For the first time we present a technique for the spatio-temporally resolved localization of liquid-gas interfaces on centrifugal microfluidic platforms based on total internal reflection (TIR) at the channel wall. The simple setup consists of a line laser and a linear image sensor array mounted in a stationary instrument. Apart from identifying the presence of (usually unwanted) gas bubbles, the here described online meniscus detection allows to measure liquid volumes with a high precision of 1.9%. Additionally, flow rates and viscosities (range: 1-10.7 mPa s) can be sensed even during rotation at frequencies up to 30 Hz with a precision of 4.7% and 4.3%, respectively

Ducrée, Jens

Hoffmann, J.

Mark, D.

Riegger, L.

von Stetten, F.

Zengerle, Roland

English

DCU Online Research Access Service

TIR-BASED DYNAMIC LIQUID-LEVEL AND FLOW-RATE SENSINGAND ITS APPLICATION ONCENTRIFUGAL MICROFLUIDIC PLATFORMS1l1,.2 1,2 3J Hoffmann +, L. Riegger -, D. Mark', F von Stetten], R. Zengerle , J Ducree1 HSG-IMIT, Villingen-Schwenningen, GERMANY2 IMTEK - University of Freiburg, Freiburg, GERMANY3BDI, Dublin City University, Dublin, IRELAND+ contributed equallyABSTRACTFor the first time we present a technique for thespatio-temporally resolved localization of liquid-gasinterfaces on centrifugal microfluidic platforms based ontotal internal reflection (TIR) at the channel wall. Thesimple setup consists of a line laser and a linear imagesensor array mounted in a stationary instrument. Apartfrom identifying the presence of (usually unwanted) gasbubbles, the here described online meniscus detectionallows to measure liquid volumes with a high precision of1.9 00. Additionally, flow rates and viscosities (range: 1 -10.7 mPa s) can be sensed even during rotation atfrequencies up to 30 Hz with a precision of 4.7 00 and 4.300, respectively.INTRODUCTIONMicrofluidic lab-on-a-chip systems make use of therescaling of hydrodynamic force ratios in miniaturizedfluidic networks. Towards the micron world, surface-to-volume ratios significantly increase, thus making surfacemediated effects such as surface tension and viscous dragprevail over bulk forces such as inertia and gravity.Lab-on-a-chip devices typically concatenatelaboratory unit operations for liquid handling such assample preparation, aliquoting, metering, and mixing on asingle substrate [1,2] to perform bio-chemical assays in aprocess-integrated, automated, miniaturized and oftenparallel fashion. However, due to their strong dependencyon often hard to control surface properties, lab-on-a-chipsystems are intrinsically prone to inconsistencies such asthe formation of gas bubbles or pockets during primingand operation. This tendency may lead to insufficientfilling of microfluidic cavities with sample or reagents. Inorder to leverage a successful transfer of academic lab-on-a-chip technology to real-world applications, it istherefore key to introduce a quality control whichmonitors the course of liquid filling.To this end, we recently presented a technique basedon TIR on the channel wall to localize liquid-gasinterfaces in microchannels at rest [3]. In thiscontribution, we present for the first time itsimplementation on a centrifugal microfluidic platform [4].Our technique is based on linear illumination anddetection elements allowing quasi-continuous meniscusdetection with a spatial resolution of 50 ptm at high speedsof rotation up to 30 Hz.SETUPThe experimental setup consists of a rotatable lab-on-a-chip substrate (also referred to as lab-on-a-disk), and adevice (called the "player") which accommodates theoptical components and spins at well defined rotationalfrequencies and acceleration rates.Disk Design and FabricationThe disk contains four pairs of channels aligned at900-offset to each other according to the symmetry of thedisk as illustrated in Figure 1.Figure 1: Schematic of the on-disk structure, showing allrelevant channels and geometric features referred to inthis section.Each channels pair is laterally shifted with respect toa central radial line. The channel under investigation andthe auxiliary channel possess a triangular cross-section,each possessing a wall inclination angle of 450 ("V-groove"). These channels are