cell counting chamber for brightfield and fluorescence imaging. The droplets needed to be arranged as a monolayer within the chamber for effective readout. This was achieved by loading 10 µl of the droplet suspension into a pipette tip and allowing the droplets to rise. The entire volume was then loaded into the chamber, starting with the pure fraction of the continuous phase to ensure the droplets were injected into the partially pre-filled chamber. After loading, the rear slit of the chamber was sealed with adhesive tape to minimize evaporation and prevent droplet motion or rearrangement during the readout process.
dPCR assay data analysis
The parameter settings of the Fluigent-MFCSTM-EZ pressure control system, as described in the Materials and Methods section, were used to generate droplets for dPCR samples. Figure 2 illustrates the observed characteristics of the generated surfactant droplets, including the droplet generation regime, size, and frequency. The average droplet size was measured to be 70 µm, with a volume of 180 pL.
Conclusion
The experiments have shown that the dSurf surfactant is suitable for scientific as well as routine digital PCR applications. The generated droplets were homogeneous in shape and size. Superior droplet stability of the dSurf surfactant system was observed during the amplification process. A few droplets have dissipated during the experiments, but this can be neglected.
The reproducibility of the experiments was also confirmed. Droplet generation with identical parameters leads to identical droplet size and quality. Summarily, dSurf can be employed as a surfactant composition for digital droplet-based assays, and therefore, for digital PCR assay.
References
1. Pohl, G. and I.-M. Shih, Principle and applications of digital PCR. Expert review of molecular diagnostics, 2004. 4(1): p. 41-47.
2. Huggett, J.F., S. Cowen, and C.A. Foy, Considerations for digital PCR as an accurate molecular diagnostic tool. Clinical chemistry, 2015. 61(1): p. 79-88.
3. Quan, P.-L., M. Sauzade, and E. Brouzes, dPCR: a technology review. Sensors, 2018. 18(4): p. 1271.
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MICROFLUIDICS in DROPLET DIGITAL PCR
Discover - Application notes
High-throughput cell DNA screening using digital PCR
Discover Droplet Digital PCR (ddPCR)
Discover
Introduction to DNA Screening Using Digital PCR
Polymerase Chain Reaction, or PCR, has an almost ubiquitous presence in biomedical sciences. Due to the ease of execution and the exquisite sensitivity offered by PCR, which covers amplification from a single template molecule up to about nine orders of magnitude, the applications of PCR are widespread. This includes targeted mutagenesis, DNA screening using digital PCR, virus diagnosis and analysis, and more. (1).
Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets make it possible to considerably reduce sample volumes, which lowers costs. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable previously inaccessible high-throughput screening applications, including single-cell and single-molecule assays (2).
Droplet digital PCR (ddPCR) is based on the amplification of single target DNA molecules in many separate droplets, which provides a new method for accurate quantification of DNA copy numbers. In a ddPCR assay, the distribution of target DNA molecules among the reactions follow Poisson statistics, which means that the majority of reactions contain either one or zero target DNA molecules. DdPCR has several advantages over more traditional qPCR methods.
- It enables the absolute quantification of target nucleic acid without the reliance on rate-based measurements and the need to use calibration curves.
- It demonstrates high sensitivity and precision for low-copy-number target nucleic acids. (2,3).
In this application note, the objective is to use Fluigent products to perform DNA screenings using digital PCR by isolating individual DNA molecules and analyzing the enzymes resulting from their expression.
How to perform high-throughput digital DNA screening
Materials: Products
Fluigent pressure-driven flow control solutions are ideal for high-throughput droplet generation for DNA screening, as the excellent flow stability offered by this technology ensures high droplet monodispersity, a controlled encapsulation rate, and experimental reproducibility.
Methods
For these studies, it is important that the droplets be generated at the correct frequency and at a uniform size. Using the MFCS™-EZ in this experiment is critical, as the device generates stable flows and precise control of the different phases.
Part one: generation of the pcr/dna droplets
To perform DNA high-throughput digital screening using digital PCR, two separate emulsions were first generated. The first one encapsulated an aqueous phase of a PCR and DNA mixture in an organic continuous phase on a dedicated chip. To do this, the initial sample is injected into the microfluidic droplet generator, where it is cut into highly-monodisperse droplets by a perpendicular stream of perfluorinated oil (figure 2). Each droplet contains either one or zero molecules of DNA. The amplification step triggers the activation or production of a fluorescent dye in the droplets containing the targeted DNA. Counting the number of fluorescent droplets is equivalent to counting the number of DNA molecules.
Part two: fusion of one pcr droplet with one ivt droplet generated on a chip
A green fluorescent marker is added to the PCR primers to make them visible at the end of the process. These droplets either contain DNA to be enhanced or are empty. An orange dye is also added to measure the size of the droplet, where the intensity of the light captured by a CCD camera is proportional to the size of the droplet.
For DNA screenings using digital PCR, after amplification, the PCR droplets must be injected into a second chip. This chip generates IVT droplets, while also synchronizing them to create one PCR droplet that is inserted between two IVT droplets. The IVT droplets contain an orange dye to differentiate the contents. Fusion is processed by the application of an electric field.
Results
Our set-up features fast start-up and an automated configuration that allows the experiment to be performed without constant adjustments, thus saving time and enabling systemic screening. By continuously measuring the lows and pressures, the performance of the fluidic system can be evaluated at a glance, providing full control of the process.
In our experimental DNA screening using digital PCR, we obtained a 1/1 fusion at an 85% to 88% success rate (no fusion: 10%, double fusion: 5%) with stable and synchronized decays.
This graph represents a count of droplets with given orange and green signals. The darker red dot signifies a large number of droplets with these characteristics, while light blue represents a low number of droplets. The green signal shows the presence of DNA in the PCR droplet, and the orange one is relevant for detecting the occurrence of fusion.
Conclusion
To perform DNA screening using digital PCR, it is essential to have high stability in order to obtain reproducible and optimal results. Flow-rate control solutions based on pressure actuation provide:
- High droplet monodispersity (even at low low-rates over long time periods.)
- Straightforward, easily automated setup
- Precise volume and low control – even with complex, multi-channel chips to minimize cross-talk between low channels.
- Ability to detect and compensate for small disruptions such as air bubbles.
Related resources
References
- Hoshino, T. and Inagaki, F. (2012) “Molecular quantification of environmental DNA using microfluidics and digital PCR,” Systematic and Applied Microbiology, 35(6), pp. 390–395. Available at: https://doi.org/10.1016/j.syapm.2012.06.006.
- Payne, E.M. et al. (2020) “High-throughput screening by droplet microfluidics: Perspective into key challenges and future prospects,” Lab on a Chip, 20(13), pp. 2247–2262. Available at: https://doi.org/10.1039/d0lc00347f.
- Markey, A.L., Mohr, S. and Day, P.J.R. (2010) “High-throughput droplet PCR,” Methods, 50(4), pp. 277–281. Available at: https://doi.org/10.1016/j.ymeth.2010.01.030.
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Features of Opto High-Speed Camera
Plug & Play solution
The high-speed microscope comes ready to use. It connects to a PC via a USB 3.1 cable. The device includes a fast camera with fixed optic parameters, allowing for highly reproducible results.
Ultra-compact & robust
With dimensions of only W140 x L193 x H40, the high-speed microscope is one of the most compact units available. It allows users to save bench space.
Microscopy for microfluidics
The digital camera and optics are optimized to fit with microfluidic applications. With 5X magnification, fast image acquisition of up to 1000 fps, high resolution (360LP/mm), and high contrast transmission though brightfield transmitted light (4000K), the microscope can track droplets and other fast-moving particles in microfluidic systems. The large FoV of (1.3 x 0.84 mm) and the working distance of 13 mm allow users to work with many types of microfluidic setups.
Optimized Microfluidic software
The OptoViewer software allows for visualization, photography, and video recording. The software offers many features including adjustable imaging settings (exposure time, framerate, gain, and more) and annotations, which are ideal for microfluidic experiments. In addition, the integrated droplet plug-in allows for live microfluidic droplet measurement and counting.
Related applications
High-speed microscope for microfluidics: “Track Droplet” software plugin
The OptoViewer software features a “Track Droplets” plugin. Thisallows for droplet/particle analysis. Single cells or microfluidic droplets can be automatically detected, tracked, velocity measured, and volume determined or read out inline. All statistics are displayed and can be saved.
- Droplet count
- Droplet per second
- Average droplet size
Combine with Fluigent Pressure – Flow Controllers for the most reliable results
In microfluidic devices, droplet size and generation rate are directly linked to the flow rates of the liquid solution phases . Fluigent pressure controllers provide highly stable flow rates (0.1% CV pressure, < 5% of measured flow rate value) with excellent response times (< 1s), producing highly homogenous droplets or particles for long periods of time.
Microscopy for droplet microfluidics
Water-in-oil and oil-in-water emulsion production
An emulsion refers to a mixture of two liquids that are normally unable to mix. It consists of small droplets of one liquid suspended within another liquid. Specifically, in oil-in-water (O/W) emulsions, tiny droplets of oil are dispersed and enclosed within the continuous water phase. In water in oil (W/O) emulsions, small droplets of water are dispersed within the continuous oil phase.
