AU2019100367A4

AU2019100367A4 - Synthesis of CoFe Cube Nanozymes and a New Method for Ferrous Ions Detection

Info

Publication number: AU2019100367A4
Application number: AU2019100367A
Authority: AU
Inventors: Qianqian Chen; Yizhe Gao; Jie Hou; Sichen Liu; Jinyan Wang; Ningtianyu Wang
Original assignee: Chen Qianqian Miss; Liu Sichen Miss; Wang Jinyan Miss; Wang Ningtianyu Miss
Current assignee: Chen Qianqian Miss; Liu Sichen Miss; Wang Jinyan Miss; Wang Ningtianyu Miss
Priority date: 2019-04-05
Filing date: 2019-04-05
Publication date: 2019-05-09
Anticipated expiration: 2027-04-05

Abstract

The invention relates to a colorimetric method for the detection of ferrous ion (Fe2) with CoFe cube nanozyme. The CoFe cube nanozyme is obtained through coprecipitation synthesisof CoCl 2 - 6H20,K3[Fe(CN)6] andC6H 5Na 3 O7 - 2H 20solution. The intrinsic oxidase-like activity of CoFe cube nanozyme is demonstrated to be dependent on pH, temperature, and concentration of nanozyme and substrate (TMB). The best detection condition as following: pH is equal to 4.0, the temperature is 20 degree centigrade, the metering of nanozyme is 30 pL and TMB concentration is 0.22 mM. Under the best conditions, the detection range of ferrous ion is from 0.01mM to 0.10 mM. The detection of low concentration of Fe2+ by nanozyme is rapid and accurate.

Description

Synthesis of CoFe Cube Nanozymes and a New Method for Ferrous Ions

Detection

FIELD OF THE INVENTION

The present invention relates to colorimetric detection of ferrous ions by CoFe cube nanozyme, which holds significance in environment, medical, biomedical, and food industrial field.

BACKGROUND OF THE INVENTION

Ferrous ion (Fe²⁺) is a metal cation. The structure that helps it to combine with free electron makes ferrous ion a strong reductant, and then can react with various oxidizer. Iron is one of the most vital components of the environmental system. It is widely distributed in this world, accounting for 4.75% of the crust content. And in groundwater and mineral, ferrous ion occupies a greater proportion. Ferrous ion also appears in human body as an important component of hemoglobin for oxygen transport. Therefore, the detection method of ferrous ions can bring great economic effect by reducing the cost in analyzing and enhancing the quality of products.

Nanozyme is a new generation of artificial analog enzyme. Like natural

2019100367 05 Apr 2019 enzyme, nanozyme is able to efficiently catalyze the substrate of enzyme under mild conditions, offering the catalytic efficiency and enzymatic reaction kinetics which are similar to natural enzyme. But it is better than natural enzyme stability, even in the strong acid/alkali (pH 2 ~ 10) or large temperature range (4 °C ~ 90 °C), still can maintain 85% catalytic activity. Besides, these advanced materials also have some unique physicochemical properties, which provide prerequisites of designing complex catalytic system. Nanozyme also have low cost of controllable preparation, structure design, adjustable components and catalytic activity. Therefore, the artificial peroxidase mimetics with good catalytic properties will possess a brilliant future in enzyme related applications. In addition to peroxidase mimetics, many kinds of hybrid materials were constructed and proved to be highly efficient catalyst systems in various organic transformation reactions. As a result, great efforts should be made to design and fabricate new nanocomposites with peroxidase-like activity.

A fluorescence detection method using nanoprobe has been developed to detect Fe²⁺, which is mainly used for the redox reaction between manganese dioxide nanomaterials and ferrous ions to degrade manganese dioxide nanomaterials. And currently, atomic absorbance is also used in the detection requirements for low concentration Fe²⁺. Compared with other methods, colorimetric method has lower requirement of the

2019100367 05 Apr 2019 equipment and takes less time during the detection. This means colorimetric has a wider use in popular detection because of its low cost and high efficiency. Though test strips used for colorimetric detection is available on the market, they only can be available when the concentration of the sample is greater than 2 mg/L. And when the difference of concentration is large, while the difference of the color scale is small, it can easily lead to misjudgment. Therefore, a method that can fast and accurately detect low concentrations of Fe²⁺ is of great need.

