CN106190112A - The preparation method and applications of gold quantum dot - Google Patents

The preparation method and applications of gold quantum dot Download PDF

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Publication number
CN106190112A
CN106190112A CN201610535961.1A CN201610535961A CN106190112A CN 106190112 A CN106190112 A CN 106190112A CN 201610535961 A CN201610535961 A CN 201610535961A CN 106190112 A CN106190112 A CN 106190112A
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quantum dot
gold
stability
gold quantum
plant
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CN201610535961.1A
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王丽
高翔
陈静
窦润芝
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Northeastern University China
Northeast Normal University
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Northeast Normal University
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
    • 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
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

Abstract

The invention discloses the preparation method and applications of gold quantum dot in plant living body imaging.Golden quantum dot for plant living body imaging provided by the present invention is a kind of bovine serum albumin coated gold quantum dot, utilizes the way of high speed centrifugation diafiltration to be purified it, utilizes spectrofluorophotometer to detect its pH stability and H2O2Stability.The present invention utilizes the photoluminescent property of gold quantum dot by using gold quantum dot to process plant, plant carries out tissue tabletting and live body vibration section, carries out the living imaging of plant cell and tissue under fluorescence microscope.

Description

The preparation method and applications of gold quantum dot
Technical field
The invention belongs to materials application technical field, be specifically related to the preparation side of a kind of bovine serum albumin coated gold quantum dot Method, and it is applied to the living imaging of plant cell and tissue.
Background technology
Nano material refers to that the size of a most at least dimension is between 1-100 nm or by being less than The material of the elementary cell composition of 100 nm.Along with the quick stable development of nanometer science and technology, nano material is the most extensive It is applied in the various fields such as agricultural, medicine, food, cosmetics, article of everyday use.But producing, using and during process etc. Nano material can inevitably be transmitted in the middle of ecological environment, and may cause organism and ecological environment further can not The impact of precognition, thus the environmental behaviour of nano material and poisonous effect thereof have caused the extensive concern of research worker.Plant It is the Producer of ecosystem, can contact and absorb dispersion nano material in the environment to a great extent, so that nanometer Material is entered in animal even human body by food chain further, thus increasing people lays particular emphasis on nano-scale material pair The impact of plant.
Gold quantum dot (AuNCs) is a kind of novel fluorescent nano material, is generally made up of several to 100 gold atoms, It is smaller in size than 2 nm.They have, and fluorescence intensity is big, toxicity is low, water solublity is good, biocompatibility advantages of higher, thus is giving birth to The aspects such as thing sensing and bio-imaging are widely used.Current only animal about the application of imaging in cell of gold quantum dot at present Cell has been reported that, and for the gold research of quantum dot living imaging in plant and have no relevant report.
Summary of the invention
It is an object of the present invention to provide the preparation method and applications of a kind of gold quantum dot.
The technical scheme is that:
The golden quantum dot for plant living body imaging provided, specifically can prepare and purify according to the method comprised the steps Obtain:
(a1) by the gold chloride (HAuCl of 50 mL4) aqueous solution (10 mM, 37 DEG C) joins the bovine serum albumin of 50 mL (BSA), in aqueous solution (50 mg/L, 37 DEG C), sodium hydroxide (NaOH) aqueous solution (1 mol/ of 5 mL after stirring 5 min, is added L), 37 DEG C of water-bath 12 h.
(a2) prepared golden quantum dot 14,000 r/min is centrifuged 30 min, takes supernatant and join bag filter and (retain point Son amount be 14 kDa) in dialysis 4 days, degerming with the filtering with microporous membrane that aperture is 0.22 μm afterwards, 4 DEG C of placements are standby.
The present invention provides a kind of method detecting gold quantum dot stability.Specifically include pH stability and H2O2Stability Detection:
(1) pH Detection of Stability: the golden quantum dot after being diluted by purification is 4 with 1M HCl and 1M NaOH furnishing pH respectively, 5,6, The aqueous solution of 7,8,9,10, utilizes spectrofluorophotometer to detect its fluorescence intensity under 480 nm excitation wavelengths.
(2) H2O2Stability analysis: add appropriate H in gold quantum dot2O2, make H2O2Final concentration of 1,10,100 and 1000 mM, utilize spectrofluorophotometer detection gold quantum dot fluorescence intensity under 480 nm excitation wavelengths.
The method that the present invention also provides for the golden quantum dot of preparation is applied to plant living body imaging.Including following concrete step Rapid:
