CN103149185B - Novel high-efficiency protease activity detecting method - Google Patents
Novel high-efficiency protease activity detecting method Download PDFInfo
- Publication number
- CN103149185B CN103149185B CN201310045562.3A CN201310045562A CN103149185B CN 103149185 B CN103149185 B CN 103149185B CN 201310045562 A CN201310045562 A CN 201310045562A CN 103149185 B CN103149185 B CN 103149185B
- Authority
- CN
- China
- Prior art keywords
- quantum dot
- proteinase
- concentration
- peptide substrate
- zinc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a novel high-efficiency protease activity detecting method. The novel high-efficiency protease activity detecting method is characterized in that establishing correlativity between to-be-detected protease activity and fluorescence spectrum peak intensity of quantum dots synthesized by corresponding products in advance, designing polypeptide substrate including a special cleavage site of the protease according to the category of the to-be-detected protease, according to an optimal temperature and optimal potential of hydrogen (pH) of the protease, putting into protease content with unknown concentration, conducting enzyme digestion reaction on the same amount of polypeptide substrate, putting the same amount of precursors of synthesized quantum dots to enzyme-digested products of the polypeptide substrate, enabling the enzyme digestion reaction to generate the quantum dots under an appropriate temperature, after the reaction ends up, detecting the fluorescence spectrum of the synthesized quantum dots, and comparing the fluorescence spectrum peak intensity of quantum dots synthesized by the enzyme-digested products of the protease with unknown concentration with the correlativity in order to obtain the protease activity of the protease with concentration unknown. The novel high-efficiency protease activity detecting method is convenient to achieve, quick, high in sensitivity and low in cost. The novel high-efficiency protease activity detecting method also has general applicability.
Description
Technical field
The present invention relates to function nano material and biochemical field, be specifically related to a kind of new and effective proteinase activity detection method, the present invention is the endonuclease reaction to peptide substrate by proteinase, with the enzyme of gained, cut product and as surface ligand, synthesize the nano material quantum dot with fluorescent effect, and then according to the variation of the fluorescent effect of the quantum dot of synthesized, reflect the variation of endonuclease reaction afterproduct, thereby reflect the variation of proteinase activity.
Background technology
The specificity catalytic hydrolysis reaction of proteinase to proteins and peptides substrate, is bringing into play extremely important effect for the normal activities of biosome.Detect enzyme content and existence, be difficult to directly for example, by the amount (quality, volume, concentration) of enzyme, represent, and the ability of the conventional a certain specific chemical reaction of enzymatic represents enzyme amount, by enzymatic activity, represents.Research shows, the imbalance of proteinase activity can cause disease, as cancer, inflammation, degenerative disorders etc., this has caused scientist's extensive concern, therefore need under study for action to detect the activity of proteinase, the activity that detects proteinase aspect the medical diagnosis of proteinase activity relevant disease and commercial production all tool be of great significance.Traditional method for detecting enzymatic activity, as immunofluorescence adsorption method (being called for short ELISA), although the research of Tothill shows immunofluorescence adsorption method conveniently, its sensitivity is limited; Although it is high that Lamore S. D. proposes the detection sensitivity of mass spectrum and gel electrophoresis method, need expensive instrument and equipment, and can not be quantitative; Though the research of Lee J. S. etc. shows that the sensitivity of FRET (fluorescence resonance energy transfer) method is high, due to the limitation of substrate, makes it not possess universality.Therefore, how to design the problem that a kind of novel protein method for detecting enzymatic activity efficient, convenient and swift, that cost is low becomes the present invention's research.
Summary of the invention
The object of the invention is to provide a kind of new and effective proteinase activity detection method, and its object is to solve the problem that the sensitivity of current proteinase detection method is limited, cost is higher and do not possess general applicability.
For achieving the above object, the technical solution used in the present invention is: a kind of new and effective proteinase activity detection method, by two parts, formed,
First:
Set up in advance the correlationship between the fluorescence spectrum peak intensity of the proteinase activity of the required detection quantum dot synthetic with corresponding product, the foundation of described correlationship is comprised of the following step:
The first step, the kind for the proteinase of required detection, designs one section of peptide substrate that contains this proteinase specificity cleavage site;
The kind of described proteinase refers to any one in the trypsase, Proteinase K, pepsin, fibrin ferment, papain, clostridiopetidase A, matrix metalloproteinase, cysteine proteinase, chymotrypsin, aminopeptidase, carboxypeptidase with specificity cleavage site;
In described peptide substrate, comprise one section by the peptide chain of 2 ~ 10 Amino acid profiles, in described peptide chain, containing can be by peptide bond and two halfcystine with mercapto groups of described proteinase specificity cutting, wherein, the two ends of peptide chain be respectively one with the halfcystine of mercapto groups;
Second step, on the basis of the first step, under the optimum temperature and optimum pH condition of the proteinase of required detection, to the proteinase that respectively drops into concentration known in the peptide substrate of many groups same amount, each is organized between the proteinase of concentration known has concentration gradient, then carry out endonuclease reaction, obtain respectively organizing enzyme and cut product, wherein, maximum concentration in the proteinase of many group concentration known is 0.5% ~ 10 % of described peptide substrate volumetric molar concentration, time of each group endonuclease reaction of setting is identical and be less than or equal to the time that proteinase 100% enzyme of maximum concentration is cut peptide substrate, the volumetric molar concentration of the mercapto groups containing in the described peptide substrate dropping into is between 0.5 ~ 10mmol/L,
The 3rd step, to the described enzyme of respectively organizing, cut the presoma with metallic ion and nonmetallic ion for the synthesis of quantum dot that drops into same amount in product, and at 25 ~ 100 ℃, react 0.5 ~ 1 hour, each group reaction finishes rear manufacturing quantum dot, wherein, in the described presoma dropping into the volumetric molar concentration of metallic ion be the mercapto groups that contains in described peptide substrate volumetric molar concentration 85 ~ 120%, the volumetric molar concentration of nonmetallic ion is 20 % ~ 100 % of metallic ion volumetric molar concentration;
The presoma of described metallic ion is selected from any one or any two kinds in cadmium compound, lead compound, silver compound, zinc compound, indium compound, mercury compound, copper compound, manganese compound, and the presoma of described nonmetallic ion is selected from sulfocompound, selenium-containing compound, containing any one or any two kinds in tellurium compound, phosphorus-containing compound, arsenical; But the presoma of the presoma of any two metal ion species and any two kinds of nonmetallic ions is applied to the reaction of a synthetic quantum dot when different;
