CN105717103A - Method used for detecting content of adenosine triphosadenine in breast cancer cell with colorimetric biosensor and constructed based on gold nanoparticles - Google Patents

Method used for detecting content of adenosine triphosadenine in breast cancer cell with colorimetric biosensor and constructed based on gold nanoparticles Download PDF

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CN105717103A
CN105717103A CN201610055701.4A CN201610055701A CN105717103A CN 105717103 A CN105717103 A CN 105717103A CN 201610055701 A CN201610055701 A CN 201610055701A CN 105717103 A CN105717103 A CN 105717103A
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atp
aunps
breast cancer
cancer cell
content
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CN105717103B (en
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周学敏
江郭一
沈心
徐磊
李晓旭
王芮
刘春雨
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Nanjing University
Nanjing Medical University
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Abstract

The invention discloses a method used for detecting the content of adenosine triphosadenine in a breast cancer cell with a colorimetric biosensor and constructed based on gold nanoparticles. The method includes the following steps that AuNPs is sucked into a centrifuge tube, an ATP breast cancer cell extract diluted with a PKS buffer solution by 40-60 times is added, incubation is conducted for 3-7 min to obtain a mixture, 4-sulfydryl phenylboronic acid is added into the mixture, the PKS buffer solution is added again to react for 10-15 min, a color change is observed, an ultraviolet spectrophotometer is adopted, the absorption rate of A520/A683 and the content of ATP are fitted linearly, a standard curve is drawn through an external standard method, and then the content of a sample is measured. The new colorimetric biosensor analysis method used for detecting the content of adenosine triphosadenine in the breast cancer cell is easy to operate, low in price and sensitive in response and has important significance in diagnosis and treatment on clinic cancers.

Description

The colorimetric bio sensor detection method of content of adenosine triphosphate in breast cancer cell built based on Jenner's grain of rice
Technical field
The invention belongs to technical field of analysis and detection, be specifically related to a kind of colorimetric sensor built based on AuNPs and detect the content detection new method of tumor markers adenosine triphosphate in breast cancer cell for easy, rapid, susceptiveness.
Background technology
Adenosine triphosphate (Adenosine triphosphate, ATP) is a kind of nucleotide[1], also known as adenosine triphyosphate.ATP is the direct sources of energy needed for in-vivo tissue cell all life activity, store and transmission chemical energy, participate in protein, fat, sugar and the anabolism of nucleotide, reparation and the regeneration of the various cell of body can be promoted, strengthen cell metabolic activity, all have stronger specific aim to treating various diseases.And the content of ATP also has close relationship with numerous disease such as anemia, hypoglycemia, cardiovascular disease and cancer etc. in cell, a kind of method therefore developing efficient and sensible goes detection ATP molecule to have very important meaning.
Traditional ATP detection method has electrophoresis method[2], high performance liquid chromatography[3]And tracer method[4]Deng.These methods also exist operating process complexity, reagent somewhat expensive, can not pinpoint the problem detected the most rapidly, therefore, build a kind of inexpensive ATP detection method accurate, sensitive, easy particularly necessary.
Colorimetry operation is very simple, and result can with the naked eye be readily observed, and majority of case is also without the instrument using precision.AuNPs is owing to having higher specific absorbance, small-size effect[5]Can be as the sensing element of colorimetric sensor Deng light property, showing as finely disseminated AuNPs aqueous solution presents claret, and owing to surface plasma absorbing wavelength is elongated, the AuNPs of gathering presents blueness or purple.
4-mercaptophenyl boronic acid (4-Mercaptophenylboronic acid, MPBA) contains a sulfydryl and a boric acid base group being connected in para-position.MPBA and double alcohol radical characteristic reactions show as boric acid base group and derivant thereof can be with 1 in the range of pH value is 6-10, and 2-or 1,3-pentanediol carries out reversible reaction and forms 5 yuan stable or 6 ring borates[6-7].Having 1 in ATP structure, the double alcohol radical of 2-, therefore, the characteristic reaction of boric acid and double alcohol is the method for an alternative detection ATP.
Summary of the invention
The present invention provides a kind of colorimetric bio sensor method of the Sensitive Detection of adenosine triphosphate in breast cancer cell built based on Jenner's grain of rice, and the method is easy, rapid, susceptiveness is high.
