CN105699462A

CN105699462A - Method for quantitative detection of L-cysteine

Info

Publication number: CN105699462A
Application number: CN201410714046.XA
Authority: CN
Inventors: 刘爱骅; 侯传涛
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2014-11-28
Filing date: 2014-11-28
Publication date: 2016-06-22
Anticipated expiration: 2034-11-28
Also published as: CN105699462B

Abstract

The invention relates to a method for detection of a physiological matter, and in particular, relates to a method for quantitative detection of L-cysteine by using a glucose/oxygen biofuel cell. A self powder supply property of the glucose/oxygen biofuel cell is utilized, at the same time, an open circuit electric potential of the cell is inhibited in the presence of Cu<2+>, Cu-S bonds are formed by the L-cysteine and Cu<2+>, and according to the change of the open circuit electric potential along with the concentration of the L-cysteine, the L-cysteine in a to-be-measured sample is quantitatively detected. With utilization of the principle, a linear relationship between the change quantity of the open circuit electric potential and the concentration of the L-cysteine is established, and high-sensitivity detection of the L-cysteine is realized. The method has the advantages of simple operation, high sensitivity and good specificity, and is applicable in detection of the L-cysteine in the fields of food industry, physiological processes and clinical medicine.

Description

A kind of method of detection by quantitative Cys

Technical field

The present invention relates to the detection method of a kind of physiologically substance, specifically refer to a kind of method utilizing glucose/oxygen biological fuel cell quantitative assay Cys。

Background technology

Cys is a kind of important aminoacid, is present in numerous protein, glutathion。It has very important function in many bioprocesss, and its many important diseases such as contents level exception and hepar damnification, skin lesion, senile dementia and cardiovascular disease are related。Additionally Cys is widely used in food processing industry, is mainly used in baked goods, as the required composition of flour-dough improver。No matter therefore the detection by quantitative of Cys is to physiological process and clinical diagnosis, or to food processing industry, it is respectively provided with highly important meaning。

At present, the common method of detection by quantitative Cys has spectrophotography, high performance liquid chromatography, gas chromatography-mass spectrography and electrochemical method etc.。But these methods all have its weak point, for instance, although the precision comparison of spectrophotography, high performance liquid chromatography and gas chromatography-mass spectrography detection is high, but its expensive equipment, sample treatment is loaded down with trivial details, and technology requires height。The electrochemical sensor that development in recent years is got up, such as the range of linearity of electrode detection Cys and the detection limit respectively 0.5-20 μM and 0.02 μM of graphene oxide/gold nano particle modification, the range of linearity of the electrode detection Cys that Zinc oxide nanoparticle is modified and detection limit respectively 0.2-20 μM and 0.05 μM。Although this electrochemical sensor have employed relatively inexpensive instrument, but its technology requires that higher defect is inevitable。And electrochemical workstation is difficult to microminiaturization。Therefore, a kind of simple, sensitive, special Cys detection method is set up imperative。

Summary of the invention

Present invention aim at providing a kind of method based on glucose/oxygen biological fuel cell high sensitivity quantitation detection Cys。

For achieving the above object, the technical solution used in the present invention is:

A kind of method of detection by quantitative Cys, the confession utilizing glucose/oxygen biological fuel cell is electrical, simultaneously at Cu²⁺There is the lower OCP suppressing battery, and at Cys and Cu²⁺Form Cu-S key, realize the detection by quantitative of Cys in testing sample with the change of Cys concentration according to OCP。The glucose dehydrogenase (FAD-GDH) that described glucose/oxygen biological fuel cell relies on flavin adenine dinucleotide (FAD) acts on biological anode, and laccase acts on biological-cathode。

Technical solution of the present invention is further preferred that, at Cu²⁺Existence under, utilize Cu²⁺Suppress the activity of biological anode FAD-GDH, reduce the OCP of glucose/oxygen bio-fuel, then Cys and Cu in recycling testing sample²⁺Forming Cu-S key, and reduce its inhibitory action, being added the change of front and back OCP by Cys, thus reaching the purpose of detection by quantitative Cys。

The biological anode of described glucose/oxygen biological fuel cell adopts vitamin K₃For electron mediator, biological-cathode then adopts 2, and 2 '-azino-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) is electron mediator。

The effect of the present invention is:

1. the present invention utilizes the biological anode based on FAD-GDH and the biological-cathode based on laccase, and biological anode and biological-cathode are respectively adopted vitamin K₃It is electron mediator with ABTS, thus having constructed high-performance glucose/oxygen biological fuel cell。Utilize the detection to Cys that this battery is quantitative simultaneously；Wherein, battery maximum power density is 98 μ Wcm^-2, OCP 0.78V。The method measuring Cys is highly sensitive, has wider detection range (20nM-3 μM) and relatively low detection limit (10nM)。And the aminoacid of non-sulfydryl is to detecting almost without interference。

2. the present invention utilizes Cys and Cu²⁺Between formed Cu-S key effect, reduce Cu²⁺Inhibitory action to battery open circuit current potential, utilizes the self-powered of biological fuel cell to realize the indirect quantification detection of Cys simultaneously。The sensitivity of detection can be significantly improved。

3. the method that the glucose/oxygen biological fuel cell constructed by the present invention establishes highly sensitive detection Cys。The method is highly sensitive, specificity good, simple to operate, cost is low, can be used for the monitoring of Cys in the fields such as food service industry, physiological process and clinical diagnose。

