Label-free biosensing with functionalized nanopipette probes

Abstract

Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research.

Fig. 1.

Nanopipette-based biosensing platform. (A) Measurement setup. Up to 2 nanopipette electrodes were used to record the current amplitude, in which case one nanopipette (labeled channel 1) was designed to detect antigens specific to the antibodies immobilized on its tip surface, whereas the other nanopipette (channel 2) served as a control. (B) Schematic representation of sensing strategies. Interaction between free analytes in solution and the nanopipette tip modulates the ionic flow through the 50-nm pore. Electrostatic interaction occurs when the analytes and the surface are both charged, affecting the rectification property of a quartz nanopipette. Specific interaction occurs when a recognition element immobilized on the nanopipette tip surface distinguishes its target molecules from others present in the sample solution.

Fig. 2.

Modulation of the current rectification property caused by polyelectrolytes. (A) Onto the negatively charged quartz surface, free poly-L-lysine (PLL) added to the bath (10⁻⁴% wt/vol) inverted the polarity of current rectification to positive values. Rinsing with buffer (hatched areas) canceled this effect, reviving the original negative rectification (which became less prominent, however). Applied voltage, ±500 mV. (B) Current recorded at t = 0 at the onset of the first PLL addition (indicated by an arrow in panel A).

Fig. 3.

Streptavidin-FITC detection by the nanopipette probe functionalized with biotinylated BSA. (A) After the addition of free streptavidin-FITC molecules at t = 0, at a concentration of 40 μg/ml, an increase in the negative current amplitude was recorded by the probe nanopipette (solid lines), whereas essentially no change was observed with the control nanopipette (dashed lines). Applied voltage, ±500 mV. (B) Microscopic images of the tips of the 2 nanopipettes captured after the electrical measurement. The tip with biotinylated BSA exhibited much stronger fluorescence (Right) than the tip with nonbiotinylated BSA (Left). DIC, differential interference contrast. (Scale bar, 10 μm.)

Fig. 4.

Validation of the antibody conjugation onto the nanopipette tip surface. Microscopic images of the tips with biotinylated and nonbiotinylated IgG are shown. To clarify the difference of fluorescence intensity, we processed a pair of identical FITC images by applying 2 different lookup tables to them. The FITC upper row shows the bright fluorescence from the biotinylated-IgG nanopipette tip on the right, whereas the lower row emphasizes the very weak fluorescence (comparable to the background level) from the nonbiotinylated IgG tip on the left. DIC, differential interference contrast. (Scale bar, 10 μm.)

Fig. 5.

Cancer biomarker detection by the nanopipette probe functionalized with corresponding IgG molecules. (A) IL-10 detection (4 μg/ml). Applied voltage, ±200 mV. (B) VEGF detection (4 μg/ml). Applied voltage, ±200 mV. In both cases, an immediate decrease in current was recorded by the probe nanopipette (either anti-IL-10 or anti-VEGF, solid lines) after the addition at t = 0, which was significantly larger than that recorded by the control nanopipette (antiferritin bound, dashed lines).