(1) gold quantum dot processes plant: we select the seed do not sprouted to carry out gold quantum dot process, and corn seed is first with 70% Ethanol Treatment 2 min, then by sterile water wash 5 times.By the golden quantum dot (50,100,200,400 mg/L) of variable concentrations with 5 The amount of mL/ ware joins (containing monolayer filter paper) in culture dish, and each culture dish puts into 10 seeds, each process 3 equably Repeating, blank ultra-pure water processes.Being positioned in phjytotron by culture dish, design temperature is 25 DEG C, and humidity is 80%, illumination is that 24 h are dark.
(2) corn root cell tabletting, tissue slice and fluorescence microscopy are observed: expose after terminating, used by corn plant root Ultrapure water, cuts the tip of a root (about 0.5 mm length) and carries out tabletting process and see under fluorescence microscope (NIKON, Japan) Examine.Take root maturation zone 5% Agarose embedding simultaneously, right with LEICA VT1000 S vibratome (Lai Ka company, Germany) It carries out crosscutting.Crosscutting rear maize tissue sample is directly at fluorescence microscopy Microscopic observation.
Accompanying drawing explanation
Fig. 1 is form and the sign of gold quantum dot, and wherein a figure is the high resolution transmission electron microscopy image of gold quantum dot；B schemes Grain size distribution for gold quantum dot；C figure is that the UV-Vis of gold quantum dot absorbs (black line) and fluorescence spectrum (red line, excitation wave A length of 480 nm)；D figure is the photo of gold quantum dot under daylight and uviol lamp (365 nm).The golden quantum dot of preparation is rounded Granule, mean diameter is 2.05 ± 0.41 nm, has absworption peak under 480 nm excite at 638 nm, and at uviol lamp (365 Nm) in cerise under.
Fig. 2 is the pH stability of gold quantum dot, wherein a figure be pH be the golden quantum dot photo in the sunlight of 4-10；B schemes For golden quantum dot that pH the is 4-10 photo under uviol lamp (365 nm)；C figure be pH be that the golden quantum dot of 4-10 is when difference Between the fluorescence spectrum (excitation wavelength is 480 nm) of section.Gold quantum dot fluorescence intensity when pH is 8 is maximum, along with pH increase or Reducing, the fluorescence intensity of gold quantum dot gradually weakens.As pH > 7 time, fluorescence intensity increases over time first to rise and declines, As pH, < when 7, fluorescence intensity increases over time after first undulatory property declines and tends to be steady.
Fig. 3 be gold quantum dot variable concentrations hydrogen peroxide (1,10,100,1000mM) in fluorescence curve after 1 day, H2O2Can make gold quantum dot fluorescence cancellation, concentration is the biggest, and gold quantum dot fluorescence intensity is the most weak, and it is strong to increase over time fluorescence Degree is gradually lowered.The H of final concentration of 100mM and 1000mM2O2The gold complete cancellation of quantum dot fluorescence can be made.
Fig. 4 is the living imaging of Semen Maydis, and wherein a figure is that ultra-pure water processes the 3rd day root cell tabletting (40 × thing of Semen Maydis Mirror)；B figure is that 400 mg/L gold quantum dots process the 3rd day root cell tablettings (40 × object lens) of Semen Maydis, therefrom it can be seen that 400 There is red fluorescent material in the tip of a root exfoliative cyte of Semen Maydis after the process of mg/L gold quantum dot, illustrate that gold quantum dot can penetrate jade The cell wall of rice cell and cell membrane, enter in tip of a root exfoliative cyte.C figure is that 400 mg/L gold quantum dots process Semen Maydis the 3rd day The sectional view (4 × object lens) of root；D figure is the sectional view (20 × object lens) that 400 mg/L gold quantum dots process the 3rd day root of Semen Maydis. Autofluorescence is blueness, and gold quantum dot fluorescence takes on a red color.In figure it can be seen that the Zea mays root maturation zone processed at gold quantum dot Cortical cell there is also a small amount of red fluorescent material, illustrate that gold quantum dot can enter corn root.
Detailed description of the invention
Further illustrating the present invention in following specific embodiment, this does not limit the scope of the invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Reagent: gold chloride (AuCl3·HCl·4H2O, analytical pure) purchased from Shanghai reagent one factory, bovine serum albumin (BSA, Analytical pure) purchased from the intelligent Science and Technology Ltd. of Beijing Ke Er, agarose (Biowest Agarose) is purchased from gene company limited, hydrogen Sodium oxide (NaOH), concentrated hydrochloric acid (HCl), concentrated nitric acid (HNO3), hydrogen peroxide (H2O2) it is analytical pure, purchased from Beijing Chemical Plant.
Material: the corn hybrid seed Zheng Dan 958 that test material provides for Beijing DeNong seeds Co., Ltd, germination percentage >= 95%, preserve before using in the environment being dried at room temperature for dark.
Instrument and apparatus: DF-101Z heat collecting type constant-temperature heating magnetic stirring apparatus (Yu Hua Instrument Ltd. of Henan Province), The intelligent growth cabinet of RXZ (south of the River, Ningbo instrument plant), table model high speed centrifuge (SIGMA company, Germany), Milli-Q surpass Water purification machine (Millipore company, the U.S.), SW-CJ-1F clean bench (SuZhou Antai Air Tech Co., Ltd.), Thermo NanoDrop 1000 spectrophotometer (Nanodrop, the U.S.), Cary Eclipse spectrofluorophotometer are (in watt An company, U.S.), SX-300 high-pressure steam sterilizing pan (KAGOSHIMA SEISAKUSYO company, Japan), nylon66 fiber JTSF pin Cartridge type membrane filter (aperture 0.22 μm, Tianjin Jin Teng experimental facilities Instrument Ltd.), single use aseptic injection (molecular cut off is 14 kDa, raw work biological engineering stock for device (band pin, Shanghai Mishawa Medical Industry Co., Ltd.), bag filter Part company limited, Shanghai), JEOL JEM-2100F high resolution transmission electron microscopy (HR-TEM, Jeol Ltd., Japan), Zetasizer Nano ZS(Malvern Instr Ltd., Britain), fluorescence microscope (NIKON, Japan), LEICA VT1000 S vibratome (Lai Ka company, Germany), SB5200DT ultrasonic washing unit (Ningbo new sesame biotechnology Limited company), pipettor (Eppendorf company, Germany), electronic balance (Mettler Toledo Inc., Shanghai), Pordigy inductively coupled plasma atomic emission spectrum (LEEMAN, the U.S.) etc..