Accompanying drawing explanation

The detection mechanism figure of the glucose that Fig. 1 provides for the embodiment of the present invention/oxygen biological fuel cell and Cys。It is Cys and Cu²⁺After effect, can effectively reply battery open circuit current potential, it is achieved the indirect quantification detection of Cys。

The power density curve chart of the glucose that Fig. 2 provides for the embodiment of the present invention/oxygen biological fuel cell。

The canonical plotting of the Cys detection that Fig. 3 provides for the embodiment of the present invention。

Detailed description of the invention

The object of the invention, function and advantage will in conjunction with the embodiments, are described further with reference to accompanying drawing。

Embodiment 1

Constructing of glucose/oxygen biological fuel cell

1. the preparation of biological anode

By 4 μ L multi-walled carbon nano-tubes (2mg/mL), 5 μ L vitamin Ks₃With 10 μ LFAD-GDH solution (5mg/mL) drop coatings successively on glass-carbon electrode, dried dropping 5 μ LNafion solution (0.05wt%)。Drying at room temperature, prepares the biological anode based on FAD-GDH。

2. the preparation of biological-cathode

4 μ L multi-walled carbon nano-tubes (2mg/mL) and 10 μ L laccase solution (5mg/mL) drop coatings successively and dry on glass-carbon electrode。Then drip 2 μ L glutaraldehyde solution (2wt%), and with 4 DEG C at dry prepare the biological-cathode based on laccase。

Biology anode and biological-cathode are inserted 5mL Acetic acid-sodium acetate buffer (pH5.5) assembles biological fuel cell, 30mM glucose makes fuel, and add 2mMABTS as electron mediator, utilize CHI660E type electrochemical workstation, adopt linear sweep voltammetry (LSV), sweeping speed with 1mV/s to scan 0V from OCP and draw i-E curve, obtain the power density curve (referring to Fig. 2) of battery after conversion, namely battery maximum power density is 98 μ Wcm^-2, OCP 0.78V。

Embodiment 2

Utilize the glucose/oxygen biological fuel cell constructed, at Cu²⁺Existence under, it is achieved the high-sensitivity detection of Cys:

The battery of above-described embodiment adds the Cu of 4 μMs²⁺, the OCP of battery is then detected with circuit tester。It is subsequently added the Cys of variable concentrations and records corresponding OCP value。Last with Cys concentration for abscissa, the variable quantity of OCP is vertical coordinate, drawing standard curve, straight line can be obtained, obtain its slope, be the linear relationship (referring to Fig. 3) of the working curve slope of Cys, the variable quantity of OCP and Cys concentration。

The working curve of Cys as in figure 2 it is shown, as seen from the figure, the R of this working curve²Value reaches 0.997, and Linear Quasi is right very well。

Namely detection range is 20nM-3 μM, and relatively low detection is limited to 10nM。

Embodiment 3

The mensuration of Cys content in certain brand Cys composite sheet local:

1. sample treatment: take the Cys composite sheet of appropriate certain brand local, grind and be dissolved in ultra-pure water, be filtered processing with the disposable filter membrane of 0.22 μm, be diluted constant volume with 100mM Acetic acid-sodium acetate buffer (pH5.5)。

The detection process of 2.L-cysteine: with containing 30mM glucose, 2mMABTS 5mL Acetic acid-sodium acetate buffer (pH5.5) for end liquid assembling fuel cell, and in end liquid add 4mMCu²⁺Record the OCP OCP of system₁, in end liquid, then it is sequentially added the Cys sample of different volumes, and surveys its OCP OCP respectively₂。

The calculating of 3.L-semicystinol concentration: take the variable quantity (OCP of OCP₂–OCP₁) dilution ratio in standard curve range is calculated, substitute into the computing formula that standard curve draws, finally it is multiplied by corresponding extension rate, thus obtaining the content of Cys in the Cys composite sheet of certain brand is 43.1 ± 2.8 (w/w, %)。Cys cubage formula is:

Above in conjunction with accompanying drawing, the preferred embodiments of the disclosure and embodiment are described in detail; but the present invention is not limited to the above-described embodiment and examples; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention。

Claims

1. the method for a detection by quantitative Cys, it is characterised in that: the confession utilizing glucose/oxygen biological fuel cell is electrical, simultaneously at Cu²⁺There is the lower OCP suppressing battery, and at Cys and Cu²⁺Form Cu-S key, realize the detection by quantitative of Cys in testing sample with the change of Cys concentration according to OCP。

2. by the method for the detection by quantitative Cys described in claim 1, it is characterized in that: the glucose dehydrogenase (FAD-GDH) that described glucose/oxygen biological fuel cell relies on flavin adenine dinucleotide (FAD) acts on biological anode, and laccase acts on biological-cathode。

3. by the method for the detection by quantitative Cys described in claim 1 or 2, it is characterised in that: at Cu²⁺Existence under, utilize Cu²⁺Suppress the activity of biological anode FAD-GDH, reduce the OCP of glucose/oxygen bio-fuel, then Cys and Cu in recycling testing sample²⁺Forming Cu-S key, and reduce its inhibitory action, being added the change of front and back OCP by Cys, thus reaching the purpose of detection by quantitative Cys。

4. by the method for the detection by quantitative Cys described in claim 3, it is characterised in that: the biological anode of described glucose/oxygen biological fuel cell adopts vitamin K₃For electron mediator, biological-cathode then adopts 2, and 2 '-azino-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) is electron mediator。