Alar
Ainla
,
Gavin D. M.
Jeffries
,
Ralf
Brune
,
Owe
Orwar
and
Aldo
Jesorka
*
Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296, Göteborg, Sweden. E-mail: aldo@chalmers.se; Fax: +46 31 772 6120; Tel: +46 31 772 6112
First published on 17th January 2012
Microfluidics has emerged as a powerful laboratory toolbox for biologists, allowing manipulation and analysis of processes at a cellular and sub-cellular level, through utilization of microfabricated features at size-scales relevant to that of a single cell. In the majority of microfluidic devices, sample processing and analysis occur within closed microchannels, imposing restrictions on sample preparation and use. We present an optimized non-contact open-volume microfluidic tool to overcome these and other restrictions, through the use of a hydrodynamically confined microflow pipette, serving as a multifunctional solution handling and dispensing tool. The geometries of the tool have been optimised for use in optical microscopy, with integrated solution reservoirs to reduce reagent use, contamination risks and cleaning requirements. Device performance was characterised using both epifluorescence and total internal reflection fluorescence (TIRF) microscopy, resulting in ∼200 ms and ∼130 ms exchange times at ∼100 nm and ∼30 μm distances to the surface respectively.
Recently, several research groups have reported a new class of devices, which can deliver liquid into an open volume while simultaneously rerouting the liquid back into the chip. This is achieved by means of closely spaced adjacent channels under positive and negative pressure, respectively, creating a hydrodynamically confined flow (HCF) volume.3–5 This volume, fluidically connected to the device tip, can be positioned to stimulate and analyse a single cell or other object of interest, without affecting the surrounding liquid.
Hydrodynamic confinement of one miscible solution inside another is possible due to convective recirculation, counteracting diffusion, which otherwise would mix the two solutions. The balance of convection and diffusion in such an arrangement is described by the dimensionless Péclet number (Pé). A larger Pé leads to a greater level of confinement, establishing a sharper, more defined boundary between the two liquids.
The idea of recirculation probes originates from physiology, where push–pull cannulae have been used for in vivo sampling since 1961.6 These probes remain popular for sampling of neurotransmitters.7,8 Hydrodynamic confinement within an open volume was first demonstrated in a picolitre ‘fountain-pen’ made of two co-axial pipettes, where the inner capillary is used for injection of reagent, and the outer one for aspirating the reagent back into the pipette.9 Similarly, theta tube capillaries have been applied for recirculation to stimulate cells with lysis buffer and collect the lysate for analysis.10 Both types of delivery require fragile tips that can be costly and difficult to fabricate.
Later, microfabrication in silicon and polydimethylsiloxane (PDMS) was used to create microfluidic probes, which circulate the liquid in a thin cleft, formed between the face of the probe tip and the surface under investigation.3,5,11–13 A similar principle has been exploited for use in an electrochemical probe, fabricated from polyethylene terephthalate (PET) and used to analyse dry surfaces.14 In this case the recirculation provides and refreshes an electrolyte droplet in front of the working electrode. Other free-standing probes have been employed for studying chemotaxis15 and to deliver genetic material16 in optical microscopy experiments. The open volume flow recirculation principle has also been applied for stimulating brain slices.17
We have previously reported the concept of a HCF microfluidic pipetting device, constructed by sealing channel grooves with a thin membrane.4 This configuration enables recirculation while allowing for angle adjustment and repositioning of the device. It closely resembles the utility of glass micropipettes, which are typically used in microscopy experiments. This particular feature strongly influenced the decision to coin our concept a ‘pipette’.
Herein we describe the construction and characterization of a multifunctional pipette, developing the initial concept into a research-ready device, optimized to fit into practically any micromanipulation environment. The pipette has been given a sharp elongated shape, facilitating the application within spatially confined microscopy setups, typically found in bioscience environments, where crowding with probes and manipulation equipment is commonplace. We therefore optimized the geometry to reduce the impact on the microscope sample stage, leaving sufficient space for integrating eight on-chip wells, which can store and provide solutions. We have also created a self-aligning holder, designed for robust clamping of the pipette and providing each well with individual pressure control. The pipette and its interfacing have been optimized for both performance and convenience of use by bioscientists without particular microfluidics expertise.
