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Associate Professor Wang Yan, Harbin University of Commerce, et al.: Detection of Cu2+ by differential pulsed voltammetry by methane oxidoxin-coupled lipase biosensor

 Last Update: 2023-01-05
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According to the World Health Organization, the mass concentration standard of Cu²⁺ in drinking water is not higher than 2.
0 mg/L, and when^{the cumulative amount} of Cu2+ in serum exceeds 0.
035 mmol/L, copper poisoning
will be caused.
The existing Cu²⁺ detection methods include spectroscopy, inductively coupled plasma mass spectrometry, ion chromatography, spectrophotometry, etc.
, most of which have some limitations and disadvantages, such as long time-consuming, low sensitivity, cumbersome operation, expensive instruments and relying on laboratory professionals for testing, which are not conducive to the practical application
of on-site rapid detection.
Electrochemical analysis is recognized as a rapid, sensitive and accurate trace elemental analysis method, which is simple to operate and inexpensive, which is its outstanding advantage
that has attracted much attention.
At present, the electrochemical method has low sensitivity and selectivity for Cu2⁺ detection, and the differential pulsed voltammetry (DPV) method has higher sensitivity than cyclic voltammetry, and the current response signal has a proportional relationship with the total amount of analyte to be analyzed by surface electrolysis, which can be quantitatively detected
.

Methane oxidizin (Mb) is a biomolecule secreted by methane oxidizing bacteria during the growth process, is a protein active peptide with high copper affinity, free sulfhydryl groups and amine groups in Mb can be bonded with gold nanometers (AuNPs) through Au-S and Au-N bonds, and Mb specifically captures copper in the environment and has strong specificity
to^Cu2+.

Wang Yan, Zhao Ning, Wang Yue and others from the Key Laboratory of Food Science and Engineering, Harbin University of Commerce investigated the electrochemical response of the sensor construction by cyclic voltammetry on Mb-coupled lipase biosensor, and used DPV to achieve specific qualitative and quantitative detection of Cu2⁺, the principle of which is shown
in Figure 1 。 Based on layer-by-layer assembly technology, Mb/Lipase@AuNPs-Gold Electrode was constructed, and the hydrolysis reaction of lipase catalyzed trioleate was based on the reaction model, with triolein as the substrate molecule, the biosensor could detect the current signal generated when hydrolyzing the substrate, and use the characteristics of Mb to specifically capture Cu2+ to achieve Cu2⁺ The enrichment phenomenon around lipase inhibits the catalytic activity of lipase, resulting in a rapid decrease in current intensity, and the rapid qualitative and quantitative detection
of Cu2+ is realized by investigating the difference of current signal before and after Cu2⁺ is added.
The lipase biosensor constructed in this study can solve the problems of low sensitivity, specificity and high detection limit of Cu2+ by electrochemistry, and provide a new idea and research basis
for the rapid quantitative detection of Cu2⁺ in situ.

01Analysis of the mechanism of immobilized lipase construction

Transmission electron microscopy characterizes immobilized lipase

AuNPs solution and immobilized lipase were observed under transmission electron microscopy, as shown in Figure 2A, the AuNPs particles in AuNPs solution were uniformly dispersed and uniform in size, and the particle size of AuNPs was not changed in Figure 2B immobilized lipase solution because lipase was fixed on AuNPs particles by covalent bonding, but the AuNPs particle distribution was relatively tight, which could prove that lipase fixation was successful
.

Spectral characterization of immobilized lipases

As shown in Figure 3A, a new characteristic absorption peak appeared at 520 nm after AuNPs combined with lipase, and the characteristic absorption peak was redshifted after the immobilized lipase was successfully prepared, and the amino acid ultraviolet characteristic absorption peak around 280 nm decreased, and the results showed that lipase had been successfully fixed on AuNPs, which increased the polymerization degree between AuNPS particles and lipase
molecules.
The fluorophore in porcine pancreatic lipase can undergo intermolecular interactions with quencher such as excited state reactions, molecular rearrangement, energy transfer, formation of ground state complexes and molecular collisions, etc.
, which can trigger the quenching
of fluorescence.
When the excitation wavelength is 280 nm, tryptophan and tyrosine are excited
at the same time.

