CN106568749A - Method used for detecting trypsin using aminated graphene quantum dot/BSA complex probe - Google Patents
Method used for detecting trypsin using aminated graphene quantum dot/BSA complex probe Download PDFInfo
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- 239000002096 quantum dot Substances 0.000 title claims abstract description 89
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical class [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 84
- 239000000523 sample Substances 0.000 title claims abstract description 74
- 102000004142 Trypsin Human genes 0.000 title claims abstract description 31
- 108090000631 Trypsin Proteins 0.000 title claims abstract description 31
- 239000012588 trypsin Substances 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 26
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 44
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 44
- 238000001514 detection method Methods 0.000 claims abstract description 34
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 238000012360 testing method Methods 0.000 claims abstract description 8
- 229910021389 graphene Inorganic materials 0.000 claims description 77
- 238000005576 amination reaction Methods 0.000 claims description 66
- 239000000243 solution Substances 0.000 claims description 48
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 16
- 230000005284 excitation Effects 0.000 claims description 11
- 239000008055 phosphate buffer solution Substances 0.000 claims description 11
- 238000002189 fluorescence spectrum Methods 0.000 claims description 10
- 229910021529 ammonia Inorganic materials 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000011835 investigation Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- -1 Graphite alkene Chemical class 0.000 claims description 3
- 239000004809 Teflon Substances 0.000 claims description 3
- 229920006362 Teflon® Polymers 0.000 claims description 3
- 229910002804 graphite Inorganic materials 0.000 claims description 3
- 239000010439 graphite Substances 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000004044 response Effects 0.000 claims description 2
- 238000012956 testing procedure Methods 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 150000001336 alkenes Chemical class 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 201000010099 disease Diseases 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 208000016222 Pancreatic disease Diseases 0.000 abstract 1
- 208000024691 pancreas disease Diseases 0.000 abstract 1
- 206010033645 Pancreatitis Diseases 0.000 description 10
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 7
- 206010033647 Pancreatitis acute Diseases 0.000 description 5
- 201000003229 acute pancreatitis Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000005540 biological transmission Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000006862 quantum yield reaction Methods 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000007306 functionalization reaction Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000510 noble metal Inorganic materials 0.000 description 2
- 229950000845 politef Drugs 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- BFKJFAAPBSQJPD-UHFFFAOYSA-N tetrafluoroethene Chemical compound FC(F)=C(F)F BFKJFAAPBSQJPD-UHFFFAOYSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 240000005589 Calophyllum inophyllum Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000001027 hydrothermal synthesis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- 239000002086 nanomaterial Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
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- 238000005424 photoluminescence Methods 0.000 description 1
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- 238000000197 pyrolysis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
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- 238000006268 reductive amination reaction Methods 0.000 description 1
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- 230000028327 secretion Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
Classifications
-
- 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"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/067—Pancreatitis or colitis
Abstract
The invention provides a method used for detecting trypsin using an aminated graphene quantum dot/BSA complex probe. The method comprises following steps: 1, preparation of aminated graphene quantum dot; 2, preparation of the aminated graphene quantum dot/ bovine serum albumin complex probe; 3, fluorescence testing; and 4, detection of trypsin. The aminated graphene quantum dot/BSA complex probe possesses following advantages: a preparation method is simple; fluorescence stability is high; operation in detection of trypsin is convenient and rapid, and is high in sensitivity; application prospect in the field of disease diagnosis and treating is promising; and the novel method is provided for rapid high efficiency detection of the content of trypsin of patients with pancreatic diseases.
Description
Technical field
The present invention relates to a kind of amination graphene quantum dot/BSA combined probes detect tryptic method, belong to glimmering
Light probe material tests field.