These emulsion techniques, O/W and W/O are extensively utilized in various OEM and research settings to create droplets, hydrogel beads, polymer beads, or wax beads.
Fluigent has published several application notes on this topic, introducing cutting-edge technology that involves the use of a high-speed microscope. This tool enables researchers to observe the production of high-quality monodispersed droplets.
Figure 1: Water-in-oil droplets generated by Fluigent controllers and chips, visualized through the microscope.
Read our application notes:
Double emulsion production
In addition to its use in single-emulsion experiments, our fast microscope for microfluidics can also be utilized in setups involving double emulsions. The creation of double emulsions hold great promise in various academic and industrial fields. Examples include fragrance manufacturing in the cosmetics industry, food applications, drug delivery in pharmaceutics, and more.
The application note “Double Emulsion Generation” introduces Fluigent’s Double Emulsion Production Pack: a comprehensive system equipped with a microscope. This system enables the production of monodisperse double emulsions. It features the RayDrop, developed and manufactured by Secoya, to facilitate one-step production of double emulsions using a single device.
Figure 2: System setup for double emulsions
Read our application note:
Microparticles Synthesis
Fluigent technology enables the production of highly monodispersed microparticles or microcapsules. Several materials are used depending on the final application, including alginates, PLGA, or agarose microparticles. To achieve a high level of uniformity, the RayDrop is utilized as part of a microfluidic setup that includes the digital high-speed microscope.
Figure 3: System for PLGA microparticle production.
See our application notes for microparticles production:
“I see great potential for our collaboration. As Opto, we can support FLUIGENT in optics, microscopy, automation, and software. Our small and compact microscopy solutions are perfect for the combination with microfluidics (compact, cost-efficient, scalable, high quality made in Germany).
The greatest benefit for the users is our free AI- based Software. It enables the recognition and tracking of objects like particles or droplets. This input is further used to control the pump. This is what makes our common package unique.”
Daniel Kraus | Product Manager Biophotonics at Opto GmbH | Project Manager at SpectroNet
Specifications Microscope
Magnification | 5X |
FoV [mm] | 1.3 x 0.84 |
Resolution [LP|mm] | 360 |
Working Distance WD [mm] | 13 |
Object Space Resolution [μm/Pixel] | 0.7 |
Depth of Field DoF [mm] | 0.008 |
Sensor | IMX392LQR-C| 2.35 MP | Colour | 166 fps* *At maximum resolution. By reducing pixels area, fps can go up to ~1000fps |
Dimensions [mm] | W140 x L193 x H40 |
Weight [g] | 1400 |
Interface | USB3.1 Gen 1 Type C |
Illumination | Transmitted light | white| 4000K |
Control Software | OptoViewer 2.0 |
Certifications | CE / RoHS / WEEE-Reg.-No. DE 68564667 |
Markus Riedi, CEO Opto GmbH
Expertise & resources
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Accessories
I. Droplet-based microfluidics
Microfluidic generation of droplets has attracted a lot of interest, enabling high throughput experiments by generating millions of micro-reactors and chambers in a few seconds. This method produces highly monodispersed droplets of very small volumes (μL to fL) of fluids with high frequency (up to hundreds of kHz), providing better control of processes like mixing, encapsulation, sorting, and sensing. Microfluidic-based droplets have many diverse and varied applications, such as particle synthesis3 and chemical analysis4. Highly controlled droplet production also enables single-cell analysis or drug testing 5,6.
Microfluidic-based droplet generation and control allow for:
- Highly monodispersed (<2% size variation) droplet production, with potentially high generation rate (up to hundreds of kHz)
- Highly reproducible complex structures (water-in-oil-in-water emulsions, multiple encapsulations…)
- Single droplet manipulation as an individual pL scale biochemical reactor
- Miniaturization of production and bioanalytical devices
With these characteristics, droplet microfluidics has a large value, including bio(chemical) analysis, and nano and microscale generation of materials7. Several chip designs exist to generate droplets. A common design is the T-junction, where the dispersed phase is injected from a channel that is perpendicular to the channel carrying the continuous phase (figure 1).
Droplet microfluidic concepts, components, and processes are now being adopted and leveraged by end-users to enable new science and innovation. Real-world success is now evidenced through a range of mainstream commercial products that are applied to key biological and healthcare-related problems (e.g., 10X Genomics, Drop-seq, and nucleic acid quantification via Droplet DigitalTM PCR systems)8.
Today, it is possible to produce multiple emulsions with complex droplet morphologies. Production of multi-cored droplets (droplets that contain a controlled number of inner droplets at one or more hierarchical levels), Janus droplets (i.e. biphasic or triphasic droplets with two or three physically and chemically distinct surface domains) is now possible using droplet microfluidics9 (figure 2).
Figure 2: Types of multiple emulsions from the simple encapsulation (a), double emulsion (b), and so on (c,d) to bi- and tri-phasic structures (e to h), multiple encapsulations (I to k), and hybrid microjet achievable using droplet-based microfluidics9
A microfluidic chip for the production of highly reproducible PLGA microparticles
In recent years, biodegradable microspheres/microparticles have gained widespread importance in the delivery of bioactive agents10. The copolymers of Poly (D, L-lactic-co-glycolic acid) (called PLGA)/poly (lactic acid) (called PLA) microparticles are one of the most successful new drug delivery systems (DDS) in labs and clinics. Because of their biocompatibility and biodegradability, they can be used in various areas, such as long-term release systems, vaccine adjuvant, and tissue engineering11.
In PLGA microparticle production for drug release and delivery, microparticle size is a key parameter as it is directly related to the microparticles degradation rate and the drug release rate12. Although PLGA microparticle synthesis appears to be a successful drug delivery system, the current processes and tools to produce PLGA microparticles have many limitations, such as wide microparticle size distribution, poor repeatability, and aggressive chemical preparation conditions11. To solve these problems, droplet-based microfluidics offers an efficient method for improvement.
Fluigent provides the Raydrop: a new breakthrough technology leading to outstanding particle size monodispersity and production flexibility. The Raydrop works as a co-flow focusing principle (figure 3). The nozzle and outlet capillary are aligned in a continuous phase chamber, the dispersed phase comes through the nozzle to create the microparticles into the continuous phase and exits by the outlet insert. Using this system, PLGA microparticles ranging from 15 to 50 µm diameters have been successfully generated.
The PLGA microparticle production station allows excellent reproducibility and significantly improved monodispersity (CV < 2%) as compared to other methods on the market. It allows one to continuously produce PLGA microparticles (up to 10 000 droplets/s) without unwanted interruption for long-term experiments.
Single-cell mRNA-seq sequencing using droplets: Drop-seq technology
The production of highly monodispersed emulsions or more complex structures (water-in-oil-in-water emulsions, multiple encapsulations…) makes microfluidics an excellent tool for single-cell analysis or single-cell culture. The technique allows for droplet-based single-cell RNA-sequencing, such that one can characterize complex tissues with many cell types and states under diverse conditions. One of the pioneering methods is Drop-Seq technology, which entraps a single cell and a single primer-barcoded bead in each droplet (figure 4).
Cells are thus separated into nanoliter-sized aqueous droplets, with a different barcode associated with each cell’s mRNAs. The primers on beads contain a barcode consisting of three sequences. One sequence is for PCR amplification and is common to all the beads. The second sequence consists of hundreds of individual primers that also share the same ‘‘cell barcode’’. Finally, the third part has different unique molecular identifiers (UMI), enabling mRNA transcripts to be digitally counted13 (figure 5). The droplets are sequenced altogether, allowing quick profiling of thousands of individual cells from a heterogeneous population. The power of this technology resides in the fact that during sequencing, one can distinguish where the original information came on a cell to cell basis. This allows one to make a gene expression map of the cell, or even to distinguish cell populations within a tissue.
II. Microfluidic cell culture
Microfluidic cell culture has significant advantages over macroscopic culture in flasks, dishes, and well-plates14 (figure 6). The microfluidic chip fabrication process allows great flexibility in the design of microfluidic devices, permitting one to understand and control interactions between cells, substrates, and the surrounding medium, physically as well as biochemically15.
This technology offers new possibilities to accurately reproduce the cellular environment and enables the analysis of biological processes that were not accessible before. Morphology-wise, chips can be structured at the cell scale to reproduce the mechanical constraints experienced by cells. Biochemically, stable gradients can be implemented with a high spatial resolution (typically, micrometer resolution).
Finally, constant perfusion enables the continuous renewal of nutrients and oxygen to promote cell growth and maintain optimal activity during long-term cell culture. Cost reduction due to volume reduction is also a major benefit15.
Development and characterization of a microfluidic model of the tumor microenvironment
The physical microenvironment of tumors is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products, and oxygen. This tumor microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient and oxygen-rich conditions. Ayuso et al. presented an easy to use microfluidic device that can mimic the three-dimensional architecture of multicellular spheroids, while at the same time generating a visible, live “tumor slice” that allows easy monitoring of cells in different regions of the microenvironment in real-time as well as their response to different drugs17 (figure 7).
In this study, tumor cell behavior in different regions of the microdevice was studied and analyzed in conjugation with measurements of hypoxia and glucose concentrations across the device. The differential cellular response to several well-known drugs in different parts of the microdevice emphasizes the potential of this technology for analyzing the impact of microenvironmental parameters on drug response.
