CN105717103B - Detection method of content of the colorimetric bio sensor based on Jenner's grain of rice structure for atriphos in breast cancer cell - Google Patents
Detection method of content of the colorimetric bio sensor based on Jenner's grain of rice structure for atriphos in breast cancer cell Download PDFInfo
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Abstract
A kind of detection method of content the invention discloses colorimetric bio sensor based on Jenner's grain of rice structure for atriphos in breast cancer cell, this approach includes the following steps：It draws in AuNPs to centrifuge tube, is added and dilutes 40-60 times of ATP breast cancer cell extracting solution with PKS buffer solutions, hatching 3-7min obtains mixture, it is added in 4- mercaptophenyl boronic acids to mixture, adds PKS buffer solutions reaction 10-15min, observe color change, using ultraviolet specrophotometer, with A520/A683It absorbs ratio and carries out linear fit with ATP concentration, external standard method draws standard curve, then carries out the measurement of sample concentration.A kind of colorimetric bio sensing analysis new method easy to operate, cheap, ATP contents in response Sensitive Detection breast cancer cell is constructed in the present invention, it is significant to the diagnosis and treatment of clinical cancer.
Description
Technical field
The invention belongs to technical field of analysis and detection, and in particular to it is a kind of based on AuNPs structure colorimetric sensor be used for
Easy, rapid, tumor markers atriphos in sensitivity detection breast cancer cell content detection new method.
Background technology
Atriphos (Adenosine triphosphate, ATP) is a kind of nucleotide[1], also known as adenosine three
Phosphoric acid.ATP is the direct sources of energy needed for in-vivo tissue cell all life activity, and storage and transmission chemical energy participate in egg
The anabolism of white matter, fat, sugar and nucleotide can promote the reparation and regeneration of the various cells of body, enhancing cell metabolism to live
Property, there is stronger specific aim to treating various diseases.And in cell the content of ATP also with many diseases such as anaemia, low blood
Sugar, angiocardiopathy and cancer etc. have close relationship, therefore a kind of method of efficient and sensible of development removes detection ATP molecules
There is very important meaning.
Traditional ATP detection methods have electrophoresis[2], high performance liquid chromatography[3]And tracer method[4]Deng.These sides
Method there is operating process complexity, reagent somewhat expensive, the problem of rapid detection in real time cannot be pinpointed, therefore, build a kind of standard
Really, sensitive, easy inexpensive ATP detection methods are particularly necessary.
Colorimetric method operation is very simple, and result can be with the naked eye readily observed, and majority of case does not need yet
Use accurate instrument.AuNPs is due to higher absorptivity, small-size effect[5]Equal light properties can be used as colorimetric sensing
The sensing element of device shows as well dispersed AuNPs aqueous solutions and claret is presented, since surface plasma absorbing wavelength becomes
Long, blue or purple is presented in the AuNPs of aggregation.
4- mercaptophenyl boronic acids (4-Mercaptophenylboronic acid, MPBA) are containing there are one sulfydryls and one to be connected in
The boric acid base group of contraposition.It is within the scope of 6-10 that MPBA shows as boric acid base group and its derivative in pH value with double alcohol radical characteristic reactions
Reversible reaction can be carried out with 1,2- or 1,3- pentanediols forms stable 5 yuan or 6 membered ring borates[6-7].Have in ATP structures
There are the bis- alcohol radicals of 1,2-, therefore, the characteristic reaction of boric acid and double alcohol is the method for an alternative detection ATP.
Invention content
The present invention provides a kind of colorimetric bio sensor built based on Jenner's grain of rice for triphosphoric acid in breast cancer cell
The method of the Sensitive Detection of adenosine, this method is easy, rapid, sensitivity is high.
This method by Au-S key covalent bonds, is not adding ATP using the sulfydryl and AuNPs of 4- mercaptophenyl boronic acids (MPBA)
When, boric acid base group occurs the condensation of itself three molecular dehydration as crosslinking agent and causes the aobvious blue of AuNPs reunions；ATP is added, when, ATP
With boric acid base group special esterification, which occurs, for cis- vicinal diamines structure in molecule makes AuNPs disperse and aobvious red.
