CN102384902A - Enzyme chip based on quantum dot fluorescence detection, preparation method and application - Google Patents
Enzyme chip based on quantum dot fluorescence detection, preparation method and application Download PDFInfo
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- CN102384902A CN102384902A CN2010102699351A CN201010269935A CN102384902A CN 102384902 A CN102384902 A CN 102384902A CN 2010102699351 A CN2010102699351 A CN 2010102699351A CN 201010269935 A CN201010269935 A CN 201010269935A CN 102384902 A CN102384902 A CN 102384902A
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Abstract
The invention relates to an enzyme chip based on quantum dot fluorescence detection, a preparation method and application, which manufactures substrates of the enzyme chip through a mask photoetching. A plurality of mini-chambers are obtained on the substrates. A reaction system formed by a water-solubility quantum dot, enzyme oligomer and coenzyme or formed by the water-solubility quantum dot, enzyme and the coenzyme is loaded in each mini-chamber which is used for detection and arranged on the substrates provided with the plurality of mini-chambers. Or one reaction system formed by the water-solubility quantum dot, the enzyme oligomer and the coenzyme or formed by the water-solubility quantum dot, the enzyme and the coenzyme is respectively loaded in different mini-chambers which are arranged on the substrate and used for detection. By means of the reaction of the enzyme to be detected and the enzyme oligomer or the reaction of the enzyme and the enzyme oligomer to be detected, fluorescence strength of the water-solubility quantum dot changes. High-selectivity quantitative detection of activity of the enzyme to be detected and/or density of the enzyme oligomer to be detected can be achieved through detection of the quantity of the change of the fluorescence strength of the water-solubility quantum dot.
Description
Technical field
The invention belongs to field of biological detection, particularly relate to enzyme chip, detect enzyme chip and the above-mentioned enzyme chip production method and the application of enzymatic activity based on the enzyme chip of quantum dot fluorescence detection of enzymatic reactions substrate and based on quantum dot fluorescence based on quantum dot fluorescence multienzyme joint-detection.
Background technology
In numerous early stage fast detecting means, biochip be development in recent years the most rapidly and be considered to have most a kind of detection means of industrialization prospect, it has very significantly advantage, integrated, parallel, fast detecting or the like.The enzyme chip is a kind of of biochip, and it is that association reaction through enzyme molecule and corresponding substrate detects, and therefore has high selectivity, low expense, handling safety, is convenient to advantage such as on-site measurement.Because the various physiological change of human body are a lot of relevant with enzyme reaction, so the enzyme chip can be used for the detection of multiple disease.
It is the index that often has in the blood testing that gamma glutamyltransferase (GGT), lactic dehydrogenase (LDH) and alkaline phosphatase enzymatic activitys such as (ALP) detect, and has very important clinical meaning.For example: the major function of gamma glutamyltransferase is to participate in the metabolism of body internal protein, and extensively is present in each tissue of human body and the organ, and is the abundantest with kidney, secondly is that pancreas liver etc. is located.The synthetic GGT of liver increases under cholestasis, inflammation etc. stimulate, and therefore most of disease in the liver and gallbladder serum GGT activity can have increase in various degree.When alcoholic cirrhosis and biliary cirrhosis, GGT obviously raises, and can reach normal 5～10 times.Heavy type hepatitis, end-age cirrhosis are because microsome destroys, enzymic synthesis reduces GGT thereby decline.Intrahepatic cholestasis, extrahepatic biliary passages that a variety of causes causes block, and GGT all obviously increases, can reach 400 international units/liter more than.During liver cancer, because the contrary differentiation of cancer cell, similar embryonic period, embryonic phase, the generation of this enzyme increases, so serum GGT also is moderate or highly raises, also often whether transfers this enzyme the reference index of liver cancer to as observing chronic hepatitis companion patient