CN109187470A - A kind of mediated with aptamers mixes silver-colored carbon dots catalysis H2O2The method of fluorescence spectrometry lead is reacted with TMB - Google Patents
A kind of mediated with aptamers mixes silver-colored carbon dots catalysis H2O2The method of fluorescence spectrometry lead is reacted with TMB Download PDFInfo
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- CN109187470A CN109187470A CN201811052716.0A CN201811052716A CN109187470A CN 109187470 A CN109187470 A CN 109187470A CN 201811052716 A CN201811052716 A CN 201811052716A CN 109187470 A CN109187470 A CN 109187470A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
Silver-colored carbon dots catalysis H is mixed with aptamers mediation the invention discloses a kind of2O2The method that fluorescence spectrometry lead is reacted with TMB includes the following steps: the lead standard solution system of (1) preparation known concentration, and it is F that the fluorescence peak intensity value at its 404nm is measured by excitation wavelength of 275nm；(2) placebo solution system is prepared, also measuring its fluorescence peak intensity value is F0；(3) Δ F=F-F is calculated0；(4) it is worked curve with concentration of the Δ F to lead；(5) sample solution is prepared, measuring its fluorescence peak intensity value is FSample, calculate Δ FSample=FSample‑F0；(6) according to working curve, the concentration of the lead in sample is calculated.This method mixes silver-colored carbon dots (CD using lead adaptation precursor reactant mediationAg) catalytic action, realize with mixing silver-colored carbon dots catalysis reaction fluorescence method quantitative determination lead, this method is easy, quickly, selective good, high sensitivity.
Description
Technical field
The present invention relates to analytical chemistry field, silver-colored carbon dots catalysis H is mixed in specifically a kind of aptamers mediation2O2It is reacted with TMB
The method of fluorescence spectrometry lead.
Background technique
Aptamers are a kind of single-stranded oligonucleotides that can be specifically combined with higher affinity with all kinds of target molecules, are had
Easily modify, be easily-synthesized, easy characteristics such as storage, can specifically various ligands such as conjugated protein, organic matter, metal ion,
It is applied in biochemical analysis.Such as: it is a kind of to prepare modification Pb (II) aptamer fluorescence probe on graphene quantum dot, lead to
The Photo-induced electron transfer between graphene quantum dot and graphene oxide is crossed, realizes the fluorescence shutdown of graphene quantum dot,
In the presence of Pb (II), is formed and modify Pb (II) aptamer-Pb (II) G- tetrad structure on graphene quantum dot, and from
Surface of graphene oxide is detached from, and fluorescence is opened, and establishes Pb (II) in Fluorometric assay water, detection range 9.9-435nmol/
L, detection limit reach 0.6nmol/L, this method light requirement induces and need graphene oxide, and process is complicated；One kind being based on the DNA of Pb (II)
Splitting action unlocks double-stranded DNA, forms mononucleotide aptamers, and be wrapped in gold nano surface, enables gold nano stable dispersion
In high concentration sodium chloride medium, system resonance scattering value is reduced, and can detect the lead ion of 16.7-666.7nmol/L；A kind of use
Aptamers-ferroheme system absorption spectrometry detects isocarbophos, blood red to be known as a strong absworption peak in polar organic solvent,
It in conjunction with random dna and can be piled into nanoparticle by π-π, under isocarbophos effect, assemble, and cause system
Absorption spectrum strength reduction, detection range are 0.5-40 μ g/L, and detection rising limit reaches 0.2 μ g/L.
Fluorescent spectrometry have good selectivity with higher sensitivity, be a kind of common detection method.In recent years,
Application of the nano materials such as quantum dot, nano-cluster with fluorescence in fluorescence analysis obtains greater advance.Existing literature report
A kind of water soluble fluorescence 3, the gold nanoclusters (l-DOPA-AuNC) of 4- dihydroxy -1- phenylalanine (l-DOPA) protection,
The unique coordination induced fluorescence quenching of Fe (III) ion and catechol group on the surface l-DOPA-AuNCs, ascorbic acid
(AA) fluorescence of 1-DOPA-AuNCs can be restored, therefore can be used to detect Fe (III) ion and AA, detection range are respectively
0.48-3.33nmol/L and 32-156 μm of ol/L；A kind of combination with two aptamer probes and target protein start with each
Hybridization between the acomplementary connector sequence of aptamer connection causes to make the jag rich in G close to silver nanoclusters (AgNCs)
Significant fluorescence enhancement.In this way, it determines the detection of mankind α-fibrin ferment and is limited to 1nmol/L, linear dynamic range
For 5nmol/L-2 μm of ol/L.Only have not yet to see the report that silver-colored carbon dots fluorescence analysis is mixed in the regulation of non-marked aptamers.