The method utilizes the sulfydryl of 4-mercaptophenyl boronic acid (MPBA) with AuNPs by Au-S key covalent bond, and when not adding ATP, boric acid base group occurs self three molecular dehydration condensation to cause the aobvious blueness of AuNPs reunion as cross-linking agent；Add ATP, time, in ATP molecule, cis vicinal diamines structure and boric acid base group occur special esterification to make AuNPs dispersion show redness.
The absworption peak of AuNPs is at 520nm, and Polymer absorption peak is 683nm at wavelength, with A520/A683Absorbing ratio and carry out linear fit with ATP concentration, its range of linearity is 8.0-100 μM, and detection is limited to 0.12 μM.
We establish a kind of simplicity, the colorimetric sensing new method of selective ATP quick, high first based on AuNPs resistant to aggregation characteristic, and achieve the sensitive quick content detection of ATP in T47D breast cancer cell, have potential using value in terms of clinical tumor diagnosis and treatment.
It is an object of the invention to be accomplished by:
A kind of colorimetric sensor built based on Jenner's grain of rice (AuNPs) detects the detection method of content of tumor markers adenosine triphosphate in breast cancer cell for easy, rapid, susceptiveness, and the method comprises the following steps:
Drawing AuNPs in centrifuge tube, add and dilute 40-60 times of ATP breast cancer cell extracting solution with PKS buffer, hatching 3-7min obtains mixture, add 4-mercaptophenyl boronic acid in mixture, add PKS buffer reaction 10-15min, observe color change, use ultraviolet spectrophotometer, with A520/A683Absorbing ratio and carry out linear fit with ATP concentration, external standard method draws standard curve, then carries out the mensuration of sample concentration.
AuNPs is prepared by citric acid reducing process.The concrete preparation process of AuNPs is as follows: take 25mM gold chloride 5mL, adds 120mL distilled water, and 120 DEG C are heated with stirring to boiling, the most quickly it is poured into 12.5mL sodium citrate (38.8mM, 1%), continues stirring reaction 30min, claret AuNPs obtained, is cooled to room temperature.AuNPs concentration can be 2.7nM, i.e. draws 1.35mL 3nM AuNPs solution, and reaction cumulative volume is 1.5mL.3nM is that AuNPs mother solution dilutes 4 times.
MPBA is 3-7 μM as AuNPs cross-linking agent, the concentration of MPBA, i.e. draws 6 μ L 1mM MPBA solution, and reaction cumulative volume is 1.5mL.
In the present invention, MPBA cis pair of alcohol of boric acid base group identification ATP, reaction generates borate structure, and ATP plays AuNPs resistant to aggregation characteristic.After adding MPBA, react 10 minutes observation colors and change and carry out ultraviolet detection.The ATP that act as of MPBA identifies molecule
Chrominance response system is KH2PO4-NaOH (PKS) buffer salt；PKS buffer ionic strength range be 75-125mM, PKS buffer system pH scope be 6.5-7.5；Range of reaction temperature is 0-25 DEG C.
ATP concentration can be 100 μMs, i.e. draws 100 μ L 1.5mM ATP solution, and reaction cumulative volume is 1.5mL.Before not adding MPBA, ATP brooding time is 5 minutes.
The preparation process of ATP breast cancer cell extracting solution is as follows: T47D cell counts through cell counting count board, containing cell number 8.3 × 10 in the T47D cell sample of every 3mL6Individual, take the celliferous culture fluid of 3mL, be centrifuged 7 minutes under the rotating speed of 3000 revs/min, with the centrifugal gained cell of phosphate buffered solution washing, and it is scattered in again in the PKS buffer solution of 200 μ L, then, cell is placed on ultrasonic 20min in the mixture of ice and water of 0 DEG C, so that cell breakage, the most again at 4 DEG C, under the conditions of 18000rpm, ultrafiltration is centrifuged 20min, removes cell debris homogenate, take clear liquid, and be resuspended in 200 μ L PKS buffer obtaining ATP breast cancer cell extracting solution.
Amicon Ultra-0.5 ultra-filtration centrifuge tube is used to be centrifuged.In order to avoid the interference of protein in cell, Amicon Ultra-0.5 ultra-filtration centrifuge tube is used for the centrifugal to remove isolating protein of ATP extracting solution.