We provide extensive characterization of the pipette performance, and show exemplary circuitry for on-chip switching between three solutions, which we applied to sequentially deliver capsaicin and calcium to single adherent cells in a surface-adhered collective. The concept is highly flexible, novel circuits and functions can be quickly introduced by using new microstructure molds and adapting the control software accordingly. The holder itself is reusable, allowing for rapid adaptation at low cost.
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Fig. 1 The multifunctional pipette: components, construction and setup. (A) The PDMS microfluidic pipette tip. (B) The holder, shown in a closed state holding the tip. An elevated angle is also displayed to highlight the manifold's interfacing structure, which contacts the tip. (C) Pneumatic interface between the holder and the tip is formed with sharp flanges protruding from the manifold, which are pushed into the PDMS surrounding the well. Each well has an individual connection, through which pressure can be controlled. (D) Deformation associated with pressure change within the well. (E) Implemented solution to (D), minimising the effects through the use of an isolation well. Exemplary circuitry of a fast 3-solution switch, showing the driving mechanism (F) and the microfluidic switching junction near the tip (G). Microscopy images of a fluidic switching junction (H) and a recirculation volume (I) loaded with coloured water. |
Characteristic | Symbol | Value | Unit |
---|---|---|---|
Channel height | H | 20 | μm |
Channel widths | W | 20 and 40 | μm |
Flow conductance of channels | |||
Supply/waste/recirculation (well → tip) | G | 37 | nL s−1 bar−1 |
Outlet (switch → tip) | G o | 500 | nL s−1 bar−1 |
The upper part of the holder consists of a hinged manifold, which provides pressure connectivity to each of the wells. The underside of this manifold contains sharp flanges (Fig. 1C), which are pushed against the PDMS pipette, sealing it to the manifold when the holder is closed. This interface has been tested to withstand pressures in excess of 2 bar, more than double the maximum operation pressure projected. The manifold connects each well individually with the pressure control unit through a 1 m long and 1 mm I.D. length of Nalgene® PVC tubing, which we determined to be optimal to obtain the fastest pressure switching (see Fig. S2†). Supply pressures were controlled with an in-house developed pressure and valve actuation controller (Fig. S3†), allowing vacuum control within the range 0.00–0.60 ± 0.01 bar, and channel pressure control for two channels in the range between 0.00 and 0.90 ± 0.01 bar (at 95% confidence level). Pressure and vacuum controllers were calibrated with a high precision digital manometer LEX1 (Keller AG, Winterthur, Switzerland). Flow switching is achieved through a valve controller, actuating pressure in the wells with the following specifications: electronic time resolution, 1 ms; delay between the electronic signal and valve opening, 23 ± 2 ms; pressure rise time, 6.0 ± 0.7 ms; delay between the electronic signal and valve closing, 6 ms and pressure fall time, 6 ± 1 ms. All experiments were controlled via an in-house programmed graphical user interface, developed in Microsoft Visual Studio 2008.NET (C++).
Solution switching near the substrate surface (∼100 nm) was further characterized using TIRF microscopy with a Leica HCX PL APO 63× 1.67 NA oil TIRF objective, utilising 488 nm Sapphire 488-150 CW CDRH laser excitation (Coherent, Santa Clara, USA).
Two levels of pneumatic pressure are supplied by the attached pressure control device, and fast switching is achieved through the use of miniature solenoid valves (Fig. S3†). Two pressure levels are needed to avoid backflow from the switch to any of the supply channels, which would cause unwanted mixing of these solutions. For this switching principle to function properly, the pressures applied need to meet certain conditions. The flow rates in the two inactive (switched off) channels have to be smaller than the flow rates to one of the waste channels. In this case only one solution is directed to the output,
2G(P1 − p) < (p + V)G |
Crucially, there must be no backflow from the switch to the wells in the “switched off” state, requiring p < P1. Finally, for typical recirculation settings, the total inflow has to be twice the outflow, requiring VG ≈ pG0.
