Preparation and binding study of a complex made of DNA-treated single-walled carbon nanotubes and antibody for specific delivery of a “molecular heater” platform

Figures & data

Figure 1 Procedure for immobilization of streptavidin on the DNA-SWNT surface, and binding of biotinylated IgG to immobilized streptavidin to prepare the DNA-SWNT antibody complex. (A) Suspension of DNA-SWNT antibody complex.

Abbreviations: Ab, antibody; SWNT, single-walled carbon nanotubes; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride.

Table 1 Binding ratio of the SWNT antibody and the DNA-SWNT antibody complexes calculated from quartz crystal microbalance data

	SWNT (carbon atoms)	SA molecules	IgG molecules
SWNT-Ab complex
Amount (ng)	7.30 (0.71) × 10^15	3.81 (0.25) × 10^11	3.43(0.26) × 10^11
Ratio	1.92 × 10^4	1	0.91
DNA-SWNT-Ab complex
Amount (ng)	7.35 (0.70) ×10^15	3.86 (0.29) ×10^11	3.34 (0.28) × 10^11
Ratio	1.89 × 10^4	1	0.87

Notes: Carbon atom numbers were calculated from Avogadro’s constant. The binding ratio is presented with the number of binding SA molecules assumed as 1. Values in parentheses denote standard deviation (n = 7).

Abbreviations: SA, streptavidin; Ab, antibody; SWNT, single-walled carbon nanotubes.

Figure 2 Typical frequency changes for (A) streptavidin immobilized to DNA-SWNT according to the quartz crystal microbalance sensor, and (B) binding of biotinylated anti-human IgG to the streptavidin-immobilized DNA-SWNT according to the quartz crystal microbalance sensor.

Abbreviation: SWNT, single-walled carbon nanotubes.

Figure 3 Selective binding of the SWNT antibody complex to target protein in the presence or absence of DNA. Either human IgG or rabbit IgG was adhered to the bottom of a microplate well. Initial rates of oxidized TMB production are plotted against the concentration of the SWNT antibody complex. (A) SWNT was not treated with DNA. (B) SWNT was treated with DNA, and the buffer contained 1 mg/mL DNA.

Note: ***P < 0.01, compared with the corresponding value for rabbit IgG as the target protein (n = 4).

Abbreviations: TMB, 3,3′,5,5′-tetramethylbenzidine; SWNT, single-walled carbon nanotubes.

Figure 4 Binding of the SWNT antibody complex to its specific target. (A) Typical saturation curve. (B) Lineweaver–Burk plots. The data derived from the saturation curve were fitted to the dashed line (see text). The line represents 1/V_max (obtained from ordinate intercept) and 1/K_m (obtained from abscissa intercept). 1/v = 0.007373 (1/[SWNT]) + 0.000812, R² = 0.999. V_max,SWNT = 1231, K_m,SWNT = 9.09 (μg/mL). (C) Hill plots. Slope of the line represents n_H = 0.959.

Abbreviations: TMB, 3,3′,5,5′-tetramethylbenzidine; SWNT, single-walled carbon nanotubes.

Table 2 Binding affinity and capacity of SWNT-Ab and IgG itself to the target molecule in the absence of DNA

	nH	Km,SWNT(μg/mL) or Km,IgG (ng/mL)	Km,IgG(SWNT) or Km,IgG (mol/L)	pKm,IgG(SWNT) or pKm,IgG	Vmax,SWNT or Vmax,IgG (fmol/min)	Vmax,SWNT/Vmax,IgG
SWNT-Ab	0.99 ± 0.001	9.95 ± 1.47	(3.90 ± 0.58) × 10^−8	7.41 ± 0.06*	1255 ± 126	0.97 ± 0.04
IgG	0.99 ± 0.001	54.7 ± 3.1	(3.65 ± 0.20) × 10^−10	9.44 ± 0.02	1302 ± 132	1

Notes:

The K_m values based on the concentration of IgG (K_m,IgG(SWNT)) were calculated according to Lineweaver–Burk plots and quartz crystal microbalance data. The V_max for SWNT-Ab (V_max,SWNT) was normalized with the corresponding data for IgG itself (V_max,IgG) to obtain the relative V_max value (V_max,SWNT/V_max,IgG). n_H, Hill coefficient; pK_m,IgG(SWNT), −log K_m,IgG(SWNT).

* P < 0.001 compared with the IgG group, n = 3.

Abbreviations: Ab, antibody; SWNT, single-walled carbon nanotubes.

Figure 5 Binding of IgG to its specific target. (A) Typical saturation curve. (B) Lineweaver–Burk plots. The data derived from the saturation curve were fitted to the dashed line (see text). The line represents 1/V_max (obtained from ordinate intercept) and 1/K_m (obtained from abscissa intercept). 1/v = 0.041700 (1/[IgG]) + 0.000719, R² = 0.999. V_max,IgG = 1391, K_m,IgG = 58.0 (ng/mL). (C) Hill plots. Slope of the line represents n_H = 0.957.

Abbreviation: TMB, 3,3′,5,5′-tetramethylbenzidine.

Table 3 Binding affinity and capacity of DNA-treated SWNT-Ab and IgG itself to the target molecule in the presence of DNA

	nH	Km,SWNT(μg/mL) or Km,IgG (ng/mL)	Km,IgG(SWNT) or Km,IgG (mol/L)	pKm,IgG(SWNT) or pKm,IgG	Vmax,SWNT or Vmax,IgG (fmol/min)	Vmax,SWNT/Vmax,IgG
DNA-	0.99 ± 0.001	6.23 ± 0.48	(2.38 ± 0.19) × 10^−8	7.63 ± 0.03*	1355 ± 94	1.04 ± 0.05
SWNT-Ab IgG	0.99 ± 0.001	57.2 ± 4.5	(3.81 ± 0.30) × 10^−10	9.42 ± 0.03	1307 ± 123	1

Notes:

The K_m values based on the concentration of the IgG (K_m,IgG(SWNT)) were calculated according to the Lineweaver–Burk plots and quartz crystal microbalance data. The V_max for SWNT-Ab (V_max,SWNT) was normalized with the corresponding data for IgG itself (V_max,IgG) to obtain the relative V_max value (V_max,SWNT/V_max,IgG). n_H, Hill coefficient; pK_m,IgG(SWNT), −log K_m,IgG(SWNT).

* P < 0.001 compared with the IgG group, n = 3.

Abbreviations: Ab, antibody; SWNT, single-walled carbon nanotubes.

Figure 6 Schematic illustration of the DNA-SWNT antibody complex and binding to its target protein.

Abbreviations: Ab, antibody; SWNT, single-walled carbon nanotubes.