T47D breast cancer cell incubation step is as follows: T47D breast cancer cell cultivate in cell cultivate containing 10% hyclone (FBS) and the DMEM culture fluid of Pen .-Strep of 100IU/mL, couveuse condition is 5%CO2, 37 DEG C.
In cancerous cell extracting solution of the present invention, ATP concentration detecting step can be specific as follows: draws in AuNPs to the 2mL centrifuge tube that 1.35mL dilutes 4 times, adds 50 times of ATP cell extract 100 μ L of dilution and hatches 5min.Add 6 μ L MPBA (1mM) afterwards in mixture, add 44 μ LPKS buffer (100mM, pH 7.5) to 1.5mL, react 10min.
In above-mentioned steps, in the case of ATP is non-existent, the sulfydryl of MPBA and AuNPs are by Au-S key covalent bond, and the boric acid base group self-condensation of three molecules is sloughed trihyarol and is formed boron oxygen six annulus, in PKS buffer medium, AuNPs occurs significantly to assemble and aobvious blueness, determines MPBA reunion saturated concentration；
The phosphate backbone being firstly added in ATP, ATP structure carries negative charge, adds the electrostatic repulsion in AuNPs suspension, does not make AuNPs assemble；Being subsequently added the MPBA having determined that concentration, preferential with cis-2 with the reaction of double alcohol by boric acid base group, 3-ribose ATP reaction generates borate, and protection AuNPs is not assembled by self dehydrating condensation.
During AuNPs colorimetric bio sensing characterizes, UV-Vis sign utilizes Shimadzu UV-2450 type ultraviolet spectrophotometer (Shimadzu Corporation of Japan) to measure, spectral scan scope 400nm-800nm, step-length 0.1nm；TEM characterizes on the copper mesh that three kinds of samples drop in cladding carbon film respectively, the most naturally dries.Measure with FEI transmission electron microscope (Tecnai G12 Spirit Bio TWIN), accelerating potential 200V；DLS characterizes and with Zetasizer Nano ZS particle size measuring instrument (Malvern company of Britain), three kinds of samples is measured granule hydration mean diameter respectively, and design temperature is 25 DEG C.
AuNPs resistant to aggregation colorimetric sensor is as follows with detection limit detecting step for ATP standard curve: adding 100 μ L variable concentrations in 1.35mL AuNPs is 0.01 μM, 0.1 μM, 1 μM, 10 μMs, 100 μMs, the ATP solution hatching 5min of 1mM, add 6 μ L MPBA (1mM) and with PKS buffer solution (100mM, pH7.5) constant volume is to 1.5mL, reacts 10min.Seen from naked eyes, the color increasing system solution along with ATP concentration gradually becomes redness from purple.Absorb the concentration Criterion curve of ratio and ATP according to A520/A683, the range of linearity is 8 μMs-100 μMs, and detection is limited to 0.12 μM.
In said method, the content of ATP is 51.30 ± 1.2 μMs (n=3), and average recovery is between 99.5%～102.3%, and RSD is less than 4.2%.
Beneficial effects of the present invention compared with the prior art:
1) the double hydroxyl of ATP is utilized to become the specific reaction of ester to hinder MPBA to self condense with MPBA boronate first, play the AuNPs resistant to aggregation characteristic of ATP, achieve the macroscopic quick analysis of ATP using AuNPs as colorimetric probe, simple to operate, with low cost, it is simple to fixed point detection in real time.
2) this method is used successfully to breast carcinoma T47The sensitive quick detection of ATP in D cell, the response rate is high, and experimental error is little.
3) analysis and research that colorimetric bio sensor detection ATP is biomolecule built based on AuNPs provide new approaches, have potential using value in terms of clinical disease diagnosis and treatment.
Accompanying drawing explanation
Fig. 1 is that AuNPs resistant to aggregation detects ATP principle schematic
Fig. 2 is that the UV-Vis of colorimetric sensing Cleaning Principle in embodiment 1 characterizes (a.AuNPs b.AuNPs/MPBA c.AuNPs/ATP/MPBA).The AuNPs being in fine dispersion state in solution is claret, has stronger absworption peak (curve a) at 520nm.When not adding ATP in AuNPs, the color of solution becomes blue from redness, it is seen that the absorption intensity at 520nm reduces, and at 683nm, (curve b) shows that MPBA makes AuNPs there occurs gathering to the absworption peak of the polymer that appearance is new.After adding ATP, solution colour becomes aubergine from redness, it is seen that absworption peak disappearance (the curve c), it was demonstrated that ATP has resistant to aggregation effect of polymer.
