Estimated molecular structure of a carbon nanotube molecular heater based on binding properties to a target protein

Preparation of DNA-functionalized SWNT/IgG (anti-human IgG antibody) complexes

DNA treatment of SWNT to achieve stable dispersibility and streptavidin binding to the DNA-functionalized SWNT was carried out in accordance with the method described previously. Briefly, SWNT (400 mg, CG100; South West Nanotechnology, Norman, OK, USA) was poured into a mixture of 300 mL of sulfonic acid and 100 mL of nitric acid, and the mixture was sonicated for 7 h. This acid-treated SWNT was collected by filtration (0.2 μm pore size polytetrafluoroethylene membrane). The DNA-functionalized SWNT was prepared by sonication of a mixture of acid-treated SWNT (10 mg) and 300 bp of double-stranded DNA (100 mg, derived from salmon testis; Maruha-Nichiro Corporation, Tokyo, Japan) in 10 mL of deionized water for 1 h. Streptavidin was attached covalently onto the DNA-functionalized SWNT via an N-hydroxysuccinimide and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride coupling reaction to yield stable amide linkages. The DNA-functionalized SWNT/IgG complexes were prepared using a streptavidin-biotin reaction of the DNA-functionalized SWNT/streptavidin and biotinylated IgG.

Three different types of biotinylated IgG (anti-human IgG antibody, generated in donkey; Rockland Immunochemicals, Gilbertsville, PA, USA) were purchased from Medical & Biological Laboratories (Nagoya, Japan). These biotinylated IgGs had different spacer arm lengths between the IgG antibody molecule and the biotin site, corresponding to 13.5 Å, 29 Å, and 56 Å. Schematic chemical structures of these biotinylated IgG are shown in Figure 1.

The streptavidin-bound DNA-functionalized SWNT complex (3 mg) was dissolved in 3 mLl of distilled water, and 300 μg of the biotinylated IgG added. The mixture was gently stirred for 2 h at 5°C. The resulting solution was filtered using a poly­tetrafluoroethylene membrane (0.2 μm pore size) to collect the DNA-functionalized SWNT/IgG (anti-human IgG antibody) complex. The filtered complex was adjusted to a concentration of 500 μg/ml (based on amount of SWNT) in phosphate-buffered saline (containing 0.01 % n-dodecyl-b-D-maltoside [DDM]; Dojindo, Kumamoto, Japan) using ultraviolet absorbance. These DNA-functionalized SWNT/IgG complexes with spacer arms of 13.5 Å, 29 Å, and 56 Å in length were coded as DNA-functionalized SWNT/IgG complex (S), (M), and (L), respectively.

Measurement of binding ratio using a quartz crystal microbalance

The quartz crystal microbalance (QCM, Affinix C4; Initium, Chigasaki, Japan) contained a gold-coated quartz sensor with a fundamental frequency of 27 MHz. A frequency decrease of 1 Hz in the QCM system was calibrated as an increase in 0.03 ng (21, 22).

The QCM sensor was exposed to 100 μL of phosphate-buffered solution containing 1 μg of Protein G (recombinant; Thermo Scientific, Rockford, IL, USA) for 1 h at room temperature to reduce nonspecific binding of antibody molecules to the sensor surface. After rinsing with phosphate-buffered solution to remove unbound Protein G, the sensor was exposed to 500 μL of phosphate-buffered solution, and a human IgG solution (Chrom Pure, whole molecule; Jackson Immuno Research Laboratories, West Grove, PA, USA) was injected (final concentration 50 μg/ml). The amount of human IgG immobilized on the QCM sensor was determined from the frequency shift.

The human IgG-coated sensor was exposed to 500 μL of TE buffer containing 0.15 M NaCl and 0.01% DDM, and the DNA-functionalized SWNT/IgG complex (S) (final concentration 50 μg/ml) was injected. The amount of complex bound to the human IgG antibody was determined from the frequency shift. The amounts of DNA-functionalized SWNT/IgG complex (M) and (L) bound to the human IgG antibody were determined in the same manner as described above.

Detection of binding of the complexes to target protein

Ninety-six well polystyrene microplates (BD Biosciences, Franklin Lakes, NJ, USA) were incubated overnight at 4°C with 100 μL of 10 μg/ml human IgG (Chrom Pure, whole molecule; Jackson Immuno Research Laboratories). After washing with phosphate-buffered saline (PBS), the wells were blocked with 3% bovine serum albumin (BSA) dissolved in PBS containing 0.01% DDM for 30 min. Then, the solution was replaced with different concentrations of either biotinylated anti-human IgG antibodies (generated in donkey; Rockland Immunochemicals) or a complex of streptavidin-bound SWNT molecules and biotinylated IgG antibodies (SWNT/IgG complex), each of which was dissolved in PBS containing 1% BSA, 0.01% DDM, and DNA (1 mg/ml) (buffer 1). Three types of biotinylated IgG antibodies were used, which possessed spacers of different lengths between biotin and IgG. The SWNT-IgG complex was functionalized using DNA. After incubation at 37°C for 60 min to 90 min, the wells were washed 3 times with 0.05% Tween 30-containing PBS (buffer 2).

