AU2018101303A4

AU2018101303A4 - Synthesis of Rh-Cu Nanozyme and Application for the Detection of Ascorbic Acid and Tannic Acid

Info

Publication number: AU2018101303A4
Application number: AU2018101303A
Authority: AU
Inventors: Shicheng Hu; Jiashu Huang; Aoling Li; Feifan Li; Peilun Li; Hong Wang
Original assignee: Li Feifan Miss
Current assignee: Li Feifan Miss
Priority date: 2018-09-06
Filing date: 2018-09-06
Publication date: 2018-10-18
Anticipated expiration: 2026-09-06

Abstract

In this invention, a colorimetric method for the detection of ascorbic acid (AA) and tannic acid (TA) was disclosed. copper-rhodium nanomaterial (Rh-Cu nanozyme) can be an ideal oxidizer toward 3,3',5,5'-tetramethylbenzidine (TMB). The catalytic activity of Rh-Cu nanozyme depends on several variables such as concentration of enzyme, concentration of TMB, pH, temperature and so on. Ascorbic acid (AA) and tannic acid (TA) process strong reduction identity, which can directly reduce the oxidized TMB (oxTMB) to TMB, resulting color of the solution faded from blue to colorless. Thus, the quantitative presence of AA and TA can be obviously detected by the extent of changed color. In order to optimize the performance of this system to the biggest degree, the best conditions of variables were studied. With all these optimal conditions, this detection system can be an inexpensive and simple method to identify TA and AA in many cases, which will also be potentially beneficial to other technologies. The results present a range of AA detection from 5.48 pmol/L to 52.1 pmol/L, and a range of TA detection from 0.14 mmol/L to 0.739 mmol/L. With this particular sensitive nanozyme, a new method of detection can be applied into multiple fields of industries, including food industry and pharmacy.

Description

invention, a colorimetric method for the detection of ascorbic acid (AA) and tannic acid (TA) was disclosed, copper-rhodium nanomaterial (Rh-Cu nanozyme) can be an ideal oxidizer toward 3,3’,5,5’-tetramethylbenzidine (TMB). The catalytic activity of Rh-Cu nanozyme depends on several variables such as concentration of enzyme, concentration of TMB, pH, temperature and so on. Ascorbic acid (AA) and tannic acid (TA) process strong reduction identity, which can directly reduce the oxidized TMB (oxTMB) to TMB, resulting color of the solution faded from blue to colorless. Thus, the quantitative presence of AA and TA can be obviously detected by the extent of changed color. In order to optimize the performance of this system to the biggest degree, the best conditions of variables were studied. With all these optimal conditions, this detection system can be an inexpensive and simple method to identify TA and AA in many cases, which will also be potentially beneficial to other technologies. The results present a range of AA detection from 5.48 qmol/L to 52.1 qmol/L, and a range of TA detection from 0.14 mmol/L to 0.739 mmol/L. With this particular sensitive nanozyme, a new method of detection can be applied into multiple fields of industries, including food industry and pharmacy.

2018101303 06 Sep 2018

DESCRIPTION

TITLE

Synthesis of Rh-Cu Nanozyme and Application for the Detection of Ascorbic Acid and Tannic Acid

FIELD OF THE INVENTION

The invention relates to the colorimetric detection of ascorbic acid and tannic acid by using rhodium-copper alloy nanoparticles, which is important for agriculture industry, analytical chemistry, medical and biomedical fields.

BACKGROUND OF THE INVENTION

Ascorbic acid (AA), found in fruits and vegetables, provides plenty of applications such as avoiding leukoderma, reducing the probability of cancer, and supplementing best antiscorbutic element for human body. The structure of ascorbic acid is similar to glucose—polyhydroxy compound, where the 3 adjacent enol hydroxy groups in the molecule are easily dissociated from the two adjacent enol hydroxy groups and release H⁺. Strong reducibility which AA processes is significant and can be utilized in other cases. Tannic acid (TA) is light brownish yellow amorphous powder that has particular odor and extremely astringent taste. Being a convergent agent, tannic acid precipitates proteins then forms insoluble complexes with alkaloids, glucosides and heavy metals. TA is soluble in water, ethanol and glycerol and TA solution with the presence of ferrous salt solution would appear in black and blue.