Background technology
Acute pancreatitis (AP) are a kind of fatal diseases, are classified as persistence pancreatitiss disease.Overall acute pancreatitis
The mortality rate of patient records the case number made a definite diagnosis also in sharp increase 7% or so.However, the diagnosis and treatment of AP remain one
Clinical signature of the very big challenge in default of the ripe biomarker of special disease and research.Research and development are a kind of true in biopsy
Before examining can effective, quick, reliable Screening Diagnosis AP be necessary.Trypsin is a kind of digestion in pancreas
Enzyme, what content was mainly controlled by the trypsin inhibitor of pancreatic secretion, the trypsin of overexpression can cause multiple protein
The final destroying cells of overexpression of enzyme.The trypsin of overexpression generally detected from the urine of pancreatitiss patient, therefore
Detection by quantitative in urine specimen is considered as the noinvasive labelling of pancreatitiss screening.Enzyme linked immunosorbent assay is trypsin detection
A kind of common method, it is however, this method is expensive, time-consuming and usually require that sample size is more.Sample preanalysis are purified
Process can cause the loss of sample, therefore can sacrifice the accuracy and repeatability of testing result.Based on the detection method of fluorescence it is
The method of another protease detection by quantitative.In past 10 years, the method based on the fluoroscopic examination of bioanalysiss is wide
It is general for detecting proteolytic enzyme, protein, nucleic acid, small molecule etc., while this method is also provided and a kind of effectively spends fast
The method of speed detection enzyme.In all of fluorescent nano material, quantum dot is widely used in bio-sensing.
Quantum dot is a kind of zero dimensional nanometer materials that fluorescence can be produced under excitation, and its radius is less than or is close to
Bohr radius.Due to the particle diameter very little of quantum dot, there is quantum confined effect, continuous energy level structure becomes with molecular characterization
Level structure, so as to launch strong fluorescence.Due to the dimensional effect and dielectric effect of quantum dot, the luminous performance of quantum dot
Go out unique property.The characteristic of quantum dot light emitting mainly has：With wider excitation spectrum, can be adjusted by size Control
Save fluorescence, there is longer fluorescence lifetime with higher light stability.Quantum dot has abundant marker color, higher
Luminous intensity and the advantages of longer observing time, become and can substitute organic fluorescent dye and visit as fluorescence
Pin, is applied in bio-imaging, biological detection and environment measuring.But as semiconductor-quantum-point has certain biology poison
Property, the expensive application limited in terms of its biology of noble-metal nanoclusters.By contrast, some hypotoxic quantum dots are such as
Silicon quantum dot, carbon quantum dot and graphene quantum dot have broad application prospects in biological detection.Wherein Graphene quantum
Point (Wang Y, Zhang L, Liang R P, et al.Using graphene quantum dots as
photoluminescent probes for protein kinase sensing[J].Analytical chemistry,
2013,85(19):9148-9155.) due to extensive research that its unique physico-chemical property causes in terms of biological detection.
(Hu L, Han S, Parveen S, the et al.Highly sensitive fluorescent detection of such as Hu
trypsin based on BSA-stabilized gold nanoclusters[J].Biosensors and
Bioelectronics,2012,32(1):297-299.) report using BSA stable gold nanoclusters detection trypsin, plus
After entering trypsin, the fluorescence intensity of BSA stable gold nanoclusters substantially weakens, while the biocompatibility of quantum dot increases
By force, tryptic concentration can be detected according to the change of fluorescence intensity.But nano metal is in noble metal in this method,
Testing cost is high.(Poon C Y, Li Q, Zhang J, the et al.FRET-based modified graphene such as Poon
quantum dots for direct trypsin quantification in urine[J].Analytica chimica
acta,2016,917:A kind of FRET (fluorescence resonance energy transfer) detection trypsin of utilization graphene quantum dot is reported 64-70.)
Method, but the selection of FRET (fluorescence resonance energy transfer) pair and connection remain a big problem of FRET (fluorescence resonance energy transfer).Cause
This, works out easy, inexpensive tryptic detection method important for the detection of the relevant diseases such as pancreatitiss has
Research Significance.
Graphene quantum dot extensive concern as its unique water solublity and fluorescent stability are caused, but due to which
Size is little, fluorescence quantum yield is relatively low, typically can strengthen its functionalization or modification in biological detection in practical application
Its fluorescence quantum yield (Liu C, Zhang P, Tian F, et al.One-step synthesis of surface
passivated carbon nanodots by microwave assisted pyrolysis for enhanced
multicolor photoluminescence and bioimaging[J].Journal of Materials
Chemistry,2011,21(35):13163-13167.), reduce the Fluorescence-quenching for causing because quantum dot itself is reunited.