The preparation of embodiment 1 gold medal quantum dot and purification
Experiment preparation and contain the glass container of reagent all by chloroazotic acid immersion treatment and clean dry.Gold chloride by 50 mL (HAuCl4) aqueous solution (10 mM, 37 DEG C) joins bovine serum albumin (BSA) aqueous solution (50 mg/L, 37 DEG C) of 50 mL In, add sodium hydroxide (NaOH) aqueous solution (1 mol/L) of 5 mL, 37 DEG C of water-bath 12 h after stirring 5 min.Will system Golden quantum dot 14,000 r/min obtained is centrifuged 30 min, takes supernatant and joins in bag filter (molecular cut off is 14 kDa) saturating Analysing 4 days, degerming with the filtering with microporous membrane that aperture is 0.22 μm afterwards, 4 DEG C of placements are standby.
The sign of embodiment 2 gold medal quantum dot
Drop on copper mesh after taking a small amount of gold ultrasonic 30 min of quantum dot solution, dried with JEOL JEM-2100F high score under room temperature Distinguish that it is characterized by transmission electron microscope (HR-TEM, Jeol Ltd., Japan).Utilize Nano Measure soft Part calculates gold quantum point grain diameter size.Utilize Zetasizer Nano ZS laser particle analyzer (Malvern Instr Ltd., English State) record gold quantum dot zeta potential.Golden quantum dot (400 mg/L) after being diluted by purification irradiates with the ultraviolet light of 365 nm to be seen Examine its fluorescence color, and survey it with Cary Eclipse spectrofluorophotometer (Varian Associates, Inc. (US) 611 Hansen Way, Palo Alto, California 94303, U.S.A., the U.S.) and excite at 480 nm Fluorescence spectrum under wavelength, utilizes Thermo NanoDrop 1000 spectrophotometer (Nanodrop, the U.S.) to detect its UV- Vis absorption spectrum.
Result such as Fig. 1, the rounded granule of golden quantum dot of preparation, mean diameter is 2.05 ± 0.41 nm, at 480 nm Excite down at 638 nm, have absworption peak, and in cerise under uviol lamp (365 nm).Zetasizer Nano ZS is utilized to swash It is-14.70 ± 0.96 that light particle size analyzer records the zeta potential of gold quantum dot, shows that gold quantum dot is electronegative.
The stability analysis of embodiment 3 gold medal quantum dot
One, pH stability analysis
By purification dilute after golden quantum dot respectively with 1M HCl and 1M NaOH furnishing pH be 4,5,6,7,8,9,10 water-soluble Liquid, utilizes spectrofluorophotometer to survey its fluorescence intensity under 480 nm excitation wavelengths.
Result such as Fig. 2, gold quantum dot fluorescence intensity when pH is 8 is maximum, along with increase or the reduction of pH, gold quantum dot Fluorescence intensity gradually weakens.As pH>7 time, fluorescence intensity increases over time first to rise and declines, and as pH,<when 7, fluorescence is strong Degree increases over time after first undulatory property declines and tends to be steady.
Two, H2O2Stability analysis
Appropriate H is added in gold quantum dot2O2, make H2O2Final concentration of 1,10,100 and 1000 mM, utilize fluorescence spectrophotometer light Degree meter detection gold quantum dot fluorescence intensity under 480 nm excitation wavelengths.
Result is as it is shown on figure 3, H2O2Can make gold quantum dot fluorescence cancellation, concentration is the biggest, and gold quantum dot fluorescence intensity is the most weak, And increase over time fluorescence intensity and be gradually lowered.The H of final concentration of 100mM and 1000mM2O2Gold quantum dot fluorescence can be made complete Full cancellation.
Embodiment 4
1. the exposure of Semen Maydis is processed by gold quantum dot
Corn seed (Zheng Dan 958, Beijing DeNong seeds Co., Ltd) is first with 70% Ethanol Treatment 2 min, then uses sterile water wash 5 times.The golden quantum dot (50,100,200,400 mg/L) of variable concentrations is joined in culture dish (containing single with the amount of 5 mL/ wares Metafiltration paper), 10 seeds put into equably by each culture dish, and each process 3 repetition, blank ultra-pure water processes.Will Culture dish is positioned in growth cabinet, and design temperature is 25 DEG C, and humidity is 80%, and illumination is that 24 h are dark.
2. corn root cell tabletting, tissue slice and fluorescence microscopy are observed
After exposure terminates, by corn plant root ultrapure water, cut the tip of a root (about 0.5 mm length) carry out tabletting process and Observe under fluorescence microscope (NIKON, Japan).Take root maturation zone 5% Agarose embedding simultaneously, use LEICA VT1000 It is carried out crosscutting by S vibratome (Lai Ka company, Germany).
Tabletting and section result such as Fig. 4.Tabletting result shows: gold quantum dot can penetrate the cell wall of maize cell with thin After birth, enters in tip of a root exfoliative cyte.Section result shows in the cortical cell of the Zea mays root maturation zone that gold quantum dot processes Can also be observed that a small amount of red fluorescent material, illustrate that gold quantum dot can enter corn root.