Figure 7: Microdevice operation of the tumor microenvironment. (A) 3 microdevices in a Petri dish containing a central culture chamber (detailed in C) and 6 channels. (B) One microdevice is filled with (yellowish) collagen hydrogel flowing to the microchamber from the right middle channel and blue-colored water perfused through the two lateral microchannels. (C) Culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the center of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumors. (D) Cellular monitoring with fluorescent dye in the microdevice17.
III. Organ on a chip
Many efforts are devoted to the development of cancer metastasis models that can help in understanding the disease and the development of innovative therapeutic strategies. Current in vitro and in vivo cancer models are incapable of satisfactorily predicting the outcome of various clinical treatments on patients18. Therefore, new methods and approaches for drug discovery and health research are being developed. The concept of mimicking the organ-level function of human physiology or disease using cells inside a microfluidic chip was first published in 2004. In 2010 that the term organ-on-a-chip (OOAC) was invented by Ingber, et. al., who developed a microfluidic chip to capture organ-level functions of the human lung19. Microfluidics enables one the unique ability to control the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals in a more physiologically relevant context19. The manipulation of the micro-liter volumes of liquids has made these models a platform where scaling, and dynamic crosstalk between cells can be achieved. Microfluidic chips can now use geometries and structures to permit the use of physiological length scales, concentration gradients, and the mechanical forces generated by fluid flow to mimic the in vivo microenvironment experienced by cells20. These biomimetic platforms overcome many drawbacks encountered with conventional tissue culture models. OOAC engineering has attracted enormous interest and attention from the pharmaceutical industry, regulatory agencies, and even national defense agencies. This is demonstrated by the increase of OOAC research papers and by the emergence of at least 28 organ-on-a-chip companies in less than seven years21.
A microfluidic chip to reconstitue organ-level lung functions
To demonstrate that it is possible to engineer a microsystem that replicates the complex physiological functionality of living organs, Huh et al. developed a multifunctional microdevice that reproduces key structural, functional, and mechanical properties of the human alveolar-capillary interface, which is the fundamental functional unit of the living lung19. The device consisted of compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with the extracellular matrix and human alveolar epithelial cells and human pulmonary microvascular endothelial cells is cultured on opposite sides of the membrane (figure 8). Air is subsequently introduced into the compartment to create an air-liquid interface to mimic the lining of the alveolar air space19
Using this organ on a chip platform, the authors demonstrated that breathing motions, simulated by the platform, might greatly accentuate the proinflammatory activities of silica nanoparticles and contribute substantially to the development of acute lung inflammation19. This behavior could not be determined using existing in vitro models.
IV. Particle/cell sorting
Isolating and sorting cells from complex, heterogeneous mixtures represents a critical task in many areas of biology, biotechnology, and medicine. Cell sorting is often used to enrich or purify cell samples into well-defined populations to enhance efficiency in research and development applications22. Cell detection is generally performed using optical methods such as FACS (Fluorescent Activated Cell Sorting), where cells pass through a laser beam and the light scattered is characteristic to the cells and their components. Cells are subsequently sorted according to the scattered emission. It is an automated and robust solution for cell sorting and was considered a gold standard for cell sorting. The immense contributions of current commercial cell sorting platforms using FACS are tempered by several significant and persistent limitations, including limited sample throughput and processing speeds that make the generation of clinical-scale samples very difficult.
As opposed to conventional instrumentation, microfluidic chips are affordable, easy to use, and smaller. These chips make use of a wide range of techniques to sort cells, with specific speeds and efficiencies. The dimensions of the chip also allow a wide range of cell sorters to exist, from large volume cell sorters to precise single-cell sorters. Moreover, microfluidic cell sorting can be combined with additional fluidic operations within a single chip for complete lab-on-a-chip applications, diagnostic and therapeutic purposes. Microfluidic cell sorting is a promising application both in academic and industrial labs. Cell sorting is based on determining a specific cell parameter that can differ from one cell type to another, such as cell size, shape, density, surface markers. In general, a heterogeneous cell solution is injected through a microfluidic chip.
The chip is designed such that cells with different properties experience a different amount of force (that could originate from inertia, channel geometry, external sources …) while similar cells undergo equal forces. The differentiated cells are subsequently pushed into different streamlines and exit the chip from different outlets. Microfluidic cell sorting can be categorized into three distinct groups: fluorescent label-based, bead-based, and label-free cell sorting22. Label-free cell sorting in microfluidic devices is perhaps the most studied of the three approaches as it encompasses active systems (i.e., systems that rely on the use of external fields cells for sorting), and passive systems that do not rely on fluorescent labels or beads. Instead, these methods rely on the inherent differences in cellular morphology between cell groups22.
Inertial microfluidics for continuous particle separation in spiral microchannels
Kuntaegowdanahalli et al. developed a simple inertial microfluidic device that achieves continuous multi-particle separation using the principle of Dean-coupled inertial migration in spiral microchannels.
The dominant inertial forces coupled with the Dean rotational force due to the curvilinear microchannel geometry, cause particles to occupy a single equilibrium position near the inner microchannel wall. The position at which particles equilibrate is dependent on the ratio of the inertial lift to Dean drag forces.
Using this concept, they demonstrated a spiral lab-on-a-chip for size-dependent focusing of particles at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture23.
A major benefit of this system is its high throughput (e.g., 1.5 mL/min) without sheath flow or sequential cell manipulation, which is useful for processing native biological fluids and flow cytometry.
V. Micromixers
Micromixers are important components of lab-on-chip devices for applications in drug delivery, sequencing, amplification, and biochemical reactions. Based on the actuation method, micromixers can be broadly classified as passive and active. In passive mixing, no external sources are used. Thus mixing typically relies on the microfluidic chip geometry and on fluid properties. Under laminar flow, which is the typical fluid regime in microfluidics, mixing mostly happens through diffusion. This property allows performing mixing using lamination: two or more liquids are flowing in parallel, allowing for diffusion to happen and thus permitting to precisely tune mixing between liquids. For improved and faster mixing, chaotic advection can be generated by modifying the microfluidic chip geometry, such as by altering the shape of the channel for splitting, folding, stretching, or breaking the flow of fluid.
Active mixing occurs by using an external perturbation. Many active mixing methods exist. Dielectrophoresis mixing makes use of an electric field to move particles toward or away from an electrode, creating a chaotic advection. Acoustic wave energy can also be used to mix fluids: strong acoustic waves can be produced, and interfere with each other as they propagate in the fluid, generating advection24. As the diffusion coefficient of a liquid depends on the temperature, mixing can also be enhanced by increasing the microfluidic chamber temperature25.
Submillisecond organic synthesis using a serpentine-shaped microfluidic chip
In chemical synthesis, it is important to explore the synthetic pathways of an intermediate. To fully observe these pathways, control over its lifetime and mixing time is required.
The reaction mixture had to be well-mixed within the lifetime of the reactive intermediate. An efficient means of prolonging the lifetime is to lower the reaction temperature (-78°C to -100 °C), above the melting point of many organic solvents. Using microfluidic devices, mixing can be extremely fast, with mixing times unattainable by batches26. However, mixing time is increased at low temperatures, as the solvent viscosity exponentially increases.
To circumvent this issue, the authors used a three-dimensional serpentine-shaped microfluidic chip, allowing improved mixing by chaotic advection. Using this microfluidic chip, submillisecond mixing was performed.
VI. Microvalves
In the past 10 years, efforts have been devoted to the development of microfluidic platforms capable of performing several assays using programmable fluidic operations within an array of microvalves. Similar to programmable logic circuits where multiple electronic computing routines are executed on a single microdevice, programmable microfluidic platforms have been implemented27, allowing one to perform fluidic operations such as mixing, sampling, washing, and reacting automatically within a single chip by modifying the sequence/order of fluidic operations using the software. The primary advantage of microvalves over their macroscale counterparts is the significantly reduced dead volume, which is important in many applications that require precise flow control at small flow rates28. They are useful in biological and chemical applications, such as quantitative metabolic biomarker and genetic analysis29,30, protein-based biomarker detection31, or small molecule chemical and environmental analysis32. These platforms usually consist of a 2D array of microvalves that permit flow regulation, on/off switching and sealing of liquids, gases, or vacuums33. Several microvalves have been developed using pneumatic, electrokinetic and electrochemical actuators. Among these mechanisms, pneumatic actuation is often recognized as the most reliable method due to the simplicity of fabrication, ease of use, scalability, reliability, and a high degree of accuracy. Pneumatically actuated microvalves utilize the deflection of an elastomer (typically PDMS) membrane to control fluid flow34.
A fully integrated multilayer microfluidic chemical analyze for automated sample processing, labelling, and analysis
Capillary zone electrophoresis (CZE), is a powerful tool for chemical analysis and is widely used for environmental monitoring, astrobiology, and biosensing32. CZE assays usually require complex and manual sample processing.
Commercial platforms for automated CZE have been implemented to address this concern, but are expensive, and large, thus hardly field deployable in challenging environments. Microfluidic systems allow miniaturization, automation, and reduction in sample volume requirements for chemical and biochemical sensing.
Using membrane microvalve technology, it is possible to automate metering, transporting, routing, and mixing operations. Kim et al. introduced a microfluidic platform consisting of pneumatically actuated “lifting gate” microvalves integrated with a glass CZE microchip, providing extremely low dead volumes between components.