Described quantum dot is binary quantum dot, be specially cadmium telluride, cadmium selenide, cadmium sulfide, vulcanized lead, lead selenide, silver sulfide, silver selenide, zinc sulphide, zinc selenide, zinc telluridse, cadmium phosphide, Cadmium arsenide, indium phosphide, any one in indium arsenide, or described quantum dot is ternary quantum dots, be specially cadmium mercury tellurium, cadmium mercury selenium, cadmium mercury sulphur, zinc-mercury tellurium, zinc-mercury selenium, zinc-mercury sulphur, zinc cadmium tellurium, zinc cadmium selenium, zinc cadmium sulphur, zinc tellurium selenium, zinc tellurium sulphur, zinc selenium sulfur, cadmium tellurium selenium, cadmium tellurium sulphur, cadmium selenium sulphur, zinc-copper selenium, zinc-manganese selenium, copper indium sulphur, any one in copper indium diselenide,
The 4th step, the quantum dot that in the 3rd step, each group generates is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of described quantum dot, again according to the concentration known of the proteinase of many group concentration known described in second step, finally obtain the correlationship between the fluorescence spectrum peak intensity of the protease concentration quantum dot synthetic with corresponding product, i.e. correlationship between the fluorescence spectrum peak intensity of the proteinase activity quantum dot synthetic with corresponding product;
Second portion:
For any one in trypsase, Proteinase K, pepsin, fibrin ferment, papain, clostridiopetidase A, matrix metalloproteinase, cysteine proteinase, chymotrypsin, aminopeptidase, carboxypeptidase, proteinase activity detection method is comprised of following steps:
The first step, design peptide substrate
For detected proteinase kind, design identical peptide substrate in one section of first step with described first;
Second step, carries out endonuclease reaction
Design after peptide substrate, under the optimum temperature and optimum pH condition of detected proteinase, the detected proteinase to dropping into unknown concentration in peptide substrate, then carries out endonuclease reaction, obtains enzyme and cuts product; Wherein, the time of endonuclease reaction is identical with the endonuclease reaction time in the second step of described first, and the amount of described peptide substrate is identical with the amount of peptide substrate in the second step of described first;
The 3rd step, synthetic quantum dot
To the enzyme in second step, cut the presoma dropping in product for the synthesis of in the 3rd step of the described first of quantum dot, and under identical temperature and time conditions, react in the 3rd step with described first, manufacturing quantum dot after finishing, wherein, in the described presoma of input in the amount of metallic ion and nonmetallic ion and the 3rd step of described first in presoma the amount of metallic ion and nonmetallic ion identical;
The 4th step, obtains the activity of the proteinase of detected unknown concentration
The quantum dot generating in the 3rd step is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of this quantum dot, again the correlationship obtaining in this fluorescence spectrum peak intensity and described first is compared, finally obtain the proteinase activity of detected unknown concentration.
Related content in technique scheme is explained as follows:
1, in such scheme, maximum concentration in the proteinase of many group concentration known is 0.5% ~ 10 % of described peptide substrate volumetric molar concentration, the time of endonuclease reaction is less than or equal to the time that proteinase 100% enzyme of maximum concentration in the second step of described first is cut peptide substrate, that is to say when the proteinase of known variable concentrations and the peptide substrate of same amount carry out endonuclease reaction, if the proteinase of maximum concentration has not also reacted with peptide substrate, the proteinase of other low concentrations is just certain does not so have and peptide substrate complete reaction, the amount that the enzyme that carries out endonuclease reaction generation by the proteinase of known variable concentrations and the peptide substrate of same amount is cut product is different, and then the fluorescence spectrum peak intensity of cutting the synthetic quantum dot of product with enzyme is also different.
2, in such scheme, described optimum temperature refers to temperature when enzymic catalytic reaction speed reaches a maximal value with temperature rising, and optimal pH refers to the pH value of solution when enzymatic activity is the highest, the too high or too low decline that all can cause enzymatic activity of pH.Each proteinase has optimum temperature and optimal pH separately, and those of ordinary skills can easily obtain optimum temperature and the optimal pH of each proteinase.
3, in such scheme, in the second step of described first and the second step of described second portion, described pH value adopts pH buffer solution to regulate, and pH buffer solution is selected from any one in trishydroxymethylaminomethane-hydrochloric acid buffer solution (claim not only Tris-HCl buffer solution), ammonium hydrogencarbonate buffer solution, phosphate buffered solution (but also claiming PBS buffer solution); In above-mentioned pH buffer solution, can also contain volume fraction and be 0.1 ~ 1% serum, refer to that the volume of serum in above-mentioned pH buffer solution accounts for 0.1 ~ 1% of overall solution volume.
Design concept of the present invention is: quantum dot (English name quantum dots, QDs) be the semiconductor nanocrystal that a kind of yardstick was less than or was similar to exciton bohr particle diameter, there is unique optical property, the continuous distribution such as exciting spectrum width, emission wavelength can regulate by particle size and constituent, and fluorescence quantum yield is high, life-span is long, thereby in fluorescence labeling imaging, luminescent device, the fields such as photoelectric material have a wide range of applications.Adopt containing the amino acid of sulfydryl or the method for polypeptide preparation quantum dot also very ripe, as Wing-Cheung Law adopts the synthetic CdTe quantum dot of halfcystine (English name L-cysteine), Ying-Fan Liu adopts the synthetic CdTe quantum dot of glutathione (English name GSH).
Kimihiro Susum research finds that the character of the sulfhydryl compound part that the stability of quantum dot is surperficial with it is closely related.Use the compound part that contains two sulfydryls than the compound part containing single sulfydryl, quantum dot is carried out after surface ligand exchange, quantum dot shows stronger stability, and two sulfydryl parts have different impacts from single sulfydryl part to quantum dot.So, can infer, with the part of two sulfydryls and single sulfydryl, carry out synthetic quantum dot, can there is different fluorescent effects.Otherwise the variation of the fluorescent effect by synthetic quantum dot, just can infer the variation of the number of the sulfydryl containing in its synthetic ligands compound.Suppose that ligand compound is the polypeptide that contains specific restriction enzyme site, and these restriction enzyme site both sides are respectively containing a sulfydryl, so along with the rising of enzyme concentration, carry out after endonuclease reaction, polypeptide quantity containing two sulfydryls in the product obtaining reduces gradually, and the polypeptide quantity that contains single sulfydryl increases gradually, and the fluorescence of synthetic quantum dot also changes thereupon, thereby can the anti-variation that pushes away enzyme concentration by the change in fluorescence of quantum dot, enzymatic activity is effectively detected.
The present invention is in existing technical foundation, a kind of new and effective proteinase activity detection method has been proposed, the substrate of the specificity restriction enzyme site of the enzyme that contains required detection by design, after the proteinase effect with variable concentrations, resulting different product synthesizes the nano material quantum dot with different fluorescent effects as surface ligand, the i.e. variation of the fluorescent effect of the quantum dot by synthesized, reflect the variation of cutting product as the enzyme of part, thereby finally reflect the variation of proteinase activity.