with liver cirrhosis clinically.Lactic dehydrogenase is a kind of glycolytic ferment, and lactic dehydrogenase is present in all histiocytic kytoplasms of body, and is wherein higher with kidney content.After the acute myocardial infarction outbreak, LDH is active in the early stage blood serum raises; LDH can raise when hepatitis, acute hepatocellular injury and Skeletal muscle injury.Suffering from sick LDH such as activity rheumatic heart disease, acute viral myocarditis, hemolytic anemia, renal necrosis also can raise.Alkaline phosphatase is a kind of phosphomonoesterase, extensively is present in tissue and the body fluid, with bone, liver, mammary gland, intestinal mucosa, kidney, in the placenta.Obstructive jaundice, cirrhosis, hepatonecrosis, alkaline phosphatase obviously raise (parenchymal jaundice then raises not obvious); Alkaline phosphatase also obviously raises when primary and secondary carcinoma of liver, increases relevant with liver cell synthetic alkali acid phosphatase in the cancerous tissue or around the cancerous swelling; Other tumours such as breast cancer, lung cancer, oophoroma, osteocyte knurl, osteosarcoma etc., when alkaline phosphatase increased, prompting had liver and shifts.Therefore the single or joint-detection of gamma glutamyltransferase (GGT), lactic dehydrogenase (LDH), alkaline phosphatase (ALP) can realize the early diagnosis of cancer (S.L.Liang, D.W.Chan, Clinica Chimica Acta 2007,381,93-97).
Because enzyme reaction has very strong selectivity; Zymoprotein often be easy to obtain than antibody, antigen or gene protein etc. and biologically active relatively stable; The substrate of enzyme reaction generally all is micromolecular organism, and easy acquisition, low price are so structure detects the chip of enzyme mark with more convenient and inexpensive; Be easy to promote the universalness and the family oriented of major disease early diagnosis, thereby improve the cure rate of major disease.
In the detection of biochip, detecting through fluorescent marker method is the method for using always.At present, using the most general fluorescent material is organic dyestuff, in most of the cases; Because their excitation spectrum is all narrower; So be difficult to excite various ingredients simultaneously, and its fluorescent characteristics is composed broad, and it is asymmetric to distribute; This makes troubles for the fluorescence of distinguishing different material, therefore will detect comparatively difficulty of various ingredients simultaneously.The most serious defective of organic dyestuff still is that photochemical stability is poor, and the fluorescent photon par that photobleaching and photodissociation can be sent each dye molecule is few, and photolytic product tends to again biosome is produced lethal effect.In recent years, quantum dot is because its special optics and electrical properties have become one of biochemical hot research fields.Compare with organic fluorescent dye, quantum dot has following advantage: (1) fluorescent emission is more stable, and its luminescent lifetime can reach the ms level, and is difficult for by photobleaching; (2) fluorescent emission intensity is high, and the spectrum peak is narrow, the peak shape symmetry; (3) emission wavelength red shift regularly with the increase of particle diameter changes particle diameter and just can obtain multicolor luminous; (4) its PLE almost is continuous absorbing more than the threshold value, helps multi-wavelength excitation.Thereby quantum dot is expected to be substituted with organic dye and is applied to biochip.Quantum dot has been applied to assembling (S.Sabella, G.Vecchio, R.Cingolani, R.Rinaldi, the P.P.Pompa of biochip at present; Langmuir, 2008,24,13266-13269), once there was the report assembling to utilize the fluorescence principle to detect the enzyme chip (C.B.Cohen of protein kinase; E.C.Dixon, S.Jeong, T.T.Nikiforov, Analytical Biochemistry; 1999,273,89-97), but used fluorescent material is an organic dyestuff.Though the report that detects of quantum dot fluorescence was on the increase in recent years, the research that is used to detect the enzyme disease markers does not also appear in the newspapers.
Summary of the invention
The object of the present invention is to provide the enzyme chip that detects based on quantum dot fluorescence, this enzyme chip can single detection or is detected two or more enzymatic activitys simultaneously and/or the concentration of enzyme reaction substrate.