The functionalization of graphene quantum dot and modification simultaneously has great importance for the enhancing quanta point biological compatibility.Amination amount
A kind of structure of the Graphene that son point terminates with new amino, with good fluorescence controllability (Tetsuka H,
Asahi R,Nagoya A,et al.Optically Tunable Amino‐Functionalized Graphene
Quantum Dots[J].Advanced materials,2012,24(39):5333-5338.).BSA be a kind of water solublity very
Good protein molecule, can be with amination graphene quantum dot as noncovalent interaction power is compound, the combined probe tool of formation
There are good water solublity and biocompatibility, can be good at for biological detection.Up to the present, amidized Graphene amount
Son is put the research for biological detection and is not also had been reported that.
Change in view of the fluorescence intensity of combined probe has specific function for the detection of trypsin amount, and this is
The trypsin amount of simple detection pancreatitiss patient provides a class new method.
The content of the invention
The present invention provides a kind of amination graphene quantum dot/bovine serum albumin combined probe is used for trypsin detection
Method.
The technical solution used in the present invention is：Amination graphene quantum dot is prepared for by the method for Hydrothermal Synthesiss first
(af-GQDs), then by af-GQDs it is combined with bovine serum albumin (BSA), prepares bovine serum albumin/amination graphene quantum
Strengthening occur in point combined probe (af-GQDs/BSA), the fluorescence of combined probe, and fluorescence quantum yield increases.Finally with af-
GQDs/BSA combined probes are fluorescent detection probe, for tryptic detection, have investigated which to tryptic detection effect
Really.With the increase of trypsin amount, the fluorescence of combined probe strengthens rapidly, can draw pancreas according to the change of fluorescence intensity
The content of protease.The combined probe has good fluorescent stability, for tryptic detection with easy to operate and
High sensitivity, has broad application prospects in terms of the Clinics and Practices of disease.
The present invention is achieved through the following technical solutions：
(amination graphene quantum dot/bovine serum albumin is combined a kind of amination graphene quantum dot/BSA combined probes
Probe) tryptic method is detected, comprise the steps：
The preparation of step 1, amination graphene quantum dot (af-GQDs)：
Prepare graphene oxide (GO) first, prepare the GO solution of 1.0mg/mL, measure GO solution, 3 that 5mL prepares～
The ammonia and 5mL redistilled water mix homogeneously of 9mL mass fractions 28%, is transferred them to after continuing stirring 30min rapidly poly-
Constant temperature thermal response is carried out in tetrafluoroethene autoclave, room temperature is cooled to, using the teflon membrane filter sucking filtration of 0.22um,
Obtain filtrate；Filtrate is heated into 1h at 100 DEG C and removes unnecessary ammonia；Resulting solution is used into the dialysis bag dialysis 24h of 10kDa,
Amination graphene quantum dot is obtained after vacuum lyophilization；
The preparation of step 2, amination graphene quantum dot/bovine serum albumin combined probe：
The amination graphene quantum dot solution for preparing the phosphate buffer solution and 0.1mg/mL of pH=8 is stand-by；Violent
Measure amination graphene quantum dot solution 4mL, phosphate buffer solution 4mL and BSA solution 2mL under conditions of stirring respectively, mix
Close, wherein the concentration of BSA solution is 1mg/mL, is placed in Constant temperature hatch at 37 DEG C in the solution for mixing, you can obtain amination
Graphene quantum dot/bovine serum albumin combined probe；Combined probe solution is placed at 4 DEG C and preserves stand-by；
Step 3, fluorometric investigation：
The amination graphene quantum dot solution of 0.1mg/mL prepared by step 2 carries out fluorescence spectrum test, obtains
The a length of Ex=340nm of optimum excitation wave, optimal launch wavelength are Em=431nm；Testing procedure 2 is obtained at identical conditions
Amination graphene quantum dot/bovine serum albumin combined probe, when excitation wavelength be Ex=340nm, the transmitted wave of combined probe
A length of Em=445nm；The fluorescence emission wavelengths of the amination graphene that compares quantum dot, the fluorescence emission wavelengths of combined probe are sent out
Red shift is given birth to, and the fluorescence intensity of combined probe has strengthened；
Step 4, tryptic detection：
Amination graphene quantum dot/bovine serum albumin combined probe the 2mL for preparing is measured, adds the pH=8's of 2mL
Phosphate buffer solution mix homogeneously, adds the trypsin solution 1mL of variable concentrations, and mix homogeneously is hatched at 37 DEG C, inspection
Survey its launch wavelength and fluorescence intensity when excitation wavelength is Ex=340nm.
