Detection and difference analysis of the enzyme activity of colloidal gold nanoparticles with negatively charged surfaces prepared by different reducing agents

doi:10.21203/rs.3.rs-496828/v1

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Detection and difference analysis of the enzyme activity of colloidal gold nanoparticles with negatively charged surfaces prepared by different reducing agents

https://doi.org/10.21203/rs.3.rs-496828/v1

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Research on the activity of nanoenzymes has always been a focus of nanomaterials. In this study, several reducing agents with different structures were used to prepare colloidal gold with a negative charge and similar size by controlling the temperature and pH. The affinity analysis of the substrate H 2 O 2 and TMB showed that the activity of colloidal gold nanoenzymes prepared by different reducing agents was in the order of Cc, Hq, Rs, Vc, Ga, Sc, Sm, St. The rule is that the enzyme activity of colloidal gold reduced by benzene ring is higher than that of colloidal gold enzyme reduced by linear chain. Finally, we discussed the activity of colloidal gold peroxidase from the number and position of isomers and functional groups, and demonstrated that the nanoenzyme activity affected the surface activity of colloidal gold, the elimination of hydroxyl radical and TMB binding efficiency.

Biological Chemistry

Decision Sciences

colloidal gold

negative charge

nanoenzyme

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Table1: Proportion of reagents for preparing colloidal gold

Reducing agent concentration	ddWH₂O	HAucl₄(1%)	NaOH(0.1M)	K₂CO₃(0.1M)	HCL(0.1M)	Reaction conditions
▲72.7 µL Hq（30mM）	●9.696 mL	■81.6 µL	★150 µL			50℃ Stir
▲100ul Cc（30mM）	●9.62 mL	■100 µL		★100 µL		50℃ Stir
▲60 µL Rs (1%)	●9.74 mL	■100 µL		★20 µL		Room temperature
▲20 µL Ga(1%)	●9.73 mL	■100 µL	★150 µL			Room temperature
▲400 µL Vc（1.98×10^-3M）	●9.52 mL	■100 µL			★100 µL	0℃ Stir
▲100 µL St(1%)	●9.72 mL	■100 µL	★100 µL			50℃ Stir
■100 µL Dm（2%）	●9.8 mL	★100 µL				Boil and stir
■100 µL Sc(2%)	●9.8 mL	★100 µL				Boil and stir

Table 2: Nano-enzyme activity determination verification

Reactant Vial number	NaAc-HAc buffer (mL)	Nanozyme (mL)	TMB (µL)	H₂O₂(µL)
Vial 1	2	1.2	40	363.7
Vial 2	2.364	1.2	40	0
Vial 3	3.2	0	40	363.7

Table 3: K_m and V_max with H₂O₂ as substrate

Type of reducing agent	Cc	Hq	Rs	Ga	Vc	Sc	Sm	St
K_m(mM)	0.249	0.497	0.378	0.226	0.849	1.634	2.75	2.382
V_max	6.596	6.057	6.12	5.385	6.897	4.228	3.785	6.369

Note: K_m is the Michaelis constant, V_max is the maximal reaction velocity

Table 4: K_m and V_max with TMB as substrate

Type of reducing agent	Cc	Hq	Rs	Vc	Ga	Sc	Sm	St
K_m(mg/ml)	0.0074	0.0153	0.0105	0.003	0.0059	0.0026	0.0008	0.0016
V_max	6.689	4.496	5.01	6.039	3.495	1.866	1.306	2.729

Note: Km is the Michaelis constant, V_max is the maximal reaction velocity

Table 5: The results of enzyme activity measurement

Type of reducing agent	Cc	Hq	Rs	Vc	Ga	Sc	Sm	St
Total	4ml
The amount nanozyme added	500 µL
nanozyme concentration ( mg·ml^-1)	0.0816	0.1	0.1	0.1	0.1	0.1	0.1	0.1
b(IU)	0.058	0.023	0.018	0.012	0.011	0.004	0.0023	0.0022
a(IU·mg^-1)	1.415	0.464	0.378	0.242	0.219	0.084	0.047	0.044

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Review #1 received at journal
13 May, 2021
Review #2 received at journal
12 May, 2021
Reviewer #3 agreed at journal
11 May, 2021
Reviewer #2 agreed at journal
11 May, 2021
Reviewers invited by journal
09 May, 2021
Reviewer #1 agreed at journal
09 May, 2021
Editor invited by journal
28 Apr, 2021
Editor assigned by journal
27 Apr, 2021
Submission checks completed at journal
27 Apr, 2021
First submitted to journal
24 Apr, 2021

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Detection and difference analysis of the enzyme activity of colloidal gold nanoparticles with negatively charged surfaces prepared by different reducing agents

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