In new and effective proteinase activity detection method of the present invention, although also utilize the variation of the fluorescent effect of quantum dot to reflect the variation of proteinase activity, be different from the FRET (fluorescence resonance energy transfer) method that adopts quantum dot to carry out completely.The core of the proteinase activity detection method in the present invention is that with the peptide substrate of same amount and the proteinase of different content, carrying out different enzymes after endonuclease reaction cuts product and synthesize the quantum dot with different fluorescent effects, thus the anti-activity that pushes away proteinase; And the method for FRET (fluorescence resonance energy transfer) is that the specific quantum dot that is connected with specific peptide substrate is after the enzyme effect with different content, the fluorescent effect of quantum dot changes, thereby infers the activity of enzyme.
Because technique scheme is used, advantage and effect that the present invention compared with prior art has are: specificity, quantum dot fluorescence and the being closely related property of surface ligand compound and the economy of synthetic quantum dot and the convenience of cutting substrate due to enzyme, make proteinase activity detection method of the present invention compared to traditional proteinase activity detection method, as the methods such as immunofluorescence adsorption method, mass spectrum, gel electrophoresis and FRET (fluorescence resonance energy transfer) have higher economic and practical, higher sensitivity and the higher features such as convenience.And proteinase activity detection method of the present invention has general applicability, only need be according to the kind of the proteinase of required detection, restriction enzyme site in corresponding change peptide substrate, without changing amino acid and other parts that contains sulfydryl in peptide substrate, also without the building-up process that changes quantum dot, just can realize the active detection to different types of proteinase.That is to say, proteinase activity detection method provided by the invention is convenient and swift, highly sensitive, cost is low, and has general applicability.
Accompanying drawing explanation
Accompanying drawing 1 is a kind of new and effective proteinase activity detection method process flow diagram of the present invention;
Accompanying drawing 2 is the fluorescence comparison diagram of the embodiment of the present invention one;
Accompanying drawing 3 is the first mass spectrogram of the embodiment of the present invention one;
Accompanying drawing 4 is the second mass spectrogram of the embodiment of the present invention one;
Accompanying drawing 5 is the fluorescence spectrum figure of the quantum dot of the embodiment of the present invention one
Accompanying drawing 6 is the fluorescence spectrum peak intensity of quantum dot and the relevant criterion curve of proteinase activity of the embodiment of the present invention one;
Accompanying drawing 7 is the ultraviolet absorpting spectrum of the quantum dot of the embodiment of the present invention one;
Accompanying drawing 8 is the first Electronic Speculum collection of illustrative plates of the quantum dot of the embodiment of the present invention one;
Accompanying drawing 9 is the second Electronic Speculum collection of illustrative plates of the quantum dot of the embodiment of the present invention one;
Accompanying drawing 10 is the fluorescence spectrum peak intensity of quantum dot and the relevant criterion curve of proteinase activity of the embodiment of the present invention two;
Accompanying drawing 11 is the fluorescence spectrum peak intensity of quantum dot and the relevant criterion curve of proteinase activity of the embodiment of the present invention three;
Accompanying drawing 12 is the fluorescence spectrum peak intensity of quantum dot and the relevant criterion curve of proteinase activity of the embodiment of the present invention four.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment mono-: a kind of new and effective proteinase activity detection method
Proteinase activity detection method flow process can be shown in Figure 1.The present embodiment is trypsase (English name: trypsin) activity test method.
First:
Set up in advance the correlationship between the fluorescence spectrum peak intensity of the tryptic activity quantum dot synthetic with corresponding product, the foundation of described correlationship is comprised of the following step:
The first step, for trypsase, design one section of peptide substrate that contains this proteinase specificity cleavage site, in described peptide substrate, comprise one section by the peptide chain of 4 Amino acid profiles, in described peptide chain, contain peptide bond and two (symbols: C), its specificity restriction enzyme site is aminoterminal (chemical formula: NH of the halfcystine with mercapto groups that can be cut by trypsase specificity
2-) be lysine (symbol: K), c-terminus (chemical formula: COOH-) be glycocoll (symbol: the peptide bond G), the two ends of peptide chain are respectively one, and with the halfcystine of mercapto groups, the two ends of peptide chain are also respectively connected with a glycocoll, and this peptide substrate is NH
2-GCKGCG-COOH, its product after by tryptic digestion is NH
2-GCK-COOH and NH
2-GCG-COOH.
Second step, on the basis of the first step, under tryptic optimum temperature and optimal pH condition, to the proteinase that respectively drops into concentration known in the peptide substrate of many groups same amount, then carry out endonuclease reaction, obtain respectively organizing enzyme and cut product, wherein, tryptic optimum temperature is 37 ℃ of the physiological temps of human body, and optimum pH is 7 ~ 9.Sulfhydryl content in input peptide substrate is 1.15 mmol/L, tryptic gradient concentration is set as 0 μ mol/L, 0.2 μ mol/L, 1 μ mol/L, 2 μ mol/L L, 5 μ mol/L, 10 μ mol/L, 20 μ mol/L, 40 μ mol/L, in pH=8.2 and containing in the ammonium bicarbonate buffers of volume fraction 0.1 % serum, under 37 ℃ of water bath condition, react 2 hours.Time of each group endonuclease reaction of setting is identical and be less than the time that trypsase 100% enzyme of maximum concentration is cut peptide substrate.
The 3rd step, cuts to the described enzyme of respectively organizing the caddy (chemical formula: CdCl that drops into 1.15 mmol/L in product
2) precursor and 0.30 mmol/L sodium hydrogen telluride (chemical formula: NaHTe) precursor, react 1 hour to synthesize CdTe quantum dot at 100 ℃.
The 4th step, carries out fluorescence spectrometry to the CdTe quantum dot that in the 3rd step, each group generates, and obtains the fluorescence spectrum peak intensity of described quantum dot, then by organizing tryptic concentration known, drawing standard curve more.The synthetic fluorescence spectrum peak intensity of CdTe quantum dot and the relevant criterion curve of trypsase gradient concentration of product of the trypsin hydrolysis peptide substrate of gradient concentration, shown in Figure 6.
Second portion:
For trypsase, described tryptic activity detection method is comprised of following steps:
The first step, design peptide substrate
Design identical peptide substrate in one section of first step with described first.
Second step, carries out endonuclease reaction
Design after peptide substrate, under the reaction conditions in the second step of described first, the trypsase to dropping into unknown concentration in peptide substrate, then carries out endonuclease reaction, obtains enzyme and cuts product.Wherein, the temperature of endonuclease reaction, pH condition, the time of endonuclease reaction and the amount of peptide substrate are all identical with the second step of described first.
The 3rd step, synthetic quantum dot
To the enzyme in second step, cut the presoma dropping in product for the synthesis of in the 3rd step of the described first of quantum dot, and under identical temperature and time conditions, react in the 3rd step with described first, after finishing, generate CdTe quantum dot, wherein, in the described presoma of input in the amount of metallic ion and nonmetallic ion and the 3rd step of described first in presoma the amount of metallic ion and nonmetallic ion identical.
