Hybrids of a genetically engineered antibody and a carbon nanotube transistor for detection of prostate cancer biomarkers

Abstract

We developed a novel detection method for osteopontin (OPN), a new biomarker for prostate cancer, by attaching a genetically engineered single-chain variable fragment (scFv) protein with high binding affinity for OPN to a carbon nanotube field-effect transistor (NT-FET). Chemical functionalization using diazonium salts is used to covalently attach scFv to NT-FETs, as confirmed by atomic force microscopy, while preserving the activity of the biological binding site for OPN. Electron transport measurements indicate that functionalized NT-FET may be used to detect the binding of OPN to the complementary scFv protein. A concentration-dependent increase in the source-drain current is observed in the regime of clinical significance, with a detection limit of approximately 30 fM. The scFv-NT hybrid devices exhibit selectivity for OPN over other control proteins. These devices respond to the presence of OPN in a background of concentrated bovine serum albumin, without loss of signal. On the basis of these observations, the detection mechanism is attributed to changes in scattering at scFv protein-occupied defect sites on the carbon nanotube sidewall. The functionalization procedure described here is expected to be generalizable to any antibody containing an accessible amine group and to result in biosensors appropriate for detection of corresponding complementary proteins at fM concentrations.

Figure 1. Functionalization scheme for OPN attachment

First, sp3 hybridized sites are created on the nanotube sidewall by incubation in a diazonium salt solution. The carboxylic acid group is then activated by EDC and stabilized with NHS. ScFv antibody displaces the NHS and forms an amide bond (surface amine-rich lysine residues responsible for this bond are depicted in red), and OPN binds preferentially to the scFv in the detection step. The OPN epitope is shown in yellow, and the C-and N-terminuses are in orange and green respectively.

Figure 2

a) AFM image of scFv antibodies covalently bound to carbon nanotubes, with a typical density of 4–5 attachment sites per micrometer of nanotube length. Scale bar is 1 μm. Height scale is 7 nm. b) Histogram of the heights of the white features bound to the nanotube in Fig 2a shows a maximum at ~ 2.5 nm, consistent with the expected height of scFv antibodies. Secondary maxima at 5 nm and 7.5 nm are attributed to small aggregates of scFv antibodies. The total number of features analyzed is 180.

Figure 3

a) I–V_g characteristics for a single device after successive functionalization steps. Exposure to OPN at a concentration of 30 ng/mL (912 pM) caused the ON state current to increase from 29 nA (green curve) to 37 nA (orange curve), an increase of 27%. b) Similar data for exposure to OPN at a concentration of 10 pg/mL (304 fM). The ON state current increases from 276 nA to 307 nA, an increase of 11%.

Figure 4

Measured sensor responses over a wide range of concentrations of osteopontin (OPN). The solid line is a fit using a modified Hill-Langmuir expression that includes an offset response of 4% due to the buffer itself (see main text). A clear signal is still present at OPN concentrations of 1 pg/mL.

Figure 5

Summary of data from control experiments. Devices exposed to neat PBS buffer showed a response of +4%. Exposure to bovine serum albumin (BSA) at 450 ng/mL gave a null response. Devices prepared with the anti-HER2 scFv antibody in place of anti- OPN scFv and exposed to 90 ng/mL OPN also gave a null response. (Inset) Devices prepared with anti-OPN scFv antibodies and exposed to a mixture of 90 ng/mL OPN and 450 ng/mL BSA background protein gave a response identical to that expected for 90 ng/mL OPN in plain buffer.