Considering these conditions, and the physical performance limits of our pressure controller, we can derive a set of suitable operation parameters, which are summarized in Table 2. The number of solutions to be switched can be increased, which in the case of our system is limited to seven, as at least one well has to be reserved for recirculation and waste.
Parameter | High flow rate | Medium flow rate | Low flow rate |
---|---|---|---|
P 2/bar | 0.9 | 0.5 | 0.3 |
P 1/bar | 0.2 | 0.06 | 0.035 |
V/bar | 0.4 | 0.19 | 0.094 |
Outflow I0/nL s−1 | 13.5 | 6.5 | 4.9 |
Run time/min | 16 | ∼30 | ∼45 to 120 |
Pressure control in elastic devices, such as PDMS, requires attention to possible elastic deformation, which might compromise positioning precision (Fig. 1D). To mitigate motion of the tip during pressure pulsing, we have added a deformation isolation well to the tip and introduced additional flanges toward the front of the manifold (Fig. 1E). These measures reduce unwanted motion during most operations to less than 4 μm in the axial direction (pressure 0.5 bar) (Fig. S4†).
In an attempt to facilitate a more unified design, both the molding tools and the holder have a universal geometry, while the internal circuitry of the pipette tip can be re-designed to address specific experimental needs. This allows very rapid customization, requiring only fabrication of a new channel molding master.
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Fig. 2 Characterising solution exchange times. (A) Experimental setup using TIRF microscopy, to monitor solution exchange dynamics near the sample surface. The same setup can be used for epifluorescent measurements of a deeper region, through modification of the excitation angle. (B) Fluorescent intensity measurements of periodic solution exchange near a glass surface using TIRF. (C) Concentration rise time near the surface with high and medium flow rates (∼10 and ∼5 nL s−1, respectively). |
To elucidate the factors influencing the solution exchange time, we carried out a theoretical evaluation, based on analytical models and finite element simulations (COMSOL). The results are summarized in Table 3, Fig. 3, and in the ESI (Fig. S6–S9†). The largest contribution arose from the flow rate rise time in the supply channel, which is due to the elasticity of PDMS, especially at the end section of the channel, where one of the walls is a thin, unsupported and easily deformable PDMS layer (Fig. S6†). Channel deformability leads to compliance, the hydraulic analogue of capacitance.21 When coupling this with hydraulic resistance of the channel, a low-pass RC filter is formed, dampening rapid pressure changes, leading to slower flow rate changes towards the end of the channel (Fig. S7†). Thereafter the concentration pulse passes through the outlet channel, where it is subject to dispersion,22 which broadens the pulse (Fig. S8†).
Component | τ/ms | Scaling ∝ |
---|---|---|
Solenoid valves | 2 | |
External tubing | 4 | |
Inertia | 0.5 | w 2 l −1 |
Supply channel | 30 | w −1 l 2 d −1 |
Dead volume of the switch | 2.5 | w 3 Q −1 |
Outlet channel | ∼70 | w 2 l 0.5… 1 Q −0.5… −1 |
Outlet to cell | ∼40 | |
Sum | ∼150 |
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Fig. 3 Results of a computer simulation to model the fluidic exchange times close to the sample surface. (A) Concentration rise time near the surface (100 nm above) and at the channel output. (B) Contributing factors and influence to the solution exchange time, based on experimental results, simulations and calculations. |
Once the solution has exited the channel, it has to reach the surface-immobilized cell. Since flow near the surface is restricted due to friction, a short final distance has to be passed mainly by means of diffusion, which occurs at a slower rate (Fig. 3A). Other factors, such as filling the dead volume of the switch, inertia of the liquid and external pressure control, have only minor contributions (Table 3 and Fig. 3B). To improve the switching speed, the outlet channel can be shortened. Enlarging the channel dimensions, which increases the flow rate, would also increase the speed (Fig. S9†). This will reduce the experimental run time as the liquid in the supply wells will be consumed at an accelerated rate. A practical solution in many experiments, since short and long time-scale processes are typically not studied simultaneously, would be to minimize flow rates during setup and positioning (“stand-by mode”), then to only increase supply pressures when high switching speeds are desired. We have implemented such structured modes of operation into the application software. A steeper positioning angle, which would direct the flow closer to the surface, and fabrication in hard materials would be other feasible options to reduce solution exchange times.