The absorption peak of AuNPs is in 520nm, and Polymer absorption peak is 683nm in wavelength, with A520/A683Absorb ratio and ATP
Concentration carries out linear fit, and the range of linearity is 8.0-100 μM, and detection is limited to 0.12 μM.
We establish a kind of colorimetric biography of easy, quick, highly selective ATP for the first time based on AuNPs resistant to aggregation characteristic
Feel new method, and realize the sensitive quick content detections of ATP in T47D breast cancer cells, has in terms of clinical tumor diagnosis and treatment latent
Application value.
The purpose of the present invention is what is be accomplished by the following way：
Colorimetric sensor of the one kind based on Jenner's grain of rice (AuNPs) structure detects breast cancer for easy, rapid, sensitivity
The detection method of content of tumor markers atriphos in cell, this approach includes the following steps：
It draws in AuNPs to centrifuge tube, is added and dilutes 40-60 times of ATP breast cancer cell extracting solution with PKS buffer solutions, incubate
Change 3-7min and obtain mixture, be added in 4- mercaptophenyl boronic acids to mixture, adds PKS buffer solutions reaction 10-15min, see
Color change is examined, using ultraviolet specrophotometer, with A520/A683It absorbs ratio and carries out linear fit with ATP concentration, external standard method is painted
Standard curve processed, then carry out the measurement of sample concentration.
AuNPs is prepared by citric acid reduction method.The specific preparation process of AuNPs is as follows：25mM gold chloride 5mL are taken,
120mL distilled waters are added, 120 DEG C are heated with stirring to boiling, are then quickly poured into 12.5mL sodium citrates (38.8mM, 1%), after
Continuous to be stirred to react 30min, obtained claret AuNPs is cooled to room temperature.AuNPs concentration can be 2.7nM, that is, draw
1.35mL 3nM AuNPs solution, reaction total volume are 1.5mL.3nM is that AuNPs mother liquors dilute 4 times.
MPBA a concentration of 3-7 μM of MPBA, that is, draws 6 μ L 1mM MPBA solution, reaction is overall as AuNPs crosslinking agents
Product is 1.5mL.
In the present invention, MPBA boric acid base groups identify that the cis- double alcohol of ATP, reaction generate boric acid ester structure, and ATP plays AuNPs
Resistant to aggregation characteristic.It is added after MPBA, reacts 10 minutes observation color changes and carries out ultraviolet detection.MPBA's act as ATP
Identify molecule
Chrominance response system is KH2PO4- NaOH (PKS) buffer salt；PKS buffer solution ionic strength ranges are 75-125mM,
PKS buffer systems pH ranging from 6.5-7.5；Range of reaction temperature is 0-25 DEG C.
ATP concentration can be 100 μM, that is, draw 100 μ L 1.5mM ATP solution, and reaction total volume is 1.5mL.It is not added
Before MPBA, ATP brooding times are 5 minutes.
The preparation process of ATP breast cancer cell extracting solutions is as follows：T47D cells are counted by cell counting board, per 3mL
Contain cell number 8.3 × 10 in T47D cell samples6It is a, take the celliferous culture solutions of 3mL, under 3000 revs/min of rotating speed from
The heart 7 minutes washs centrifugation gained cell with phosphate buffer solution, and it is scattered in the PKS buffer solutions of 200 μ L again
In, then, cell is placed on ultrasound 20min in 0 DEG C of mixture of ice and water, so that clasmatosis, then again at 4 DEG C,
Ultrafiltration centrifuges 20min under the conditions of 18000rpm, removes cell fragment homogenate, takes clear liquid, and is resuspended in 200 μ L PKS bufferings
ATP breast cancer cell extracting solutions are obtained in liquid.
