41Molecular Biotechnology – Practice | Bi7430c
MICROFLUIDICS & LAB ON A CHIP
location: INBIT, Kamenice 34, ground floor, rooms 0.22 and 0.23
lecturer: Mgr. David Kovář, Ph.D.(kovard@mail.muni.cz)
Mgr. Michal Vašina (michal.vasina@recetox.muni.cz)
Jan Česnek (242752@mail.muni.cz)
I. WORKFLOW
– on-demand droplet generation
– droplet microfluidics andmicroscopy
– screening of reaction conditions on the chip
– capillary microfluidic platform
II. MOTIVATION
Microfluidics can be defined as the science and technology manipulating and analysing fluid
flow in sub-millimeter dimensions. It is becoming important technology for many emerging
applications and disciplines, especially in the fields of chemistry, biology and medicine.
Concrete application examples are biosensor devices for molecular diagnostics, polymerase
chain reaction chips, high-throughput screening, controlled drug delivery systems, drug
discovery methods, forensic analysis instruments, and so on (1).
III. THEORETICAL BACKGROUND
III-A. On-demand droplet generation
A nice technique for droplet generation, when multiple distinct samples are necessary, is the
on-demand generation using the commercial microfluidic device, Mitos Dropix from Dolomite
Microfluidics, UK. The scheme of the droplet generation principle is shown in Figure 1.
Figure 1 Droplet on demand – a constant suction driven flow results in the creation of a segmented
flow. The timing of the ‘up’ or ‘down’ position of the sampling hook dictates the droplet volume and
spacing volume respectively. The transverse position of the hook dictates the selection of the sampling
well. (Source: https://www.dolomite-microfluidics.com/wp-content/uploads/mitos-dropix-droplet-
splitting-application-note-1.pdf)
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III-B. On-chip droplet formation
Formation of water in oil droplets in microfluidic chip has several benefits when compared to
standard technology. Amongst such benefits belong low volume of reagents consumed, chip
modularity, low cost and simple fabrication. When all pros combined properly one may
encounter drop costs of screening million fold [3,4].
In this practice students will put hands on microfluidic chip technology. Prepared chips are to
be used for water in oil droplet generation [5]. An example will follow with encapsulation of
single E.coli BL21 DE3 cells to droplets. Finally, there will be observation of single cells in
emulsions generated.
III-C. Screening of reaction conditions suitable for luciferase enzyme on the chip
Microfluidic devices are suitable for time-efficient and strictly precise mixing of solutions.
Therefore, microfluidic chip allows to test big number of possible combinations in short time
and it can be used for semi-automated screening of reaction conditions such as optimal pH,
concentration of effectors or additional salts. Concentration of reaction components inside
droplets are managed by setting proper flowrates of individual solutions. Gradient of pH can
be established in droplets by mixing buffer acid (tricine) and buffer base (bis-tris propane =
BTP) in proper ratios.
Renilla reniformis luciferase (RLuc) is oxidoreductase which emits blue light during conversion
of its substrate coelenterazine (CTZ). The aim of this practical part is to test the activity of
RLuc on a glass chip in different pH and approximately find out the pH optimum for this
enzyme. Ultra-sensitive camera will be used for detection of enzymatic bioluminescence.
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III-D. Capillary microfluidic platform
Technical setup (Figure 2) can be described separately as an optical, mechanical and
microfluidic part. A precise calibration is one of the primary processes. First, the concentration
of HCl must be determined by acid-base titration with potentiometric detection. Once we
know the concentration of the acid, the titration of the working buffer must be performed to
know the dependency between the proton concentration (pH respectively) and the
fluorescence intensity of the probe. Working buffers with three different pH values are used
for the “on-platform” calibration of the proton-sensitive assay – the buffer with initial pH
≈8.00 and several mixtures adjusted by titration with HCl to desired pH, respectively. The
fluorescence intensity of the pH-sensitive probe must be measured at all check-points (loops)
and for all ratios of reaction mixture and acid. Please note, that the fluorescent dye is
temperature sensitive and therefore the calibration must be measured for all the
combinations.
Figure 2 A photography of the capillary-based microfluidic platform with highlighted optical (O),
mechanical (M) and microfluidic (µFlu) parts.
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IV-A. PROTOCOL (On-demand droplet generation and fusion)
1. Prepare 1mL of 50 μM HPTS solution out of 1 mM HPTS stock solution. Using the 6 mM
HCl stock solution, prepare five HCl calibration solutions of concentrations equally
distributed between 1.5mM and 0.3mM. Use the just prepared 50 μM HPTSsolution for
dilution. Plan the pipetting so that you finally have at least 40 μL of each solution.
2. Insert 20 μLof the 50 μMHPTS solution and of each calibration solution into separate
wells of the rack. Insert also 20 μL of the test-enzyme sample into another well.
Markdown the numbers of thewells.
3. Start the microfluidic pump at 10 μL/min in a withdraw mode.
4. Write the sequence of droplets to be generated into the Dropix software. The order of samples is up to
you, but it should have some logic. Just follow the rule that each aqueous phase droplet should be 150 nL
and the oil phase in between the droplets should be 300 nL. Also, 10 droplets should be generated per
each of the sample (per each well).
5. Start the droplet generation onDropix.
6. When finished wait a while, until you visually see that the generated droplets in the
tubing.