All the procedures, including buffer filling, labeling, and dilution, can be automated. The microchip was used to analyze diverse compound classes, such as amino acids, and oxidized biomarker compounds, like aldehydes/ketones and carboxylic acids in less than 30 min.
VII. Wearable microfluidics: an emerging application
An emerging field is the use of microfluidic concepts for wearable device applications36. Here, microfluidics presents several key value propositions. The microstructures store or handle fluids and are the core of the sensing device. Using microchannels, precise liquid amounts can be manipulated, allowing for highly accurate and reliable devices and being useful for bodily fluids that are often secreted or extracted in limited quantities. Also, wearable microfluidic devices could also stock a specific drug for precise dispensing at controlled intervals. Innovations in flexible microfluidics and electronics have led to numerous applications. Typically, a wearable microfluidic device will collect a fluid, transfer it to a localized site where detection or measurement is performed. Blood and sweat are common analytes as they provide insights into physiological states such as temperature, pH, and hydration36. Wearable microfluidics finds applications in the pharmaceutical, food, sportswear, and cosmetic fields.
A wearable microfluidic device for the capture, storage and colorimetric sensing of sweat
As mentioned previously, sweat is an analyte of interest because of its rich content of important biomarkers. It is easy to collect compared to blood. In situ quantitative analysis of sweat is of great interest for monitoring physiologic health status (for example, hydration state) and for the diagnosis of disease37.
Existing systems for sweat collection and analysis are confined to laboratories, where standard analytical technologies can be performed. Though highly precise, the analysis is time-consuming and costly.
To address this issue, Kho et al. developed a soft wearable microfluidic system than can directly harvest sweat from pores on the surface of the skin37.
The device routes the sample to different channels and reservoirs for multiparametric sensing of markers of interest, with options for wireless interfaces to external devices for image capture and analysis.
The device can measure total sweat loss, pH, lactate, chloride, and glucose concentrations by colorimetric detection using wireless data transmission. As it is a simple, low-cost, and fast analysis point of care device, it could be used to accumulate data from individual users over time, and this could serve as an analytical approach for interpreting trends in marker concentrations, potentially providing warning signs when performing physical activity.
Conclusion
Since the introduction of microfluidics, the scope of applications has kept extending over the years. The first applications were focused on analytical chemistry, but today the field of life science and specifically point of care is in the core of microfluidics. We have presented applications where microfluidics show great advantages compared to conventional systems. The importance of these applications was illustrated by showing research papers related to these applications. Some important applications were introduced here. Microfluidics covers a wide range of applications such as microreactors, bioprinting, or fuel cells, and many more.
References
- Seemann, R., Brinkmann, M., Pfohl, T. & Herminghaus, S. Droplet based microfluidics. Reports Prog. Phys. 75, (2012).
- Paquin, F., Rivnay, J., Salleo, A., Stingelin, N. & Silva, C. Droplet Control Technologies for Microfluidic High Throughput Screening (µHTS). Muhsincan Sesen,a Tuncay Alan,a and Adrian Neild∗a 10715–10722 (2017) doi:10.1039/b000000x.
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- Jullien, M.-C., Tsang Mui Ching, M.-J., Cohen, C., Menetrier, L. & Tabeling, P. Droplet break in a low capillary T-junction. in 19th Mechanical French Congress (AFM, Maison de la Mécanique, 39/41 rue Louis Blanc-92400 Courbevoie, 2009).
- Yu, L., Chen, M. C. W. & Cheung, K. C. 2010 Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing. Lab Chip 10, 2424–2432 (2010).
- N.Shembekar, C.Chaipan, R. U. & C. A. M. 2016 Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics. Lab Chip (2016) doi:10.1039/C6LC00249H.
- Shang, L., Cheng, Y. & Zhao, Y. Emerging Droplet Microfluidics. Chem. Rev. 117, 7964–8040 (2017).
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- Vladisavljević, G. T., Al Nuumani, R. & Nabavi, S. A. Microfluidic production of multiple emulsions. Micromachines 8, (2017).
- Soppimath, K. S. & Aminabhavi, T. M. Ethyl acetate as a dispersing solvent in the production of poly(DL-lactide-co-glycolide) microspheres: Effect of process parameters and polymer type. J. Microencapsul. 19, 281–292 (2002).
- Qi, F., Wu, J., Li, H. & Ma, G. Recent research and development of PLGA / PLA microspheres / nanoparticles : A review in scienti fi c and industrial aspects. (2018).
- Anderson, J. M. & Shive, M. S. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv. Drug Deliv. Rev. 64, 72–82 (2012).
- Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).
- Halldorsson, S., Lucumi, E., Gómez-Sjöberg, R. & Fleming, R. M. T. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 63, 218–231 (2015).
- Yeo, L. Y., Chang, H. C., Chan, P. P. Y. & Friend, J. R. Microfluidic devices for bioapplications. Small 7, 12–48 (2011).
- Coluccio, M. L. et al. Microfluidic platforms for cell cultures and investigations. Microelectron. Eng. 208, 14–28 (2019).
- Ayuso, J. M. et al. Development and characterization of a microfluidic model of the tumour microenvironment. Sci. Rep. 6, 1–16 (2016).
- Caballero, D. et al. Organ-on-chip models of cancer metastasis for future personalized medicine: From chip to the patient. Biomaterials 149, 98–115 (2017).
- Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science (80-. ). 328, 1662–1668 (2010).
- Bhise, N. S. et al. Organ-on-a-chip platforms for studying drug delivery systems. J. Control. Release 190, 82–93 (2014).
- Zhang, B., Korolj, A., Lai, B. F. L. & Radisic, M. Advances in organ-on-a-chip engineering. Nat. Rev. Mater. 3, 257–278 (2018).
- C. Wyatt Shields IV, Dr. Catherine D. Reyes, and P. G. P. L. Microfluidic Cell Sorting: A Review of the Advances in the Separation of Cells from Debulking to Rare Cell Isolation. Lab Chip (2015) doi:10.1039/c4lc01246a.
- Kuntaegowdanahalli, S. S., Bhagat, A. A. S., Kumar, G. & Papautsky, I. Inertial microfluidics for continuous particle separation in spiral microchannels. Lab Chip 9, 2973–2980 (2009).
- Vivek, V., Zeng, Y. & Kim, E. S. NOVEL ACOUSTIC-WAVE MICROMIXER.
- Ward, K. & Fan, Z. H. Mixing in microfluidic devices and enhancement methods. J. Micromechanics Microengineering 25, (2015).
- Kim, H. et al. Submillisecond organic synthesis: Outpacing Fries rearrangement through microfluidic rapid mixing. Science (80-. ). 352, 691–694 (2016).
- Thorsen, T., Maerkl, S. J. & Quake, S. R. Microfluidic large-scale integration. Science (80-. ). 298, 580–584 (2002).
- Aditya Aryasomayajula, Pouriya Bayat, Pouya Rezai, P. R. S. Microfluidic Devices and Their Applications. Springer vol. 50 (2017).
- Jensen, E. C., Bhat, B. P. & Mathies, R. A. A digital microfluidic platform for the automation of quantitative biomolecular assays. Lab Chip 10, 685–691 (2010).
- Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).
- Erik C. Jensen, Yong Zeng, Jungkyu Kim, and R. A. M. Microvalve Enabled Digital Microfluidic Systems for High Performance Biochemical and Genetic Analysis. 15, 455–463 (2011).
- Kim, J., Jensen, E. C., Stockton, A. M. & Mathies, R. A. Universal microfluidic automaton for autonomous sample processing: Application to the mars organic analyzer. Anal. Chem. 85, 7682–7688 (2013).
- Oh, K. W. & Ahn, C. H. A review of microvalves. J. Micromechanics Microengineering 16, (2006).
- Kim, J., Stockton, A. M., Jensen, E. C. & Mathies, R. A. Pneumatically actuated microvalve circuits for programmable automation of chemical and biochemical analysis. Lab Chip 16, 812–819 (2016).
- Chin, V. I. et al. Microfabricated platform for studying stem cell fates. Biotechnol. Bioeng. 88, 399–415 (2004).
- Yeo, J. C., Kenry & Lim, C. T. Emergence of microfluidic wearable technologies. Lab Chip 16, 4082–4090 (2016).
- Ahyeon Koh, Daeshik Kang, Yeguang Xue, Seungmin Lee, Rafal M. Pielak, Jeonghyun Kim, Taehwan Hwang, Seunghwan Min,1 Anthony Banks, Philippe Bastien, Megan C. Manco, Liang Wang, Kaitlyn R. Ammann, Kyung-In Jang, Phillip Won Seungyong Han, Roozbeh Ghaffari, J. A. R. A soft, wearable microfluidic device for the capture, storage, and colorimetric sensing of sweat. Sci. Transl. Med. 26, 39–46 (2017).
THE MISSION OF FLUIGENT IS TO DEVELOP SOLUTIONS TO SUPPORT RESEARCHERS AND INDUSTRIALS CONDUCTING MICROFLUIDIC EXPERIMENTS UNDER EXCELLENT CONDITIONS.