It is centrifuged using Amicon Ultra-0.5 ultra-filtration centrifuge tubes.In order to avoid the interference of protein in cell,
Amicon Ultra-0.5 ultra-filtration centrifuge tubes are used for the centrifugation of ATP extracting solutions to remove isolating protein.
T47D breast cancer cell incubation steps are as follows：Cell culture is containing 10% tire in T47D breast cancer cell cultures
The DMEM culture solutions of the Pen .- Strep of cow's serum (FBS) and 100IU/mL, incubator condition are 5%CO2, 37 DEG C.
ATP concentration detecting steps can be specific as follows in cancer cell extracting solution of the present invention：It draws 1.35mL and dilutes 4 times
In AuNPs to 2mL centrifuge tubes, it is added and dilutes 50 times of 100 μ L hatchings 5min of ATP cell extracts.6 μ L MPBA are added later
In (1mM) to mixture, 44 μ LPKS buffer solutions (100mM, pH 7.5) are added to 1.5mL, react 10min.
In above-mentioned steps, in the case where ATP is not present, the sulfydryl of MPBA with AuNPs by Au-S key covalent bonds, and
And three the boric acid base group self-condensation of molecule slough trihyarol and form six annulus of boron oxygen, AuNPs occurs bright in PKS buffer mediums
Aobvious aggregation and aobvious blue, determine MPBA reunion saturated concentrations；
It is firstly added ATP, the phosphate backbone in ATP structures carries negative electrical charge, increases the electrostatic in AuNPs suspension
Repulsion does not make AuNPs assemble；It is then added and has determined that the MPBA of concentration, react excellent by boric acid base group and pair alcohol
It is first to generate borate with cis- -2,3- ribose ATP reactions, protect AuNPs not assemble by itself dehydrating condensation.
UV-Vis characterizations utilize Shimadzu UV-2450 type ultraviolet specrophotometers in AuNPs colorimetric bios sensing characterization
(Japanese Shimadzu Corporation) measures, spectral scanning range 400nm-800nm, step-length 0.1nm；TEM characterizations drip three kinds of samples respectively
On the copper mesh of cladding carbon film, naturally dry in air.With FEI transmission electron microscopes (Tecnai G12 Spirit Bio TWIN)
It measures, accelerating potential 200V；Three kinds of samples are used Zetasizer Nano ZS particle size measuring instruments (Britain by DLS characterizations respectively
Malvern companies) particle hydration average grain diameter is measured, set temperature is 25 DEG C.
AuNPs resistant to aggregation colorimetric sensors are as follows for ATP standard curves and detection limit detecting step：In 1.35mL
Be added in AuNPs 100 μ L various concentrations be 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, the ATP solution of 1mM hatch 5min, then
6 μ L MPBA (1mM) are added and with PKS buffer solutions (100mM, pH7.5) constant volume to 1.5mL, react 10min.Visually it is visible with
The color for the increase system solution of ATP concentration gradually becomes red from purple.Ratio and ATP are absorbed according to A520/A683
Concentration establishes standard curve, and the range of linearity is 8 μM -100 μM, and detection is limited to 0.12 μM.
In the above method content of ATP be 51.30 ± 1.2 μM (n=3), sample recovery rate 99.5%~102.3% it
Between, RSD is less than 4.2%.
Beneficial effects of the present invention compared with the prior art：
1) it hinders MPBA to self condense at the idiosyncrasy of ester with MPBA boronates using the bis- hydroxyls of ATP for the first time, plays ATP's
AuNPs resistant to aggregation characteristics realize the macroscopic quick analysis of ATP using AuNPs as colorimetric probe, easy to operate, at
This is cheap, convenient for fixed point detection in real time.
2) this method is used successfully to breast cancer T47The sensitive quick detection of ATP in D cells, the rate of recovery is high, and experimental error is small.
3) the colorimetric bio sensor detection ATP based on AuNPs structures provides new think of for the analysis and research of biomolecule
There is potential application value on road in terms of clinical disease diagnosis and treatment.
