CN105842210A - Thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer - Google Patents

Thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer Download PDF

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CN105842210A
CN105842210A CN201610167375.6A CN201610167375A CN105842210A CN 105842210 A CN105842210 A CN 105842210A CN 201610167375 A CN201610167375 A CN 201610167375A CN 105842210 A CN105842210 A CN 105842210A
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thrombin
quantum dot
nps
dots
biological
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CN105842210B (en
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梁汝萍
匡岚
邱建丁
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Nanchang University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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"
    • G01N2021/6432—Quenching

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Abstract

Belonging to the technical field of optical sensing, the invention discloses a thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer. A thrombin aptamer is employed as the template to prepare bio-dots, the bio-dots are mixed with a to-be-tested thrombin sample, then the mixed solution is added into an Au NPs solution, at the moment, the fluorescence intensity of the bio-dots is directly correlated with the concentration of thrombin, and the concentration of thrombin is judged according to the fluorescence intensity of bio-dots.

Description

Based on biological quantum dot and Au NPs The thrombin detection method of FRET (fluorescence resonance energy transfer)
Technical field
The present invention relates to a kind of based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), belong to optical sensing technical field.
Background technology
Thrombin generally exists in human body, is a kind of endo protease playing thrombin function in physiology and pathological process, Fibrinogen can be changed into fibrin.Thrombin also has the character of similar hormone, plays an important role during thrombosis and platelet activation.Therefore, thrombin plays key player in many cardiovascular disease, has inflammation and the effect of tissue repair on modulating vascular wall.Thrombin is also used as therapeutic agent and biomarker for diagnosing multiple disease, such as pulmonary metastases and relevant dysfunction of blood coagulation etc..Therefore, major disease early diagnosis and related drugs are researched and developed significant by the thrombin detection method setting up simple and sensitive.
Biological quantum dot (bio-dots) is a kind of novel fluorescence quantum dot risen recently, and it synthesizes with DNA for template under the conditions of low-temperature hydrothermal, and method is simple, green.Bio-dots has relatively low cytotoxicity, stable photoluminescent property, good water solublity and biocompatibility, in addition, bio-dots not only has the photoluminescent property similar to carbon quantum dot, also maintaining the basic function of DNA profiling, the above characteristic of bio-dots is that its extensively application in bio-sensing field is laid a good foundation.
FRET (fluorescence resonance energy transfer) (FRET) is the radiationless energy transfer between a kind of energy donor and receptor, is a kind of energy transfer technique relevant to distance.Golden nanometer particle (Au NPs) has the optical property that the strongest distance is relevant, and has high quencher efficiency in the widest wave-length coverage, is therefore widely used as the receptor of FRET (fluorescence resonance energy transfer).But, there is not yet the correlational study of FRET between Au NPs and bio-dots, also have no its application in thrombin detects.
Summary of the invention
It is an object of the invention to provide a kind of based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), the method has highly sensitive and that selectivity is good feature to the detection of thrombin.
The present invention is achieved like this, a kind of based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), it is characterized in that, with thrombin aptamer for the biological quantum dot of template preparation, biology quantum dot is mixed with thrombin testing sample, then mixed solution is joined in Au NPs solution, now, the biological fluorescence intensity of quantum dot is proportionate with the concentration of thrombin, judges the concentration of thrombin according to the fluorescence intensity of biological quantum dot.
The present invention is by the following technical solutions:
(1) preparation of biological quantum dot: thrombin aptamer ultra-pure water is dissolved, solution is transferred in politef autoclave, reacts 12 h in 100 ° of C, make thrombin aptamer biology quantum dot；
(2) based on biological quantum dot and Au NPs FRET (fluorescence resonance energy transfer) detection thrombin: in phosphate buffer, biology quantum dot is mixed with thrombin, 1 h is hatched at 37 ° of C, mixed solution is joined in Au NPs solution, utilize fluorescence spectrophotometer to measure the fluorescence intensity of biological quantum dot.
In said method, described phosphate buffering liquid concentration is 10 mM, and pH is 7.4, containing 50 mM NaCl, 5 mM KCl and 4 mM MgCl2；Described biological quantum dot concentration is 80 nM.
The solution have the advantages that: the present invention uses hydrothermal reaction at low temperature to prepare biological quantum dot with thrombin aptamer for template, quantum dot is combined with Au NPs and makes Au NPs assemble, the absorption spectrum of Au NPs overlaps with the emission spectrum of quantum dot simultaneously, makes quantum dot and Au NPs occur FRET (fluorescence resonance energy transfer) to cause quantum dot fluorescence to weaken；When sample exists thrombin, two aptamers binding sites of thrombin make it make more Au NPs combining quantum dot assemble as bridge, a large amount of gatherings cause the absorption spectrum generation red shift of Au NPs, thus reduce with the emission spectrum lap of quantum dot, FRET (fluorescence resonance energy transfer) effect between quantum dot and Au NPs is reduced, causes the fluorescence of quantum dot not to be quenched；The fluorescence intensity of quantum dot is proportionate with the concentration of thrombin, realizes the detection to thrombin accordingly, and the method has stable, highly sensitive and that selectivity is good advantage.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of biological quantum dot.