Technological advances for vaccine development
First-generation vaccines are based on whole organisms, in either live attenuated or killed forms. Second-generation vaccines consist of protein components derived from the organism. Today, third-generation vaccines are being developed. These nucleic acid vaccines are based on the genetic material of the infectious organism. DNA vaccines are examples of third generation vaccines. Clinical trials for DNA vaccines to prevent HIV are underway.
During the COVID-19 pandemic, the first vaccines to reach clinical trials were based on viral vector and nucleic acid technologies. One of the most promising vaccine candidates was based on mRNA.
Scientific advances are becoming even more critical in the success of fundamental vaccine research, vaccine development, or diagnostics. Microfluidic methods can be used to improve vaccine research and development.
Microfluidics in vaccine research and development
Efficient delivery of mRNA and DNA into target cells in vivo is a major challenge. Today, many vaccines are encapsulated into nanoparticles, specifically lipid nanoparticles (LNP) such as liposomes, allowing for improving efficient delivery. The first COVID-19 vaccines to reach the market are mRNA vaccines encapsulated in LNP.
Size and size distribution are essential properties that determine the clinical successes of the nanocarriers. Recently, improvements have been made with the development of microfluidic production methods, in which LNPs formation occurs within a confined microenvironment. These methods have demonstrated higher control over the physical properties of the end product, particularly in terms of liposome size and size distribution.
Microfluidic application example
Vaccine adjuvant production
In vaccine development, adjuvants are used to amplify the recipient’s specific immune responses against pathogen infection. A new generation of adjuvants is being developed to meet the demands of improved antigen specific responses with less toxicity. For example, Konishi et al. reported the first synthesis of a series of saponins, a vaccine adjuvant, using a microfluidic mixer3.
High throughput screening
Flow cytometry for single single-virus analysis shows high potential in virology, vaccine development, or antiviral research. However, it is hard to perform such analysis as Fluorescence-activated cell sorting (FACS), the standard technique in modern biology, does not allow single-virus measurements due to the small size of viruses.
The Fluigent double emulsion production station is a robust and complete system for producing outstanding monodispersed double emulsion in one single device.
Droplet-based microfluidics combined with next-generation sequencing can allow for screening virus particles in a high throughput manner. For instance, Chaipan et al. developed a droplet-based microfluidic platform to screen single virus particles for optimal antigenic features of vaccine candidates. They demonstrated its use by screening HIV particles4.
The Drop-Seq protocol, is a high throughput method that enables the sequencing of the mRNA from a large number of cells.
Virus detection and analysis
Polymerase chain reaction (PCR) and other PCR related methods such as quantitative PCR (qPCR) amplify and quantify DNA and RNA for subsequent analysis. In contrast to standard PCR, digital PCR (dPCR) divides the sample into subsamples of pico to nanoliter range, allowing for a more reliable collection and sensitive measurement of nucleic acid amounts.
For example, Ahrberg et al. developed an easy-to-use microfluidic dPCR device to amplify and quantify complementary deoxyribonucleic acid (cDNA) samples for the H7N9 influenza5. In another study, researchers from the California Institute of Technology and MIT developed a microfluidic dPCR approach to physically link single bacterial cells harvested from a natural environment with a viral marker gene for examining virus-bacterium interactions in many environments6.
In-Vitro cell analysis
Microfluidics provides unique capabilities to control the cellular microenvironment and present cells with mechanical and biochemical signals in a more physiologically relevant context. Microfluidic chips offer an ideal microenvironment to study molecular- and cellular-scale activities that underlie human organ function and identify new therapeutic targets in vitro. For instance, Markus et al. used microfluidic cell culture chambers to analyze the hosting of varicella-zoster virus in human embryonic stem cell-derived neurons7.
References
- World Health Oganization. Global vaccine market report. 14 p (2019).
- Luthando Dziba, Isabel Sousa Pinto, Judith Fisher, K. T. IPBES Workshop on biodiversity and pandemics. (2020).
- Konishi, N. et al. Synthesis of Bisdesmosidic Oleanolic Acid Saponins via a Glycosylation-Deprotection Sequence under Continuous Microfluidic/Batch Conditions. J. Org. Chem. 82, 6703–6719 (2017).
- Chaipan, C. et al. Single-Virus Droplet Microfluidics for High-Throughput Screening of Neutralizing Epitopes on HIV Particles. Cell Chem. Biol. 24, 751-757.e3 (2017).
- Ahrberg, C. D., Lee, J. M. & Chung, B. G. Microwell Array-based Digital PCR for Influenza Virus Detection. Biochip J. 13, 269–276 (2019).
- Arbel D. Tadmor, Elizabeth A. Ottesen, Jared R. Leadbetter, and R. P. Probing Individual Environmental Bacteria for Viruses by Using Microfluidic Digital PCR. Science (80-. ). 23, 1–7 (2012).
- Markus, A., Lebenthal-Loinger, I., Yang, I. H., Kinchington, P. R. & Goldstein, R. S. An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons. PLoS Pathog. 11, 1–22 (2015).
Fluid considerations when integrating fluidics into your system
How will pulsatile flow affect system performance?
Droplet-based applications: Droplet digital PCR and cell encapsulation for single-cell analysis usually make use of droplets generated by microfluidic technology. These applications require liquid handling systems that produce flow with very low to no pulsation to obtain homogeneous droplets, which is vital for the success of these applications.
Flow cytometry and surface plasmon resonance (SPR) are based on real-time analytical measurements of samples passing by a detection area. They require a fluid handling delivery system with a stable flow rate to deliver fluids through the fluidic channels.
Live cell imaging: In live-cell imaging applications with fluid perfusion, pulsatile flow leads to variable shear stress imposed on cells, and may impact cell viability. Liquid flow should be stable and controlled.
In some applications, a steady flow rate is a prerequisite for reliable operation after fluidic system integration:
For these applications, pressure-based fluid delivery can deliver flow rates in the microliter- or nanoliter-per-minute range with a stable flow rate (high precision and accuracy) and no pulsation.
Do you require fast settling times?
Some applications call for alternating between multiple different flow rates in a short period of time. This is common for flow cytometry, cell sorting, or fluorescent activated cell sorting (FACS). In the latter, 3 flow rate modes are usually available: slow, medium, and fast, and one can switch from one to another.
For fluidic system integration in applications like these, consider using a fluid delivery solution that allows for rapid response time (a few seconds). Pressure-based flow control has the advantage of having a quasi-immediate response. A step of several bars can be made within milliseconds, and the liquid flow rate reaction is equally fast, allowing the system to reach typical microfluidic flow rates in less than one second. This cannot be achieved by most motors used in syringe pumps, as revealed by the graph below.
Is sterility required?
Many biological applications require a sterile environment, including cell culture under perfusion, immunostaining, organoid culture, organ on a chip, drug discovery, single-cell analysis and cell cytometry, among others. When using fluids such as culture media, PBS, buffers, blood or plasma, every component in the fluidic path should be disposable or sterilizable.
For these applications, pressure-based liquid delivery systems that do not contact fluids or have sterilizable components are ideal. As for liquid handling without flow rate monitoring, using pressure allows the system to use disposable tubing only between the inlet reservoirs and the application. At the fluidic system integration stage, however, you may want to plan for the ability to monitor or control the flow rate. The question of sterility arises when choosing the flow rate metering solution. Most flow sensors are too costly to be considered as disposables, yet are in contact with the fluids. A non-intrusive flow sensing technology is a particularly interesting way to tackle this issue.
Do you need to handle multiple fluid streams?
Multiple streams are useful for operations that perform several tests simultaneously, such as drug screening where different drug candidates are tested, or personalized medicine applications that make use of tumor biopsy studies. It is also sometimes convenient and cost-effective to separate a fluid originating from one liquid delivery system into two distinct paths.
It’s important to choose the right liquid delivery system when designing for multiplexing. Depending on your choice, an increased number of systems may be highly expensive, and fluidic system integration can be cumbersome. Using fluidic valves or quake valves can also be an interesting alternative, as they allow for fluid management with a reduced number of fluid delivery systems.
What are the volumes to be dispensed?
The volumes to be injected are highly application-dependent. For instance, in cell biology applications such as cellular imaging or dynamic cell culture, fluids may be injected over a period of several days. In these applications, high reservoir volumes (up to 1 L) are required. Another option could be recirculating media or reagents with a peristaltic pump, a recirculation valve or a fully integrated solution. Conversely, drug screening applications may make use of expensive liquids, and only very small amounts of fluids should be used (< 100 µL).
Depending on the liquid delivery method used in the fluidic system, reservoir size can affect precision and accuracy. For example, when using syringe pumps, smaller syringes maximize accuracy for small volumes but require frequent refilling for larger volumes. Larger syringes will increase capacity, but will lose accuracy and become pulsatile at lower flow rates. Using a peristaltic pump for fluid recirculation is relatively uncomplicated.
Do you need to control or measure flow rate?
For most liquid delivery applications, the user wants to set a desired flow rate and have a solution to reach it via fluidic system integration through use of a feedback loop algorithm. Syringe or peristaltic pumps give easy access to the real time volumetric flow rate, but pressure controllers do not.
To overcome this, the user can add a flow sensor to the configuration that can either be connected to the pressure controller for flow rate control, or used for monitoring only. There are several flow sensing technologies based on different approaches, each with their respective advantages and limitations. The best choice for a given application depends on your requirements with regard to response time, chemical compatibility, flow rate range, cost, footprint and other parameters. For microfluidic applications in particular, one must also be cautious about channel and fluid interactions, the formation of bubbles, and multi-phase flows, although these can be neglected at a larger scale.