Description of the drawings
Fig. 1 is that AuNPs resistant to aggregation detects ATP principle schematics
Fig. 2 is that the UV-Vis of colorimetric sensing testing principle in embodiment 1 characterizes (a.AuNPs b.AuNPs/MPBA
c.AuNPs/ATP/MPBA).The AuNPs in fine dispersion state is in claret in solution, there is stronger absorption at 520nm
Peak (curve a).When ATP is not added in AuNPs, the color of solution becomes au bleu from red, it is seen that the absorption intensity at 520nm
It reduces, (curve b) shows that MPBA makes AuNPs be assembled to the absorption peak of the new polymer of appearance at 683nm.Work as addition
After ATP, solution colour becomes aubergine from red, it is seen that absorption peak disappearance (the curve c), it was demonstrated that ATP has anti-of polymer
Aggtegation.
Fig. 3 be embodiment 1 in colorimetric sensing testing principle transmission electron microscope and dynamic optical scanning characterization (a, d.AuNPs b,
e.AuNPs/MPBA c,f.AuNPs/ATP/MPBA).Scheme the visible AuNPs uniform particle sizes of a transmission electron microscopes and in preferably dispersion
State, figure d dynamic light scatterings prove that average grain diameter is 25nm, it was demonstrated that AuNPs has been generated.Not plus when ATP, the visible AuNPs of figure b
Occur obviously to reunite blocking, figure e dynamic light scatterings show that average grain diameter increases to 255nm, it was demonstrated that MPBA makes AuNPs reunite.Add
When entering ATP, the visible less aggregations of AuNPs of figure c, figure f proves that average grain diameter is reduced to 40nm.Transmission electron microscope and dynamic optical scanning knot
Fruit is consistent, proves that ATP plays the role of resistant to aggregation in conjunction with uv-spectrogram (Fig. 2).
Fig. 4 is Raman Characterization (the a.AuNPs b.AuNPs/MPBA of colorimetric sensing testing principle in embodiment 1
c.AuNPs/ATP/MPBA).The AuNPs of dispersion has the compound containing sulfydryl as substrate almost without Raman signal, AuNPs
There is very strong surface light enhancing Ramam effect (SERS), therefore combines after MPBA causes AuNPs to reunite then it can be seen that very
Strong Raman absorption peak greatly enhances Raman scattering intensities this is because surface plasmons couples to form Raman hot spot.
Wherein 1335cm in MPBA solids-1、2555cm-1、3045cm-1The B-O keys at place, S-H keys, the Raman absorption peak disappearance of-OH are (bent
Line a), in 457cm-1And 1473cm-1(curve b) shows MPBA successes to the neighbouring Raman absorption characteristic peak for oxygen bridge B-O-B occur
It is assembled into the surfaces AuNPs and six circular ring structures that dehydrating condensation forms B-O-B has occurred.Add (curve after object ATP
C), solution is in dispersity, and Raman signal greatly weakens, and further illustrates the resistant to aggregation effect of ATP.
Fig. 5 is the canonical plotting that the colorimetric bio sensor built in embodiment 2 detects ATP, from left to right, ATP
Concentration is respectively 8,10,12,20,40,60,80,100 μM, and abscissa is ATP concentration, and ordinate is absorbance ratio.Absorptance
(A683/A520) it is gradually increased with the increase of ATP concentration.In 8 μM -100 μM, the concentration of the absorption ratio and ATP of AuNPs
It is in a linear relationship, equation of linear regression y=0.1232x+2.9024, R=0.9902.The detection of ATP can be calculated with 3 σ methods
It is limited to 0.12 μM.
Fig. 6 be in embodiment 3 the colorimetric bio sensor that builds to the detection of ATP in T47D breast cancer cell samples with plus
The sample rate of recovery is investigated.Actual sample T47D breast cancer cells (~107It is a, dilute 50 times) in ATP content be 51.30 ± 1.2 μ
M (n=3), for sample recovery rate between 99.5%~102.3%, RSD is less than 4.2%.