In this report, we prepared CoFe cube nanozyme by a simple method. CoFe cube nanozyme can efficiently catalyze oxygen to produce free radical or promote electron transfer. Besides, this type of nanozyme has the beneficial of fast, timely, sufficient reaction and high efficiency, which is beneficial to environmental monitoring, food industrial and clinical medicine detection. It plays an important role and provides a new method and material for analysis and detection of Fe²⁺.

SUMMARY OF THE INVENTION

Nanozyme is a new generation of artificial analog enzyme. Similar to natural enzyme, nanozyme is able to efficiently catalyze the enzyme substrates under mild conditions. Compared to natural enzyme, nanozyme has a better stability, even in strong acid/alkali or large temperature range,

2019100367 05 Apr 2019 the material still can maintain a relatively high catalytic activity. Besides, the unique physicochemical structure of this kind of material also provides prerequisites of designing complex catalytic system. In addition, nanozyme also have low cost of controllable preparation, structure design, adjustable components and catalytic activity, which can provide potential economic benefits. Therefore, the nanozyme should be regarded in a high position.

The object of the invention is to provide a colorimetric method by using nanomaterials to detect Fe²⁺, which are convenient to synthesis. In order to solve problems mentioned above, this invention provides a new method by using CoFe cube nanozyme to detect Fe²⁺. In this invention, CoFe cube nanozyme is synthesized by a simple coprecipitation method. It has been observed that variables of catalysis include pH, concentration of substrate, temperature, etc. are approved by various experiments. Finally, the chromogenic reaction of Fe²⁺ succeeded. And it has been demonstrated that CoFe cube nanozyme has chemical stability and it can conveniently detect Fe²⁺ with high efficiency.

Preparation of CoFe Cube Nanozymes:

0.5 mM C0CI2 · 6H2O and 1 mM CeHsNasCF · 2H2O were dissolved in mF deionized water, denoted as solution A. 0.25 mM K₃[Fe(CN)6]

2019100367 05 Apr 2019 were dissolved in 20 mL deionized water, denoted as solution B. Under rapid stirring, solution B was added slowly into the solution A, continuing to stir for 24 hours. After being centrifuged, washed and dried, purple powder could be obtained, which was recorded as sample CoFePBA-1. Keeping other conditions unchanged, only the amount of K₃[Fe(CN)6] substance was changed to ImM, preparing sample, which was recorded as sample CoFePBA-2.

Machines

Transmission electron microscopy (TEM) images of CoFe cube nanozymes were obtained by a transmission electron microscope (FEI Tecnai G2 20 S-TWIN) operating at an accelerating voltage of 200 kV. Five pH Meter, Thermo shaker were also used. Centrifuge used centrifugal force to separate liquid and solid particles or liquid and liquid mixtures. Magnetic stirrer used the principle of homogeneity and reciprocal attraction of the magnetic field, the magnetic field was used to push the magnetic stirrer placed in the container for circumferential operation, thereby it used to stir the CoFe cube nanozyme.

Temperature

0.4 mL of phosphate buffer (pH 4.0) was added into a 1.5 mL centrifuge tube, then that 30 pL sample CoFePBA-2 was added in to the same

2019100367 05 Apr 2019 container. Then 10 pL TMB was added into the tube which was kept under constant temperature (0, 20, 30, 40, 50, 60 and 70 °C) for 8 minutes, the absorbance could be determined at 650 nm. These steps were repeated for 3 times.

The concentration of nanozyme

0.4 mL of phosphate buffer (pH = 4.0) was added to the 1.5 mL centrifuge tube, then sample CoFePBA-2 with different volume 10.0, 20.0, 30.0, 40.0, 50.0 pL and 5.0 pL of TMB were added. The solution was kept reacting at the temperature of 20 °C, then color changes were observed. After 10 minutes, the solution could be detected by identifying light absorbance at 650 nm. These steps needed to be repeated 3 times.