Fig. 2 is (a) 80 nM biology quantum dot, (e) 80 nM biology quantum dot+Au NPs and the emission spectrum of (g) 80 nM biology quantum dot+80 nM thrombin+Au NPs, (b) biological quantum dot, (c) Au NPs, (d) 80 nM biology quantum dot+Au NPs and the uv-visible absorption spectra of (f) 80 nM biology quantum dot+80 nM thrombin+Au NPs.
Fig. 3 is (A) Au NPs, (B) Au NPs+80 nM biology quantum dot and the transmission electron microscope picture of (C) Au NPs+80 nM biology quantum dot+80 nM thrombin.
Fig. 4 is the fluorescence spectrum figure of (A) Au NPs+ biology quantum dot+variable concentrations thrombin, (B) linear relationship curve.
Fig. 5 be Au NPs+80 nM biology quantum dot (Blank) and respectively with thrombin (Tb), bovine serum albumin (BSA), horseradish peroxidase (HRP), acetylcholinesterase (AchE), trypsin Try), Chymetin (α-CT), hemoglobin (Hb) effect after maximum absorption band situation of movement (Δ λmax)。
Detailed description of the invention
The present invention is further elaborated with specific embodiment below in conjunction with the accompanying drawings, and the present invention is not limited to this；
Embodiment 1
(1) preparation of biological quantum dot: thrombin aptamer (TBA) ultra-pure water is dissolved, solution is transferred in politef autoclave, is heated to 100 ° of C and reacts 12 h, make thrombin aptamer biology quantum dot (TBA-dots).
(2) preparation of Au NPs: by the HAuCl of 2 mL4(1% wt) and 48 mL ultra-pure water mix homogeneously, transfer to be heated to boiling in conical flask and be stirred vigorously；Rapidly joining 1 mL sodium citrate (5% wt) solution after solution boiling, solution colour, from the faint yellow peony that slowly becomes, continues stirring 5 min, is cooled to room temperature, is prepared as Au NPs solution, save backup at 4 ° of C.
Using transmission electron microscope to characterize the pattern of TBA-dots, result is as shown in Figure 1.As seen from Figure 1, TBA-dots is spherical in shape and even particle size distribution, and mean diameter is about 1.6 nm.Use fluorescence spectrum and uv-visible absorption spectra that the optical property of TBA-dots is characterized.From Figure 2 it can be seen that when a length of 320 nm of optimum excitation wave, the maximum emission peak of TBA-dots is positioned at 425 nm(curve a) and does not moves with the change of excitation wavelength.TBA-dots has strong ultraviolet characteristic absorption peak at 260 nm, and (curve b), Au NPs has strong ultraviolet characteristic absorption peak (curve c) at 520 nm absorption band at 320-420 nm；After adding TBA-dots in Au NPs, a large amount of amino that surface is contained make TBA-dots can be assembled into Au NPs surface formation " shell " encapsulated by structures by Au-N key effect and Au NPs, golden nanometer particle is caused to be assembled, Au NPs absworption peak at 520 nm weakens, and at 660 nm, occur in that new absworption peak (curve d), simultaneously because the emission spectrum of the absorption spectrum of Au NPs and TBA-dots overlaps, and Au NPs has high quencher efficiency in the widest wave-length coverage so that the fluorescence of TBA-dots weakens (curve e) significantly.When there is thrombin in sample, thrombin contains two aptamers binding sites can make more Au NPs combining TBA-dots assemble as bridge, causes the further red shift of absworption peak (curve f), simultaneously the absworption peak reduction at 520 nm of Au NPs；Now, the absorption spectrum of Au NPs reduces with the emission spectrum lap of quantum dot, causes the FRET (fluorescence resonance energy transfer) effect between the Au NPs of TBA-dots and Severe aggregation to reduce, and the fluorescent weakening degree of TBA-dots declines (curve g).
Embodiment 2
Based on biological quantum dot and Au NPs FRET (fluorescence resonance energy transfer) detection thrombin
At phosphate buffer, (concentration 10 mM, pH 7.4, containing 50 mM NaCl, 5 mM KCl, 4 mM MgCl2In), the TBA-dots of 80 nM is mixed with the thrombin of variable concentrations, 1 h is hatched at 37 ° of C, mixed solution is added in the Au NPs solution of 2.3 nM, fluorescence spectrophotometer is utilized to measure fluorescence intensity (excitation voltage 700 V of TBA-dots, slit width 10 nm, excitation wavelength 320 nm, wavelength scanning range 350-570 nm).
From Fig. 3 A, Au NPs, there is good dispersibility；After adding TBA-dots in Au NPs solution, TBA-dots is assembled into Au NPs surface by Au-N key effect and forms " shell " encapsulated by structures and Au NPs, Au NPs is caused to assemble (Fig. 3 B), simultaneously, owing to the absorption spectrum of Au NPs overlaps with the emission spectrum of TBA-dots, TBA-dots Yu Au NPs occurs FRET to make TBA-dots fluorescent weakening；When sample exists thrombin, thrombin can be combined with two TBA-dots, as bridge further TBA-dots parcel Au NPs distance, more Au NPs is made to assemble (Fig. 3 C), cause the further red shift of maximum absorption band of Au NPs that the FRET between TBA-dots and Au NPs is weakened, TBA-dots fluorescence is not quenched (Fig. 4), and fluorescence intensity is proportionate with concentration of thrombin, can detect thrombin accordingly.From fig. 4, it can be seen that along with the increase of concentration of thrombin, the fluorescence of TBA-dots gradually strengthens, this fluorescence intensity and concentration of thrombin are in good linear relationship in the range of 8-160 nM, and detection is limited to 6.5 nM, it is achieved that quickly detecting with susceptiveness thrombin.
In order to prove the selectivity that thrombin is detected by this method, with Au NPs+ biology quantum dot for blank (Blank), choose bovine serum albumin (BSA), horseradish peroxidase (HRP), acetylcholinesterase (AchE), trypsin Try), Chymetin (α-CT) and hemoglobin (Hb) be that interfering component is investigated.As seen from Figure 5, when only acting on thrombin (Tb), the maximum absorption band of Au NPs just can occur obvious red shift, maximum absorption band movement value (Δ λmax) shuffling reaches 68 nm；And when adding other interfering component in Au NPs+ biology quantum dot, Δ λmaxMobile the least.Result above shows, the inventive method has good selectivity to thrombin detection.