The 4th step, obtains the tryptic activity of detected unknown concentration
CdTe quantum dot in the 3rd step is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of quantum dot, again the relevant criterion curve obtaining in this fluorescence spectrum peak intensity and described first is compared, finally obtain the tryptic activity of detected unknown concentration.
Fig. 2 represents the CdTe quantum dot that the reacted product of maximum concentration tryptic digestion in peptide substrate and gradient concentration is synthetic and only has relatively photo of the fluorescence of the synthetic CdTe quantum dot of peptide substrate under uviol lamp.Way is under the irradiation of the common uv lamp of 365 nm, with general camera, directly takes pictures.Wherein, in Fig. 2, the EP pipe belt table on the right is the quantum dot generating after the trypsase cutting peptide substrate of maximum concentration in gradient concentration, and its fluorescence is the strongest; The EP pipe belt table on the left side be peptide substrate completely by trypsase cutting (or trypsinase concentration is 0) time, directly synthetic quantum dot does not have fluorescence.This picture has represented the highs and lows of the typical curve that need to do intuitively.
Fig. 3 and Fig. 4 represent that the trypsase of gradient concentration cuts the mass spectrogram of afterproduct to peptide substrate enzyme, way is the trypsase of 200 μ L to be sheared to the product of front and back to peptide substrate in 1200LC-6410B/QQQ/MS liquid phase mass spectrograph (manufacturer: upper test U.S. Agilent company), be 15 minutes the analysis time of each sample.Wherein, specific charge 524 is NH
2-GCKGCG-COOH, specific charge 307 is NH
2-GCK-COOH.In Fig. 3, molecular weight 524 is the peptide substrate NH2-GCKGCG-COOH of enzyme before cutting, molecular weight 307 product after cutting for peptide substrate NH2-GCKGCG-COOH is digested in Fig. 4, after this explanation trypsase and peptide substrate effect, really can cut peptide substrate, therefore by such cutting, the different enzymes that obtain are cut product, could synthesize the strong and weak different quantum dot of fluorescence.
Fig. 5 represents the fluorescence spectrum figure of the CdTe quantum dot that the product of trypsin hydrolysis peptide substrate of different gradient concentrations is synthetic, way is to using the ultraviolet light of 365 nm as excitation source, a result that adopts AvaSpec-ULS2048-USB2 fiber spectrometer (manufacturer: Dutch Avantes company) to test.Wherein, transverse axis represents wavelength, and unit is nanometer (nm), and the longitudinal axis represents the peak intensity of relative fluorescence spectrum.As can be seen from Figure 5, after the trypsase cutting peptide substrate of variable concentrations, the variation of the fluorescence spectrum of synthetic quantum dot, from top to bottom, rising along with trypsinase concentration, the peak intensity of the fluorescence spectrum of the quantum dot obtaining is also to strengthen gradually, for setting up enzyme concentration-quantum dot fluorescence strength criterion curve, provides foundation.
On the basis of above confirmatory experiment, obtained the synthetic fluorescence spectrum peak intensity of CdTe quantum dot and the relevant criterion curve of trypsase gradient concentration of product of the trypsin hydrolysis peptide substrate of different gradient concentrations, shown in Figure 6, the conversion that fluorescence spectrum in Fig. 5 is carried out, transverse axis is trypsinase concentration, unit is μ mol/L, and the longitudinal axis is the peak intensity numerical value of fluorescence spectrum and the ratio of background (background is the peak intensity that enzyme concentration is the fluorescence spectrum of 0 o'clock) of corresponding CdTe quantum dot.
The ultraviolet absorpting spectrum that Fig. 7 represents the synthetic CdTe quantum dot of the reacted product of maximum concentration tryptic digestion in peptide substrate and gradient concentration, only have the synthetic CdTe quantum dot of peptide substrate and only have the synthetic quantum dot of maximum concentration trypsase in gradient concentration, transverse axis represents wavelength, unit is nanometer (nm), and the longitudinal axis represents absorbance.Way is in CARY50 ultraviolet-visible pectrophotometer (manufacturer: upper measurement U.S. Varian company); The ultra-violet absorption spectrum of Fig. 7 can react wherein particle size and the concentration of quantum dot, and more past the right in figure (being that transverse axis is larger), the particle diameter of quantum dot is larger; More past top (being that the longitudinal axis is larger), the concentration of quantum dot is larger.From this picture, can find out, (enzyme concentration is 0 o'clock) synthetic quantum dot while only having peptide substrate, be labeled as Peptide+CdTe, on transverse axis the right (i.e. 350 ~ 600 nm), locate, longitudinal axis numerical value (being absorbance) is very low, the particle diameter that shows quantum dot is wherein less than normal, and the quantum dot of large particle diameter is wherein on the low side in other words; Contrary after adding the trypsase of maximum concentration, synthetic quantum dot, is labeled as Pepetide+Trypsin+CdTe, it is at 350 ~ 600 nm places, absorbance obviously uprises, and shows that quantum point grain diameter is wherein bigger than normal, and the quantum dot of large particle diameter is wherein on the high side in other words.And the quantum dot that the most important thing is large particle diameter just can send fluorescence, the quantum dot of small particle diameter can not send fluorescence, thus Fig. 7 from a side for detection method of the present invention provides theoretical foundation.When not adding trypsase, do not add the precursor of synthetic quantum dot yet, it is in same area, and absorbance is 0 substantially, i.e. basic not absorption; When only there being the trypsase of maximum concentration, do not add peptide substrate, do not add the precursor of synthetic quantum dot yet, it is in same area, and absorbance is substantially also 0, i.e. basic not absorption.These two in contrast, proves that the variation in 350 ~ 600 nm region absorbances above does not change because of peptide substrate, neither change because of trypsase, therefore also just can infer the reason of only having synthetic different quantum dot to be only variation.
Fig. 8 represents the Electronic Speculum collection of illustrative plates of the CdTe quantum dot that the reacted product of the tryptic digestion of maximum concentration in peptide substrate and gradient concentration is synthetic, and Fig. 9 represents to only have the Electronic Speculum collection of illustrative plates of the synthetic CdTe quantum dot of peptide substrate.Way is to take the JEM-2100 of 200KV transmission electron microscope (manufacturer: Japanese JEOL company) is lower, obtains Electronic Speculum collection of illustrative plates.Fig. 8 and Fig. 9 have illustrated the content of picture 7 more intuitively.Fig. 8 is that peptide substrate has added synthetic quantum dot after the trypsase of maximum concentration in gradient concentration, obviously than large in Fig. 9, (in two width pictures, that the upper left corner is high-resolution figure to grain diameter, more intuitively reflect this change of size), thereby mutually confirm with Fig. 7, illustrate and add the trypsase of maximum concentration and the trypsase that concentration is 0, synthetic quantum point grain diameter has significant change, thereby for change in fluorescence provides foundation, also for Criterion curve provides foundation.Fig. 7, Fig. 8 and Fig. 9 have also reacted from a side: along with the lifting of enzyme concentration, after cutting peptide substrate, the fluorescence of quantum dot strengthens gradually, and its basic reason is that the mean grain size of the quantum dot of synthesized is progressively increasing, and the quantum dot proportion of large particle diameter is raising gradually in other words.