Claims (3)

1. the preparation method of gold quantum dot, is characterized in that comprising the steps:
(1) by the gold chloride HAuCl of 50 mL4Aqueous solution 10 mM, 37 DEG C of bovine serum albumin BSA joining 50 mL are water-soluble Liquid 50 mg/L, in 37 DEG C, adds sodium hydroxide NaOH aqueous solution 1 mol/L of 5 mL, 37 DEG C of water-baths after stirring 5 min 12 h；
(2) prepared golden quantum dot 14,000 r/min being centrifuged 30 min, taking supernatant and joining molecular cut off is 14 kDa Dialysing 4 days in bag filter, degerming with the filtering with microporous membrane that aperture is 0.22 μm afterwards, 4 DEG C of placements are standby.
2. the method for detection gold quantum dot stability, is characterized in that including pH stability and H2O2The detection of stability:
(1) pH Detection of Stability: the golden quantum dot after being diluted by purification is 4 with 1M HCl and 1M NaOH furnishing pH respectively, 5,6, The aqueous solution of 7,8,9,10, utilizes spectrofluorophotometer to detect its fluorescence intensity under 480 nm excitation wavelengths；
(2) H2O2Stability analysis: add appropriate H in gold quantum dot2O2, make H2O2Final concentration of 1,10,100 and 1000 MM, utilizes spectrofluorophotometer detection gold quantum dot fluorescence intensity under 480 nm excitation wavelengths.
3. gold quantum dot application in terms of the living imaging of plant cell and tissue.
CN201610535961.1A 2016-07-10 2016-07-10 The preparation method and applications of gold quantum dot Pending CN106190112A (en)

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Application publication date: 20161207