Another object of the present invention is to provide a kind of enzyme chip production method that detects based on quantum dot fluorescence, quantum dot and enzyme detection architecture are structured on the substrate that adopts the making of mask lithography method, to obtain the described enzyme chip that detects based on quantum dot fluorescence.
A purpose more of the present invention provides the application of the enzyme chip that detects based on quantum dot fluorescence, comprehensive detection when utilizing the enzyme chip that detects based on quantum dot fluorescence to carry out enzymatic activity and/or the enzyme reaction substrate concentration of multienzyme.
The enzyme chip that detects based on quantum dot fluorescence of the present invention be one have a plurality of microchambers on-chip each microchamber that is used for detecting be mounted with the reaction system that constitutes by water-soluble quantum dot, enzyme reaction substrate and coenzyme; Or be mounted with the reaction system that constitutes by water-soluble quantum dot, enzyme and coenzyme, or be mounted with reaction system that constitutes by water-soluble quantum dot, enzyme reaction substrate and coenzyme and the reaction system that constitutes by water-soluble quantum dot, enzyme and coenzyme respectively at the said on-chip different microchamber that is used for detecting.
In the described reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme, the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10
-8～6.5 * 10
-4Mol/L, the volumetric molar concentration of enzyme reaction substrate is 8 * 10
-5～6 * 10
-2Mol/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L.
In the described reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme, the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10
-8～6.5 * 10
-4Mol/L, the concentration of enzyme is 1.2 * 10
3～7.5 * 10
4Individual unit of activity/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L.
Described microchamber is (can with reference to CN200710177651.8) who adopts that the mask lithography method forms on substrate, and preferred described microchamber is that each length of side all is the square microchamber of 0.2～50mm, and the quantity of said microchamber is restriction not.One deck high molecular polymer is arranged at described microchamber bottom.
Described high molecular polymer is at least a in the group formed of gelatin, xanthans, cross-linked polyacrylamide, agar and carragheen.
Described substrate is quartz or glass sheet.
Described water-soluble quantum dot is a prior art products, as preparing with reference to CN 200810101429.4 disclosed methods.The fluorescent emission wavelength of described water-soluble quantum dot is positioned at 400～700 nanometers, and quantum yield is greater than 20%.
Described water-soluble quantum dot is CdTe quantum dot, CdTe/CdS quantum dot, CdTe/CdS/ZnS quantum dot, CdSe/ZnS quantum dot, ZnTe quantum dot or ZnSe quantum dot preferably.
At least a in the group that described enzyme reaction substrate is selected from gamma-glutamyl-3-carboxyl-paranitroanilinum, glycylglycine, gamma-glutamyl-a naphthylamines, γ one glutamy paranitroanilinum, n-butyl lactate, L-lactic acid, pyruvic acid, phosphoric acid p-nitrophenyl, paranitrophenol disodium hydrogen phosphate, disodium phenyl phosphate, L-alanine, KG, acecoline, Choline Chloride, glucose, form nitro phenyl phosphate, sodium pyrophosphate and cholesterol.
Described coenzyme is NADH oxidation state (NAD
+) or its go back ortho states (NADH).
Described enzyme is gamma glutamyltransferase, lactic dehydrogenase, glutamic-pyruvic transaminase, cholinesterase, choline oxidase, glucose oxidase, cholesterol oxidase or alkaline phosphatase.