As shown in Figure 3B, when the excitation wavelength λex is 280 nm, after adding AuNPs to the enzyme solution, the characteristic emission peaks of tryptophan and tyrosine at λex/λem=280/342 nm are significantly reduced, the fluorophore is quenched, the emission wavelength of lipase is slightly redshifted, from 342 nm redto 345 nm, when the immobilized lipase is successfully prepared, the emission wavelength is redshifted to 351 nm again, which indicates that both amino acid residues have been exposed.
Immobilized lipase preparation was successful to form kinyl complexes, resulting in longer
wavelengths.

The infrared pattern of lipase and immobilized lipase changes is shown in Figure 3C, there is a strong polar O—H expansion vibration on unsaturated carbon at 3 437 cm-1, a new three-bond at 2 319 cm-1 and a cumulative double bond region C≡N are stretched, and the C=C telescopic vibration of 1 638 ^cm-1 is significantly enhanced, which may be due to the bonding of —SH and the C=O of carboxylic acid in AuNPs solution.
Here, the characteristic absorption peak is enhanced, 980 cm-1 may be C—O telescopic vibration, 872 cm-1 = CH2 out-of-plane osway, 607 cm-1 and 531 cm-1 may be C—S and C—N, 525 ^cm-1 is -S—S—The telescopic vibration is significantly weakened.
It was shown that the disulfide bond in lipase was coupled to AuNPs particles to form an Au-S ion covalent bond.

In view of the importance of disulfide bonds in maintaining the structural stability of proteins, and the results of ultraviolet and fluorescence spectroscopy, the preparation of immobilized lipase was successful
.

02Mb coupled lipase biosensor construction method screening

In the electrolyte solution, only the gold electrode of AuNPs solution was modified: because AuNPs had the ability to amplify the current signal, the conductivity of the electrode was enhanced, and the ability to capture the electrical signal was more sensitive than that of the bare gold electrode, and the current value increased by 8.
802 μA; The Mb-coupled lipase biosensor constructed in three ways had an increasing current signal by electrodeposition, immersion and drip coating, which reduced the current signal by 17.
861 μA compared with the bare electrode current signal, 21.
992 μA by immersion method compared with the bare electrode current signal, and 27.
093 μA by electrodeposition method, and the results are shown in Figure 4, indicating that the electrodeposition method has the best
effect on constructing lipase biosensor.

In Fig.
5, the enzyme biosensors prepared by three methods to assemble Mb-coupled immobilized lipase were detected in the reaction system with trioleate as substrate, respectively: the current signal value of electrodeposition method was 18.
449 μA, the current signal value of immersion method was 12.
908 μA, the current signal value of drip coating method was 11.
426 μA, and the peak current detection of lipase biosensor constructed by electrodeposition, immersion method and drop coating method decreased sequentially, and both results showed that the electrodeposition method modified immobilized lipase with the best effect.
Therefore, in this study, the lipase biosensor
was constructed by electrodeposition.
The electrodeposition method can achieve the ideal modification of the electrode according to the design order, and the target is orderly and relatively dense, and the stability of the modification is higher
than that of the dropping method and the immersion method.

03Mb coupled lipase biosensor construction characterization

As shown in Figure 6A, modifying AuNPs on the surface of the bare electrode in the electrolyte solution amplifies the current signal, and the electrical signal gradually weakens
as the number of modified lipase layers increases.
As shown in Figure 6B, the arc of the solid resistance-imaginary resistance curve in the detection by AC impedance method gradually increases, showing that the surface resistance of the sensor is increasing, and it also proves that the enzyme biosensor is
successfully constructed 。 In the detection system with glyceryl trioleate as the substrate, the current intensity transmitted is very weak, and the bare electrode can only capture a weak redox electrical signal; As the number of enzyme layers of the lipase biosensor constructed by electrodeposition method increases, the redox reaction gradually strengthens, and the electrical signal intensity increases, but when the number of layers is 4 layers, the electrical signal in the detection system decreases sharply, the result is shown in Figure 7, the more layers will form steric hindrance effect, the active centers cover each other and bury, and affect the induction effect of the sensor on the current signal, and the formation of Au-S bond covalent bond and strength weaker than its own weight will also cause fracture; However, if the number of modification layers is too small, the signal transmission efficiency, sensor response surface and effective transducer do not reach saturation, so the sensitivity of detection is too low, so 3 layers are selected as the optimal number of modified lipase layers
.