7. Start the detection on the LabView detectionwindow.
8. Observe the droplets being detected – both with the naked eye and on the detector.
IV-B. PROTOCOL (On-chip droplet formation)
Solutions and reagents:
– HFE-7500 or FC-40 oil
– PicoSurf-2 surfactant
– Trichloro(1H,1H,2H,2H-perfluorooctyl)silane
– 1.5M NaCl
– deionized H2O
– E.coli BL21 DE3 (calculate the concentration!)
– Percoll ®
– Isopropanol
Equipment:
– Chemyx Fusion 200 syringe pumps or precise neMesys syringepumps
– gas-tight syringes (variousvolumes)
– PTFE tubing
– Microfluidic chips – variousdesigns
– glass slide
– Inverted microscope
1. load syringes with HFE-7500 oil and 150 mM NaCl, 25 % (v/v) Percoll and properly
diluted cells
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2. attach PTFE tubing to the syringe and remove anybubbles
3. put syringes into the syringe pumps, lock tight and set proper syringe diameter
4. connect syringe via tubing into thechip
5. set liquid flow – 300 µL.h-1
for oil phase and 30 µL.h-1
for the aqueous phase
6. observe droplet formation under microscope at variousmagnification
7. verify cell occupation in emulsions on invertedmicrosope
IV-C. PROTOCOL (Screening of reaction conditions
suitable for luciferase enzyme on the chip)
Solutions and reagents:
– HFE-7500 or FC-40 oil
– PicoSurf-2 surfactant
– 50mM bis-tris propane = BTP (pKA = 9.0) + 150mM NaCl solution
– 50mM tricine (pKA = 8.1) + 150mM NaCl solution
– Renilla reniformis luciferase (RLuc8) solution (diluted in mixture of BTP and tricine 1:9)
– coelenterazine (CTZ) solution (diluted in mixture of BTP and tricine 1:9)
– deionized H2O
– isopropanol or ethanol
Equipment:
– glass microfluidic chip
– ultra-sensitive camera
– neMesys syringepumps
– gas-tight syringes (variousvolumes)
– FEP tubing
– pH meter
1. load syringes with HFE-7500 oil, RLuc8, CTZ, 50mM BTP and 50mM tricine
2. remove any bubbles from the syringes, attach FEP tubing to the syringe and fill tubing
with solutions
3. put syringes into the syringe pumps, lock tight and set proper syringe diameter
4. connect syringe via tubing into thechip
5. set liquid flow – 4 µL.min-1
for oil phase and 1 µL.min-1
for the aqueous phases
6. observe droplet formation under microscope
7. change flowrates of BTP and tricine to establish different pH in droplets
8. take images of the chip by FluorCam software for every flowrate settings
9. measure pH of bis-tris propane and tricine mixtures (in tested ratios) by pH meter
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IV-D. PROTOCOL (Enzyme kinetic on the microfluidic platform - calibration)
Solutions and reagents:
– FC-40 oil
– 1.0M NaCl
– 50 mM Tricine
– 50 mM BisTrisPropane
– deionized H2O
Equipment:
– neMesys precise pump
– gastight syringes (variousvolumes)
– PTFE tubing
– Microfluidic platform
– CCD spectrophotometer
1. Prepare 5 mL of 50 μM HPTS solution (use the 10 mM HPTS stock and UB1 buffer) and
0.5 mL of 2 mM HCl solution (use 100 mM stock solution and DW)
2. Fill the gastight syringes (HPTS, HCl, FC-40) and mount them into pump
3. Start the pumps at flowrates (Oil 20 µL.min-1
/ HPTS 8 µL.min-1
) – generating plugs
4. Increase the flowrate of HCl for increment of 0.05 µL.min-1
till 0.5µL.min-1
4. Collect data and plot the calibration curves
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V. HOMEWORK
1. Calculate the volumes to be pipetted to prepare all reaction solutions according to the
protocol IV-A.
2. Estimate volume in pico-/femto- litres for monodisperse droplet formed at channel
having dimensions 5, 10 and 20 µm, respectively (assume square cross-section forms
spherical droplets of the samediameter).
3. For calculated droplet volumes estimate approximate cell density in x.10y
per mL, to put
single cell per droplet. There are approximately 1.108
cells in medium with OD600 0.5.
Cultivated cells have OD600 4.8. In case that grown culture has insufficient density, calculate
the factor for thickening cell media to sufficient level. Account for 10 % pipetting error.
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VI. LITERATURE
1. Dittrich, P. S., and Manz, A. (2006) Lab-on-a-chip: microfluidics in drug discovery., Nat.
Rev. Drug Discov. 5,210–218.
2. F. Gielen et al., “A fully unsupervised compartment-on-demand platform for precise
nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition,” Anal.
Chem., vol. 85, no. 9, pp. 4761–4769,2013.
3. Mazutis, L., Gilbert, J., Ung, W. L., Weitz, D. a, Griffiths, A. D., and Heyman, J. a. (2013)
Single-cell analysis and sorting using droplet-based microfluidics., Nat. Protoc. 8, 870–
91.
4. Yin, H., and Marshall, D. (2012) Microfluidics for single cell analysis., Curr. Opin.
Biotechnol. 23, 110–9.
5. Teh, S.-Y., Lin, R., Hung, L.-H., and Lee, A. P. (2008) Droplet microfluidics., Lab Chip, The
Royal Society of Chemistry 8,198–220.