Specific implementation mode
Explanation is further explained to the present invention by the following examples：
Drug and reagent：ATP (5 '-triphosphate of Adenosine, Shanghai Sheng Gong bioengineering Co., Ltd), 4-
Mercaptophenyl boronic acid (C6H7BO2S, Sa En chemical technology (Shanghai) Co., Ltd.), gold chloride (HAuCl4, Chinese medicines group chemical reagent
Co., Ltd), two citric acid monohydrate trisodiums (C6H5NaO7H2O, photochemistry Co., Ltd., Factory of Guangdong China), three (methylol) ammonia
Methylmethane (C4H11NO3, Sinopharm Chemical Reagent Co., Ltd.), sodium dihydrogen phosphate (NaH2PO4, analysis is pure, the examination of Nanjing chemistry
Agent Co., Ltd), disodium hydrogen phosphate (Na2HPO4, analyze pure, Shanghai Ling Feng Chemical Co., Ltd.s), potassium dihydrogen phosphate (KH2PO4,
The Shantou, Guangdong city chemical plant Xi Long), dipotassium hydrogen phosphate (K2HPO4, the Shantou, Guangdong city chemical plant Xi Long), sodium hydroxide (NaOH, south
Capital chemical reagent Co., Ltd), hydrochloric acid (HCl, Liyang east chemical reagent Co., Ltd), experimental water be ultra-pure water.
Instrument：Accurate a ten thousandth electronic balance：Shimadzu AUW220D, Japanese Shimadzu Corporation；Precision 100,000/
One electronic balance：Shimadzu AUW220D, Japanese Shimadzu Corporation；Ultraviolet-visible spectrometer：Shimadzu UV-2450, day
This Shimadzu Corporation；PHS-3C precisions PH meters：Shanghai Dao scientific instrument Co., Ltd；Nano particle size instrument：ZS90, Britain
Malvern companies；Transmission electron microscope：FEI(Tecnai G12 Spirit Bio TWIN)；Raman spectrometer：DXR
Smart, Thermo Fischer Scient Inc. of the U.S..
Embodiment 1
The preparation method of AuNPs is as follows：All glass containers used of testing are all with freshly prepd HN03-HCl(1：
3, V/V) solution soaked overnight, then with secondary water rinse, in air drying for standby.Take 25mM gold chlorides (HAuCl4) 5mL in
In 250mL round-bottomed flasks, 120mL distilled waters are added, are heated to boiling for 120 DEG C by constant temperature water bath magnetic stirring apparatus, then soon
Speed is poured into 12.5mL sodium citrates (38.8mM, 1%), continues to be stirred to react 30min, obtained claret AuNPs natural coolings
It is put into refrigerator (4 DEG C) and preserves after to room temperature.The grain size of the AuNPs of preparation is 13nm, there is maximum absorption band at 520nm, purple
A concentration of 12nM of outer measurement.AuNPs is in the electrostatic repulsion masterpiece that preferable monodisperse status is due to the citrate on surface
With the Van der Waals force between resistance AuNPs so that surface plasmon absorption peak aobvious claret at 520nm.
In the case where ATP is not present, the sulfydryl of MPBA (4 μM) and AuNPs are by Au-S key covalent bonds, and three points
The boric acid base group self-condensation of son sloughs trihyarol and forms six annulus of boron oxygen, makes AuNPs that the aobvious blue of apparent aggregation occur.When
In the presence of ATP (100 μM), the phosphate backbone in ATP structures carries negative electrical charge, increases the electrostatic reprimand in AuNPs suspension
Power.Borate preferentially is generated with cis- -2,3- ribose ATP reactions with reacting for double alcohol by boric acid base group, protection AuNPs is obstructed
It crosses itself dehydrating condensation to assemble, makes the aobvious red of AuNPs dispersions.
Embodiment 2
The standard curve of ATP：It draws in 1.35mL AuNPs to 2mL centrifuge tubes, the volume that various concentration is added is 100 μ L
ATP (extracting solution) hatches 5min.It is added later in 6 μ L MPBA (1mM) to mixture, adds 44 μ LPKS buffer solutions
(100mM, pH 7.5), reaction 10min observation color changes simultaneously carry out ultraviolet specrophotometer measurement.