Fig. 3 is transmission electron microscope and dynamic optical scanning sign (a, d.AuNPs b, e.AuNPs/MPBA c, f.AuNPs/ATP/MPBA) of colorimetric sensing Cleaning Principle in embodiment 1.Scheming AuNPs uniform particle sizes seen from a transmission electron microscope and be in preferable dispersity, figure d dynamic light scattering proves that mean diameter is 25nm, it was demonstrated that AuNPs has generated.When not adding ATP, AuNPs seen from figure b occurs substantially to reunite in bulk, and figure e dynamic light scattering display mean diameter increases to 255nm, it was demonstrated that MPBA makes AuNPs reunite.When adding ATP, the less gathering of AuNPs seen from figure c, figure f proves that mean diameter is reduced to 40nm.Transmission electron microscope is consistent with dynamic optical scanning result, proves that ATP serves the effect of resistant to aggregation in conjunction with uv-spectrogram (Fig. 2).
Fig. 4 is the Raman Characterization (a.AuNPs b.AuNPs/MPBA c.AuNPs/ATP/MPBA) of colorimetric sensing Cleaning Principle in embodiment 1.Scattered AuNPs is almost without Raman signal, AuNPs has the strongest surface light as substrate to the compound containing sulfydryl and strengthens Raman effect (SERS), therefore combine after MPBA causes AuNPs to reunite then it can be seen that the strongest Raman absorption peak, this is owing to surface plasmons coupling forms Raman focus, strengthens Raman scattering intensities greatly.Wherein 1335cm in MPBA solid-1、2555cm-1、3045cm-1Place B-O key, S-H key ,-OH Raman absorption peak disappear (curve a), at 457cm-1And 1473cm-1Near occur oxygen bridge B-O-B Raman absorption characteristic peak (curve b), show MPBA be successfully assembled into AuNPs surface and there occurs dehydrating condensation formed B-O-B six circular ring structures.After adding object ATP, (curve c), solution is dispersity, and Raman signal greatly weakens, and further illustrates the resistant to aggregation effect of ATP.
Fig. 5 is the canonical plotting that ATP is detected by the colorimetric bio sensor built in embodiment 2, and from left to right, ATP concentration is respectively 8,10,12,20,40,60,80,100 μMs, and abscissa is ATP concentration, and vertical coordinate is absorbance ratio.Absorptance (A683/A520) gradually strengthens with the increase of ATP concentration.In 8 μMs-100 μMs, the absorption ratio of AuNPs is linear with the concentration of ATP, and equation of linear regression is y=0.1232x+2.9024, R=0.9902.The detection that can calculate ATP by 3 σ methods is limited to 0.12 μM.
Fig. 6 is that the detection of ATP in T47D breast cancer cell sample is investigated by the colorimetric bio sensor built in embodiment 3 with average recovery.Actual sample T47D breast cancer cell (～107Individual, dilute 50 times) in the content of ATP be 51.30 ± 1.2 μMs (n=3), average recovery is between 99.5%～102.3%, and RSD is less than 4.2%.
Detailed description of the invention
By the following examples the present invention is further explained explanation:
Medicine and reagent: ATP (Adenosine 5 '-triphosphate, Shanghai Sheng Gong biological engineering company limited), 4-mercaptophenyl boronic acid (C6H7BO2S, Sa En chemical technology (Shanghai) Co., Ltd.), gold chloride (HAuCl4, Chemical Reagent Co., Ltd., Sinopharm Group), two citric acid monohydrate trisodium (C6H5NaO7H2O, photochemistry Co., Ltd., Factory of Guangdong China), three (methylol) aminomethane (C4H11NO3, Chemical Reagent Co., Ltd., Sinopharm Group), sodium dihydrogen phosphate (NaH2PO4, analytical pure, Nanjing Chemistry Reagent Co., Ltd.), disodium hydrogen phosphate (Na2HPO4, analytical pure, Shanghai Ling Feng Chemical Co., Ltd.), potassium dihydrogen phosphate (KH2PO4, Xi Long chemical plant, Shantou, Guangdong city), dipotassium hydrogen phosphate (K2HPO4, Xi Long chemical plant, Shantou, Guangdong city), sodium hydroxide (NaOH, Nanjing Chemistry Reagent Co., Ltd.), hydrochloric acid (HCl, east, Liyang chemical reagent company limited), experimental water be ultra-pure water.