However, the conventional methods for detecting AA and TA encountered some obstacles such as being expensive, sophisticated and inefficient. One of the methods of detecting ascorbic acid is 2,6-dichlorophenol indophenol titration, which is simple for operation and highly efficient. But the reagent is expensive and poorly selective for detecting AA. The detection methods for tannic acid currently are spectrophotometry, titration, protein precipitation, electrochemical analysis and so on. All of those methods process high efficiency and accuracy, but almost all require complex operations and considerably professional equipment. Not a single low-cost and effective method is proposed so far.

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Nanozymes are a class of mimic enzymes that have both the unique properties of nanomaterials and catalytic functions. Nanozymes are characterized by high catalytic efficiency, stability, economy and large-scale preparation. They are widely used in medicine, chemical, food, agriculture, and the environment. Different from natural enzymes, this new kind of enzyme won’t loss its function within high temperature or low pH value.

Lately, research on nanozymes and related technologies has extended the horizon of knowledge. Nanozymes, because of considerably tiny size, process as same as a natural enzyme, and does not contain the limitation of natural enzymes. This particular feature provides many extensions of current science and technology and new applications. Due to that this technology just been released, there aren’t many nanozymes been created and putted into a series of testing. Based on these reasons, the purpose of processing this experiment is mainly to discover a new and better way of detecting ascorbic acid and tannic acid also to create a brand new nanozyme.

SUMMARY OF THE INVENTION

The purpose of this invention is to create a method to detect AA and TA by utilizing materials that process unique identities such as high efficiency, good stability, simple to prepare and lowcost to manufacture. Since nanomaterials have distinct properties that do not appear on other regular materials, investigating Rh-Cu nanozyme for ascertaining AA and TA will be prospecting and beneficial.

Preparation of Rh-Cu nanoparticles includes following steps:

mg polyvinyl pyrrolidone (PVP) was added to a flask. Then, 3.84 mL distilled water was added. The mixture was put in the ultrasonic until the PVP was dissolved. Then, the RhC'l·, solution (25 mmol/L) and the C’uCT (25 mmol/L) solution were added. The specific combinations of differ ratio of reagents showing below were made accordingly. The flask was put in an ice-bath, stirring for 5 min.

Cu=l	Cu: 0.16 mL	Rh: 0
Cu:Rh=5:l	Cu: 0.133mL	Rh: 0.0267mL
Cu:Rh=3:l	Cu: 0.12mL	Rh: 0.04mL
Cu:Rh=l:l	Cu: 0.08mL	Rh: 0.08mL

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E:Cu:Rh=l:3 Cu: 0.04mL Rh:0.12mL

F: Cu:Rh=l:5 Cu: 0.0267mL Rh: 0.133mL

G: Rh=l Cu: 0 Rh: 0.16mL

0.25 mL NaBEE solution (1 mg/mL ) was added to the flask 15 seconds per drop. After the solution was added completely, the mixture was placed in ice-bath continually, stirring for 2 hours.

The detection of TA and AA includes following steps:

0.9 mL of phosphate buffer with pH of 3.5 was poured into a 1.5 mL centrifugal tube. Then

0.12 mL of the best nanozyme sample F and 0.075 mL of TMB with concentration of 0.82 mM were added to the tube. After an 11-min reaction under the best temperature of 25 °C, the liquid in the tube was distributed equally to three 1.5 mL centrifugal tubes. Then TA with different concentration was added to each tube. After 3 min, the color change was observed and absorbance of the solution in the tubes at 652 nm was detected. The experimental combination below was repeated using the same method respectively.

Volume/pL	TA concentration in the tube after adding/mM
0	0
35	0.192
40	0.219
45	0.246
55	0.301
65	0.356
95	0.520
115	0.630
125	0.680
135	0.739

The detection of AA was similar to that of TA. The solution (including buffer solution, nanozyme and TMB) for the detection of AA was distributed after an 8-min reaction, then AA with different concentration was added to each tube. After 4 min, the color changes were observed and absorbance of the solutions in the tubes at 652 nm was detected. The experimental combination below was repeated with the same procedure.