The selectivity of ATP：It draws in 1.35mL AuNPs to 2mL centrifuge tubes, 100 μM of GTP, 100 μM of UTP, 100 is added
μM CTP, three kinds of ATP analogs and 100 μM of ATP hatch 5min, are added later in 6 μ L MPBA (1mM) to mixture, then add
Enter 44 μ LPKS buffer solutions (100mM, pH 7.5), reaction 10min observation color changes simultaneously carry out ultraviolet specrophotometer measurement.
AuNPs colorimetric bios sensor of the present invention is detected by the following method：
Color change：The shooting observation of Nikon D3100 cameras is utilized after reacting 10min.
UV, visible light spectrophotometer：Detect spectral scanning range 400nm-800nm, step-length 0.1nm.
Selectivity of the colorimetric bio sensor built in embodiment 2 to ATP：100 μM of GTP, 100 μM of UTP are investigated,
100 μM of CTP, three kinds of ATP analogs and 100 μM of ATP are to the resistant to aggregation effect of MPBA-AuNPs, after three kinds of analogs are added
AuNPs solution becomes rapidly blue, the absorption peak of polymer occurs, shows that three kinds of analogs are acted on without resistant to aggregation.And phase is added
After the ATP of concentration, AuNPs solution in the aobvious red of dispersity, and A520/A683 absorb ratio be significantly higher than three kinds it is similar
Object, the results showed that this method has higher specificity to the detection of ATP.
Embodiment 3
Cell culture：T47D breast cancer tumor cells cell strains are provided by American Type Culture collection warehousing, are then put
Enter in Tissue Culture Flask and cultivate, cell is in the Pen .- Strep containing 10% fetal calf serum (FBS) and 100IU/mL
It is cultivated in DMEM culture solutions, incubator condition is 5%CO2, 37 DEG C.
The extraction of adenosine triphosphate atp in cancer cell：T47D cells (T47D per 3mL after cell counting board counts
Contain cell number about 8.3 × 10 in cell sample6It is a), the celliferous culture solution of certain volume is taken, in turning for 3000 revolutions per minute
The lower centrifugation of speed 7 minutes washs centrifugation gained cell with phosphate buffer solution, and it is scattered in the PKS of certain volume again
In buffer solution.Then, cell is placed on mixture of ice and water (ultrasound 20min in 0 DEG C, so that clasmatosis.Then it centrifuges again
(18000rpm, 20min, 4 DEG C) leaves clear liquid, and be resuspended in 200 μ L PKS buffer solutions to remove cell fragment homogenate
In.In order to avoid the interference of protein in cell, Amicon Ultra-0.5 ultra-filtration centrifuge tubes are used for the centrifugation of ATP extracting solutions
To remove isolating protein.
ATP extracting solutions after ultrafiltration centrifugation dilute 50 times with PKS buffer solutions.
The sample detection of ATP：It draws in 1.35mL AuNPs to 2mL centrifuge tubes, above-mentioned PKS buffer solutions dilution 50 is added
100 μ L of ATP extracting solutions again, hatching 5min obtain mixture, are added in 6 μ L MPBA (1mM) to mixture, add later
44 μ LPKS buffer solutions (100mM, pH 7.5) react 10min, observe color change and carry out ultraviolet specrophotometer measurement, with
A520/A683It absorbs ratio and carries out linear fit with ATP concentration, external standard method draws standard curve, then carries out the measurement of sample concentration.
Bibliography
[1]Wang W.Isothermal amplified detection of ATP using Au nanocages
capped with a DNA molecular gate and its application in cell lysates[J]
.Analyst,2015,140(5):1672-1677.
[2]Ke Chen,Dacheng Wang,Lukasz Kurgan.Systematic investigation of
sequence and structural motifs that recognize ATP[J].Computational Biology
and Chemistry,2015,56:131-141.