Embodiment bis-: a kind of new and effective proteinase activity detection method
Proteinase activity detection method flow process can be shown in Figure 1.The present embodiment is clostridiopetidase A (English name: collagenase) activity test method.
First:
Set up in advance the correlationship between the fluorescence spectrum peak intensity of the collagenase activity quantum dot synthetic with corresponding product, the foundation of described correlationship is comprised of the following step:
The first step, for clostridiopetidase A, design one section of peptide substrate that contains this proteinase specificity cleavage site, in described peptide substrate, comprise one section by the peptide chain of 4 Amino acid profiles, in described peptide chain, contain peptide bond and two halfcystine (symbols: C) with mercapto groups that can be cut by clostridiopetidase A specificity, its specificity restriction enzyme site is proline-halfcystine-Gly-Pro (symbol: (symbol: C) peptide bond afterwards of halfcystine P-C-G-P), the two ends of peptide chain be respectively one with the halfcystine of mercapto groups, one end of peptide chain is also connected with a proline, this peptide substrate is NH
2-PCGPC-COOH, its product after being cut by clostridiopetidase A enzyme is NH
2-PC-COOH and NH
2-GPC-COOH.
Second step, on the basis of the first step, under the optimum temperature and optimal pH condition of clostridiopetidase A, to the clostridiopetidase A that respectively drops into concentration known in the peptide substrate of many groups same amount, then carry out endonuclease reaction, obtain respectively organizing enzyme and cut product, wherein, the optimum temperature of clostridiopetidase A is 37 ℃ of the physiological temps of human body, and optimal pH is 7.4.Sulfhydryl content in input peptide substrate is 0.5mmol/L, the gradient concentration of clostridiopetidase A is set as 0 μ mol/L, 0.01 μ mol/L, 0.2 μ mol/L, 1 μ mol/L, 2 μ mol/L, 5 μ mol/L, 10 μ mol/L, 25 μ mol/L, in the phosphate buffered solution of pH=7.4, under 37 ℃ of water bath condition, react 2 hours.Time of each group endonuclease reaction of setting is identical and be less than the time that clostridiopetidase A 100% enzyme of maximum concentration is cut peptide substrate.
The 3rd step, cuts to the described enzyme of respectively organizing the silver nitrate (chemical formula: AgNO that drops into 0.6 mmol/L in product
3) sodium sulphide (chemical formula: Na of precursor and 0.3 mmol/L
2s) precursor, reacts 0.5 hour to synthesize Ag at 100 ℃
2s quantum dot.
The 4th step, to the Ag that in the 3rd step, each group generates
2s quantum dot carries out fluorescence spectrometry, obtains the fluorescence spectrum peak intensity of described quantum dot, then by the concentration known gradient of organizing clostridiopetidase A, drawing standard curve more.The synthetic Ag of product of the clostridiopetidase A hydrolyzed peptide substrate of gradient concentration
2the fluorescence spectrum peak intensity of S quantum dot and the relevant criterion curve of clostridiopetidase A gradient concentration, shown in Figure 10, be the conversion that fluorescence spectrum is carried out, wherein, transverse axis is clostridiopetidase A concentration, and unit is μ mol/L, and the longitudinal axis is corresponding Ag
2the peak intensity numerical value of the fluorescence spectrum of S quantum dot and the ratio of background (it is the fluorescence spectrum of 0 o'clock that background is enzyme concentration).
Second portion:
For clostridiopetidase A, described collagenase activity detection method is comprised of following steps:
The first step, design peptide substrate
Design identical peptide substrate in one section of first step with described first.
Second step, carries out endonuclease reaction
Design after peptide substrate, under the reaction conditions in the second step of described first, the clostridiopetidase A to dropping into unknown concentration in peptide substrate, then carries out endonuclease reaction, obtains enzyme and cuts product.Wherein, the temperature of endonuclease reaction, pH condition, the time of endonuclease reaction and the amount of peptide substrate are all identical with the second step of described first.
The 3rd step, synthetic quantum dot
To the enzyme in second step, cut the presoma dropping in product for the synthesis of in the 3rd step of the described first of quantum dot, and under identical temperature and time conditions, react in the 3rd step with described first, finish rear generation Ag
2s quantum dot, wherein, in the described presoma of input, in the 3rd step of the amount of metallic ion and nonmetallic ion and described first, in presoma, the amount of metallic ion and nonmetallic ion is identical.
The 4th step, obtains the activity of the clostridiopetidase A of detected unknown concentration
To the Ag in the 3rd step
2s quantum dot carries out fluorescence spectrometry, obtains the fluorescence spectrum peak intensity of quantum dot, then the relevant criterion curve obtaining in this fluorescence spectrum peak intensity and described first is compared, and finally obtains the activity of the clostridiopetidase A of detected unknown concentration.
Embodiment tri-: a kind of new and effective proteinase activity detection method
The present embodiment is fibrin ferment (English name: thrombin) activity test method.
First:
Set up in advance the correlationship between the fluorescence spectrum peak intensity of the thrombin activity quantum dot synthetic with corresponding product, the foundation of described correlationship is comprised of the following step:
The first step, for fibrin ferment, design one section of peptide substrate that contains this proteinase specificity cleavage site, in described peptide substrate, comprise one section by the peptide chain of 4 Amino acid profiles, in described peptide chain, contain peptide bond and two (symbols: C), its specificity restriction enzyme site is aminoterminal (NH of the halfcystine with mercapto groups that can be cut by fibrin ferment specificity
2-) be arginine (R), c-terminus (COOH-) be glycocoll (G) between peptide bond, the two ends of peptide chain be respectively one with the halfcystine of mercapto groups, this peptide substrate is NH
2-CRGC-COOH, its product after being cut by fibrin ferment enzyme is NH
2-CR-COOH and NH
2-GC-COOH.
Second step, on the basis of the first step, under the optimum temperature and optimal pH condition of fibrin ferment, to the fibrin ferment that respectively drops into concentration known in the peptide substrate of many groups same amount, then carry out endonuclease reaction, obtain respectively organizing enzyme and cut product, wherein, the optimum temperature of fibrin ferment is 37 ℃ of the physiological temps of human body, and optimal pH is 7 ~ 9.Sulfhydryl content in input peptide substrate is 10 mmol/L, the gradient concentration of fibrin ferment is set as 0 μ mol/L, 0.01 μ mol/L, 0.02 μ mol/L, 0.05 μ mol/L, 1 μ mol/L, 5 μ mol/L, 20 μ mol/L, 50 μ mol/L, in containing in the trishydroxymethylaminomethane-hydrochloric acid buffer solution (claiming again Tris-HCl buffer solution) of volume fraction 0.5 % serum of pH=8.3, under 37 ℃ of water bath condition, react 2 hours.Time of each group endonuclease reaction of setting is identical and be less than the time that fibrin ferment 100% enzyme of maximum concentration is cut peptide substrate.