The present invention is the substrate through mask lithography fabrication techniques enzyme chip; On said substrate, obtain a plurality of microchambers; And be mounted with one by the reaction system of water-soluble quantum dot and enzyme reaction substrate (coenzyme is further arranged sometimes) formation or the reaction system that constitutes by water-soluble quantum dot and enzyme (coenzyme is further arranged sometimes) at the microchamber that each is used for detecting, or be mounted with one respectively by the reaction system of water-soluble quantum dot and enzyme reaction substrate (coenzyme is further arranged sometimes) formation and the reaction system that constitutes by water-soluble quantum dot and enzyme (coenzyme is further arranged sometimes) at the said on-chip different microchamber that is used for detecting; Reaction through enzyme to be detected and enzyme reaction substrate or the reaction through enzyme and enzyme reaction substrate to be detected; Measure the influence of the photoluminescent property of above-mentioned reaction pair water-soluble quantum dot, thereby to enzymatic activity to be detected and/or treat the detection by quantitative of the high selectivity of detection of enzymatic reactions concentration of substrate.Specifically be in containing the reaction system of water-soluble quantum dot, or further also contain in the reaction system of coenzyme, add a certain amount of enzyme to be detected or enzyme reaction substrate to be detected.When detecting enzymatic activity; Reaction system is made up of water-soluble quantum dot and enzyme reaction substrate; Sometimes also further contain coenzyme in the reaction system, when adding enzyme to be detected, the reaction of enzyme to be detected and enzyme reaction substrate will take place like this; This reaction can make the fluorescence intensity of water-soluble quantum dot change, and the variable quantity of the fluorescence intensity through water-soluble quantum dot is realized the detection by quantitative to the high selectivity of enzymatic activity to be detected; When the detection of enzymatic reactions concentration of substrate; Reaction system is made up of water-soluble quantum dot and enzyme; Sometimes also further contain coenzyme in the reaction system, when adding enzyme reaction substrate to be detected, the reaction of enzyme and enzyme reaction substrate to be detected will take place like this; This reaction can make the fluorescence intensity of water-soluble quantum dot change, and the variable quantity of the fluorescence intensity through water-soluble quantum dot realizes treating the detection by quantitative of the high selectivity of detection of enzymatic reactions concentration of substrate.When the enzyme chip that detects based on quantum dot fluorescence,, so just can detect a plurality of systems to be detected simultaneously because a system to be detected (enzyme to be detected or enzyme reaction substrate to be measured) correspondence is loaded in a microchamber that is used for detecting; And, therefore can realize detection simultaneously to enzymatic activity to be detected and enzyme reaction substrate concentration to be measured because enzyme to be detected can be loaded in the said on-chip different microchamber that is used for detecting respectively with enzyme reaction substrate to be measured.
The enzyme chip production method that detects based on quantum dot fluorescence of the present invention may further comprise the steps:
(1) preparation of macromolecule polymer solution: it is to obtain macromolecule polymer solution in 40～80 ℃ the water that high molecular polymer is dissolved in water temperature, and the concentration of macromolecule polymer solution is 0.5～10wt%;
(2) making of substrate: (length of side of preferred square microchamber is 0.2～50mm) on the substrate as the enzyme chip, to obtain a plurality of microchambers through the mask lithography technology; And the macromolecule polymer solution of implantation step (1) preparation in each microchamber, cool to room temperature (during the macromolecule polymer solution cool to room temperature, form one deck high polymer layer, have water absorptivity) in the microchamber bottom; Macromolecule polymer solution used herein all is when high temperature (40～80 ℃), to inject, and when cool to room temperature, forms high polymer layer in the microchamber bottom, and after following step (8) was injected solution, high molecular polymer will water absorption and swelling, can't overflow.