illuminated by a laser fromthe top, i.e. from a direction perpendicular to the plane ofthe disk. According to Snell's law TIR occurs for aperpendicular incidence in case the ratio of refractiveindices of the bulk material of the disc and the mediuminside the channel exceeds 1.41 at the location of theinclined channel wall. This condition is fulfilled for a gasfilled channel segment, but not in the presence of a liquid.Said channels have a depth of 1.5 mm and a width of3 mm. The four inner endings of the channels near thecenter of rotation are flanked by an inlet reservoir. Theradially outer side of the channel leads to a rectangularchannel measuring 400 ptm in width, 100 ptm in depth,and - 4 mm in length, referred to as the "microchannel".The other four V-grooves are auxiliary channels which arepermanently filled with air.The 2-D fluidic design of the disk is generated by aCAD software. It provides the code for a micromillingmachine which creates the corresponding 2.5-D design978-1-4244-2978-3/09/$25.00 ©2009 IEEE 539Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 11:47:43 UTC from IEEE Xplore.  Restrictions apply. within a cyclo olefin colpolymer (COC) substrate (refract-tive index n = 1.49). The total time from design to thefabricated disk takes approximately 4 hours (depending onthe complexity of the fluidic design).Optical and Read-out ComponentsThe optical measurement components are a line laserand a linear image sensor array. The laser diode emits atthe peak-wavelength Apeak = 650 nm having a maximumoptical power of 24 mW (Roithner RLLH 650-24-3). Thehousing contains this diode as well as a special line opticsgenerating the probe line. The used CMOS linear imagesensor (Hamamatsu S8377) is a compact sensor featuringa built-in timing generator as well as signal processingcircuit. The active area has a length of 25.6 mmsubdivided into 512 individual pixels (pixel pitch: 50 ptm,pixel height: 500 ptm).An electrical controlling unit (Spectronics Devices) isused to address the operating mode of the sensor as wellas to transfer optical data to a PC. The set comprises twosmall size printed circuit boards (PCB): a small 50 mm x20 mm PCB holding the sensor incorporating a pre-amplifier and A/D converter electronics and a secondPCB with the control electronics, a 1-MByte data storagememory for saving 1000 scans and a USB PC-interface.The processing time for one scan takes 2 pts, only, whichallows the here required high-speed data acquisition.OPERATING PRINCIPLEFigure 1-A illustrates the functional principle for theTIR-based meniscus detection while the experimentalsetup is depicted in Figure 1-B:A b)hiquid - aiSnoWls LawFigure 2: (A) Setup of TIR-based meniscus detection. (B)Photo ofthe experimental setup. The beam is issuedfroma line laser onto the rotating substrate and reflected backto a linear image sensor. Legend: a) line laser b) linearimage sensor c) channel under investigation d) auxiliarychannel.Optical PathwayThe beam emitted by the line laser (a) impinges on anauxiliary, permanently air-filled channel (d). Due to the450 inclination of its respective wall, the beam is deflectedby 900 into the plane of the substrate. After a defineddistance corresponding to the spacing between the laserand the detector, the beam impinges on the V-groovedsegment of the channel under investigation (c). If aprobed segment is filled with air, a second TIR redirectsthe laser beam by 900 towards the linear image sensor (b)placed perpendicular to the plane of the disk. Thesegments of the channel which are filled by liquid changethe local refractive index and rule out TIR.The emitting power of the line laser is set to a value(5 mW), thus always driving those pixels of the sensorreceiving high intensity into saturation. This way adiscrete, binary signal is obtained where 1 indicates anempty section, 0 a liquid-filled section of the channel (c).This works up to frequencies of 30 Hz. At higherfrequencies, the received intensity is too low to push thesensor into saturation.Measurements under RotationThe azimuthal position of the channels on the diskand the sensor position are synchronized by using theTTL-signal provided by the player once per revolution asa command-signal for the