Optimization of Cu²+ detection system by 04DPV method

Determination of substrate mass concentrations catalyzed by lipase biosensors

As shown in Figure 8A, the current peaks around 0.
2 V, as shown in Figure 8B, the mass concentration of 2 g/100 mL trioleic acid glycerol dissolved in Tirs-HCl buffer solution when the electrical signal intensity is up to 8.
795 μA, the number of enzymes on the lipase biosensor is constant, the number of active sites of the enzyme catalytic activity center is also certain, when the substrate mass concentration is too high, the active site of the enzyme's catalytic activity center that can transmit electrical signals will be masked and attenuated.
At the same time, the electric double diffusion coefficient of the detection system will decrease, and the current signal captured by the sensor at the same time will be multiplied
.
When the mass concentration of the substrate is too low, the enzymatic reaction does not reach saturation, at this time, although the diffusion coefficient of the electric double layer of the detection system is greater than the coefficient when the mass concentration of glycerol trioleate is too large, the mass concentration has an effect on the ability of the enzymatic reaction on the lipase biosensor to transmit electrical signals, compared with the electrical signal intensity when the two are equilibrium, the dielectric coefficient of the electric double layer of the system reaches saturation, so trioleyl trioleate is selected to dissolve in Tirs-HCl buffer solution, and 2 g/100 mL is the optimal mass concentration of the reaction substrate
。

Lipase biosensors catalyze the determination of the optimal pH of trioleate

As shown in Figure 9A, the oxidation peak current reaches the maximum value at a potential of 0.
2 V, as shown in Figure 9B, the electrical signal intensity at pH 7.
5 is up to 15.
81 μA, the pH value has a significant effect on the catalytic activity of lipase, when the diffusion coefficient of the electric double layer interface is certain, the enzymatic effect is proportional to the electrical signal intensity, the difference in pH value will change the secondary structure and tertiary structure of the enzyme, the active amino acids and residues of the enzyme will be partially or completely inactivated, and the enzymatic reaction cannot proceed normally.
At this time, the intensity of the transmitted signal at the active site of the lipase biosensor is decayed exponentially, so the lipase catalytic system with pH 7.
5 is selected as the best detection system
.

05Cu²⁺ detection linear range, fitting curve, detection limit

As shown in Figure 10, the linear regression equation is y=0.
228x+0.
774 8 (^R2=0.
995 0), indicating that the Cu2⁺ concentration has a good linear relationship between the 1~100 nmol/L range and the current signal drop height, and the detection limit is calculated to be 0.
03 nmol/L
.

06Cu²⁺ detection system anti-interference performance

As shown in Figure 11, when Cu2^{+ solution is} added to the detection system, the current decreases in a step-like order, but 20 μL with a concentration of 5 μmol/L is added every 50 s in turn, with a concentration of 5 μmol/L Ca2+, Mg2+, ^Zn2⁺, Hg There was no change in the induced current intensity of the lipase biosensor in ^{the solution of 2}+,^Pb2+, Ba²+, and Ni2⁺, indicating that this lipase biosensor had no change in Cu in the detection range of ultra-trace amounts ^{The 2}⁺ detection is specific, and other common divalent metal and non-metal ions have no interference
with the detection system.

07Stability and service life of enzyme biosensors

As shown in Figure 12, the peak current of the Cu2⁺ response on day 14 reached 86.
96%, indicating that the lipase biosensor had good stability, and with the increase of detection times and the extension of construction time, the active site of lipase catalytic substrate gradually decreased, resulting in decreased repeatability and detection performance, so it was used
in time during the effective detection time of the sensor.