The detection of Fe²⁺ includes following steps:

The optimum components, pH value and reaction temperature were determined. 0.4 mL of the optimal pH solution was placed in a 1.5 mL centrifuge tube, and the optimal volume of the best nanozyme sample and the optimal volume of TMB were added respectively, setting in the optimal reaction temperature for 10 minutes, and Fe²⁺ in different concentrations were added. Continue the constant temperature reaction, observe the color change, and measure the absorbance of the solution at 650 nm after 2 minutes. Three samples were repeated for each group. The

2019100367 05 Apr 2019 detection limit, detection range and linear detection range of Fe²⁺ were determined.

DESCRIPTION OF DRAWINGS

Figure 1 illustrates the TEM image of CoFe Nanozyme.

Figure 2 illustrates the different proportions on the catalytic activity of CoFe Nanozyme.

Figure 3 illustrates the impacts of pH value on the catalytic activity of CoFe Nanozyme.

Figure 4 illustrates the impacts of temperature on the catalytic activity of CoFe Nanozyme.

Figure 5 illustrates the impacts of TMB substrate concentration on the catalytic activity of CoFe Nanozyme.

Figure 6 illustrates the impacts of volume on the catalytic activity of CoFe Nanozyme.

Figure 7 illustrates the the detection of absorbance for different concentration of Fe²⁺. Δ A= Ao-A, A is the absorbance of Fe²⁺; Ao is the absorbance of the standard, which without adding any substance.

DESCRIPTION OF PREFERRED EMBODIMENTS

The embodiment of the present invention will be explained in details so that the present invention can be more readily understood.

2019100367 05 Apr 2019

The present invention provides a new method to detect Fe²⁺, includes following:

The Preparation of CoFe cube nanozyme:

> 0.5 mM C0CI2 ·6Η₂Ο and ImM C6H₅Na₃O7 ·2Η₂Ο was added into 20 mL Deionized water, marked as solution A. Repeat the above operation and configure another solution A.

2> 0.25 mM K₃[Fe(CN)e] was added into 20 mL deionized water, marked as solution Β. 1 mM K₃[Fe(CN)6] was added into 20 mL deionized water, marked as solution C.

Solution A was dissolved by Magnetic mixer, then solution B was added into solution A slowly. This stirring last for 24 hours after the solution B was added. Then recording purple powder as sample CoFePBA-1 after centrifuged, washed and dried; Keeping other conditions unchanged, the solution C was added into solution A, record as a sample CoFePBA-2. TEM image of CoFe Nanozyme is shown in Figure 1.

Comparison of catalytic activity with Different Proportions:

0.4 mF of phosphate buffer (pH 4.0) was added into 1.5 mF centrifuge tube with 30 pF water or CoFe cube nanozyme samples and 10 pF TMB. To find the nanozyme sample with highest catalytic activity, the absorbance could be determined at 650 nm after 10 minutes. Each group repeated for 3 times. As experiment shown, sample CoFePBA-2 has the

2019100367 05 Apr 2019 deepest blue color, which reflects that it may have the highest catalytic activity. As showing the highest peak among all samples, Figure 2 indicated that sample CoFePBA-2 has the strongest absorbance. In order for a more apparent reaction phenomenon, sample CoFePBA-2 will be applied in the following experiments.

Impacts of pH Value:

0.4 mL of phosphate buffer with different pH value (pH 3.0-10.0) was added into a 1.5 mL centrifuge tube, adding 30 pL sample CoFePBA-2 and 10 pL TMB, the absorbance could be determined at 650 nm after 10 minutes, determining the best pH Value buffer. Each group repeated for 3 times. Figure 3 could be deduced that in the buffer solution with pH 4.0, the absorbance achieves the stage, it means that the catalytic activity of nanozyme is the strongest when it is in the environment of pH 4.0.