Important considerations when integrating fluidics into your system
Time to market?
In a fast-growing market sector, it’s crucial to minimize time-to-market and launch new technologies before the competition. To accelerate development, consider pre-built components or solutions, such as liquid handling components that can be integrated quickly into your system or prototype. Fluigent’s standard and customizable liquid handling OEM components offer flexibility and ease of integration into your device. Using a readymade solution avoids the hassle of dealing with multiple fluid handling issues and leaves you more time to focus on your core expertise and application.
If you need expertise in fluidic system integration and are looking for a company that can do it for you, choose one that’s reactive, communicates with its customers, and can respect short timelines to achieve your desired launch date. At Fluigent, we design and manufacture all of our products under one roof at our headquarters in the Paris region. Our R&D and production teams work closely with each other and with our customers to ensure maximum satisfaction.
Do you have all the resources needed for in-house development?
Handling fluidic system integration by yourself is an option when developing an automated liquid handling system. The conception, design and development of the system will require engineering expertise in the mechanical, electrical, software and manufacturing domains.
When developing a system internally, you need to be sure you have the experience and engineering knowledge to translate your proof-of-concept device into a reliable and efficient automated system. The entire product development process is at risk if you are missing any of the required resources.
Related expertises & ressources
- White Papers
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On the occasion of Journées du Patrimoine, we have been elated to have our flagship product, Flow EZ, in the Office of Roland Lescure, Minister in Charge of Industry.
One year after being selected as a representative of the Made in France and being exhibited in the Elysée palace, It was an honour for us to represent the French industry in the French ministry of Economy & Finance.
Our Digital Acquisition and Communication Manager, Doaa Fahmy paid a little visit to capture this amazing sight!
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The Products are sold and delivered by Fluigent to Customer EX-WORKS (as defined in the “Incoterms 2010” edition published by the International Chamber of Commerce) from Fluigent’s premises designated in the order confirmation. Fluigent may propose otherwise in its quotations or agree to provide otherwise in the order confirmation upon Customer’s request.
Delivery of the Product to Customer shall be deemed to have occurred, and risk of loss or damageshall pass to Customer,upon delivery to the carrieror in accordance with the Incoterm specifiedin the order confirmation as the case may be.
Delivery times will be indicated in the quotation or order confirmation depending on the Products. Delivery times are indicative. Fluigent agrees to use commercially reasonable efforts to meet the delivery dates communicated or acknowledged by it on the condition that Customer provides all necessary order and delivery information sufficiently prior to the agreed delivery date. Fluigent shall not be liable for, nor shall Fluigent be in breach of its obligations to Customer, because of any delivery made within a reasonable time before or after the stated delivery date or if it is prevented to do so by a force majeure event, as defined in Section 14.3.
Fluigent reserves the right to make delivery in instalments, all such instalments to be separately invoiced and paid for when due per invoice, without regard to subsequent deliveries.Delay in delivery of any instalment shall not relieve Customer of Customer’s obligations to accept remaining deliveries.
The quantity of any instalment of the Products, as recorded by Fluigent on the dispatch from Fluigent’s place of business, is conclusive evidence of the quantity received by Customer upon delivery, unless Customer provides conclusive evidence to the contrary. Fluigent will not be liable for any non-delivery of the Products to the delivery location, unless Customer gives written notice to Fluigent of the non-delivery within five (5) days following the date that Customer would, in the ordinary course of business, have received the Products. Fluigent’s liability for any non-delivery of the Products will be limited to replacing the Products within a reasonable time or adjusting the invoice for the Products to reflect the actual quantity delivered.
In the event of shortages Fluigent may allocate its available production and Products, in its sole discretion, among its customers and as a result may sell and deliver to Customer fewer Products than specified in the purchase order, as the case may be.
If Customer fails to take delivery, then Fluigent may deliver the Products in consignment at Customer’s costs and expenses.
6/ Inspection – Return of Products
Immediately upon Customer’s receipt of any Products shipped hereunder, Customer shall inspect the same and shall notify Fluigent in writing of any claims for shortages, defects or damages and shall hold the goods for Fluigent’s written instructions concerning disposition. If Customer shall fail to so notify Fluigent within five (5) days after the Products have been received by Customer, such Products shall conclusively be deemed to conform to the T&Cs hereof and their specifications and to have been irrevocably accepted by Customer.
Should said shortages, defects or damages notified by Customer be verified, Customer shall have the right to return such Products as shall be declared defective at Fluigent’s expense and be entitled to (i) replacement Products or (ii) when replacement will not be possible, refund of any part of the price paid for the Products found to be defective within fifteen (15) business days at no additional cost to Customer, it being specified that no indemnity or compensation whatsoever shall be due to Customer on this ground.
7/ Prices
Prices for Products shall be those specified in Fluigent’s then current Price List available on Customer’s request.
Except as may be required by applicable law, all stated prices are exclusive of any freight, handling and shipping insurance charges and any taxes, fees, duties, and levies, however designated or imposed, including but not limited to value-added and withholding taxes that are levied or based upon the prices paid upon these T&Cs. Any taxes related to the Products purchased pursuant to these T&Cs are then the responsibility of Customer (excluding taxes based on Fluigent’s net income), unless Customer presents an exemption certificate acceptable to Fluigent and the applicable taxing authorities. If any exemption certificate presented by Customer is held to be invalid, then Customer will pay Fluigent the amount of the taxes and any penalties and interest related thereto.
Applicable taxes shall, to the extent practical, be billed as a separate item on the invoice.
8/ Payment
All payments shall be in Euros (or any other currency only if the price in the quotation are indicated with this different currency).
Unless otherwise set forth in the order confirmation or agreed in writing by the Parties, payment of the price of the Product shall be due to, and received by Fluigent, prior to the shipment of the Product. Payments made by third parties in the name and/or on behalf of Customer may be accepted by Fluigent at Fluigent’s sole discretion.
In case of credit terms granted to Customer, (i) any payment received from Customer may be accepted and applied by Fluigent against any amount owed by Customer to Fluigent without prejudice to, or discharge of, any other indebtedness of Customer to Fluigent, regardless of any condition, statement, legend or notation appearing on, referring to or accompanying such payment, (ii) any sum not paid by Customer when due shall bear interest from the due date to the date of payment, such interest to run day to day and after as well as before any judgment, at a rate equal to the interest rate applied by the European Central Bank for its most recent refinancing operations plus 10 points andCustomer shall pay an indemnity for debt collection expenses (iii) in addition to all other remedies available to Fluigent (which Fluigent does not waive by the exercise of any rights hereunder), Fluigent may suspend the delivery of any Products if Customer fails to pay any amounts when due and the failure continues for five (5) days following Customer’s receipt of notice thereof and such action shall not be construed as a breach or cancellation of these T&Cs by Fluigent (iv) Fluigent shall retain title to the Product sold to Customer until payment in full for the price of the Product has been received by Fluigent, including without limitation the principal and any incidental amounts thereof and, until Customer makes payment in full to Fluigent as set forth above, Customer shall (a) expressly identify and designate any Product purchased from Fluigent under the T&Cs as subject to this reservation of title provision and shall not integrate such Product into any other product and (b) if any Product for which this reservation of title provision applies, is resold to, or used by, any third party, Customer shall inform Fluigent immediately and, subject to applicable law, Fluigent hereby reserves the right to take any legal action to replevin the Product, commencing on the day the price of the Product is due and payable in full, through the date payment thereof is received by Fluigent.
In case of credit terms granted to Customer, all orders shall be accepted under the provision that the Customer is in the position to pay the complete amount of the purchase price. If this prerequisite is no longer fulfilled, which shall be assumed if unfavourable information about the Customer‘s economic situation exists, as in case of formal declaration of bankruptcy of the Customer or any other situation of insolvency (whether legally declared or not) that may suppose a notorious change in its financial position affecting its credit worthiness or if payments are not made within the agreed payment period, then Fluigent will be entitled to claim for immediate payment, without having to be subject to the dates agreed, of all goods delivered and not yet paid by Customer.
Customer may not withhold payment of any amounts due and payable as a set-off of any claim or dispute with Fluigent, regardless of whether relating to Fluigent’s breach, bankruptcy, or otherwise.
9/ IP Right – License – Technical services
a/ IP Rights ownership
Fluigent reserve all right, title and interest in all IP Rights pertaining to in all Products, Software and Documentation provided or made available to Customer. Customer shall not contest, either directly or indirectly by assisting a third party, Fluigent’s sole and exclusive rights, including ownership rights, in and to the IP rights. Furthermore, Customer shall not challenge Fluigent’s title to the IP Rights or otherwise do or cause to be done anything which contradicts with such sole and exclusive ownership of Fluigent.