Volume/pL

AA concentration in the tube after adding/pM

0	0
15	8.22
23	12.6
31	17.0
37	20.3
44	24.1
51	27.9
60	32.9
70	38.4
85	46.6
95	52.1

DESCRIPTION OF DRAWING

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Figure 1 TEM image of Rh-Cu nanoparticles.

Figure 2 The catalytic activity of Rh-Cu nanoparticles with different proportions.

Figure 3 Effect of pH value on the activity of Rh-Cu nanozyme.

Figure 4 Effect of temperature on the activity of Rh-Cu nanozyme.

Figure 5 Effect of TMB concentration on the activity of Rh-Cu nanozyme.

Figure 6 Effect of concentration on the activity of Rh-Cu nanozyme.

Figure 7 The fitted curve of the absorbance at different concentration of TA (AAbs=Ao-A, Ao means the absorbance of the blank group, A means the absorbance of test groups).

Figure 8 The linear fitted curve of the absorbance at different concentration of TA.

Figure 9 The fitted curve of the absorbance at different concentration of AA (AAbs=Ao-A, Ao means the absorbance of the blank group, A means the absorbance of test groups).

Figure 10 The linear fitted curve of the absorbance at different concentration of AA.

Figure 11 The comparison of changing absorbance between TA and AA.

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.

The effect of different combination ratio on catalytic activity of Rh-Cu nanoparticles

0.6 mL of phosphate buffer with pH of 3.5 was added to each centrifugal tube, then 30 uL of water and nanozyme grouped from A-G was injected into centrifugal tubes respectively. Next,

2018101303 06 Sep 2018 uL of prepared TMB solution was added for reactions. Observing colorimetric changes, the nanozyme with the best performance activity was ascertained after 10 minutes. For characterization purposes, the sample was sent to undergo UV-Vis examination. This experiment was repeated 3 times.

The nanozyme containing best components proportion was showing in Figure 2. At 652 nm, the absorbance of sample F was higher than other samples, indicating that the nanozyme with ratio Cu:Rh=l:5 has the highest catalytic activity.

The effects of different pH value

0.6mL of each phosphate buffer solution with pH ranging from 3 to 10 was separately poured into ten 1.5 mL centrifugal tubes and 100 pL of sample F was then put into the tubes. Then 10 pL of TMB solution with concentration of 20 mM was put into each tube simultaneously. The color change of solutions was observed, and the absorbance of solutions at 652 nm was measured by the Microplates Reader after 10 min in order to understand how the pH of solutions affect the activity of nanozyme and to determine the best pH for reaction. The procedures above were repeated for three times.

The best pH (3.5) was detected according to Figure 3, and the rest of experiments were settled to the same optimized pH value.

The effects of the reaction temperature

0.6 mL phosphate buffer solution with pH of 3.5 was added to a centrifuge tube (1.5 mL in volume ) . Then 30 pL of sample F was added. The mixture was placed respectively in 273.15, 298.15, 303.15, 308.15, 313.15, 323.15, 333.15, 343.15 K, maintained for 8 min. Then 10 pL TMB solution was added to the mixture. The changes of the color of the reaction were observed during the thermostat reaction. After 8 min, the absorbance of the solution at 652 nm was detected to confirm the best reaction temperature. The experiment was repeated three times at each temperature.

The best temperature was showing in Figure 4. The highest peak of the curve in the figure was at 298.15K, suggesting that the best temperature for the nanozyme was 298.15K.

The effects of the concentration of the substrate

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0.6 mL of phosphate buffer solution with pH of 3.5 was added to 9 centrifuge tubes (1.5 mL in volume) . Then 30 pL of sample F was added. 2, 5, 10, 15, 20, 30, 40, 50 and 60 pL of TMB solution (20 mmol/L) were added to the mixture respectively at the best reaction temperature, in other words the concentration of the TMB was 0.060, 0.152, 0.303, 0.455, 0.606, 0.909, 1.21, 1.52 and 1.82 mmol/L respectively. The water was added to the mixture to ensure the volumes of all the groups were same. The changes of the color of the reaction were observed during the thermostat reaction. After 8 min, the absorbance of the solution at 652 nm was detected. The experiment was repeated three times for all molarity.

The best concentration of the substrate (TMB) can be deduced from Figure 5. The highest peak of the curve in the figure was at 1.52 mmol/L, meaning that the best concentration of the substrate (TMB) was 1.52 mmol/L.