Wherein, in step 1, described constant temp. heating reaction temperature is 70～150 DEG C, and the time of reaction is 4～6h.
Wherein, in step 2, at described 37 DEG C, the Constant temperature hatch time is 2～12h.
Wherein, in step 3, the fluorescence emission wavelengths of described combined probe there occurs red shift, and combined probe is glimmering
Light intensity strengthens.Possible the reason for is the interaction of the amino and carboxyl of amination graphene quantum dot solution and BSA solution
And the effect of hydrogen bond causes the defect of quantum dot surface to be repaired, and reduces the defect of quantum dot surface so that quantum dot
Fluorescence Increasing.
Wherein, in step 4, during described detection trypsin, as trypsin can decompose bovine serum albumin, destruction
The structure of combined probe, while trypsin decomposes the lysine that produces and arginine being capable of reductive amination Graphene quantum
Point, causes the fluorescence of quantum dot to strengthen rapidly.
Wherein, in step 4, described tryptic detection range is 0～100 μ g/mL.
Wherein, in step 4, at described 37 DEG C, brooding time is 15min～2h.
The beneficial effects of the present invention is：
Amination graphene quantum dot/bovine serum albumin combined probe prepared by the present invention has preparation method simple, glimmering
The characteristics of light stability is strong.To tryptic detection with easy to operate, quick and high sensitivity, the diagnosis of disease with control
Have broad application prospects in terms for the treatment of.This provides a kind of new side for the trypsin amount for rapidly and efficiently detecting pancreas patient
Method.
Description of the drawings
In Fig. 1, transmission electron microscope pictures of a for amination graphene quantum dot is schemed, figure b is amination graphene quantum dot/cattle
The transmission electron microscope picture of serum albumin combined probe；
In Fig. 2, fluorescence emission spectrums of the curve a for amination graphene quantum dot, curve b are amination graphene quantum
The fluorescence emission spectrum of point/bovine serum albumin combined probe, curve c are compound for amination graphene quantum dot/bovine serum albumin
Fluorescence emission spectrum of the probe after trypsin is added.
Specific embodiment
With reference to example is embodied as, the present invention will be further described.
Embodiment 1：
(1) preparation of amination graphene quantum dot (af-GQDs)
First according to Hummers methods (Hummers Jr W S, Offeman R E.Preparation of graphitic
oxide[J].Journal of the American Chemical Society,1958,80(6):1339-1339.) prepare
Go out graphene oxide (GO), prepare the GO solution of 1.0mg/mL, measure the GO solution 5mL for preparing, the ammonia of mass fraction 28%
Water 3mL, and the redistilled water mix homogeneously of 5mL, transfer them to rapidly politef high pressure after continuing stirring 30min anti-
In answering kettle, 4h is reacted at 70 DEG C, be cooled to room temperature, using the teflon membrane filter sucking filtration of 0.22um, obtain filtrate.Filtrate is existed
1h is heated at 100 DEG C and removes unnecessary ammonia.By resulting solution using the dialysis bag dialysis 24h of 10kDa, make after vacuum lyophilization
Obtain amination graphene quantum dot.
(2) preparation of amination graphene quantum dot/bovine serum albumin combined probe
Prepare pH be 8 phosphate buffer solution and 0.1mg/mL amination graphene quantum dot solution it is stand-by.Violent
Measure amination graphene quantum dot solution 4mL, phosphate buffer solution 4mL and BSA solution 2mL under conditions of stirring respectively, mix
Close, wherein the concentration of BSA solution is 1mg/mL, is placed in Constant temperature hatch 2h at 37 DEG C in the solution for mixing, you can obtain amino
Graphite alkene quantum dot/bovine serum albumin combined probe.Combined probe solution is placed at 4 DEG C and preserves stand-by.