Nothing in these T&Cs shall be deemed to confer upon Customer any right, title or interest whatsoever in any of the IP Rights except for those rights specifically granted in Section 9.2.For the avoidance of doubt, to the extent that Software and/or Documentation is embedded in a Product, the sale of such Product shall not constitute the transfer of ownership rights or title in such Software and/or Documentation, and all references to “sale” or “sold” of any Software or Documentation shall be deemed to mean a license in the terms set forth in Section 9.2.
b/ License
Subject to these T&Cs, including without limitation the specific limitations contained herein, and in consideration of Customer’s payment of the price of the Product, Fluigent grants Customer a limited, fully-paid, non-transferable, non-exclusive license,right to use, without any right to sublicense,the Software in machine-readable form, only in combination with or as part of the Products for which the Software has been provided, solely for so long as the Product is owned by Customer and its successors and permitted assigns.
With respect to Products, Software, Documentation, and portions thereof, Customer is not authorized to and agrees that it will not: (i) reverse engineer, decompile, decrypt, disassemble or otherwise attempt to derive the source code, ideas, technology or algorithms, except to the extent expressly authorized by statutory law; (ii) modify, alter, improve, develop, update upgrade, downgrade, translate, create derivative works; (iii) remove or alter any proprietary markings or notices; or (iv) merge, link or incorporate Software into any other software (iv) license, sublicense, distribute, pledge, lease, rent, assign, sell or commercially share the IP Rights herein (v) use the IP Rights in connection with any hazardous activity or any other activity which might result in serious property damage, death or serious bodily injury. Should Customer create any modifications or derivative works of Products, Software, Documentation or a portion thereof, Customer irrevocably assigns and agrees to assign all right, title and interest in any such modifications or derivative works to Fluigent.
No rights or licenses with respect to any Software source code are granted to Customer.
Customer’s rights under these T&Cs are conditional upon Customer not performing any actions that may require any Software, Products and/or any derivative work thereof, to be licensed under open source software license terms that may, for example, require disclosing source code, granting a license under IP Rights, such as granting a permission to develop derivative works, or granting other rights or assuming responsibilities commonly associated with open source software.
Customer shall (i) establish and maintain appropriate security measures to safeguard the IP Rights against any unauthorized access or use, (ii) mark, when applicable, the Product with such notices, including copyright notices, specified from time-to-time by Fluigent (iii) maintain effective control over the IP Rights in accordance with these T&Cs, (iv) keep a full and accurate written record of any authorized copies or disclosures of the IP Rights, as well as their location and (v) furnish Fluigent with copies of such written record without undue delay whenever so requested by Fluigent (vi) promptly give notice of any conduct which comes to its attention and which may infringe or constitute a conflicting or illegal use of the IP Rights.
If Customer is in default of any of the terms herein, Customer’s license will automatically terminate. Customer shall indemnify Fluigent against and hold Fluigent harmless from any damage or costs arising from or in connection with any violation or breach of the provisions of this Section 9.2 and Customer shall reimburse all costs and expenses incurred by Fluigent in defending any claim, demand, suit or proceeding arising from or in connection with such violation or breach, as set forth in Section 11.
For any third-party software licensed by Fluigent from other licensors that have been identified to Customer in writing in advance, such applicable licensor is a thirty-party beneficiary to the T&Cs with the right to enforce the obligations set forth herein and, for the purposes hereof, any such software shall be deemed Software.
c/ Technical services
Fluigentshall provide technical assistance to Customer and conduct corrective maintenance for the Product to correct, within a reasonable time, incidents detected by the Supplier or on Customer’s reasonable request.
The Customer Support Service can be contacted by phone [•], by email to support@fluigent.comor via the contact form on Fluigent’s website.
Upon receipt of Support request from the Customer, Fluigent undertakes to use all reasonable endeavours during the Standard Service Hours to make such support, corrections, repairs or adjustments to or replace such parts of the Products as may be necessary to restore the Products to their proper operating condition. Whether this can be achieved remotely, at Fluigent facilities or by an on-site visit by a Fluigent engineer will be determined by the Service Offering provided in the quotation or with the Product, as the case may, or by Fluigent at its own discretion in any other cases.
Fluigent also make available to the Customer, updates and new versions of the Software and Firmware, whether this relates to updates or new versions intended to implement corrective patches, integrate new functions or technical improvements in the Product.
Customer shall agree to come back, on Fluigent’s recommendation, to a previous version to avoid regression in the performance of the Products or in case of non-compatibility of updates and new versions with the parameters for the Software or the Firmware.
Progressive maintenance services may also be performed on the request of the Customer, according to the conditions set forth in the specific quotation which will be issued by Fluigent and accepted by Customer.
10/ Warranty
a/ Quality and conformity
Fluigent warrants to Customer that for a period of one (1) year following delivery of the Product to Customer, the Products, and the Software embedded, shall be free from defects in material or workmanship and shall substantially conform to Fluigent’s specifications for such Products and Software.
If a defect is reported to Fluigent during the one-year period following delivery of the Product to Fluigent, Fluigent’s sole and exclusive obligation, and Customer’s sole and exclusive right, with respect to claims under this warranty shall be limited, at Fluigent’s option, either (i) repair or replace the Product or Software or (ii) provide Customer with a refund of the portion of the applicable price paid by Customer to Fluigent for such Product.
Customer may ship Products returned under warranty claims to Fluigent’s designated facility only so long as the returns are in conformance with Fluigent’s then-current return material authorization policy and are accompanied by a duly completed return material authorization form issued by Fluigent. Where warranty adjustment is made, Fluigent will pay for freight expenses. Customer shall pay for returned Products that are not found to be defective or non-conforming together with the freight, testing and handling costs associated therewith. The non-conforming or defective Products shall become Fluigent’s property as soon as they have been replaced or credited for.
Notwithstanding the above, Fluigent shall have no obligations for breach of warranty if the alleged defect or non-conformance is found to have occurred as a result of: abnormal or unusual physical or electrical stress or environmental conditions, misuse, neglect, improper installation, accident, improper repair, alteration, modification, improper storage, improper transportation or improper handling, operation or use of the Products, after the risk of loss in the Products has passed to Customer.
The warranty term for a spare part used in repairing Productsis ninety (90) days from its installation in the Product or the remainder of the warranty term for the Product into which it is installed, whichever is longer. For the avoidance of doubt, the warranty term of a Product is not extended after its repair or replacement.
In case of replacement, Customer will pay Fluigent for a replacement part or Product when the replaced part or Product is not returned by Customer to Fluigent within ten (10) days after the date the replacement part or Product was delivered to Customer by Fluigent. Prices of the part replaced will be according to the current standard price in the Territory accessible on Fluigent’s website or communicated by Fluigent on Customer’s request.
Fluigent may provide an extension of warranty upon Customer’s request at additional cost. As the case may be, this extension of warranty will be mentioned in the order confirmation and in the Customer’s invoice. Each extension of warranty sold to Customer is related to one Serial Number (SN) of the concerned product. No extension of warranty will be accepted after the purchase of a Product.
b/ Non infringement
Fluigent, at its expense, shall: (i) defend against a claim in a legal proceeding brought by a third party against Customer that any Product or Software as furnished by Fluigent hereunder directly infringes the claimant’s patent or copyright; and (ii) hold Customer harmless against damages and costs awarded by final judgment in such proceeding (or agreed upon in a settlement to which Fluigent consents) to the extent directly and solely attributable to infringement by the Product or Software.
Fluigent shall have no obligation or liability to Customer under Section 10.2: (1) if Fluigent is not: (i) promptly notified in writing of the claim, (ii) given the sole right to control the defence and settlement of such claim, including the selection of counsel, and (iii) given full reasonable assistance and cooperation by Customer in such defence and settlement; (2) if the claim is made more than one (1) years after the date of delivery of the Product; (3) to the extent that any such claim arises from: (i) modification of the Product, (ii) design, specifications or instructions furnished by Customer, or (iii) the combination or use of the Product with any other product, software, service or technology; (4) for unauthorized use or distribution of the Product or use beyond the specifications of the Product; (5) to the extent that any such claim arises from Customer’s use, sale, offer for sale or importation of the Product after Fluigent’s notice to Customer that Customer should cease any such activity because the Product is, or is reasonably likely to become, the subject of a claim of infringement; (6) for any costs or expenses incurred by Customer without Fluigent’s prior written consent; (7) for infringement of any third party’s intellectual property rights with respect to which Fluigent has informed Customer or has published a statement that a separate license has to be obtained or that no license is granted or implied.
If any claim of infringement is brought against Fluigent as a result of Customer’s actions in connection with items (3) to (7) of this Section 10.2, Customer shall indemnify Fluigent against and hold Fluigent harmless from any damages or costs arising from or connected with such claim of infringement and shall reimburse all costs incurred by Fluigent in defending any claim, demand, suit or proceeding for such infringement, provided Fluigent gives Customer prompt notice in writing of any such suit or proceeding for infringement.
If any Product is, or in Fluigent’s opinion is likely to become, the subject of a claim of infringement, Fluigent shall have the right, without obligation and at its sole option, to: (i) procure for Customer the right to continue to use or sell such Product, (ii) replace or modify such Product in such a way as to make the modified Product non-infringing, (iii) ask Customer to return all such Products in Customer’s possession and upon such return credit Customer the sum paid to Fluigent by Customer for such Products, less appropriate depreciation.
c/ Warranty limitations
No contractor, consultant, reseller, agent or employee of fluigent is authorized to make any modifications, extensions or additions to the limited warranties hereof. except as provided in sections 10.1 and 10.2 hereof, Fluigent’s product and software are provided “as is”, and all other express or implied conditions, representations and warranties, including without limitation any implied warranty of merchantability, warranty of fitness for a particular purpose (even if informed of such purpose), warranty for hidden defect, or warranty arising from a course of dealing, usage or trade practice, are hereby excluded to the fullest extent allowed by applicable law.
