Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors

Abstract

Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and protein-protein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases.

Figure 1

Proteins tend to bind nonspecifically onto as-grown carbon nanotubes. (A) Schematic illustration of globular protein adsorption onto a nanotube. (B) An AFM image showing protein A (bright dot-like structures decorating the line-like nanotube) nonspecifically adsorbed on a nanotube. We have also observed a certain degree of NSB of proteins on regions of the (SiO₂) substrate free of nanotubes (data not shown). (C) QCM data (frequency shift ΔF vs. time t) revealing NSB of SA onto nanotubes at increasing protein concentrations. The NSB is irreversible upon rinsing.

Figure 2

Carbon nanotubes as electronic devices for sensing in aqueous solutions. (A) Schematic views of the electronic sensing device consisting of interconnected nanotubes bridging two metal electrode pads. An AFM image of a portion of the nanotube network (0.5 μm on a side) is shown. (B) Schematic setup for sensing in solution. (C) Conductance (G) evolution of a device for electronic monitoring of SA adsorption on nanotubes. The conductance is normalized by the initial conductance G₀. (Inset) Sensitivity to a 100-pM protein solution is shown. (D) Electrical conductance (G) vs. gate voltage (V_g) for a device in a 10-mM phosphate buffer solution. The gate voltage is applied through a Pt electrode immersed in the solution (Inset). The green (solid) and orange (broken) curves are the G–V_g characteristics for the device before and after SA binding, respectively. The shift in the two curves suggests a change in the charge environment of the nanotubes.

Figure 3

Noncovalent functionalization of nanotubes for protein resistance and water solubility. (A) Schematic of a monolayer of Tween 20 anchored on a nanotube, repelling NSB of proteins in solution. (B) An AFM image showing the absence of adsorbed proteins on a Tween-coated nanotube after exposure to a 10-nM SA solution for 60 min. (C) QCM data showing the absence of mass uptake and thus no NSB of various proteins onto a film of Tween-coated nanotubes. (Inset) The irreversible adsorption of Tween onto such a film is shown. (D) The conductance of a Tween-coated nanotube electronic device does not exhibit any change upon exposure to various protein solutions. (E) Photographs that show SWNTs coated with Tween (I) and P103 (II) forming stable suspensions in water. The suspension in III is derived from C₁₄E₈-treated SWNTs, but to obtain a stable suspension, a lower concentration of SWNTs is necessary, resulting in a lighter-color solution than those in I and II.

Figure 4

Real-time QCM and electronic sensing of specific biological recognition on nanotubes. (A) Scheme for SA recognition with a nanotube coated with biotinylated Tween. (B) QCM frequency shift vs. time curve showing that a film of nanotubes coated with biotinylated Tween binds SA specifically but not other proteins. (C) Conductance vs. time curve of a device during exposure to various protein solutions. Specific binding of SA is detected electronically. (D) Scheme for IgG recognition with a nanotube coated with a SpA–Tween conjugate. (E) QCM frequency shift vs. time curve showing a film of nanotubes coated with SpA–Tween binding human IgG specifically but not unrelated proteins. Note that 10 nM IgG concentration approaches the lower detection limit of the instrument, whereas 100 nM approaches surface saturation of the sample; thus, the response does not show a full proportionality to the concentration. (F) Conductance vs. time curve of a device during exposure to various protein solutions. Specific binding of IgG is detected electronically (some NSB is observed for 100 nM SA, but the signal is much smaller than that of IgG).

Figure 5

Specific detection of mAbs binding to a recombinant human autoantigen. (A) Scheme for specific recognition of 10E3 mAb with a nanotube device coated with a U1A antigen–Tween conjugate. (B) QCM frequency shift vs. time curve showing selective detection of 10E3 while showing rejection of the antibody 6E3, which recognizes the highly structurally related autoantigen TIAR. (C) Conductance vs. time curve of a device shows specific response to ≤1 nM 10E3 while rejecting polyclonal IgG at a much greater concentration of 1 μM (Inset).