sensor. The linear image sensorstarts to acquire optical data each time the rising flank ofthe trigger signal occurs and stops after a certainintegration time derived from the frequency of rotation.The integration time is set to a value that each revolution adisk-sector spanning over channels (c) and (d) is covered.Thereby, a consistent and stable measurement system isguaranteed requiring no alignment or signal strengthcalibration.EXPERIMENTSBubble DetectionA representative plot for the pixel intensity along achannel entrapping an air bubble under static conditions isshown in Figure 2, revealing clearly discernible edges atthe respective liquid-gas interfaces. Using proper curvefitting algorithms, the position of the meniscus can bepinpointed down to roughly 3 pixels, corresponding to150 ptm.540Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 11:47:43 UTC from IEEE Xplore.  Restrictions apply. _ IOOD/ 1 ia,_1~0 6 10 16 XChannel length [mm]Figure 3: Measuredpixel intensity plot ofa bubble withina channel captured by the linear image sensor at rest.The transition between the liquid and the gas state is verydistinct thus allowing a clear localization ofthe meniscus.Volume CalibrationThe detection of gas bubbles and liquid filling levelsare essential to assure reproducible and quantitativebioassays on LoaC systems. This section describes thecalibration of our TIR-based measurement system to meetthese requirements. Figure 3 displays a calibration curvewith five pre-metered volumes, featuring a high accuracyand a precision of 1.9 0.30 -L-I0E'5'a0U,025 -20 -15-10-10 15 20 25Reference volume [pQL30Figure 4: Calibration of the measured volumes with pre-metered volumes featuring a high precision of 1.9 %.For a fixed microchannel geometry, the theoreticalmaximum filling level is calculated with respect to thelength of the channel. In the next step, the active area ofthe linear image sensor is laterally shifted (positioned) insuch a way that it provides output characteristicscorresponding to the calculated liquid level. Once thesystem is calibrated, the measurements described in thefollowing sections are performed. For the volumecalibration, liquid-levels of five different volumes (10, 15,20, 25 and 30 ptL) are measured five times each. Eachmeasurement is conducted under rotation at 10 Hzwhereby the position of the liquid-gas interface isaveraged over 10 revolutions with one measurement perrevolution. Figure 3 shows the capability of this system toaccurately measure and meter liquid volumes duringrotation.Flow Rates and ViscositiesCentrifugally driven discharge induced by a constantrotational frequency of 10 Hz is presented in Fig. 4. Testsare based on four different glycerol solutions withdynamic viscosities in the range of 1 - 10.7 mPa s.25 -20 -c 15 -E> 10-5-~| 1.2 mPas0 2.0 mPas6.8 mPasv 10.7mPas2Time [s]3 4Figure 5: Flow rate and viscosity measurements derivedfrom a time-resolved liquid-level determination with thecalibrated system. The relative decrease of the flow ratecorrelates with increasing viscosity.25 ptL of each sample liquid are pipetted into the inletreservoir. Fluids are driven through the inclined channelby centrifugal forces and introduced into the adjoiningmicrochannel. The flow rate in the microchannel itself isindirectly measured via the movement of the liquid-gasinterface in the channel under investigation using a curve-fitting algorithm.Four different liquids with viscosities of 1.2, 2, 6.8,and 10.7 mPa s are investigated. The time resolved mea-surements for each moving liquid reveal the 1/dependence for the flow rate during the outflow. Also,with the dynamically decreasing plug position, a multi-parameter dependent flow rate can be identified. Themotion of the meniscus is affected by the spinningfrequency, interfacial interactions, microchannelgeometries, and the viscosity of the fluid.Contact-free and on-line determination of the flowrate provides an insight into liquid guidance on lab-on-a-disk systems. Furthermore, the implementation of afeedback-loop can improve the liquid guidance on-diskand contribute to a better controllable fluidic system.Figure 5 demonstrates the capability of the system tomeasure the dynamic viscosity of low-viscosity liquids.The unprecedented dynamic recordings of the flow ratesand viscosities during rotation are enabled by the linearoptical elements in contrast to the static scanning principleof our previous work [3].541II I1I;Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 11:47:43 UTC from IEEE Xplore.  