[3]Vadziuk OB.ATP-sensitive K(+)-channels in muscle cells:features
and physiological role[J].Ukr Biochem J,2014,86(3):5-22.
[4]Guo Shensheng appoints the steel tracer methods that spread out to develop with life science;J]Biology notification .2006,41 (9):
22-23。
[5]Shen Q.A simple"clickable"biosensor for colorimetric detection of
copper(II)ions based on unmodified gold nanoparticles[J].Biosens Bioelectron,
2013,41:663-668.
[6]Liu L.Highly sensitive and label-free electrochemical detection of
microRNAs based on triple signal amplification of multifunctional gold
nanoparticles,enzymes and redox-cycling reaction[J].Biosens Bioelectron,2014,
53:399-405.
[7]Xia N.Label-free and sensitive strategy for microRNAs detection
based on the formation of boronate ester bonds and the dual-amplification of
gold nanoparticles[J].Biosens Bioelectron,2013,47:461-466。
Claims (8)
1. a kind of content inspection of colorimetric bio sensor based on Jenner's grain of rice structure for atriphos in breast cancer cell
Survey method, it is characterised in that this approach includes the following steps：
It draws in AuNPs to centrifuge tube, is added and dilutes 40-60 times of ATP breast cancer cell extracting solution with PKS buffer solutions, hatch 3-
7min obtains mixture, is added in 4- mercaptophenyl boronic acids to mixture, adds PKS buffer solutions reaction 10-15min, observes face
Color change, using ultraviolet specrophotometer, with A520/A683It absorbs ratio and carries out linear fit with ATP concentration, external standard method draws mark
Directrix curve, then carry out the measurement of sample concentration；Wherein, PKS buffer solutions ionic strength range is 75-125mM, PKS buffer systems
PH ranging from 6.5-7.5.
2. detection method of content according to claim 1, it is characterised in that AuNPs is prepared by citric acid reduction method.
3. detection method of content according to claim 1, it is characterised in that the concentration range of MPBA is 3-7 μM.
4. detection method of content according to claim 1, it is characterised in that range of reaction temperature is 0-25 DEG C.
5. detection method of content according to claim 1, it is characterised in that the preparation process of AuNPs is as follows：Take 25mM chlorine
120mL distilled waters are added in auric acid 5mL, and 120 DEG C are heated with stirring to boiling, are then quickly poured into 12.5mL sodium citrates, continue to stir
It mixes and reacts 30min, obtained claret AuNPs is cooled to room temperature.
6. detection method of content according to claim 1, it is characterised in that breast cancer cell is breast cancer cell T47D.
7. detection method of content according to claim 1, it is characterised in that the preparation process of ATP breast cancer cell extracting solutions
It is as follows：The celliferous culture solutions of 3mL are taken, centrifuges 7 minutes under 3000 revs/min of rotating speed, is washed with phosphate buffer solution
Centrifugation gained cell, and it is scattered in again in the PKS buffer solutions of 200 μ L, then, cell is placed on to 0 DEG C of ice water and is mixed
Ultrasound 20min in object is closed, then again at 4 DEG C, ultrafiltration centrifugation 20min, takes clear liquid, and be resuspended under the conditions of 18000rpm
ATP breast cancer cell extracting solutions are obtained in 200 μ L PKS buffer solutions.
8. according to the method described in claim 7, it is characterized in that using Amicon Ultra-0.5 ultra-filtration centrifuge tubes carry out from
The heart.
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| A selective, sensitive and label-free visual assay of fructose using anti-aggregation of gold nanoparticles as a colorimetric probe;Foroogh Keshvari et al.;《Chinese Chemical Letters》;20160123;第27卷(第6期);第847-851页 * |
| Sensitive colorimetric visualization of dihydronicotinamide adenine dinucleotide based on anti-aggregation of gold nanoparticles via boronic acid–diol binding;Shufeng Liu et al.;《Biosensors and Bioelectronics》;20120303;第35卷(第1期);第444-445页，Supplementary data * |
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