Sequential layer analysis of protein immunosensors based on single wall carbon nanotube forests

Abstract

Electrochemical immunosensors using vertically aligned single wall carbon nanotube (SWNT) forests can provide ultrasensitive, accurate cancer biomarker protein assays. Herein we report a systematic investigation of the structure, thickness, and functionality of each layer of these immunosensors using atomic force microscopy (AFM), quartz crystal microbalance (QCM), and scanning white light interferometry (SWLI). This provides a detailed picture of the surface morphology of each layer along with surface concentration and thickness of each protein layer. Results reveal that the major reasons for sensitivity gain can be assigned to the dense packing of carboxylated SWNT forest tips, which translate to a large surface concentration of capture antibodies, together with the high quality of conductive SWNT forests.

Figure 1

Strategy for amperometric SWNT forests immunosensors. On left in the SWNT forests assembly followed by the immunosensor that has been equilibrated with analyte protein PSA (center), along with biomaterials used in the assay protocol. On right, the immunosensor has been treated with enzyme-labeled secondary antibody: HRP-Ab₂ (HRP = horseradish peroxidase). Detection involves immersing the sensor in an electrochemical cell containing buffer and mediator, applying voltage and injecting a small amount of hydrogen peroxide (H₂O₂) to activate HRP for amperometric reduction of H₂O₂.

Figure 2

Tapping mode atomic force microscope images of layers (top layers indicated) in sensor fabrication and use: (A) Nafion on freshly cleaved mica surface; (B) Nafion/FeO(OH)-FeOCl bilayer; (C) SWNT forests on Nafion/FeO(OH)-FeOCl bilayer; (D) after covalent linkage of 2 nmol mL⁻¹ primary anti-PSA antibody (Ab₁) in pH 7.0 PBS buffer + 0.05% Tween-20 followed by washing with 0.05% Tween-20, PBS buffer and water; (E) after addition of 2% BSA in 0.05% Tween-20 PBS buffer onto antibody layer and washing with 0.05% Tween-20, PBS buffer and water; (F) after addition of 40 ng mL⁻¹ PSA in calf serum and washing with 0.05% Tween-20, PBS buffer and water; (G) after addition of 4 pmol mL⁻¹ singly labeled secondary antibody (Ab₂)-HRP in 0.05% Tween-20 followed by washing with buffer solutions and water.

Figure 3

Tapping mode phase contrast AFM images of layers (top layers indicated) in sensor fabrication and use: (A) Nafion on freshly cleaved mica; (B) Nafion/FeO(OH)-FeOCl bilayer; (C) SWNT forests on Nafion/ FeO(OH)-FeOCl bilayer; (D) after covalent linkage of primary anti-PSA antibody (Ab₁) followed by washing with 0.05% Tween-20, PBS buffer and water; (E) after addition of 2% BSA in 0.05% Tween-20 PBS buffer onto antibody layer and washing with 0.05% Tween-20, PBS buffer and water; (F) after addition of 40 ng mL⁻¹ PSA in calf serum and washing with 0.05% Tween-20, PBS buffer and water; (G) after addition of 4 pmol mL⁻¹ Ab₂-HRP in PBS buffer containing 0.05% Tween-20 followed by washing with buffer solutions and water.

Figure 4

QCM frequency shifts (left) and mass per unit area (right) of layers in PSA immunosensors grown on (A) SWNT forests and (B) Au-coated quartz electrodes (control: no SWNTs): 1. Nafion; 2. Nafion/FeO(OH)-FeOCl bilayer; 3. SWNT forests on Nafion/ FeO(OH)-FeOCl bilayer; 4. 400 mM EDC and 100 mM NHSS in water; 5. after covalent linkage of 2 nmol mL⁻¹ primary anti-PSA antibody (Ab₁) in 0.05% Tween-20 PBS buffer to SWNT forests or 3-mercapto-1-propanol/ 3-mercaptopropionic acid layer in SWNT-free control sensor; 6. adsorption of 2% BSA in 0.05% Tween-20 PBS buffer onto antibody; 7. adsorption of 40 ng mL⁻¹ PSA in calf serum; 8. adsorption of 4 pmol mL⁻¹ Ab₂-HRP in PBS containing 0.05% Tween-20.

Figure 5

Thickness profile of each layer of PSA-SWNT immunoassembly determined using three-dimensional non-contact scanning white light interferometer (O) and quartz crystal microbalance (●). 1. Nafion was first deposited on the glass substrate followed by; 2. Nafion/ FeO(OH)-FeOCl bilayer; 3. SWNT forests on Nafion/ FeO(OH)-FeOCl bilayer; 4. 400 mM EDC and 100 mM NHSS in water; 5. after covalent linkage of 2 nmol mL⁻¹ primary anti-PSA antibody (Ab₁) in 0.05% Tween-20 PBS buffer to SWNT forests; 6. adsorption of 2% BSA in 0.05% Tween-20 PBS buffer onto antibody; 7. adsorption of 40 ng mL⁻¹ PSA in calf serum; 8. 4 pmol mL⁻¹ Ab₂-HRP in PBS containing 0.05% Tween-20.

Figure 6

Resonance Raman spectra (785 nm excitation) showing (A) D-band at 1320 cm⁻¹ and G-band at 1592 cm⁻¹ for Nafion/FeO(OH)-FeOCl/SWNT forests on QCM gold resonator; (B) radial breathing mode (RBM) bands for Nafion/FeO(OH)-FeOCl/SWNT forests in low frequency region at 209 cm⁻¹, 238 cm⁻¹, and 265 cm⁻¹; (C) control, bare Au.