Restrictions apply. 12,a-0C.,a.E.Fnu010,7,5,2,0,l0,0 2,5 5,0 7,5 10,0 12,5Reference viscosity [mPasjFigure 6: Dynamic viscosity measurement featuring aprecision of4.3 %0for viscosities up to 10. 7 mPa s.The previously described glycerol solutions are usedto highlight the correlation between the measuredviscosities and the reference viscosities. First, definedvolumes (25 ptL) are introduced into the channel underinvestigation and centrifugally pumped at a constantfrequency of v = 10 Hz through the connectedmicrochannel. Each flow rate is measured five times.Secondly, the viscosity is calculated taking thefollowing physical parameters into account: flow rate andposition of the passing liquid plug, temperature, angularfrequency and the dimensions of the microchannel. Aprecision of 4.3 is a good result compared to rheologymeasuring devices struggling with difficulties in the lowviscosity regime. In addition to flow rates of unknownsamples, our system reveals absolute viscosity data as asurplus.CONCLUSIONThis work investigates a novel TIR-based setup withlinear optical elements for spatio-temporally resolvedmeniscus detection. The system is well suited forcompact system integration and excels with its high-speeddata acquisition and low cost compared to conventionalimage processing systems.Due to the simple integration of the auxiliary channelstructures in the microfabrication process of the substrateas well as the uncomplicated component upgrade of thedisk-processing unit, the opto-fluidic technology can beeasily incorporated into centrifugal microfluidic systems.The system can be used for quality control, feedbackloop and calibration purposes on centrifugal lab-on-a-diskplatforms, e.g. for the on-line verification or liquid fillingor outflow, for monitoring the flow of a liquid plug, forreagent metering or viscosity measurements of unknownpatient samples. The contactless measuring principle doesnot require any pretreatment of the fluid, e.g. theintroduction of particles, and prevents any impact on thefluidic function itself.Implementation of our measurement system into real-world point-of-care devices could significantly improvethe quality of assay results by the real-time monitoring ofliquid levels, flow rates, viscosities and gas bubbleformation in liquid handling operations.REFERENCES[1] P. S. Dittrich and A. Manz, "Lab-on-a-chip:microfluidics in drug discovery," Nature ReviewsDrug Discovery, vol. 5, no. 3, pp. 210-218,Mar.2006.[2] J. Y. Park and L. J. Kricka, "Prospects for nano-and microtechnologies in clinical point-of-caretesting," Lab Chip, vol. 7, no. 5, pp. 547-549, 2007.[3] F. Bundgaard, 0. Geschke, R. Zengerle, and J.Ducree, "A simple opto-fluidic switch detectingliquid filling in polymer-based microfluidicsystems," in Proceedings of the 14th InternationalConference on Solid-State Sensors, Actuators andMicrosystems (Transducers & Eurosensors '07)Lyon, France: 2007, pp. 759-762.[4] J. Ducree, S. Haeberle, S. Lutz, S. Pausch, F. vonStetten, and R. Zengerle, "The centrifugalmicrofluidic Bio-Disk platform," J. Micromech.Microeng., vol. 17, p. S 103-S115, 2007.5425~~~~~~~~~~~~~~~~~~~~~~~~~~~lo- I//0~~~~~~~~~~~~~~~~~~~~~~~~,5 ,,-"-I_0-Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 11:47:43 UTC from IEEE Xplore.  Restrictions apply. 

A simple opto-fluidic switch detecting liquid filling in polymer-based microfluidic systems,&quot;

Lab-on-a-chip: microfluidics in drug discovery,&quot;

Prospects for nanoand microtechnologies in clinical point-of-care testing,&quot;

The centrifugal microfluidic Bio-Disk platform,&quot;

TIR-based dynamic liquid-level and flow-rate sensing and its application on centrifugal microfluidic platforms

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TIR-based dynamic liquid-level and flow-rate sensing and its application on centrifugal microfluidic platforms

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