The use of a thicker bottom layer for channel sealing can cause opposing effects, reducing, on one hand, the elasticity of the channel, while on the other hand increasing the distance between the channel outlets and surface. Based on simulations and estimations, the exchange speed in this kind of devices could be improved to ∼10 to 50 ms (increased flow rates, minimising outlet channel length to the open volume, and use of hard materials for the device). When evaluating fluidic exchange behaviour, it is also necessary to consider the nature of the desired active agent and its diffusivity in solution.
An adherent CHO cell culture, expressing hTRPV1, was used to demonstrate the use of the device for chemical stimulation of single cells, through the sequential delivery of active compounds to selected cells from within a collective. The cells were pre-treated with a cell-penetrating, mildly fluorescent species, which upon entering the cell is enzymatically converted into a calcium chelator. In the presence of calcium ions this molecule forms a highly fluorescent complex, which can be readily detected. Using the multifunctional pipette, the dye loaded cells were first treated with a 10 s pulse of capsaicin to activate the hTRPV1 channel, then washed for 5 s and treated with a 10 s pulse of Ca2+. After 50 s, the Ca2+ pulse was repeated. Cells that had been exposed to capsaicin became Ca2+ permeable, which was observed by their rapid increase in fluorescent intensity, whereas Ca2+ applied to cells not treated with capsaicin demonstrated no significant increase. A slight shape change was observed during the course of the experiment, likely due to a slight osmotic pressure imbalance.
Interestingly, we observed several different types of cellular response, as depicted in Fig. 4C. Some cells exhibited a strong response to the first calcium pulse, but not to the second (Cell 1), while others responded to both pulses (Cell 2). Varied loading efficiencies of the indicator dye within the cell population gave a wide range of initial fluorescence intensity values. One such cell was selected from the background population outside the recirculation zone (Cell 3) and demonstrated no response to the calcium pulses, indicating fluidic isolation from the targeted cells. The observed variable calcium responses may be cell population heterogeneity, or flow velocity dependent, as lateral positioning with respect to the pipette tip will determine the flow rate. Some cells situated within the centre of the recirculation zone (marked with a dashed line) exhibited behaviour similar to an internal calcium release. Capsaicin application and high flow velocities within this region may have contributed to this type of cellular response.
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Fig. 4 Chemical stimulation and response of individual hTRPV1 expressing CHO cells, through activation with capsaicin. (A) Bright field microscopy image of adherent cell culture of hTRPV1 expressing CHO cells, with the pipette tip targeting a small collective. (B) Confocal fluorescent micrograph of Calcium Green™ loaded cells after stimulation with capsaicin and subsequent exposure to Ringer's solution, spiked with 10 mM calcium chloride. (C) A graph of individual cellular responses to the application protocol, with the delivery times highlighted above. Cells 1 and 2 are within the recirculation zone and display a positive activation response. Cell 3 is a representative cell from the background outside of the stimulation zone. |
We have also demonstrated that the internal fluid processing circuitry can be contained within a recirculation device, minimizing on-chip dead volumes and decreasing the solution switching times. Hard material microfluidic probes could offer advantages when very small recirculation volumes are desired, since vertical configuration and circulation in small clefts allow for forcing liquid circulation closer to the surface and to achieve higher Péclet numbers. This is mostly favourable for chemical surface processing, but less applicable for cell cultures and tissue, as high shear stress can move, detach and destroy cellular structures. Moving away from elastomeric materials would also remove device compliance, which will allow faster solution switching.
In summary, we believe that the versatility, simplicity of design, and ease of integration of the multifunctional pipette, either as a primary or supporting device, will impact single cell and tissue culture research, most prominently in neuroscience and pharmacology, where it can amend current superfusion techniques and open new application areas.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2lc20906c |
This journal is © The Royal Society of Chemistry 2012 |