Electrochemical attosyringe

Abstract

The ability to manipulate ultrasmall volumes of liquids is essential in such diverse fields as cell biology, microfluidics, capillary chromatography, and nanolithography. In cell biology, it is often necessary to inject material of high molecular weight (e.g., DNA, proteins) into living cells because their membranes are impermeable to such molecules. All techniques currently used for microinjection are plagued by two common problems: the relatively large injector size and volume of injected fluid, and poor control of the amount of injected material. Here we demonstrate the possibility of electrochemical control of the fluid motion that allows one to sample and dispense attoliter-to-picoliter (10(-18) to 10(-12) liter) volumes of either aqueous or nonaqueous solutions. By changing the voltage applied across the liquid/liquid interface, one can produce a sufficient force to draw solution inside a nanopipette and then inject it into an immobilized biological cell. A high success rate was achieved in injections of fluorescent dyes into cultured human breast cells. The injection of femtoliter-range volumes can be monitored by video microscopy, and current/resistance-based approaches can be used to control injections from very small pipettes. Other potential applications of the electrochemical syringe include fluid dispensing in nanolithography and pumping in microfluidic systems.

Fig. 1.

Scheme of the electrochemical attosyringe.

Fig. 2.

Sequential ingress/egress of water in a DCE-filled nanopipette. (a) Initial immersion, E = +600 mV. (b) Ingress of water after the voltage was stepped to −100 mV an then to +90 mV. (c) Complete egress of water at E = +600 mV. (d) Same as b, but with a shorter step time at E = −100 mV. (e) The voltage was stepped again to −100 mV and then back to +90 mV. The aperture radius was ≈300 nm. The pipette was filled with 10 mM THATPBCl in DCE and immersed in 10 mM KF aqueous solution.

Fig. 3.

Dependence of the pipette resistance on the amount of water drawn into it. Measured values of pipette resistance (symbols) are fitted to the theory (solid line). The best fit was obtained with R_out = 1.2 GΩ. The pipette was filled with DCE containing 10 mM THATPBCl and immersed in a 100 mM MgSO₄ aqueous solution. a = 110 nm, α = 6.3°, κ_inner = 114 μS/cm, and ρ_inner^outer = 56. (Inset) The correspondent volume vs. resistance dependence calculated from Eq. 2.

Fig. 4.

Time dependences of the voltage applied to a pipette (1) and current flowing across the water/DCE interface (2) during the ingress and egress of the outer aqueous solution. The pipette was filled with DCE containing 10 mM THATPBCl and immersed in a 100 mM MgSO₄ solution. The pipette radius was 400 nm. The dashed line is the theoretical fit to the linear portions of the current response obtained with a pipette resistance, R = 550 MΩ.

Fig. 5.

Cell injection using the electrochemical syringe. (a and b) An ≈150-nm-radius pipette is positioned near the cell surface (a), and some amount of buffer solution is loaded into it (b). (c) The nanopipette is then translated toward the cell and penetrates the cell membrane. The buffer is injected inside the cell. (d) The injection was stopped before the organic solution reached the pipette tip.

Fig. 6.

Optical (a) and fluorescence (b) micrographs of immobilized MCF-10 cells. The numbers in a and b correspond to the same six cells into which BODIPY FLATP fluorescent dye was injected. (b) The picture was obtained ≈30 min after washing the cells with fresh buffer solution to remove excess dye.

Fig. 7.

Optical (a) and fluorescence (b) images of a cell field and a blown up optical micrograph of cell 2 (c). Cells 1, 2, and 3 were injected with a 10 μM EB buffer solution. The control cell was penetrated by the nanopipette without solution injection. (c) Cell 2 in the beginning of the experiment (Left) and ≈20 min later (Right). The arrow points to the membrane separation.