CN100425973C - Nano biological sensor for detecting NADH concentration and its detecting method - Google Patents
Nano biological sensor for detecting NADH concentration and its detecting method Download PDFInfo
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- CN100425973C CN100425973C CNB2006100314269A CN200610031426A CN100425973C CN 100425973 C CN100425973 C CN 100425973C CN B2006100314269 A CNB2006100314269 A CN B2006100314269A CN 200610031426 A CN200610031426 A CN 200610031426A CN 100425973 C CN100425973 C CN 100425973C
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Abstract
The present invention relates to a nanometer biosensor and a method thereof for testing NADH concentration. The present invention takes Au nanometer particles fixedly arranged on the surface of a sensor as the crystal seeds, applies the principle of catalysis increasing of Au/Ag nuclear shell mold nanometer particles and the changing rule of absorption spectrum under the reducing action of NADH, and calculates the variance of nanometer particles absorption peak in the spectrum to test the NADH concentration. The linear range for testing the NADH concentration of the present invention is 2*10<-4 > to 3.2*10<-3 > M, and the lower limit of testing is 1.56*10<-5 > M. Compared with the prior art, the present invention has the advantages of simple operation, low cost and strong anti-interference ability. The present invention better solves on-line rapid testing in a complex biological system and in the progress of degrading environmental pollutant, and provides more convenient and rapid technique for analyzing oxidoreductase activity using the NADH as coenzyme.
Description
Technical field
The present invention relates to the detection of coenzyme, be specifically related to the detection method of a kind of nano biological sensor and NADH concentration thereof.
Background technology
Nicotinamide adenine dinucleotide reduced (NADH) extensively is present in the living cells of animals and plants and microorganism, is a kind of important coenzyme in the bio-metabolic process.In hundreds and thousands of metabolic responses, NADH is an electron donor, NAD in active somatic cell
+Be electron accepter, have redox active, they play the vital role of electron transport in processes such as glycolysis, citrate cycle and photosynthesis.In vivo, NADH has participated in the process of vitamin-derived, the production process of extremely important potential material atriphos (ATP) in the biosome, and be the coenzyme of more than 250 kind of dehydrogenasa having been identified, cell growth, differentiation and energetic supersession kept.In the biological restoration process of the white-rot fungi that receives much concern in recent years to contaminated environment, quinones substance is the key intermediate species that the outer superoxide enzymatic degradation lignin of fungi born of the same parents closes generations such as aromatic series organic contaminant, NADH has played vital role as the coenzyme of quinone reductase in the fungal cell in the degree of depth metabolism of these quinones substances.Therefore, the NADH level has been represented the overall target of biosome intracellular metabolite and microbial degradation ability, the research of NADH detection method be one with great life process and the closely bound up hot research field of environmental contaminants degradation process.
NADH has absorption at the 340nm place of ultraviolet region, and produces fluorescence at the 470nm place.This optical property is often used as the NADH horizontal detection and is in the determination of activity of the dehydrogenasa of coenzyme and reductase with NAD (H).But a lot of biomolecule can cause optical damage at ultraviolet region hyperabsorption energy, and its optical damage degree is greater than visible range or infrared region.And NADH content is very low in the cell, so its absorption spectrum and fluorescence spectrum signal are very weak.Therefore, unsatisfactory based on the detection method of direct its optical effect of measurement.High performance liquid chromatography (HPLC) needs the operating personnel of expensive cost of equipment and specialty, and can not carry out online detection though the method for detection NADH is effective.Therefore develop a kind of detect effective, easy and simple to handle, cost is low and the detection technique that can carry out the NADH concentration of online detection is an important research project.
Summary of the invention
The objective of the invention is to be used in the principle and the absorption spectrum Changing Pattern of metallics catalytic growing under the NADH reducing action, a kind of nano biological sensor is provided, to solve the problems referred to above that exist in the detection of NADH concentration.
The present invention is achieved through the following technical solutions the foregoing invention purpose.
The nano biological sensor that is used to detect NADH concentration increases layer by the basalis of cultivating crystal seed as reaction and reaction that nano particle increases and constitutes, and basalis is the Au nano particle that is fixed on the slide, and reaction increases layer and is Ag in containing the reactant liquor of NADH
+Au/Ag nuclear shell type nano meter particle in the formation of Au nanoparticle surface catalytic reduction.
The slide of basalis is in pre-service after the common slide of aminopropyl trimethoxysilane amino silaneization, and the nano particle that is fixed on the slide is that diameter is that 10nm, concentration are 1 * 10
-4M, dripping quantity is the Au nano particle of 75 μ L.