Instrument: accurate ten thousand/electronic balance: Shimadzu AUW220D, Shimadzu Corporation of Japan；Accurate 100,000/electronic balance: Shimadzu AUW220D, Shimadzu Corporation of Japan；Ultraviolet-visible spectrometer: Shimadzu UV-2450, Shimadzu Corporation of Japan；PHS-3C precision PH is counted: Shanghai Dao scientific instrument company limited；Nano particle size instrument: ZS90, Malvern company of Britain；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: freshly prepd HN0 all used by what all tests were used glass container3-HCl (1:3, V/V) solution soaking overnight, then by secondary water rinse, drying for standby in atmosphere.Take 25mM gold chloride (HAuCl4) 5mL is in 250mL round-bottomed flask, add 120mL distilled water, it is heated to boiling by constant temperature water bath magnetic stirring apparatus 120 DEG C, the most quickly it is poured into 12.5mL sodium citrate (38.8mM, 1%), continuing stirring reaction 30min, claret AuNPs obtained is put into after naturally cooling to room temperature in refrigerator (4 DEG C) and is preserved.The particle diameter of the AuNPs of preparation is 13nm, has maximum absorption band at 520nm, and the concentration of ultraviolet determination is 12nM.AuNPs be preferable monodisperse status be the citrate due to surface electrostatic repulsion forces effect resistance AuNPs between Van der Waals force so that surface plasmon absorption peak is aobvious claret at 520nm.
In the case of ATP is non-existent, the sulfydryl of MPBA (4 μMs) and AuNPs are by Au-S key covalent bond, and the boric acid base group self-condensation of three molecules is sloughed trihyarol and formed boron oxygen six annulus, makes AuNPs occur significantly and assembles aobvious blueness.In the presence of ATP (100 μMs), the phosphate backbone in ATP structure carries negative charge, adds the electrostatic repulsion in AuNPs suspension.Preferential with cis-2 with the reaction of double alcohol by boric acid base group, 3-ribose ATP reaction generates borate, and protection AuNPs is not assembled by self dehydrating condensation, makes the aobvious redness of AuNPs dispersion.
Embodiment 2
The standard curve of ATP: draw in 1.35mL AuNPs to 2mL centrifuge tube, the volume adding variable concentrations is that 100 μ L ATP (extracting solution) hatch 5min.Adding 6 μ L MPBA (1mM) afterwards in mixture, add 44 μ LPKS buffer (100mM, pH 7.5), reaction 10min observes color and changes and carry out ultraviolet spectrophotometer mensuration.
The selectivity of ATP: draw in 1.35mL AuNPs to 2mL centrifuge tube, add 100 μMs of GTP, 100 μMs of UTP, 100 μMs of CTP, three kinds of ATP analog and 100 μMs of ATP hatch 5min, add 6 μ L MPBA (1mM) afterwards in mixture, add 44 μ LPKS buffer (100mM, pH 7.5), reaction 10min observes color and changes and carry out ultraviolet spectrophotometer mensuration.
By the following method AuNPs colorimetric bio sensor of the present invention is detected:
Color changes: utilize the shooting of Nikon D3100 camera to observe after reaction 10min.
UV, visible light spectrophotometer: detection spectral scan scope 400nm-800nm, step-length 0.1nm.
The colorimetric bio sensor built in embodiment 2 selectivity to ATP: investigate 100 μMs of GTP, 100 μMs of UTP, 100 μMs of CTP, three kinds of ATP analog and the 100 μMs of ATP resistant to aggregation effect to MPBA-AuNPs, after adding three kinds of analog, AuNPs solution becomes rapidly blue, the absworption peak of polymer occurs, shows that three kinds of analog do not have resistant to aggregation effect.And after adding the ATP of same concentrations, AuNPs solution is the aobvious redness of dispersity, and A520/A683 absorbs ratio and is significantly higher than three kinds of analog, and result shows that the method has higher specificity to the detection of ATP.
Embodiment 3
Cell is cultivated: T47D breast cancer tumor cells cell strain is to be provided by American Type Culture collection warehousing, it is then placed in Tissue Culture Flask cultivating, cell containing 10% hyclone (FBS) and 100IU/mL Pen .-Strep DMEM culture fluid in cultivate, couveuse condition is 5%CO2, 37 DEG C.