The 3rd step, cuts to the described enzyme of respectively organizing the zinc acetate (chemical formula: Zn (CH that drops into isopyknic 10 mmol/L in product
3cOO)
2) precursor, 1 mmol/L caddy (CdCl
2) sodium hydrogen selenide (NaHSe) precursor of precursor and 6 mmol/L, react 1 hour to synthesize ZnCdSe quantum dot at 100 ℃.
The 4th step, carries out fluorescence spectrometry to the ZnCdSe quantum dot that in the 3rd step, each group generates, and obtains the fluorescence spectrum peak intensity of described quantum dot, then by the concentration known gradient of organizing fibrin ferment, drawing standard curve more.The synthetic fluorescence spectrum peak intensity of ZnCdSe quantum dot and the relevant criterion curve of fibrin ferment gradient concentration of product of the fibrin ferment hydrolyzed peptide substrate of gradient concentration, shown in Figure 11, it is the conversion that fluorescence spectrum is carried out, wherein, transverse axis is concentration of thrombin, unit is μ mol/L, and the longitudinal axis is the peak intensity numerical value of fluorescence spectrum of corresponding ZnCdSe quantum dot and the ratio of background (it is the fluorescence spectrum of 0 o'clock that background is enzyme concentration).
Second portion:
For fibrin ferment, described thrombin activity detection method is comprised of following steps:
The first step, design peptide substrate
Design identical peptide substrate in one section of first step with described first.
Second step, carries out endonuclease reaction
Design after peptide substrate, under the reaction conditions in the second step of described first, the fibrin ferment to dropping into unknown concentration in peptide substrate, then carries out endonuclease reaction, obtains enzyme and cuts product.Wherein, the temperature of endonuclease reaction, pH condition, the time of endonuclease reaction and the amount of peptide substrate are all identical with the second step of described first.
The 3rd step, synthetic quantum dot
To the enzyme in second step, cut the presoma dropping in product for the synthesis of in the 3rd step of the described first of quantum dot, and under identical temperature and time conditions, react in the 3rd step with described first, after finishing, generate ZnCdSe quantum dot, wherein, in the described presoma of input in the amount of metallic ion and nonmetallic ion and the 3rd step of described first in presoma the amount of metallic ion and nonmetallic ion identical.
The 4th step, obtains the activity of the fibrin ferment of detected unknown concentration
ZnCdSe quantum dot in the 3rd step is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of quantum dot, again the relevant criterion curve obtaining in this fluorescence spectrum peak intensity and described first is compared, finally obtain the activity of the fibrin ferment of detected unknown concentration.
Embodiment tetra-: a kind of new and effective proteinase activity detection method
The present embodiment is a kind of in Caspase-3(cysteine proteinase) activity test method.
First:
Correlationship between the fluorescence spectrum peak intensity of the active quantum dot synthetic with corresponding product of prior model Caspase-3, the foundation of described correlationship is comprised of the following step:
The first step, for Caspase-3, design one section of peptide substrate that contains this proteinase specificity cleavage site, in described peptide substrate, comprise one section by the peptide chain of 4 Amino acid profiles, in described peptide chain, contain peptide bond and two halfcystine (symbols: C) with mercapto groups that can be cut by Caspase-3 enzyme spcificity, its specificity restriction enzyme site is the peptide bond after aspartic acid, the two ends of peptide chain be respectively one with the halfcystine of mercapto groups, the two ends of peptide chain are also respectively connected with a glycocoll, and this peptide substrate is NH
2-GCAGCG-COOH, its product after being cut by Caspase-3 enzyme is NH
2-GCA-COOH and NH
2-GCG-COOH.
Second step, on the basis of the first step, under the optimum temperature and optimal pH condition of Caspase-3, to the Caspase-3 that respectively drops into concentration known in the peptide substrate of many groups same amount, then carry out endonuclease reaction, obtain respectively organizing enzyme and cut product, wherein, the optimum temperature of Caspase-3 is 25 ℃, and optimal pH is 7.4.Sulfhydryl content in input peptide substrate is 5 mmol/L, the gradient concentration of Caspase-3 is set as 0 μ mol/L, 0.01 μ mol/L, 0.05 μ mol/L, 0.2 μ mol/L, 1 μ mol/L, 5 μ mol/L, 20 μ mol/L, 100 μ mol/L, in containing in the ammonium bicarbonate buffer solution of volume fraction 1 % serum of pH=7.4, under 25 ℃ of water bath condition, react 2 hours.Time of each group endonuclease reaction of setting is identical and be less than time of the Caspase-3 complete degestion peptide substrate of maximum concentration.
The 3rd step, cuts to the described enzyme of respectively organizing the caddy (chemical formula: CdCl that drops into 0.6 mmol/L in product
2) precursor, isopyknic 0.20 mmol/L sodium hydrogen telluride (chemical formula: NaHTe) and the sodium hydrogen selenide of 0.20 mmol/L (chemical formula: NaHSe) precursor, react 1 hour to synthesize CdTeSe quantum dot at 100 ℃.
The 4th step, carries out fluorescence spectrometry to the CdTeSe quantum dot that in the 3rd step, each group generates, and obtains the fluorescence spectrum peak intensity of described quantum dot, then by the concentration known gradient of organizing Caspase-3, drawing standard curve more.The synthetic fluorescence spectrum peak intensity of CdTeSe quantum dot and the relevant criterion curve of Caspase-3 gradient concentration of product of the Caspase-3 hydrolyzed peptide substrate of gradient concentration, shown in Figure 12, it is the conversion that fluorescence spectrum is carried out, wherein, transverse axis is Caspase-3 concentration, unit is μ mol/L, and the longitudinal axis is the peak intensity numerical value of fluorescence spectrum of corresponding CdTeSe quantum dot and the ratio of background (it is the fluorescence spectrum of 0 o'clock that background is enzyme concentration).
Second portion:
For Caspase-3, described Caspase-3 activity test method is comprised of following steps:
The first step, design peptide substrate
Design identical peptide substrate in one section of first step with described first.
Second step, carries out endonuclease reaction
Design after peptide substrate, under the reaction conditions in the second step of described first, the Caspase-3 to dropping into unknown concentration in peptide substrate, then carries out endonuclease reaction, obtains enzyme and cuts product.Wherein, the temperature of endonuclease reaction, pH condition, the time of endonuclease reaction and the amount of peptide substrate are all identical with the second step of described first.