No warranty is made that Fluigent’s product or software will meet customer’s requirements, or that the operation of Fluigent’s product or software will be uninterrupted or error-free.
certain third-party software may be provided to customer along with certain Fluigent’s software. This third party software is provided “as is” and all limitations of warranties set forth herein apply to such third party software.
11/ Liability
Customer shall defend, indemnify, and hold harmless Fluigent and its subsidiaries, Affiliates, successors, and assigns and their respective directors, officers, shareholders, and employees from and against any loss, injury, death, damage, liability, claim, deficiency, action, judgment, interest, award, penalty, fine, cost, fees (including import and export customs fees), or expense (including reasonable attorney and professional fees and costs, and the cost of enforcing any right to indemnification hereunder and the cost of pursuing any insurance providers) (“Claims”) arising out of or occurring in connection with the negligence or willful misconduct of Customer or its employees or agents, including but not limited to: (i) any misuse or modification of the Products by Customer or its employees or agents, (ii) any act (or failure to act) by Customer or its employees or agents in contravention of these T&Cs or any safety procedures or instructions that Fluigent provides to Customer or its employees or agents, or (iii) the failure to store, install, operate, or maintain the Products in accordance with Fluigent’s instructions.
Fluigent shall defend, indemnify, and hold harmless Customer and its subsidiaries, affiliates, successors, and assigns and their respective directors, officers, shareholders, and employees (collectively, “Customer Indemnitees”) from and against any Claims brought against them by any third party and arising out of or relating to (1) the infringement of any third-party’s intellectual property rights, subject to the conditions and limitations of Section 10.2 hereof; (2) damage to property or bodily injury caused by the use of the Product in accordance with Fluigent’s Documentation, if, and only if, such Product have not been altered or modified by Customer or any third party; or (3) any negligence or willful misconduct of Fluigent; excluding in any of the foregoing cases, any Claim attributable to any Customer Indemnitee’s negligence, willful misconduct or breach of its obligations hereunder.
In no event shall fluigent or its licensors be liable for indirect, special, incidental or consequential damages (including lost profits, savings or data) whether based on contract, tort, product liability, or any other legal theory, even if fluigent has been advised of the possibility of such damages (except with respect to third party claims for which indemnification to customer is provided under section 10.2 hereof). subject to the foregoing exception, in no event shall company’s or its licensors’ liability under t&cs exceed the amount paid by customer for the products and software giving rise to the claim. Notwithstanding anything to the contrary in the terms and conditions, in the case where no amount was paid, company and its licensors shall have no liability for any damages whatsoever. The existence of more than one customer claim, or customer claims involving more than one product, shall not enlarge or extend the above specified limits.
In addition, all claims from customer must be brought within one (1) year of delivery, regardless of their nature. any claim brought after that one (1) year term will be deemed invalid, customer expressly waiving its right to introduce such a claim once such one (1) year term is over.
The limitations and exclusions set forth above in this Section 11 shall only apply to the extent permitted by applicable mandatory law.
12/ Confidentiality
Customer shall, at all time, maintain as confidential all Confidential Information and shall exercise the same degree of care to protect them from disclosure that it uses to protect its own confidential information.
Customer shall not, without Fluigent’s prior written consent, disclose or make any Confidential Information available in any form to any person, except its employees, consultants, or permitted operators, whose access is necessary to enable Customer to exercise its rights under the T&Cs and who have been advised of the confidential nature of such Confidential Information.
Customer shall only use the Confidential Information as necessary to perform its obligations hereunder and shall return or destroy it at the request of Fluigent.
Customer shall be permitted to make such disclosures to the public or to any governmental authority to the extent required by a court order or if otherwise required by law, provided that Customer gives Fluigent prior written notice of the disclosure and uses reasonable legal efforts to resist disclosing the Confidential Information.
Any violation of these confidentiality obligations shall entitle Fluigent to claim for the payment of any damage, loss or expense, including legal fees and procedural costs, resulting from the breach of the obligations provided for in this Section 12.
Customer acknowledges that a breach of the obligations set forth in this Section 12 may cause irreparable harm to Fluigent, for which damages may be difficult to ascertain, and therefore Customer hereby agrees that Fluigent shall be entitled to seek equitable relief by means of mandatory injunctions. This right of equitable relief is in addition to any other rights (such as right to damages and interest) that may be available to Fluigent.
The confidentiality obligations and restrictions on use arising from this Section 12 shall remain in force for a period of five (5) years after the termination of the relationship between the Parties for any reason whatsoever.
13/ Compliance with laws
Each party hereto represents that it is duly authorized to enter into these T&Cs and represents that with respect to its performance hereunder, it will comply with all applicable national, federal, state and local laws.
If the delivery of Products under these T&Cs is subject to the granting of an export or import license by a government and/or any governmental authority under any applicable law or regulation, or otherwise restricted or prohibited due to export or import control laws or regulations, Fluigent may suspend its obligations and Customer’s rights regarding such delivery until such license is granted or for the duration of such restriction and/or prohibition, respectively, and Fluigent may even cancel the order related to such Products, without incurring any liability towards Customer.
Furthermore, if an end-user statement is required, Fluigent shall inform Customer immediately thereof and Customer shall provide Fluigent with such document upon Fluigent’s first written request; if an import license is required, Customer shall inform Fluigent immediately thereof and Customer shall provide Fluigent with such document as soon as it is available.
By accepting Fluigent’s offer and/or accepting any Products, Customer agrees that it will not deal with the Products and/or Software and/or Documentation related thereto in violation of any applicable export or import control laws and regulations.
14/ Miscellaneous
a/ Assignment
No rights or obligations of Customer hereunder or arising out of the T&Cs may be assigned without the prior written consent of Fluigent. Any such assignment without Fluigent’s prior written consent shall be null and void.
Fluigent’s duties, rights and obligations hereunder may be assigned, and Fluigent’s duties hereunder may be delegated, to any one or more of its Affiliates in whole or in part. Fluigent reserves the right to assign, and Customer acknowledges and consents to any assignment of, the accounts receivable resulting from the T&Cs to one or more third parties as part of a factoring arrangement or otherwise.
The T&Cs shall be binding upon, inure to the benefit of, and be enforceable by, the permitted successors and assigns.
b/ Notices
All payments by check, correspondence and notices hereunder shall be in writing and given by registered or certified mail, postage and registration fees prepaid, return receipt requested, or overnight mail by an internationally recognized courier service, and shall be deemed given when so mailed or sent to Customer at the address set forth in the order confirmation or such other address as either party may notify the other Party pursuant to this Section 14.2.
In case of notification by registered letter with acknowledgment of receipt, it will be deemed to have been validly notified on the day of the first presentation of the registered letter. Rejection or other refusal to accept or the inability to deliver because of changed address of which no notice was given shall be deemed to constitute receipt of the notice, consent or communication sent.
c/ Force Majeure event
Fluigent shall not be deemed to be in default of its contractual obligations whilst performance thereof is prevented by a Force Majeure Event. Events of Force Majeure are events beyond the control of the Party and which were not reasonably foreseeable and avoidable. It is expressly agreed between the Parties that Force Majeure Event shall include(without being limited to)delays and non-deliveries or non-acceptance caused by strikes, work stoppages, riots, wars, fires, acts of God, accidents, governmental orders and regulations, curtailment of or failure in obtaining sufficient electric power, lack of transportation or distributive facilities, any governmental restrictions to travel, transport and work in response to the outbreak of any coronavirus pandemic or other pandemic, and other contingencies beyond Fluigent’s reasonable control.
d/ Severability
If any provision of the T&Cs shall be held to be invalid, illegal or unenforceable, the validity, legality and enforceability of the remaining provisions shall not be affected or impaired thereby. The paragraph headings herein are for convenience only; they form no part of the T&Cs and shall not affect their interpretation.
e/ Waiver
Fluigent’s failure to enforce any term or condition of the T&Cs or to exercise any right arising hereunder shall not constitute a waiver of Fluigent’s right to enforce such terms or conditions or exercise such right thereafter. All rights and remedies under this order are cumulative and are in addition to any other rights and remedies Fluigent may have at law or in equity. Any waiver of default by Customer hereunder shall be in writing and shall not operate as a waiver of any other default or of the same default thereafter.
f/ Governing law and jurisdiction
The T&Cs shall be governed by, and construed in accordance with the law of the country where Fluigent has its head office, excluding (a) any conflicts of law rules or principles that might refer the governance or construction of the T&Cs to the laws of any jurisdiction other than the French Republic.
In case of a dispute arising from the existence, the validity, the interpretation, the performance or the termination for whatever cause of these T&Cs or based on any right arising out of these T&Cs or on the commercial relationship between the Parties, the Parties shall make every effort to reach a settlement.
If a settlement cannot be reached within three (3) months of the date of the initial notification relating to the dispute, the dispute shall be referred to the competent courts of the country where Fluigent has its head office. Such relevant courts shall also have jurisdiction on interim measures including for purposes of protective measures as well as summary procedures, ex parte procedures, impleader or multiplicity of defendants.