A multifunctional pipette.

Fabrication of the microfluidic tip Devices ( Fig. 1A ) were fabricated in PDMS (Dow Corning Sylgard 184) using standard soft-lithography techniques. Instrumental procedures and bonding conditions were applied as previously described. 4 Four pipette tips were simultaneously molded using a polycarbonate form (Hagal Machinery AB, Mölndal, Sweden). To define the pipette shape, reservoir wells and microchannels, a master was fabricated using SU-8 on a 4″ silicon wafer, with characteristics summarized in Table 1 . The molded PDMS slab was bonded to a 20 µm thin PDMS film, followed by cutting out each tip and punching out the bottom of the wells. The PDMS part was subsequently bonded to a 0.55 mm thick glass substrate (Praezisions Glas & Optik GmbH, Iserlohn, Germany) to increase the structural strength of the tip and rigidify the body of the device. The step-by-step fabrication procedure is depicted in Fig. S1 of the ESI † . Holder and control interface The holder ( Fig. 1B ) was fabricated in stainless steel (Teadusmosaiik OÜ, Tartu, Estonia) and constitutes a compromise between aqueous solution compatibility, rigidity and cost. The lower part of the holder provides a self-aligning cavity, into which the pipette tips can slide. A cylindrical 7 mm diameter rod is attached at the rear of the holder, to allow for easy interfacing with common micromanipulators. 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++).

Characterization

Solution switching within the microfluidic channels and at the pipette outlet was characterized using a 100 µM solution of fluorescein (sodium salt) in deionised water. An inverted microscope Leica DM IRB (Leica Microsystems, Wetzlar, Germany) equipped with a fluorescence lamp, a Leica 10× 0.3 NA air objective, and a Chameleon USB camera (Point Gray Research Inc., Richmond, Canada) was used for experimentation. Alternate visualization was implemented using water soluble food colour in order to highlight the component flows at the switching junction. 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).

Cell culturing and stimulation Adherent Chinese hamster ovary

(CHO) cells expressing the human transient receptor potential vanilloid 1 (hTRPV1) ion channel were cultured as previously described. 4 The cells were loaded with calcium sensitive dye, Calcium Green™-1 AM ester (Life Technologies Inc, Carlsbad, USA). At first, a 200 µM stock solution was prepared in DMSO, which was further diluted with Ringer's solution (VWR) to obtain a final concentration of 4 µM. The cells were cultured on Petri dishes equipped with a 0.15 mm thin glass bottom, suitable for confocal imaging. After removing from the incubator, the culture medium was removed and the cells were rinsed twice with Ringer's solution, followed by addition of the dye labelling solution. The cells were exposed to the dye loading solution for 1 h at room temperature, then rinsed twice with Ringer's solution, which was also used as the experimental buffer. 1 µM capsaicin (Sigma-Adrich) and 10 mM, pH 7.4 calcium chloride solution (VWR) were loaded into the multifunctional pipette, and the microscopy experiment was performed immediately thereafter. Fluorescence intensity was measured with a Leica DM IRE2 confocal microscope equipped with a Leica TCS SP confocal scanner, an oil-immersion 40× 1.25 NA Leica objective, and Leica Confocal Software (v2.61). Fluorescent intensity was measured from single cells, with excitation at 488 nm and emission collected between 505 and 600 nm.

Simulations

Simulations were performed with COMSOL Multiphysics 4.1, combining models of laminar flow (spf) and transport of dilute species (chds) in stationary and transient 3D models. In the simulations, the mechanical properties used for PDMS were: Young’s modulus 1.8 MPa and Poisson's modulus 0.45, 18 the viscosity of water was 1 mPa s and the diffusivity of fluorescein was 5 × 10 −10 m 2 s −1 . 5

Supplementary Material Movie 1 Movie 2 SI