Claims (4)

1. based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), it is characterized in that, with thrombin aptamer for the biological quantum dot of template preparation, biology quantum dot is mixed with thrombin testing sample, then mixed solution is joined in Au NPs solution, now, the biological fluorescence intensity of quantum dot is proportionate with the concentration of thrombin, judges the concentration of thrombin according to the fluorescence intensity of biological quantum dot.
The most according to claim 1 based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), it is characterised in that step is as follows:
(1) preparation of biological quantum dot: thrombin aptamer ultra-pure water is dissolved, solution is transferred in politef autoclave, reacts 12 h in 100 ° of C, make thrombin aptamer biology quantum dot；
(2) based on biological quantum dot and Au NPs FRET (fluorescence resonance energy transfer) detection thrombin: in phosphate buffer, biology quantum dot is mixed with thrombin, 1 h is hatched at 37 ° of C, mixed solution is joined in Au NPs solution, utilize fluorescence spectrophotometer to measure the fluorescence intensity of biological quantum dot.
3. as claimed in claim 2 based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), it is characterized in that in step (2), described phosphate buffering liquid concentration is 10 mM, and pH is 7.4, containing 50 mM NaCl, 5 mM KCl and 4 mM MgCl2。
4. as claimed in claim 2 based on biological quantum dot and the thrombin detection method of Au NPs FRET (fluorescence resonance energy transfer), it is characterised in that in step (2), described biological quantum dot concentration is 80 nM。
CN201610167375.6A 2016-03-23 2016-03-23 Blood coagulation enzyme assay method based on biological quantum dot and Au NPs fluorescence resonance energy transfer Expired - Fee Related CN105842210B (en)

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CN107421937A (en) * 2017-08-29 2017-12-01 浙江工业大学 The histamine detection method of FRET biology sensor based on magnetic nano particle separation
CN107607501A (en) * 2017-08-21 2018-01-19 樊之雄 A kind of biomarker multiple detection method based on fluorescent quenching
CN109932345A (en) * 2019-02-01 2019-06-25 中南民族大学 A kind of lysine detection method based on quantum dot and nanogold
CN109975260A (en) * 2019-04-10 2019-07-05 山东大学 A kind of method and its application based on nanogold fluorescence detection lysozyme
CN112858680A (en) * 2021-01-14 2021-05-28 四川大学 Fluorescence resonance energy transfer magnetic sensor for exosome detection and preparation method thereof
CN116218860A (en) * 2023-03-24 2023-06-06 江苏海洋大学 ssDNA aptamer capable of specifically recognizing sialyl Lewis acid x and application thereof

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287297A (en) * 2017-06-26 2017-10-24 浙江工业大学 The method of FRET detection oxidative damage DNA based on carbon quantum dot and gold nano grain
CN107607501A (en) * 2017-08-21 2018-01-19 樊之雄 A kind of biomarker multiple detection method based on fluorescent quenching
CN107421937A (en) * 2017-08-29 2017-12-01 浙江工业大学 The histamine detection method of FRET biology sensor based on magnetic nano particle separation
CN109932345A (en) * 2019-02-01 2019-06-25 中南民族大学 A kind of lysine detection method based on quantum dot and nanogold
CN109975260A (en) * 2019-04-10 2019-07-05 山东大学 A kind of method and its application based on nanogold fluorescence detection lysozyme
CN109975260B (en) * 2019-04-10 2021-12-24 山东大学 Method for detecting lysozyme based on nanogold fluorescence and application thereof
CN112858680A (en) * 2021-01-14 2021-05-28 四川大学 Fluorescence resonance energy transfer magnetic sensor for exosome detection and preparation method thereof
CN116218860A (en) * 2023-03-24 2023-06-06 江苏海洋大学 ssDNA aptamer capable of specifically recognizing sialyl Lewis acid x and application thereof

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