The effects of the concentration of the nanozyme

0.6 mL of phosphate buffer solution with pH of 3.5 was added to 6 centrifuge tubes (1.5 mL in volume). Then 30, 40, 60, 80, 110 and 120 pL of sample F was added respectively, in other words the concentration of the nanozyme was 0.047, 0.055, 0.083, 0.110, 0.152 and 0.166 mmol/L. The water was added to the mixture to ensure the volumes of all the groups were the same. Then 50 pL of TMB solution was added to the mixture at the best reaction temperature. The changes of the color of the reaction were observed during the thermostat reaction. After 8 min, the absorbance of the solution at 652 nm was detected. The experiment was repeated three times.

Figure 6 illustrated the fittest nanozyme molarity. 0.11 mmol/L, where the highest peak of the curve in the figure located, was considered the best concentration of the nanozyme in order to achieve optimizing performance.

Detection Ability Comparison of TA and AA at Same Concentration

0.9 mL of phosphate buffer with pH of 3.5 was poured into each of 2 centrifugal tubes. Then 0.12 mL of the best nanozyme sample F and 0.075 mL of TMB with concentration of 0.82 mM were added to each tube. After an 8-min reaction under the best temperature of 25 °C, the liquid in the tubes was distributed to 6 tubes. Then 60 pL of TA (0.2 mM) was added to three of the tubes, while AA with the same volume and concentration was added to another three tubes.

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The color change was observed and the absorbance of the solution at 652 nm was detected by the microplate reader after 4 min. The results of the AA and TA were compared and analyzed.

The changing absorbance of TA was lower than AA in Figure 11, showing that the responsiveness of TA was less-effective than AA.

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Claims

1. A colorimetric method for the detection of ascorbic acid and tannic acid by using rhodium-copper alloy nanoparticles,in which including following steps:

1.1 Preparation of rhodium-copper alloy nanoparticles:

20 mg polyvinyl pyrrolidone (PVP) was added to a flask; Then, 3.84 mL distilled water was added; The mixture was put in the ultrasonic until the PVP was dissolved; Then, the RhCl₃ solution (25 mmol/L) and the CuCl₂ (25 mmol/L) solution were added; The flask was put in an ice-bath, stirring for 5 min; 0.25 mL NaBH₄ solution (1 mg/mL) was added to the flask 15 seconds per drop; After the solution was added completely, the mixture was placed in ice-bath continually, stirring for 2 hours;

1.2 The preparation according to claim 1.1, the optimized volume ratio of CuCl₂ to RhCl₃ was 1:5;

1.3 Rh-Cu alloy nanoparticles are obtained, wherein the detection of tannic acid includes following steps:

0.9 mL of phosphate buffer with pH of 3.5 was poured into a 1.5 mL centrifugal tube; Then 0.12 mL of sample F and 0.075 mL of TMB with concentration of 0.82 mM were added to the tube; After an 11-min reaction at room temperature of 25 °C, the liquid in the tube was distributed equally to three 1.5 mL centrifugal tubes; Then TA

2018101303 06 Sep 2018 with different concentration was added to each tube; After 3 min, the color change was observed and absorbance of the solution in the tubes at 652 nm was detected; The experimental combination below was repeated using the same method respectively;

1.4 Rh-Cu alloy nanoparticles are obtained, wherein the detection of ascorbic acid includes following steps:

0.9 mL of phosphate buffer with pH of 3.5 was poured into a 1.5 mL centrifugal tube; Then 0.12 mL of sample F and 0.075 mL of TMB with concentration of 0.82 mM were added to the tube; After an 8-min reaction at room temperature of 25 °C, the liquid in the tube was distributed equally to three 1.5 mL centrifugal tubes; Then AA with different concentration was added to each tube; After 4 min, the color changes were observed and absorbance of the solutions in the tubes at 652 nm was detected; The experimental combination below was repeated with the same procedure.

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Figure 1

Figure 2

2018101303 06 Sep 2018 pH

Figure 3

Figure 4

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Figure 5

0.045 0.060 0.075 0.090 0.105 0.120 0.135 0.150 0.165 0.180 [Nanozyme](mM)

Figure 6

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Figure 7

Figure 8

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Figure 9

Figure 10

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Figure 11