To detect the SWNT/IgG complex, the wells were treated for 1 h to 1.5 h each at room temperature with mouse monoclonal anti-streptavidin antibody (×1000, S3E11; Pierce Biotechnology, Rockford, IL, USA) and then goat monoclonal anti-mouse IgG antibody (light chain-specific, cross-reacted minimally with other species) conjugated with horseradish peroxidase (HRP) (×2000, Jackson Immuno Research Laboratories), both of which were dissolved in buffer 1. The wells were washed extensively with buffer 2 after each treatment. To detect the biotinylated antibodies without SWNT, streptavidin (1 μg/ml) was firstly bound to the biotin-side chain, and then incubated with the antibodies in the above order. Prior to the HRP assay, absorbance at 450 nm was measured as a blank (AbsBlank). Thereafter, the HRP assay was performed using 3, 3’, 5, 5’-tetramethylbenzidine (TMB) as a substrate and an excess amount of H₂O₂ (TMB substrate reagent set; BD Falcon). The reagents were incubated in the wells for 0 to 15 min, followed by stopping the reaction by adding 1 M phosphoric acid. The absorbance (AbsTMB) of the reaction mixture at 450 nm was measured, being dependent on the production of oxidized TMB. The difference value, AbsTMB - AbsBlank, was calculated to obtain the amount of oxidized TMB. The initial production rate of oxidized TMB was calculated on the basis of the Lambert-Beer law, and the molar extinction coefficient at 450 nm (e450) of oxidized TMB was reported as 5.9 × 10⁴ M^-1 cm^-1.

Statistics

All values are presented as means ± S.E.M (n, number of observations). Statistical analysis was performed using an unpaired t test. A P value less than .05 was considered to be statistically significant.

Preparation of 3 types of DNA-functionalized SWNT/IgG complexes

The prepared DNA-functionalized SWNT/IgG complexes were easily dispersible in deionized water or PBS. The suspensions of these complexes were stable for at least 2 weeks at 4°C without aggregation or precipitation.

Measurement of binding ratios of DNA-treated SWNT/IgG complexes using QCM

The amount of immobilized human IgG antibody on the QCM sensor was 24.9 ± 1.24 ng (n = 7). Figure 2 plots the changes in frequency observed following immobilization of different types of complexes. Adsorption of DNA-functionalized SWNT/IgG complex (S) resulted in a decrease in frequency of 1016 Hz, whereas DNA-functionalized SWNT/IgG complex (M) and DNA-functionalized SWNT/IgG complex (L) showed larger decreases in frequency of 2800 Hz and 3577 Hz, respectively. DNA-functionalized SWNT/IgG complexes with a longer spacer lengths (107.3 ± 3.90 ng for DNA-functionalized SWNT/IgG complex (L)) showed greater amounts of human IgG antibody binding than those with a shorter spacer length (30.5 ± 3.70 ng for DNA-functionalized SWNT/IgG complex (S)).

Analysis of binding of the DNA-functionalized SWNT/IgG complex with different spacer arm lengths to a target molecule

Our previous binding analysis revealed that DNA functionalization to improve the dispersability of SWNT did not interfere with the binding affinity or capacity between the immobilized antibody and the targeted protein. Here, in the presence of DNA, we compared IgG binding for different spacer arm lengths to the target molecule.

Figure 3 shows typical data for the binding between 3 types of DNA-functionalized SWNT/IgG complexes and the target molecule. The upper panels indicate the concentration-dependent binding of the complex to its target. A striking difference in the saturation curve was seen with the smallest slope for DNA-functionalized SWNT/IgG (S) complex, which had the shortest spacer of 13.5 Å. From the Lineweaver-Burk plots of the data (Fig. 3B), the affinity constant (Km, SWNT) and maximum reaction (V_max, SWNT) were estimated (Tab. I). The estimated Hill coefficient (n_H) was close to unity, indicating that the reaction obeyed the Michaelis-Menten formula, and a reaction occurred between the DNA-functionalized SWNT/IgG complex and the target molecule (Tab. I). The Km value based on the concentration of the IgG (Km, IgG(SWNT)) for the DNA-functionalized SWNT/IgG with a spacer length of 13.5 Å was 7.7- and 11.7-fold higher than those for the 29.0 Å and 56 Å spacers, respectively (Tab. I). This suggested that a short spacer reduced the binding affinity.

To elucidate whether a bulky structure of SWNT is prerequisite for the importance of the spacer length in determining the binding affinity, we measured the binding ability between IgGs themselves with different spacer lengths and a target molecule. The n_H value was close to unity. For the Km, the IgG value was the same among the 3 types of IgG molecules even if they possessed different spacer lengths (Tab. II), indicating the importance of distance from the bulky SWNT attached to IgG.

Next, we compared the binding capacity of either the DNA-functionalized SWNT/IgG complex or IgG itself with the different spacer lengths (Tabs. I and II). The spacer length affected neither V_max nor the relative V_max (V_max, SWNT/V max, IgG) for the DNA-functionalized SWNT/IgG complex to that for IgG themselves.