The active site release treatment with buffer solution was used to achieve the purpose of multiple use of one sensor after each detection of^Cu2⁺, and the experimental results were shown in the same way for the first 10 detection of the active site release of the sensor, and the results were shown in Figure 13, with a relative standard deviation of 2.
19%; In 11~20 times, the relative standard deviation was 2.
87%; At 21~30 times, the relative standard deviation was 3.
48%; In 31~40 times, the relative standard deviation is 4.
55%; At 41~50 times, the relative standard deviation is 8.
03% (>5%), it can be seen that this lipase biosensor can maintain good detection performance when it is reused for 40 times, but the attenuation of current signal induction is significant in more than 40 times, this phenomenon may be because the lipase on the sensor is covered or even inactivated during each release and detection round-trip, the electrical signal transmission of the enzymatic reaction is missing, or the active center that could have conducted the signal is annihilated because of inactivation, Therefore, this lipase biosensor can complete about 40 repeated detection times in terms of service life, and the effect is good
.

Conclusion

Lipase biosensors were constructed by assembling AuNPs, immobilized lipase and Mb layer by electrodeposition method, and experiments showed that the sensor prepared when the number of immobilized lipase modification layers was 3 layers was the most stable.
In the detection system of lipase-catalyzed substrate trioleate, glyceryl trioleate was dissolved in 100 mL of Tirs-HCl buffer solution, and the sensor captured the current signal response value was the highest when the mass concentration was 2 g/100 mL and pH 7.
5.
The concentration of Cu2+ is 1~100 nmol/L in the linear range, the degree of fitting is the best, the linear equation is y=0.
228x+0.
774 8 (R2=0.
995 0), the detection limit is 0.
03 nmol/L (RSN=3), and the concentration of ^Ca2⁺ is 1 μmol/L in this ultratrace detection range , Mg 2+, Zn 2+, Ba 2+, Hg 2⁺, Pb ²⁺ and other common divalent metal and non-metal ions exist at the same time, Other metals have no interference with^Cu2⁺ detection; This system has excellent anti-interference ability to detect Cu²⁺; The use of DPV in the ultra-trace range can realize rapid and quantitative detection of the same ion on-site, and the detection time is only 10 s, which solves the practical problem
that Cu²⁺ detection cannot be detected quickly on site.
The method of detecting Cu2⁺ by the novel lipase biosensor established in this study has high sensitivity, specificity and stability, which lays a research foundation
for the detection of trace and ultra-trace heavy metals in food.

First author bio

Wang Yan, female, Ph.
D.
, associate professor, master supervisor, scientific research secretary, School of Food Engineering, Harbin University of Commerce, was born in March 1984 in Baishan City
, Jilin Province.
Mainly engaged in the application research of biotechnology in the creation of value-added agricultural and sideline products and rapid detection of food safety, and has achieved innovative research results
.
The value-added transformation of grain starch was realized, and new functional food additives such as medium and long chain fatty acid starch ester and ferulate starch ester and precursor chiral drug S-naproxen starch ester were prepared.
Through the development of methane oxidin functionalized gold nanoscale, the specific visualization and rapid detection of copper ions were realized, and the gold electrode was modified by using methane oxidin functionalized nanogold to realize the specific trace detection
of copper ions and organophosphorus in food.
A total of 65 academic papers were published, including 10 in SCI, 16 in EI, 1 paper in the award, 1 second prize of Heilongjiang Science and Technology Award (Nature Category), 2 first prizes of Heilongjiang University Science and Technology Award, 2 second prizes of Heilongjiang University Science and Technology Award, and 1
second prize of Heilongjiang Natural Science and Technology Academic Achievement Award 。 He has presided over or participated in 25 scientific research projects, including 3 National Natural Science Foundation of China, 1 "100 million" key research and development project of Heilongjiang Province, 1 Science Fund for Outstanding Young Scholars of Longjiang Province, 2 Natural Science Foundation of Heilongjiang Province, 1 project of Heilongjiang Applied Technology Research and Development Plan, 1 project of talent training support plan (high-level talents) of the central government to support the reform and development of local universities, 10 departmental and bureau-level projects, and 6 horizontal projects
.
Authorized 3 national invention patents, published 3 monographs and 1 textbook
.

This paper "Detection of Cu2⁺ by Methane Oxidoxin Coupled Lipase Biosensor Differential Pulsed Voltammetry" is from Food Science, Vol.
43, No.
16, 2022, pp.
351-358, authors: Wang Yan, Zhao Ning, Wang Yue, Li Hongjia, Xin Jiaying, Sun Lirui, Guan Huannan
.
DOI:10.
7506/spkx1002-6630-20210621-249
。 Click to view information about
the article.

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