Nano biological sensor detects the method for NADH concentration for to place the 3mL reaction solution of the NADH that contains variable concentrations to cultivate 8～12min sensor, sensor is taken out from reactant liquor, insert and carry out spectral scan in the detection cell, with the concentration of NADH near the absorption peak assaying reaction liquid of nano particle in spectrum 415nm.
Other concentration of component of reaction solution is:
Phosphate buffered solution (pH 6.64) 1/15M
Hexadecyltrimethylammonium chloride (CTAC) 0.003M
AgNO
3 0.0015M
1,4-benzoquinone 0.0015M
Absorption peak (I) and NADH concentration (C, 10
-4M) equation of linear regression between is:
I＝(0.1064±0.0116)+(0.0119±0.0006)×C(1)
Correlation coefficient r
2Be 0.9806
Wherein, the range of linearity of NADH concentration is 2 * 10
-4～3.2 * 10
-3M is limited to 1.56 * 10 under detecting
-5M.
Be described in further detail the present invention below in conjunction with accompanying drawing:
Description of drawings
Fig. 1 biology sensor of the present invention is made Au/Ag nano particle catalysis propagation process figure under flow process and the NADH effect;
Sensor light spectrogram under Fig. 2 variable concentrations NADH effect, wherein, NADH concentration is (a) 0, (b) 12.32 * 10
-4M, (c) 17.98 * 10
-4M, (d) 24.59 * 10
-4M and (e) 32.16 * 10
-4M;
Scanning electron microscope (SEM) figure before and after Fig. 3 Au/Ag nano particle catalysis increases, wherein, (a) before the growth, (b) after the growth;
Linear regression graph between Fig. 4 spectral absorption peak value and the NADH concentration.
Nano particle has special optics, electricity, chemical property, shows huge answering at the biological components analysis field Use value, caused people's dense research interest. Metal nanoparticle have good dispersiveness, comparatively the rule shape Looks and stable absorption spectrum, and its absorption spectrum is often used as with the big or small Development pattern variation of particle diameter and concentration Optical markings in the biology sensor produces detectable in the reactions such as DNA hybridization to be measured, antibody/antibody recognition Optical signalling. Nearest studies show that, under the catalysis of the redox active materials such as biology enzyme, and the Au nano particle (AuNP) can increase, its particle diameter and quantity change, and carry out quantitative analysis by uv-visible absorption spectra.
With the surface of common glass sheet (long 30mm, wide 9mm, thick 1mm) through Pirahna solution and H2O
2，NH
3And H2After the mixed solution preliminary treatment of O, with aminopropyl trimethoxysilane (APTMS) amino silane, be the Au nano particle (1 * 10 of 10nm with diameter-4M) 75 μ L drip and are applied to surface of glass slide and fix, and make the Au Nanoparticle Modified Biology sensor.
Place the 3 mL reaction solutions of the NADH that contains variable concentrations to cultivate 8～12min sensor, reaction solution is 1/15M PBS (pH6.64), other component is 0.003M hexadecyltrimethylammonium chloride (CTAC), 0.0015 MAgNO3With the 0.0015M 1,4-benzoquinone.
Flow process made by biology sensor and the lower Au/Ag nano particle catalysis propagation process schematic diagram of NADH effect is seen Fig. 1. NADH is reduced into hydroquinones with 1,4-benzoquinone, Ag+Under the effect of hydroquinones, generate Ag in the reduction of Au nanoparticle surface, thereby form the Au/Ag nuclear shell type nano meter particle, reactional equation is:
The present invention adopts Japanese Shimadzu UV-2550 ultraviolet-visible spectrophotometer to carry out spectrum analysis, quartz colorimetric utensil light Journey is 1cm. All working is all finished under room temperature (25 ℃). Experimental water is ultra-pure water, and (U.S. Millipore is ultrapure Water system). In the reference of ultraviolet-visible spectrophotometer and pond to be measured, all inject the 2.5mL ultra-pure water, surface deposition is had The sensor of Ag places detection cell to carry out spectral scan. The Au nano particle has absworption peak near the 560nm wavelength, and The Au/Ag nuclear shell type nano meter particle forms new absworption peak near the 415nm wavelength, and peak value is along with NADH in the reactant liquor The increase of concentration and increasing. When reactant liquor be PBS (pH 6.64,1/15M) 3mL, CTAC (0.003 M), AgNO3(0.0015M) and during 1,4-benzoquinone (0.0015M), sensor is best to the response of NADH, difference The response spectrum of concentration NADH is seen Fig. 2. As seen from the figure, near the peak value of the spectrum 415nm wavelength along with reactant liquor in The increase of NADH concentration and increasing is the absworption peak of Au/Ag nano particle.