Carbon dots as a kind of ideal fluorescent material, have chemical stability is good, synthesis material is cheap and easy to get, toxicity is low,
It is easy to the outstanding advantages such as conversion on functionalization, stable luminescence and fluorescence and is concerned, in chemical analysis and bio-sensing
With important potential using value, it is considered to be one of the ideal fluorescent marker of one kind or detection material.It has been reported that: one
Kind fluorescence richness nitrogen quantum dot (NRQDs) and Cu2+In conjunction with the method for leading to fluorescent quenching, principle is that determinand cysteine is added
(Cys) Cu is formed afterwards2+- Cys complex, NRQDs fluorescence restore, and can measure 0.15-10 μm of ol/LCys, detection limit is reachable
0.03μmol/L；A kind of New-type bifunctional N-S doping carbon dots (Cdots) fluorescence detection Fe3+, detection range 12.5-
1000nmol/L, detection limit is up to 1.72nmol/L；One kind passing through the phase between accumulation DNA base and carbon nano-particles (CNP)
Interaction and single stranded DNA (ssDNA) probe for adsorbing fluorescent marker, lead to basic fluorescent quenching, there are Hg2+When, T-Hg2+-
The hairpin structure of T induction is not adsorbed on CNP, therefore remains dye fluorescence, and detection range is 0.5-10 μm of ol/L, detection
Limit up to 10nmol/L, however carbon dots there are fluorescence quantum yields it is low, active site is few the deficiencies of.In recent years, quantum dot, graphite
The method of modifying of the other materials such as alkene is overcome certain defects of carbon dots by the methods of exotic atom doping, improves carbon dots quantum
Yield and increase active site.Ag doping carbon dots are because the optical effect of Ag shows excellent photoelectric properties, largely
These defects of carbon dots are compensated for, thus are applied in the numerous areas such as biochemical sensitive, environment measuring, such as: it is a kind of
Core duplex shell structure Au@Ag@C composite, the nanometer composite layer are amorphous carbon, and second layer Ag, core Au pass through
Electrochemical Detection, Au@Ag@C composite material is to H2O2With good electro-catalysis reproducibility, therefore can be used to detect 5.0 μm of ol/L-
4.75mmol/L H2O2, detection limit is up to 0.14 μm of ol/L；One kind restoring AgNO in the presence of carbon dots (CD)3The carbon dots of preparation are repaired
The silver nano-grain (CDs-AgNPs) of decorations is incorporated in the functional group's energy and Cu of the CD in CDs-AgNPs2+Interaction, so that receiving
Rice grain assembles and shows that the color of CDs-AgNPs dispersion becomes bronzing from orange, develops a kind of measurement Cu2+'s
Colorimetric method, detection range 0.3-8mmol/L, detection limit is up to 0.037mmol/L, using carbon dots (CD) as reducing agent and surely
Determine agent synthesis Ag/CD nanocomposite and make nano-probe, this sensor passes through engraving method colorimetric determination H2O2, detection
Range is 0.1-80 μm of ol/L, and detection limit is up to 0.03 μm of ol/L.But related non-marked aptamers mediation is mixed silver-colored carbon dots fluorescence and is visited
Needle detection lead ion has not been reported.
Lead ion is a kind of typical heavy metal contaminants, and there are many pollution sources, is present in water body, atmosphere or biota
In, it has non-biodegradable, can long-term existence in the environment, hemopoietic system, nervous system and the kidney of people can be endangered, thus
It gives rise to diseases.Lead can threaten to health, be that can lead to poisoning when the concentration of lead in blood is more than 480nmol/L, because
This, establish it is a kind of it is easy, quickly, reliably detect Pb2+Method, have critically important meaning to environmental protection and human health
Justice.Detection Pb common at present2+Method have atomic absorption spectrography (AAS) (AAS), atomic fluorescence spectrometry (FL) and inductive coupling
Plasma Mass Spectrometry (ICP-MS), spectrophotometry etc., but each has its limitation.Therefore, it is necessary to develop one kind
Quickly, convenient and highly sensitive Pb2+Analysis method.Existing literature reports: a kind of functionalized by using glutathione
Gold nano grain (GSH-GNPs) colorimetric method detects 100nmol/L Pb2+；Separately also have been reported that based on graphene quantum dot (GQD)
" unlatching " fluorescent optical sensor with gold nanoparticle (AuNP) is for detecting Pb2+, since catalysis chain and lead ion respectively induce
The combination and depolymerization of GQD and AuNP causes fluorescent quenching and fluorescence to be restored, and achievees the purpose that detect lead ion, detection range is
50-4000nmol/L, detection limit is up to 16.7 nmol/L.