Impacts of Temperature during the Reaction:

0.4 mL of phosphate buffer (pH 4.0) was added into a 1.5 mL centrifuge tube, then that 30 pL sample CoFePBA-2 was added in to the same container. Then 10 pL TMB was added into the tube which was kept under constant temperature(0, 20, 30, 40, 50, 60 and 70 °C) for 8 minutes, the absorbance could be determined at 650 nm. These steps were repeated for 3 times. As shown in Figure 4, it could be seen that in the temperature

2019100367 05 Apr 2019 of 20 Celsius degrees, the absorbance achieves the stage, which means that the catalytic activity of nanozyme is the strongest when it is in the temperature of 20 Celsius degrees. For convenience , 20 °C will be applied in the following experiments.

Impacts of TMB Concentration:

0.4 mF of phosphate buffer (pH 4.0) was put into a 1.5 mF centrifuge tube. After that, TMB with different volume of 1.0, 2.0, 3.0, 4.0, 5.0, 7.5 pF and 30.0 pF sample CoFePBA-2 were added. The solution was kept reacting at the temperature of 20 °C, then the absorbance could be determined at 650 nm after 10 minutes. These steps were repeated for 3 times. As shown in Figure 5, absorbance was similar when the concentration reached 0.21 to 0.43 mM. So, the concentration of 0.21 mM was chosen for CoFePBA-2 in the following experiments.

Impacts of CoFe Cube Nanozyme:

0.4 mF of phosphate buffer (pH 4.0) was added to the 1.5 mF centrifuge tube, then sample CoFePBA-2 with different volume 10.0, 20.0, 30.0, 40.0, 50.0 pF and 5.0 pF of TMB were added. The solution was kept reacting at the temperature of 20 °C, then color changes were observed. After 10 minutes, the solution could be detected by identifying light absorbance at 650 nm. These steps repeated 3 times. Figure 6 shown that io

2019100367 05 Apr 2019 it achieved stage when the metering of 30 gL of CoFe cube nanozyme. Therefore, metering of 30 gL was chosen for CoFe cube nanozyme in the following experiments.

Determine the range of concentration of Fe—:

0.4 mL of phosphate buffer (pH 4.0) was added to the 1.5 mL centrifuge tube with 30 pL of sample CoLePBA-2 and 5 gL of TMB. After 10 minutes, Le²⁺ with different volume 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 gL were added. Setting a sample which without Le²⁺ for comparison; observing color changes after 8 minutes, solution can be detected by identifying absorbance at 650nm. Each sample was repeated 3 times. The detection range of concentration of Ee²⁺ was the metering of 1 gL or more. When the metering achieved 9 gL, color of solution (blue) faded completely. The solution could be detected by identifying light absorbance at 650 nm. As shown in Ligure 7, the detected range of Le²⁺is from 0.01 mM to 0.10 mM. This is much more accurate than many methods for detecting Le²⁺ that are now available.

2019100367 05 Apr 2019

Claims

1. A colorimetric method for detecting ferrous ion (Fe²⁺), is characterized in including following steps:

preparation of CoFe cube nanozyme:

C0CI2 · 6H2O and CeHsNasOv’SFbO are dissolved in 20 mL deionized water, denoted as solution A. K3[Fe(CN)6] is dissolved in 20 mL deionized water, denoted as solution B;

Under rapid stirring, solution B is added slowly into the solution A, continuing to stir for 24 hours. After being centrifuged, washed and dried, purple powder could be obtained.

2. The colorimetric method for detecting ferrous ion (Fe²⁺) according to claim 1, wherein during the preparation, the optimized mole ratio of C0CI2 · 6H2O to K₃[Fe(CN)₆] is 1:2.

3. The colorimetric method for detecting ferrous ion (Fe²⁺) according to claim 1, wherein the detection of ferrous ion (Fe²⁺) includes following steps:

the optimum components, pH value and reaction temperature are determined;

0.4 mL of the optimal pH solution is placed in a 1.5 mL centrifuge tube, and the optimal volume of the best nanozyme sample and the optimal volume of TMB are added respectively, setting in the optimal reaction temperature for 10 minutes, and Fe²⁺with different concentrations are added;

continue the constant temperature reaction, observe the color change, and measure the absorbance of the solution at 650 nm after 8 minutes.