Simultaneously, the surface of glass slide that is fixed with the Au/Ag nano particle is spattered with NEC's automatic ion of JEOL JSC-1600 Penetrate instrument and spray platinum and process to strengthen electric conductivity, spray platinum thickness is about 10nm. Adopt 200 emissions of Dutch FEI Sirion SEM is carried out scanning electron microscope analysis to the sensor surface before and after cultivating with reactant liquor. Fig. 3 (a) is for cultivating Front surface of glass slide Au nano particle is with containing PBS (pH6.64,1/15M) 3mL, CTAC (b) (0.003M), AgNO3(0.0015M), 1,4-benzoquinone (0.0015M) and NADH (6.49 * 10-4M) reaction Surface of glass slide Au/Ag nano particle after liquid is cultivated. As seen from the figure, cultivating front Au nano particle diameter is a 10nm left side The right side, the growth of Au/Ag nano particle average grain diameter is 42.93nm after cultivating, it is obvious to increase phenomenon.
Find also that in experiment the sensitivity of sensor is also relevant with the fixed amount of Au nano particle on the slide, on slide When the fixed amount of Au nano particle was more big, near the absorption peak of the sensor 560nm wavelength was also more high, to identical dense The peak value of response variable quantity of the NADH of degree is also more big. But, when near the absorption of the Au nano particle 560nm wavelength When peak value is excessive, cover easily the absworption peak that the Au/Ag nuclear shell type nano meter particle forms near the 415nm wavelength, therefore, When slide corresponding to Au nano particle fixed amount when 560nm left and right sides absorption peak is 0.14Abs, the sensor response is Good, at this moment, the dripping quantity of Au nano particle is 75 μ L on the slide in the sensor preparation.
In addition, the research of cultivating the time is found that sensor peak value of response after cultivating 8min namely reaches maximum, for Obtain stable response results, we adopt 8～12min is that reactant liquor is cultivated the time.
Under above-mentioned optimum determining condition, with the absorption peak (I) of the Au/Ag nano particle of the response spectrum of sensor and corresponding NADH concentration value (C, 10-4M) do linear regression, see Fig. 4. When NADH concentration is 2 * 10-4～3.2×10
-3During M, regression equation is:
I＝(0.1064±0.0116)+(0.0119±0.0006)×C
Wherein, correlation coefficient r2Be 0.9806, be limited to 1.56 * 10 under detecting-5M。
The prepared NADH optics nano biological sensor of the present invention is easy and simple to handle, and cost is low, and antijamming capability is strong, energy Solve better the online Fast Measurement problem of NADH in complex biological system and the environmental contaminants degradation process, for analyzing NADH provides the technology of more convenient and quicker for the oxidoreductase activity of coenzyme.
Embodiment
1. the pre-service of slide
With common microslide processing growth 30mm, wide 9mm, thick 1mm is at Pirahna solution (H
2SO
4, 98% and H
2O
2, 30%, volume ratio is 4: 1) in soak 20min down in 60 ℃.After the washing, at H
2O
2, NH
3And H
2Soak 20min down in 70 ℃ in the mixed solution of O (volume ratio is 1: 1: 2).Remove physisorption with big water gaging, methyl alcohol (chromatographically pure) flushing, under 4 ℃, be stored in the methyl alcohol standby.
2. nano-sensor preparation
Join APTMS and methyl alcohol mixed solution (volume ratio is 1: 1.5), slide is placed in one soaks 18h, 35 ℃, go weak absorption with washed with methanol, place under the normal temperature dry then.With diameter is that 10nm, concentration are 1 * 10
-4M, dripping quantity are that the AuNP of 75 μ L drips that to be applied to slide simultaneously fixing, standing and drying 2h under the normal temperature, and the water flushing is stored in the water under 4 ℃.
3.NADH concentration determination
All inject the 2.5mL ultrapure water in reference cell and pond to be measured, the sensor that the surface is not deposited Ag places detection cell to carry out spectral scan.Get 3mL phosphate buffered solution (1/15M), add 0.003M CTAC, 0.0015MAgNO successively
3, 0.0015M 1,4-benzoquinone and NADH, after mixing rapidly sensor is immersed wherein, lucifuge is cultivated 8～12min under the normal temperature.Sensor is taken out from reactant liquor, insert and carry out spectral scan in the detection cell.
Concentration according to NADH in the nano particle absorption peak assaying reaction liquid in the spectrum under the top condition.