In these methods, it is most of be caused based on Nanoparticle Modified fluorescence probe and analyte response fluorescent quenching or
Fluorescence enhancement, and the expensive equipment that uses of most method and complicated for operation, in order to overcome these disadvantages, seek a kind of simple fast
Victory measurement Pb2+Method it is particularly significant.Silver-colored carbon dots catalysis H is mixed based on adaptation precursor reactant mediation2O2It is reacted with TMB and generates fluorescence production
Object, using the product as fluorescence probe, with fluorescence spectrometry Pb2+Method there is not been reported.
Summary of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, and provides and a kind of mix silver-colored carbon dots with aptamers mediation and be catalyzed
H2O2The method of fluorescence spectrometry lead is reacted with TMB.This method mixes silver-colored carbon dots (CD using adaptation precursor reactant mediationAg) urge
Change effect, i.e. CDAgH can be catalyzed2O2It aoxidizes TMB reaction and generates fluorescence-causing substance, the fluorescence-causing substance and measured object Pb2+Concentration is linear
Relationship, realization quantitative determine Pb with fluorescence method2+, easy, quick, the selective good, high sensitivity of this method.
Realizing the technical solution of the object of the invention is:
A kind of mediated with aptamers mixes silver-colored carbon dots catalysis H2O2The method that fluorescence spectrometry lead is reacted with TMB, with existing skill
Unlike art, include the following steps:
(1) it prepares the lead standard solution system of known concentration: in different scale test tubes, sequentially adding 50 μ L-150 μ
L 25mg/L CDAgSolution, 50 μ L-150 μ L 5mmol/L pH, 3.24 Tris-HCl buffer solution, 150 μ L-250 μ L
0.3085 μm of ol/L lead is adapted to liquid solution (sequence 5 '-GGTTGGTGTGGTTGG-3 '), 10 1 μm of ol/L lead mark of μ L-200 μ L
Quasi- solution mixes, and stands reaction 10min, then be separately added into 50 μ L-150 μ L 0.5mmol/L TMB solution, 50 μ L-150 μ L
100μmol/L H2O2Solution is settled to 1.5mL with secondary distilled water, after shaking up, reacts 30min in 40 DEG C of thermostat water baths,
Ice water is cooled to room temperature；
(2) prepare placebo solution system: using step (1) method not leading standard solution preparation blank control it is molten
Liquid system；
(3) it takes respectively and is poured into quartz by step (1), the lead standard solution system of (2) preparation and placebo solution system
In cuvette, in Fluorescence Spectrometer, the fluorescence spectrum that instrument parameter scans acquisition system using 275nm as excitation wavelength is set,
Measuring the fluorescence intensity level at 404nm is F, while the fluorescence intensity level for measuring placebo solution system is F0, calculate Δ F=
F-F0；
(4) it works curve according to Δ F to the concentration relationship of lead；
(5) sample solution is prepared according to the method for step (1), wherein the lead standard solution being added replaces with sample solution,
And the fluorescence peak intensity value of sample solution is measured as F by the method for step (3)Sample, calculate Δ FSample=FSample-F0；
(6) working curve according to step (4), calculates the content of sample solution lead.
Silver-colored carbon dots are mixed in the technical program (is abbreviated as CDAg) the preparation method comprises the following steps: by the glucose and 2mL0.01moL/ of 1g
L AgNO3Solution is placed in 20mL deionized water, and concussion makes to dissolve, and is then transferred in the autoclave that polytetrafluoroethylene (PTFE) is substrate,
1h is reacted in 160 DEG C of Muffle furnaces of temperature after sealing.After reaction, ice water is cooled to room temperature to get flaxen CD is arrivedAgIt is molten
Liquid takes 1mL CDAgSolution is added 150 μ L 50mmol/L KOH and is settled to 10mL, and adjusting pH is spare to neutrality, total carbon is added
Amount calculates, and concentration 1.8mg/mL is diluted to 25mg/L before use.The preparation method for mixing silver-colored carbon dots is the prior art.