The extraction of adenosine triphosphate atp in cancerous cell: T47D cell (contains cell number about 8.3 × 10 after cell counting count board counts in the T47D cell sample of every 3mL6Individual), take the celliferous culture fluid of certain volume, be centrifuged 7 minutes under the rotating speed of 3000 turns every point, be centrifuged gained cell with phosphate buffered solution washing, and it be scattered in again in the PKS buffer solution of certain volume.Then, (ultrasonic 20min in 0 DEG C, so that cell breakage cell to be placed on mixture of ice and water.The most centrifugal (18000rpm, 20min, 4 DEG C) to remove cell debris homogenate, leave clear liquid, and be resuspended in 200 μ L PKS buffer.In order to avoid the interference of protein in cell, Amicon Ultra-0.5 ultra-filtration centrifuge tube is used for the centrifugal to remove isolating protein of ATP extracting solution.
ATP extracting solution after ultrafiltration is centrifugal dilutes 50 times with PKS buffer.
The sample detection of ATP: draw in 1.35mL AuNPs to 2mL centrifuge tube, add above-mentioned PKS buffer and dilute the ATP extracting solution 100 μ L of 50 times, hatching 5min obtains mixture, add 6 μ L MPBA (1mM) afterwards in mixture, add 44 μ LPKS buffer (100mM, pH 7.5), react 10min, observe color change and carry out ultraviolet spectrophotometer mensuration, with A520/A683Absorbing ratio and carry out linear fit with ATP concentration, external standard method draws standard curve, then carries out the mensuration of sample concentration.
List of references
[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, Ren Yangang. tracer method develops [J] with life sciences. circulate a notice of .2006,41 (9): 22-23 biology.
[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. the colorimetric bio sensor content detection side of adenosine triphosphate in breast cancer cell built based on Jenner's grain of rice Method, it is characterised in that the method comprises the following steps:
Draw AuNPs in centrifuge tube, add and dilute 40-60 times of ATP breast cancer cell extracting solution with PKS buffer, Hatching 3-7min obtains mixture, in addition 4-mercaptophenyl boronic acid to mixture, adds PKS buffer reaction 10-15min, Observation color changes, and uses ultraviolet spectrophotometer, with A520/A683Absorb ratio and carry out linear fit with ATP concentration, External standard method draws standard curve, then carries out the mensuration of sample concentration.
Detection method of content the most according to claim 1, it is characterised in that AuNPs is prepared by citric acid reducing process.
Detection method of content the most according to claim 1, it is characterised in that the concentration range of MPBA is 3-7 μM.
Detection method of content the most according to claim 1, it is characterised in that PKS buffer ionic strength range is 75-125mM, PKS buffer system pH scope is 6.5-7.5；Range of reaction temperature is 0-25 DEG C.
Detection method of content the most according to claim 1, it is characterised in that the preparation process of AuNPs is as follows: take 25mM Gold chloride 5mL, adds 120mL distilled water, and 120 DEG C are heated with stirring to boiling, are the most quickly poured into 12.5mL citric acid Sodium, continues stirring reaction 30min, claret AuNPs obtained, is cooled to room temperature.
Detection method of content the most according to claim 1, it is characterised in that breast cancer cell is breast cancer cell T47D.
Detection method of content the most according to claim 1, it is characterised in that the preparation process of ATP breast cancer cell extracting solution As follows: to take the celliferous culture fluid of 3mL, it is centrifuged 7 minutes under the rotating speed of 3000 revs/min, molten by phosphate-buffered The centrifugal gained cell of liquid washing, and it is scattered in again in the PKS buffer solution of 200 μ L, then, cell is placed on 0 DEG C Mixture of ice and water in ultrasonic 20min, the most again at 4 DEG C, under the conditions of 18000rpm, ultrafiltration is centrifuged 20min, takes clear liquid, And be resuspended in 200 μ L PKS buffer obtaining ATP breast cancer cell extracting solution.
Method the most according to claim 7, it is characterised in that use Amicon Ultra-0.5 ultra-filtration centrifuge tube to be centrifuged.
CN201610055701.4A 2016-01-27 2016-01-27 Detection method of content of the colorimetric bio sensor based on Jenner's grain of rice structure for atriphos in breast cancer cell Expired - Fee Related CN105717103B (en)

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