4. measurement result
Method for biosensor of the present invention is compared with the determined by ultraviolet spectrophotometry result to the result that NADH measures, and the aqueous solution of selecting variable concentrations NADH is as sample, and the ultraviolet absorption peak wavelength is 340nm.The results are shown in following table.
As seen from table, the biology sensor of the present invention's development is accurately and effectively to the mensuration of NADH.
Claims (3)
1, a kind of nano biological sensor that is used to detect NADH concentration, it is characterized in that sensor is made of the reaction growth layer of the basalis of cultivating crystal seed as reaction and nano particle growth, basalis is the Au nano particle that is fixed on the slide, the slide of basalis is in pre-service after the common slide of aminopropyl trimethoxysilane amino silaneization, the dripping quantity of Au nano particle is 75 μ L, and reaction increases layer and is Ag in containing the reactant liquor of NADH
+Au/Ag nuclear shell type nano meter particle in the formation of Au nanoparticle surface catalytic reduction.
2, the nano biological sensor that is used to detect NADH concentration according to claim 1, the nano particle that it is characterized in that being fixed on the slide is that diameter is that 10nm, concentration are 1 * 10
-4The Au nano particle of M.
3, a kind of nano biological sensor as claimed in claim 1 detects the method for NADH concentration, it is characterized in that placing the 3mL reaction solution of the NADH that contains variable concentrations to cultivate 8～12min sensor, sensor is taken out from reactant liquor, insert and carry out spectral scan in the detection cell, concentration with NADH near the absorption peak assaying reaction liquid of nano particle in spectrum 415nm
Other concentration of component of reaction solution is:
Phosphate buffered solution pH 6.64 1/15M
Hexadecyltrimethylammonium chloride (CTAC) 0.003M
AgNO
3 0.0015M
1,4-benzoquinone 0.0015M
Absorption peak (I) and NADH concentration (C, 10
-4M) equation of linear regression between is:
I＝(0.1064±0.0116)+(0.0119±0.0006)×C
Wherein, the range of linearity of NADH concentration is 2 * 10
-4～3.2 * 10
-3M is limited to 1.56 * 10 under detecting
-5M.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100314269A CN100425973C (en) | 2006-03-30 | 2006-03-30 | Nano biological sensor for detecting NADH concentration and its detecting method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100314269A CN100425973C (en) | 2006-03-30 | 2006-03-30 | Nano biological sensor for detecting NADH concentration and its detecting method |
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| Publication Number | Publication Date |
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| CN100425973C true CN100425973C (en) | 2008-10-15 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344494B (en) | 2011-09-26 | 2015-03-11 | 华东理工大学 | Nicotinamide adenine dinucleotide gene coding fluorescent probe as well as preparation method and application thereof |
| CN104730129B (en) * | 2015-04-10 | 2017-12-08 | 江南大学 | A kind of electrochemical method based on ethyl carbamate content in double enzyme modified electrode quick detection solution |
| US10000790B2 (en) | 2015-06-28 | 2018-06-19 | The Florida International University Board Of Trustees | Materials and methods for rapid visualization of NAD(P)H |
| KR102029957B1 (en) | 2018-01-02 | 2019-10-08 | 한국화학연구원 | Electrode for biosensor for nadh measurment and manufacturing method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456679A (en) * | 2003-05-29 | 2003-11-19 | 江西特康科技有限公司 | Reagent for determining adenosine deaminase and preparing method thereof |
| CN1485617A (en) * | 2002-09-26 | 2004-03-31 | 中国科学院武汉病毒研究所 | An optical circulation amplification type biological chip detecting process |
| JP2004233289A (en) * | 2003-01-31 | 2004-08-19 | Asahi Kasei Corp | Nad sensor |
| CN1544923A (en) * | 2003-11-27 | 2004-11-10 | 中国科学院长春应用化学研究所 | Preparing method of integrated capillary electrophoresis electrochemical luminescence detecting chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1485617A (en) * | 2002-09-26 | 2004-03-31 | 中国科学院武汉病毒研究所 | An optical circulation amplification type biological chip detecting process |
| JP2004233289A (en) * | 2003-01-31 | 2004-08-19 | Asahi Kasei Corp | Nad sensor |
| CN1456679A (en) * | 2003-05-29 | 2003-11-19 | 江西特康科技有限公司 | Reagent for determining adenosine deaminase and preparing method thereof |
| CN1544923A (en) * | 2003-11-27 | 2004-11-10 | 中国科学院长春应用化学研究所 | Preparing method of integrated capillary electrophoresis electrochemical luminescence detecting chip |
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Granted publication date: 20081015 Termination date: 20110330 |