Realizing the principle of this technical program is: mixing silver-colored carbon dots CD with prepared by the technical program methodAgAs H2O2Oxygen
Change the catalyst of TMB, TMB oxidation product (being abbreviated as TMBox) generates a fluorescence peak, the peak and CD in 404nmAgIn 442nm
Fluorescence peak wavelength it is different, after lead aptamers are added, aptamers have wrapped up CDAg, it is suppressed that CDAgTo H2O2The catalysis of-TMB
Ability generates TMBox and reduces, fluorescence peak remitted its fury, with the increase of plumbum ion concentration, aptamers-lead compound of generation
And CDAgIncrease, the fluorescence peak intensity at 404nm linearly enhances, and establishes a kind of mediated with aptamers accordingly and mixes silver-colored carbon dots catalysis
H2O2It is reacted with TMB, the fluorescent method of lead is measured using reaction product as fluorescence probe.
The advantages of this method is: compared with the existing methods, this method mixes silver-colored carbon dots using adaptation precursor reactant mediation
(CDAg) catalytic action, i.e. CDAgH can be catalyzed2O2It aoxidizes TMB reaction and generates fluorescence-causing substance (TMBox), the TMBox and measured object
Pb2+Concentration is in a linear relationship, and realization quantitative determines Pb with fluorescence method2+。
Easy, quick, the selective good, high sensitivity of this method.
Detailed description of the invention
Fig. 1 is the fluorescence spectrum schematic diagram in embodiment.
In figure, a.0.33mmol/L+1.67 μ g/L CD of 3.24 Tris-HCl+8.23nmol/L lead aptamers of pHAg+33
μmoL/L TMB+6.67μmoL/L H2O b.a+6.7nmol/L Pb2+ c.a+33nmol/L Pb2+ d. a+60nmol/L
Pb2+ e.a+87nmol/L Pb2+ f.a+113nmol/L Pb2+ g.a+133nmol/L Pb2+。
Specific embodiment
The content of present invention is further elaborated with reference to the accompanying drawings and examples, but is not limitation of the invention.
Embodiment:
A method of silver-colored carbon dots fluorescence spectrometry lead being mixed with aptamers mediation, is included the following steps:
(1) it prepares the lead standard solution system of known concentration: in 6 different scale test tubes, sequentially adding 100 μ L
25mg/L CDAgSolution, 100 μ L 5mmol/L pH3.24 Tris-HCl buffer solutions, 40 μ L, 0.3085 μm of ol/L lead adaptation
Liquid solution (sequence 5 '-GGTTGGTGTGGTTGG-3 '), then it is separately added into 10 μ L, 50 μ L, 90 μ L, 130 μ L, 170 μ L, 200
1 μm of ol/L lead standard solution of μ L mixes, and stands reaction 10min, each that 100 μ L 0.5mmol/L TMB solution and 100 μ L are added
100μmol/L H2O2Solution is settled to 1.5mL with secondary distilled water, after shaking up, reacts 30min in 40 DEG C of thermostat water baths,
Ice water is cooled to room temperature；
(2) prepare placebo solution system: using step (1) method not leading standard solution preparation blank control it is molten
Liquid system；
(3) it takes respectively and is poured into quartz by step (1), the lead standard solution system of (2) preparation and placebo solution system
In cuvette, in Fluorescence Spectrometer, set instrument parameter fluorescence voltage as 350V, slit 10nm, excitation wavelength be 275nm item
The fluorescence spectrum that acquisition system is scanned under part, the fluorescence intensity level measured at 404nm is F, while measuring placebo solution body
The fluorescence intensity level of system is F0, calculate Δ F=F-F0, as shown in Figure 1；
(4) it works curve according to Δ F to the concentration relationship of lead, acquisitions equation of linear regression is Δ F=3.50C-2.75,
Wherein the unit of lead C is nmol/L, and the measurement range of linearity is 6.7-133nmol/L, and detection is limited to 2nmol/L；
(5) sample measures: it takes waste water filtering to remove suspended particulate substance, prepares sample according to the method for step (1),
The lead standard solution being wherein added replaces with sample, operates by step (2)-(4), calculates the Δ F of sampleSample=
FSample-F0；
(6) working curve according to step (4), the content for calculating sample lead is 82nmol/L；
The verifying of the technical program detection method:
The waste water in above-described embodiment step (5) is taken, the lead standard solution that concentration is 50nmol/L is added, mark-on is carried out and returns
Experiment is received, acquiring the rate of recovery is respectively 99.5%, relative standard deviation 3.9%, illustrates that the technical program method is accurate and reliable.