The 3rd step, synthetic quantum dot
To the enzyme in second step, cut the presoma dropping in product for the synthesis of in the 3rd step of the described first of quantum dot, and under identical temperature and time conditions, react in the 3rd step with described first, after finishing, generate CdTeSe quantum dot, wherein, in the described presoma of input in the amount of metallic ion and nonmetallic ion and the 3rd step of described first in presoma the amount of metallic ion and nonmetallic ion identical.
The 4th step, obtains the activity of the Caspase-3 of detected unknown concentration
CdTeSe quantum dot in the 3rd step is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of quantum dot, again the relevant criterion curve obtaining in this fluorescence spectrum peak intensity and described first is compared, finally obtain the activity of the Caspase-3 of detected unknown concentration.
Above-described embodiment is only explanation technical conceive of the present invention and feature; can be applicable in protection domain that the proteinase of detection method of the present invention all requires in the present invention; that is to say that those skilled in the art are under the inspiration of above-described embodiment; easily understand the proteinase that other have specificity cleavage site, for example the activity of Proteinase K, pepsin, papain, matrix metalloproteinase, chymotrypsin, aminopeptidase, carboxypeptidase also can enough detection methods of the present invention detect.The object of above-described embodiment is to allow person skilled in the art can understand content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalences that Spirit Essence is done according to the present invention change or modify, within all should being encompassed in protection scope of the present invention.
Claims (4)
1. a new and effective proteinase activity detection method, is characterized in that:
First:
Set up in advance the correlationship between the fluorescence spectrum peak intensity of the proteinase activity of the required detection quantum dot synthetic with corresponding product, the foundation of described correlationship is comprised of the following step:
The first step, the kind for the proteinase of required detection, designs one section of peptide substrate that contains this proteinase specificity cleavage site;
The kind of described proteinase refers to any one in the trypsase, Proteinase K, pepsin, fibrin ferment, papain, clostridiopetidase A, matrix metalloproteinase, cysteine proteinase, chymotrypsin, aminopeptidase, carboxypeptidase with specificity cleavage site;
In described peptide substrate, comprise one section by the peptide chain of 2 ~ 10 Amino acid profiles, in described peptide chain, containing can be by peptide bond and two halfcystine with mercapto groups of described proteinase specificity cutting, wherein, the two ends of peptide chain be respectively one with the halfcystine of mercapto groups;
Second step, on the basis of the first step, under the optimum temperature and optimum pH condition of the proteinase of required detection, to the proteinase that respectively drops into concentration known in the peptide substrate of many groups same amount, each is organized between the proteinase of concentration known has concentration gradient, then carry out endonuclease reaction, obtain respectively organizing enzyme and cut product, wherein, maximum concentration in the proteinase of many group concentration known is 0.5% ~ 10 % of described peptide substrate volumetric molar concentration, time of each group endonuclease reaction of setting is identical and be less than or equal to the time that proteinase 100% enzyme of maximum concentration is cut peptide substrate, the volumetric molar concentration of the mercapto groups containing in the described peptide substrate dropping into is between 0.5 ~ 10mmol/L,
The 3rd step, to the described enzyme of respectively organizing, cut the presoma with metallic ion and nonmetallic ion for the synthesis of quantum dot that drops into same amount in product, and at 25 ~ 100 ℃, react 0.5 ~ 1 hour, each group reaction finishes rear manufacturing quantum dot, wherein, in the described presoma dropping into the volumetric molar concentration of metallic ion be the mercapto groups that contains in described peptide substrate volumetric molar concentration 85 ~ 120%, the volumetric molar concentration of nonmetallic ion is 20 % ~ 100 % of metallic ion volumetric molar concentration;
The presoma of described metallic ion is selected from any one or any two kinds in cadmium compound, lead compound, silver compound, zinc compound, indium compound, mercury compound, copper compound, manganese compound, and the presoma of described nonmetallic ion is selected from sulfocompound, selenium-containing compound, containing any one or any two kinds in tellurium compound, phosphorus-containing compound, arsenical; But the presoma of the presoma of any two metal ion species and any two kinds of nonmetallic ions is applied to the reaction of a synthetic quantum dot when different;
Described quantum dot is binary quantum dot, be specially cadmium telluride, cadmium selenide, cadmium sulfide, vulcanized lead, lead selenide, silver sulfide, silver selenide, zinc sulphide, zinc selenide, zinc telluridse, cadmium phosphide, Cadmium arsenide, indium phosphide, any one in indium arsenide, or described quantum dot is ternary quantum dots, be specially cadmium mercury tellurium, cadmium mercury selenium, cadmium mercury sulphur, zinc-mercury tellurium, zinc-mercury selenium, zinc-mercury sulphur, zinc cadmium tellurium, zinc cadmium selenium, zinc cadmium sulphur, zinc tellurium selenium, zinc tellurium sulphur, zinc selenium sulfur, cadmium tellurium selenium, cadmium tellurium sulphur, cadmium selenium sulphur, zinc-copper selenium, zinc-manganese selenium, copper indium sulphur, any one in copper indium diselenide,
The 4th step, the quantum dot that in the 3rd step, each group generates is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of described quantum dot, again according to the concentration known of the proteinase of many group concentration known described in second step, finally obtain the correlationship between the fluorescence spectrum peak intensity of the protease concentration quantum dot synthetic with corresponding product, i.e. correlationship between the fluorescence spectrum peak intensity of the proteinase activity quantum dot synthetic with corresponding product;
Second portion:
For any one in trypsase, Proteinase K, pepsin, fibrin ferment, papain, clostridiopetidase A, matrix metalloproteinase, cysteine proteinase, chymotrypsin, aminopeptidase, carboxypeptidase, proteinase activity detection method is comprised of following steps:
The first step, design peptide substrate
For detected proteinase kind, design identical peptide substrate in one section of first step with described first;
Second step, carries out endonuclease reaction
Design after peptide substrate, under the optimum temperature and optimum pH condition of detected proteinase, the detected proteinase to dropping into unknown concentration in peptide substrate, then carries out endonuclease reaction, obtains enzyme and cuts product; Wherein, the time of endonuclease reaction is identical with the endonuclease reaction time in the second step of described first, and the amount of described peptide substrate is identical with the amount of peptide substrate in the second step of described first;
The 3rd step, synthetic quantum dot
To the enzyme in second step, cut the presoma dropping in product for the synthesis of in the 3rd step of the described first of quantum dot, and under identical temperature and time conditions, react in the 3rd step with described first, manufacturing quantum dot after finishing, wherein, in the described presoma of input in the amount of metallic ion and nonmetallic ion and the 3rd step of described first in presoma the amount of metallic ion and nonmetallic ion identical;
The 4th step, obtains the activity of the proteinase of detected unknown concentration
The quantum dot generating in the 3rd step is carried out to fluorescence spectrometry, obtain the fluorescence spectrum peak intensity of this quantum dot, again the correlationship obtaining in this fluorescence spectrum peak intensity and described first is compared, finally obtain the proteinase activity of detected unknown concentration.