(3) fluorometric investigation
The amination graphene quantum dot solution of the 0.1mg/mL of preparation is carried out into fluorescence spectrum test, what is obtained is optimal sharp
It is Ex=340nm to send out wavelength, and optimal launch wavelength is Em=431nm.Amination graphene quantum is tested at identical conditions
Point/bovine serum albumin combined probe, when excitation wavelength is Ex=340nm, the launch wavelength of combined probe is Em=445nm.Phase
Compare the fluorescence emission wavelengths of amination graphene quantum dot, the fluorescence emission wavelengths of combined probe there occurs red shift, and multiple
The fluorescence intensity for closing probe strengthens.
(4) tryptic detection
Amination graphene quantum dot/bovine serum albumin combined probe the 2mL for preparing is measured, the pH for adding 2mL is 8
Phosphate buffer solution mix homogeneously, adds the trypsin solution 1mL that concentration range is 0～100ug/mL, mix homogeneously,
Hatch 15min at 37 DEG C, detect its launch wavelength and fluorescence intensity when excitation wavelength is Ex=340nm, fluorescent emission ripple
Long position is basically unchanged, and fluorescence intensity strengthens, and trypsin enhances the fluorescence of combined probe.
Embodiment 2：
(1) preparation of amination graphene quantum dot (af-GQDs)
Graphene oxide (GO) is prepared according to Hummers methods first, the GO solution of 1.0mg/mL is prepared, is measured and prepare
GO solution 5mL, the ammonia 36mL of mass fraction 28%, and the redistilled water mix homogeneously of 5mL, after continuing stirring 30min
Transferred them in politef autoclave rapidly, 6h is reacted at 150 DEG C, be cooled to room temperature, using the poly- of 0.22um
Tetrafluoroethene filter membrane sucking filtration, obtains filtrate.Filtrate is heated into 1h at 100 DEG C and removes unnecessary ammonia.Resulting solution is used into 10kDa
Dialysis bag dialysis 24h, amination graphene quantum dot is obtained after vacuum lyophilization.
(2) preparation of amination graphene quantum dot/bovine serum albumin combined probe
Prepare pH be 8 phosphate buffer solution and 0.1mg/mL amination graphene quantum dot solution it is stand-by.Violent
Measure amination graphene quantum dot solution 4mL, phosphate buffer solution 4mL and BSA solution 2mL under conditions of stirring respectively, mix
Close, wherein the concentration of BSA solution is 5mg/mL, is placed in Constant temperature hatch 12h at 37 DEG C in the solution for mixing, you can obtain amino
Graphite alkene quantum dot/bovine serum albumin combined probe.Combined probe solution is placed at 4 DEG C and preserves stand-by.
(3) fluorometric investigation
The amination graphene quantum dot solution of the 0.1mg/mL of preparation is carried out into fluorescence spectrum test, what is obtained is optimal sharp
It is Ex=340nm to send out wavelength, and optimal launch wavelength is Em=429nm.Amination graphene quantum is tested at identical conditions
Point/bovine serum albumin combined probe, when excitation wavelength is Ex=340nm, the launch wavelength of combined probe is Em=447nm.Phase
Compare the fluorescence emission wavelengths of amination graphene quantum dot, the fluorescence emission wavelengths of combined probe there occurs red shift, and multiple
The fluorescence intensity for closing probe strengthens.
(4) tryptic detection
Amination graphene quantum dot/bovine serum albumin combined probe the 2mL for preparing is measured, the pH for adding 2mL is 8
Phosphate buffer solution mix homogeneously, adds the trypsin solution 1mL that concentration range is 0～100ug/mL, mix homogeneously,
Hatch 2h at 37 DEG C, detect its launch wavelength and fluorescence intensity when excitation wavelength is Ex=340nm, fluorescence emission wavelengths position
Put and be basically unchanged, fluorescence intensity strengthens, trypsin enhances the fluorescence of combined probe.