Sequence table
<110>Guangxi Normal University
<120>silver-colored carbon dots catalysis is mixed in a kind of aptamers mediation
H<sub>2</sub>o<sub>2</sub>the method of fluorescence spectrometry lead is reacted with TMB
<141> 2018-08-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> DNA
<400> 1
ggttggtgtg gttgg 15
Claims (1)
1. silver-colored carbon dots catalysis H is mixed in a kind of aptamers mediation2O2The method of fluorescence spectrometry lead is reacted with TMB, characterized in that
Include the following steps:
(1) it prepares the lead standard solution system of known concentration: in different scale test tubes, sequentially adding 50 μ L-150 μ
L25mg/L CDAgSolution, 50 μ L-150 μ L 5mmol/L pH3.24Tris-HCl buffer solutions, 150 μ L-250 μ L0.3085 μ
Mol/L lead is adapted to liquid solution (sequence 5 '-GGTTGGTGTGGTTGG-3 '), 10 1 μm of ol/L lead standard solution of μ L-200 μ L,
It mixes, stands reaction 10min, then be separately added into 50 μ L-150 μ L 0.5mmol/L TMB solution, 50 100 μm of ol/ of μ L-150 μ L
L H2O2Solution is settled to 1.5mL with secondary distilled water, after shaking up, 30min is reacted in 40 DEG C of thermostat water baths, ice water is cooling
To room temperature；
(2) prepare placebo solution system: using the method for step (1), leading standard solution does not prepare placebo solution body
System；
(3) it takes respectively and is poured into quartz cuvette by step (1), the lead standard solution system of (2) preparation and placebo solution system
In ware, in Fluorescence Spectrometer, the fluorescence spectrum that instrument parameter scans acquisition system using 275nm as excitation wavelength is set, measurement
Fluorescence intensity level at 404nm is F, while the fluorescence intensity level for measuring placebo solution system is F0, calculate Δ F=F-F0；
(4) it works curve according to Δ F to the concentration relationship of lead；
(5) sample solution is prepared according to the method for step (1), wherein the lead standard solution being added replaces with sample solution, and pressed
The fluorescence peak intensity value of the method measurement sample solution of step (3) is FSample, calculate Δ FSample=FSample-F0；
(6) working curve according to step (4), calculates the content of sample solution lead.
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| CN109187468A (en) * | 2018-09-10 | 2019-01-11 | 广西师范大学 | It is a kind of to mediate nitrating carbon dots to be catalyzed H with aptamers2O2The method of fluorescence spectrum method for measuring isocarbophos is reacted with TMB |
| CN109884029A (en) * | 2019-03-01 | 2019-06-14 | 吉林大学 | Silver/graphite alkene quantum dot nano enzyme, SERS detection kit and application |
| CN110471098A (en) * | 2019-08-27 | 2019-11-19 | 广东医科大学 | Radon dose of radiation detection method based on carbon quantum dot biological sensor |
| CN113267529A (en) * | 2021-05-12 | 2021-08-17 | 江西师范大学 | Temperature type biosensor and method for detecting target aptamer by using temperature type biosensor |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187468A (en) * | 2018-09-10 | 2019-01-11 | 广西师范大学 | It is a kind of to mediate nitrating carbon dots to be catalyzed H with aptamers2O2The method of fluorescence spectrum method for measuring isocarbophos is reacted with TMB |
| CN109187468B (en) * | 2018-09-10 | 2020-11-20 | 广西师范大学 | Aptamer-mediated nitrogen-doped carbon dot catalysis H2O2Method for measuring isocarbophos by reaction with TMB (tetramethylbenzidine) through fluorescence spectrometry |
| CN109884029A (en) * | 2019-03-01 | 2019-06-14 | 吉林大学 | Silver/graphite alkene quantum dot nano enzyme, SERS detection kit and application |
| CN110471098A (en) * | 2019-08-27 | 2019-11-19 | 广东医科大学 | Radon dose of radiation detection method based on carbon quantum dot biological sensor |
| CN113267529A (en) * | 2021-05-12 | 2021-08-17 | 江西师范大学 | Temperature type biosensor and method for detecting target aptamer by using temperature type biosensor |
| CN113267529B (en) * | 2021-05-12 | 2022-10-25 | 江西师范大学 | Temperature type biosensor and method for detecting target aptamer by using temperature type biosensor |
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