2. method according to claim 1, is characterized in that: in the second step of described first and the second step of described second portion, described pH value adopts pH buffer solution to regulate.
3. method according to claim 2, is characterized in that: described pH buffer solution is selected from any one in trishydroxymethylaminomethane-hydrochloric acid buffer solution, ammonium hydrogencarbonate buffer solution, phosphate buffered solution.
4. method according to claim 3, is characterized in that: in described pH buffer solution, contain volume fraction and be 0.1 ~ 1% serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045562.3A CN103149185B (en) | 2013-02-05 | 2013-02-05 | Novel high-efficiency protease activity detecting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045562.3A CN103149185B (en) | 2013-02-05 | 2013-02-05 | Novel high-efficiency protease activity detecting method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103149185A CN103149185A (en) | 2013-06-12 |
| CN103149185B true CN103149185B (en) | 2014-12-10 |
Family
ID=48547389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310045562.3A Expired - Fee Related CN103149185B (en) | 2013-02-05 | 2013-02-05 | Novel high-efficiency protease activity detecting method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103149185B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103552995B (en) * | 2013-09-29 | 2016-03-02 | 苏州大学 | A kind of method preparing near-infrared quantum dots |
| CN104007268B (en) * | 2014-06-05 | 2015-09-23 | 青岛科技大学 | A kind of preparation method and application thereof detecting the biology sensor of MMP-2 |
| CN105136699B (en) * | 2015-08-25 | 2018-03-16 | 四川大学 | Protease collagen hydrolysate vigour-testing method and its application based on undenatured dyeing skin foundation cream thing |
| CN107778317B (en) * | 2016-08-26 | 2019-10-08 | 华中师范大学 | Methoxyl group fluorescein derivative and preparation method thereof and carboxypeptidase y detection method and detection kit |
| CN108191838B (en) * | 2016-12-08 | 2019-10-08 | 华中师范大学 | Half cyanines class fluorescence probe and its preparation method and application for detecting carboxypeptidase y |
| CN106645074B (en) * | 2017-03-10 | 2020-04-14 | 泉州师范学院 | Direct fluorescence spectrum detection method for cystine content in cystine tablets |
| CN107843631B (en) * | 2017-12-25 | 2019-08-23 | 安阳师范学院 | Protease detection electrochemical sensor and preparation method and detection method |
| CN108562564B (en) * | 2018-03-29 | 2021-04-02 | 青岛大学 | Carbon quantum dot for detecting activity of inulase, and preparation method and application thereof |
| CN113189074A (en) * | 2021-05-11 | 2021-07-30 | 深圳技术大学 | Ultrahigh-sensitivity protease digital detection method |
| CN114275806B (en) * | 2021-12-07 | 2023-09-19 | 中国科学院深圳先进技术研究院 | Cadmium zinc selenium quantum dot, preparation method and application thereof, and ALP detection method |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011517934A (en) * | 2008-03-03 | 2011-06-23 | カンザス ステイト ユニバーシティ リサーチ ファウンデーション | Protease assay |
| CN102081040B (en) * | 2009-11-27 | 2012-05-30 | 中国科学院理化技术研究所 | Method for detecting enzyme activity by quantum dot fluorescence |
| CN102516995B (en) * | 2011-11-22 | 2013-12-18 | 武汉因诺维生物科技有限公司 | Method for preparing water-phase CdS quantum dots in oil phase |
| CN102879454A (en) * | 2012-09-27 | 2013-01-16 | 常州大学 | Method for detecting enzymatic activity by means of florescence and capillary electrophoresis on basis of quantum dot-polypeptide compound |
-
2013
- 2013-02-05 CN CN201310045562.3A patent/CN103149185B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103149185A (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103149185B (en) | Novel high-efficiency protease activity detecting method | |
| Bao et al. | Nanoparticle-free synthesis of fluorescent gold nanoclusters at physiological temperature | |
| Thakar et al. | Efficient emission from core/(doped) shell nanoparticles: applications for chemical sensing | |
| Shang et al. | Sensitive detection of cysteine based on fluorescent silver clusters | |
| US8080229B2 (en) | Water soluble nanocrystalline quantum dots | |
| Wang et al. | Development of QDs-based nanosensors for heavy metal detection: A review on transducer principles and in-situ detection | |
| Liu et al. | The synthesis and application of I–III–VI type quantum dots | |
| Altıntas et al. | Highly efficient Cd-free alloyed core/shell quantum dots with optimized precursor concentrations | |
| Yuan et al. | A fluorescence switch sensor used for D-Penicillamine sensing and logic gate based on the fluorescence recovery of carbon dots | |
| Hua et al. | Size-dependent electrochemiluminescence behavior of water-soluble CdTe quantum dots and selective sensing of l-cysteine | |
| Zhang et al. | A novel “off-on-off” fluorescent-nanoprobe based on B, N co-doped carbon dots and MnO2 nanosheets for sensitive detection of GSH and Ag+ | |
| Williams et al. | Characterizing the influence of TOPO on exciton recombination dynamics in colloidal CdSe quantum dots | |
| CN104726085A (en) | Core-shell structured quantum dot composite nanocrystalline fluorescence probe and preparation method thereof | |
| CN108663418B (en) | Amplify building " Z " type photoelectricity aptamers analysis method based on DNAWalker signal | |
| Gui et al. | Retracted Article: Upconversion luminescent logic gates and turn-on sensing of glutathione based on two-photon excited quantum dots conjugated with dopamine | |
| Karami et al. | Intrinsic dual-emissive carbon dots for efficient ratiometric detection of Cu2+ and aspartic acid | |
| CN107253961B (en) | It is a kind of can ratio test cysteine water soluble fluorescence sensor preparation and application | |
| Petryayeva et al. | Optimization and changes in the mode of proteolytic turnover of quantum dot–peptide substrate conjugates through moderation of interfacial adsorption | |
| CN103332674A (en) | Method for hydrothermally synthesizing carbon quantum dots on basis of tartaric acid and organic amine | |
| Song et al. | Highly sensitive and selective detection of phosphate using novel highly photoluminescent water-soluble Mn-doped ZnTe/ZnSe quantum dots | |
| Zhou et al. | A novel “off-on-off” fluorescent sensor based on inner filter effect for ultrasensitive detection of protamine/trypsin and subcellular colocalization | |
| Zhu et al. | Highly selective and sensitive detection of glutathione over cysteine and homocysteine with a turn-on fluorescent biosensor based on cysteamine-stabilized CdTe quantum dots | |
| Rombach-Riegraf et al. | Blinking effect and the use of quantum dots in single molecule spectroscopy | |
| CN101948685B (en) | Synthesis and application of citrate-covered water soluble CdS nanomaterial | |
| Guo et al. | An electrostatic repulsion strategy for a highly selective and sensitive “switch-on” fluorescence sensor of ascorbic acid based on the cysteamine-coated CdTe quantum dots and cerium (iv) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141210 Termination date: 20200205 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |