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.biomolecules ͉ biosensor ͉ immunoassay ͉ current rectification ͉ nanopore N anopipettes, characterized by the submicron to nanoscale size of the pore at the tip, are of great interest because of their unique physicochemical properties and potential for various biomedical and biological applications. By pulling a single glass capillary, one can easily and cost-effectively create a pair of nanopipettes that can be used for molecular deposition onto a solid surface (1, 2), for delivery to the surface of a single cell (3) and its inner compartments (4, 5), or for biomolecular sensing as described hereafter. These applications can be optimized by an enhanced understanding of the physical and chemical interactions at the pore region, which has been a subject of theoretical studies (6). Advances in both technical and theoretical fronts will further demonstrate the utility of nanopipettebased devices for many purposes.Biomolecule sensing with a nanopipette probe has been performed with and without the aid of optical methods. Fluorescence-based pH sensing (7) shows the submicron spatial resolution and millisecond time resolution of such sensors. Fully-electrical detection of DNA-conjugated gold nanoparticles (8) uses resistive pulses caused by the translocation of fairly large (10 nm) particles, the underlying principle identical to that of nanopore biosensors (9) and DNA sequencers (10). Unlike other nanostructure-based chemical sensors (11), which often require access to semiconductor facilities, nanopipette biosensors can be created and tailored at the bench, thereby reducing turnaround time. Nanopipettes also have enormous potential for detecting a small number of molecules from a tiny amount of clinical samples or live single cells, a feature useful for medical diagnostics and molecular and cellular biology research.A key challenge for nanopipette biosensors is adapting to applications where specific molecules can be targeted. One approach would be to separate the sensing and actuating functions, an idea embodied by an engineered ion channel fused with a surface receptor protein responsible f...

Resistive-pulse and rectification sensing with glass and carbon nanopipettes

“…In most reported resistive-pulse experiments, selective detection of biomolecules was achieved by functionalizing the nanopore (e.g. by immobilizing antibodies on its surface [16]). By contrast, in [40] the analyte (IgY) was selectively captured offline using peptide-modified AuNPs.…”

“…The very small orifice radius (e.g. a < 10 nm [11,12]) also allows nanopipettes to be used for resistivepulse detection of single biomolecules [13] and current rectification sensing [14][15][16][17].…”

Experiment and Simulation of Ion Transport through Nanopipettes of Well-Defined Conical Geometry

“…[16][17][18][19] Nanopipette studies that utilize ion current rectification as related to metal ions, 20,21 small molecules, 22 polymers 23 and biomolecules 24,25 have been described. Observations of ion current rectification were reported as early as the 1960s for small diameter pipettes, 26,27 but more recent interest in current rectification at nanofabricated pores has sparked renewed interest. [28][29][30][31][32][33][34] Ion transport mechanisms and rectification behaviors of synthetic nanopores have been studied widely with numerical simulation.…”

Advances and Perspectives in Chemical Imaging in Cellular Environments Using Electrochemical Methods

“…Nanopipettes functionalized with specific recognition elements are a promising developing area of research that could yield biosensors capable of imaging at the single cell level [101]. In particular, aptamer-functionalized nanopipettes demonstrate reversible response to a target, not readily observed with antibody-modified nanopipettes [102]. Pourmand and coworkers [103] used the ion current through an aptamer functionalized STING sensor nanopipette to demonstrate reversible and quantitative detection of thrombin.…”