纳米颗粒模拟酶及应用(5)

研究了纳米颗粒的多酶活性,包括过氧化物酶和过氧化氢酶,以及在葡萄糖高灵敏检测中的应用.

Zhang et al.A

Prussian Blue Modi ed Ferritin as Peroxidase Mimetics and Its Applications in Biological Detectioncontrol(TMB+H2O2) ferritin PB-Ft NPs

Absorbance (650 nm)

Absorbance (415 nm)

2.1 1.8 1.5 1.2 0.9 0.6 0.3 0.0 0

B

0.35 0.30 0.25 0.20 0.15 0.10 0.05 control(ABTS+H2O2) ferritin PB-Ft NPs

C

a

b

c

d

100 200 300 400 500 600

0

100 200 300 400 500 600

Time (sec)

Time (sec)

Fig. 4. PB-Ft NPs show peroxidase-like activity. A (a), (b), Reaction systems catalyzed without and with PB-Ft NPs using ABTS as chromogenic substrate. c, d Reaction systems catalyzed without and with PB-Ft NPs using TMB as chromogenic substrate. (B), (C), UV-vis absorbance-time curves of reaction systems catalyzed by PB-Ft NPs in the same buffer (HAc-NaAc solution, pH= 3 6) using TMB and ABTS as chromogenic substrates. The substrate oxidation reaction taking PBS (black lines) and HoSF (blue lines) instead of PB-Ft NPs was used as contrasts.

(a) 0.00450concentration of H2O2 is 41.43 mM 0.00375 0.00300

(b) 0.00070.0006 0.0005 concentration of H2O2 is 2.07 mM

V(s–1)

0.00225 0.00150 0.00075 0.00000 0 400 800 1200 1600 Km=1.594mM Vmax=0.0061s–1

V(s–1)

0.0004 0.0003 0.0002 0.0001 0.0000–100 0 100 200 300 400 500 600 700 800 Km=0.532mM Vmax=0.0011s–1

TMB concentration (µM)(c) 0.0009 0.0008 0.0007 (d) 0.0008 0.0007 0.0006

ABTS concentration (µM)

RESEARCH ARTICLE

concentration of TMB is 1651.38µM

concentration of ABTS is 723.24µM

V(s–1)

V(s–1)

0.0006 0.0005 0.0004 0.0003 0 50 100 150 200 250 Km=11.984 mM Vmax=0.00072s–1

0.0005 0.0004 0.0003 0.0002 0.0 0.5 1.0 1.5 2.0 2.5 3.0 H2O2 concentration (mM) 3.5 4.0 Km=0.536mM Vmax=0.00077s–1

H2O2 concentration (mM)

Fig. 5. Steady-state kinetic assay of PB-Ft NPs. The velocity (V of the reaction was measured using 10 L PB-Ft NPs in 200 L HAc-NaAc buffer (pH= 3 6) at 25 C. (a) The concentration of H2 O2 was 41.43 mM and the TMB concentration was varied. (b) The concentration of H2 O2 was 2.07 mM and the ABTS concentration was varied. (c) The concentration of TMB was 1651.38 M and the H2 O2 concentration was varied. (d) The concentration of ABTS was 723.24 M and the H2 O2 concentration was varied. The reaction velocity was calculated from the initial slopes of absorbance versus time curves.

3.3. Glucose Detection Hydrogen peroxide is the main product in the glucose oxidase catalyzing reaction in the presence of glucose and oxygen.39 On the b

asis of the peroxidase-like activity of PB-Ft NPs presented before, we designed a colorimetric glucose detection method by detecting the H2 O2 generated in GOx catalyzed oxidation of glucose. As mentioned above, PB-Ft NPs displayed higher af nity to H2 O2 whenJ. Nanosci. Nanotechnol. 12, 1–8, 2012

the chromogenic substrate was ABTS (Table I), so we measured the color change from ABTS to measure the concentration of glucose indirectly. Because GOx could be denatured in pH lower than 4.0, the detection was performed in two separate steps as described in experiment. As shown in Figure 7, the glucose can be detected as low as 0.39 mol/L with the linear range of 0.39 mol/L to 6.25 mol/L. Because of high peroxidase-like activity and strong af nity to H2 O2, the developed detection method 5

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