In Fig. 1, transmission electron microscope pictures of a for amination graphene quantum dot is schemed, figure b is amination graphene quantum dot/cattle
The transmission electron microscope picture of serum albumin combined probe；From figure a and figure b, amination graphene quantum dot size is uniform, is combined
In probe, amination graphene quantum dot is adsorbed onto on bovine serum albumin and quantum dot degree of scatter is preferable.
In Fig. 2, fluorescence emission spectrums of the curve a for amination graphene quantum dot, curve b are amination graphene quantum
The fluorescence emission spectrum of point/bovine serum albumin combined probe, curve c are compound for amination graphene quantum dot/bovine serum albumin
Fluorescence emission spectrum of the probe after trypsin is added.As shown in Figure 2, compared with amination graphene quantum dot, it is combined
The fluorescence of probe occurs red shift and fluorescence intensity to be strengthened.After adding trypsin, the fluorescence intensity of combined probe increases rapidly
By force.
Claims (5)
1. a kind of amination graphene quantum dot/BSA combined probes detects tryptic method, it is characterised in that include as
Lower step：
The preparation of step 1, amination graphene quantum dot：
Graphene oxide GO is prepared first, is prepared the GO solution of 1.0mg/mL, is measured GO solution, 3～9mL matter that 5mL is prepared
The ammonia and 5mL redistilled water mix homogeneously of amount fraction 28%, transfers them to rapidly polytetrafluoroethyl-ne after continuing stirring 30min
Constant temperature thermal response is carried out in alkene autoclave, room temperature is cooled to, using the teflon membrane filter sucking filtration of 0.22um, must be filtered
Liquid；Filtrate is heated into 1h at 100 DEG C and removes unnecessary ammonia；Resulting solution is used into the dialysis bag dialysis 24h of 10kDa, vacuum
Amination graphene quantum dot is obtained after lyophilization；
The preparation of step 2, amination graphene quantum dot/bovine serum albumin combined probe：
The amination graphene quantum dot solution for preparing the phosphate buffer solution and 0.1mg/mL of pH=8 is stand-by；It is being stirred vigorously
Under conditions of measure amination graphene quantum dot solution 4mL, phosphate buffer solution 4mL and BSA solution 2mL respectively, mix, its
The concentration of middle BSA solution is 1mg/mL, is placed in Constant temperature hatch at 37 DEG C in the solution for mixing, you can obtain amination graphene
Quantum dot/bovine serum albumin combined probe；Combined probe solution is placed at 4 DEG C and preserves stand-by；
Step 3, fluorometric investigation：
The amination graphene quantum dot solution of 0.1mg/mL prepared by step 2 carries out fluorescence spectrum test, and what is obtained is optimal
Excitation wavelength is Ex=340nm, and optimal launch wavelength is Em=431nm；The amino that testing procedure 2 is obtained at identical conditions
Graphite alkene quantum dot/bovine serum albumin combined probe, when excitation wavelength is Ex=340nm, the launch wavelength of combined probe is
Em=445nm；The fluorescence emission wavelengths of the amination graphene that compares quantum dot, the fluorescence emission wavelengths of combined probe there occurs
Red shift, and the fluorescence intensity enhancing of combined probe；
Step 4, tryptic detection：
Amination graphene quantum dot/bovine serum albumin combined probe the 2mL for preparing is measured, the phosphoric acid of the pH=8 of 2mL is added
Buffer solution mix homogeneously, adds the trypsin solution 1mL of variable concentrations, and mix homogeneously is hatched at 37 DEG C, detects which
Launch wavelength and fluorescence intensity when excitation wavelength is Ex=340nm.
2. a kind of amination graphene quantum dot/BSA combined probes detect tryptic method, it is characterised in that step 1
In, described constant temp. heating reaction temperature is 70～150 DEG C, and the time of reaction is 4～6h.
3. a kind of amination graphene quantum dot/BSA combined probes detect tryptic method, it is characterised in that step 2
In, at described 37 DEG C, the Constant temperature hatch time is 2～12h.
4. a kind of amination graphene quantum dot/BSA combined probes detect tryptic method, it is characterised in that step 4
In, described tryptic detection range is 0～100 μ g/mL.
5. a kind of amination graphene quantum dot/BSA combined probes detect tryptic method, it is characterised in that step 4
In, at described 37 DEG C, brooding time is 15min～2h.
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