(3) prepare quantum dot solution: water-soluble quantum dot is dispersed in obtains quantum dot solution in the phosphate buffer, the volumetric molar concentration of the water-soluble quantum dot in the quantum dot solution is 1.3 * 10
-7～2.5 * 10
- 3Mol/L; Wherein: the concentration of preferred employed phosphate buffer is 0.01 mol, and the pH value is 6.8;
(4) prepare enzyme reaction substrate solution: enzyme reaction substrate is dissolved in obtains enzyme reaction substrate solution in the phosphate buffer, the volumetric molar concentration of the enzyme reaction substrate in the enzyme reaction substrate solution is 6 * 10
-4～3 * 10
-1Mol/L; Wherein: the concentration of preferred employed phosphate buffer is 0.01 mol, and the pH value is 6.8;
(5) prepare enzyme solutions: enzyme is dissolved in obtains enzyme solutions in the phosphate buffer, the concentration of the enzyme in the enzyme solutions is 3.6 * 10
4～2.8 * 10
6Individual unit of activity/L; Wherein: the concentration of preferred employed phosphate buffer is 0.01 mol, and the pH value is 6.8;
(6) prepare coenzyme solution: coenzyme is dissolved in obtains coenzyme solution in the phosphate buffer, the volumetric molar concentration of the coenzyme in the coenzyme solution is 0～5 * 10
-1Mol/L; Wherein: the concentration of preferred employed phosphate buffer is 0.01 mol, and the pH value is 6.8; In detection architecture of the present invention, particularly, need coenzyme to being in the reaction system that constitutes of n-butyl lactate, L-lactic acid or pyruvic acid by water-soluble quantum dot and enzyme reaction substrate or water-soluble quantum dot and enzyme are in the reaction system that constitutes of lactic dehydrogenase;
(7) solution with step (3), step (4) and step (6) preparation mixes; Add the phosphate buffer dilution and obtain the reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme, wherein: the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10 in the reaction system
-8～6.5 * 10
-4Mol/L, the volumetric molar concentration of enzyme reaction substrate is 8 * 10
-5～6 * 10
-2Mol/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L; Or
The solution of step (3), step (5) and step (6) preparation is mixed, add the phosphate buffer dilution and obtain the reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme, wherein: the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10 in the reaction system
-8～6.5 * 10
-4Mol/L, the concentration of enzyme is 1.2 * 10
3～7.5 * 10
4Individual unit of activity/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L; Wherein: the concentration of preferred employed phosphate buffer is 0.01 mol, and the pH value is 6.8;
(8) reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme that step (7) is prepared joins the on-chip microchamber that is used for detecting that step (2) makes, and assembling obtains being used to detect the enzyme chip based on the quantum dot fluorescence detection of enzymatic activity; Or
The reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme that step (7) is prepared joins the on-chip microchamber that is used for detecting that step (2) makes, and assembling obtains being used for the enzyme chip based on the quantum dot fluorescence detection of detection of enzymatic reactions concentration of substrate; Or
The reaction system that constitutes by water-soluble quantum dot, enzyme reaction substrate and coenzyme that step (7) is prepared; With join the on-chip different microchamber that is used for detecting that step (2) makes respectively by water-soluble quantum dot, enzyme and reaction system that coenzyme constitutes; Assembling obtains being used to detect the enzyme chip based on quantum dot fluorescence multienzyme joint-detection of enzymatic activity and detection of enzymatic reactions concentration of substrate, the enzyme chip that promptly detects based on quantum dot fluorescence.
The application of the enzyme chip that detects based on quantum dot fluorescence of the present invention joins enzyme to be detected on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme in a plurality of microchambers; Or
Enzyme reaction substrate to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme in a plurality of microchambers; Or
Enzyme to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme in a plurality of microchambers, enzyme reaction substrate to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme in a plurality of microchambers;
Reaction (preferred reaction 5～30 minutes) at normal temperatures; Adopt fluorospectrophotometer to detect the enzyme chip, the variable quantity of the fluorescence intensity through water-soluble quantum dot obtains the fluorescence spectrum of enzyme chip, and the variation of the fluorescence intensity through analysis of fluorescence spectrum records enzymatic activity to be detected and/or enzyme reaction substrate concentration to be detected.
Enzyme chip of the present invention can be used for the detection of physical signs such as gamma glutamyltransferase, lactic dehydrogenase, glutamic-pyruvic transaminase, alkaline phosphatase, choline, glucose or cholesterol, thereby carries out the early detection and the diagnosis of relevant diseases such as angiocardiopathy, diabetes, disease in the liver and gallbladder and cancer.
During described detection enzymatic activity; Enzyme to be detected joined on the substrate (also further contain coenzyme sometimes in the reaction system) in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot and enzyme reaction substrate in a plurality of microchambers, the variable quantity of the fluorescence intensity through water-soluble quantum dot is realized the detection by quantitative to the high selectivity of enzymatic activity to be detected.
During the concentration of described detection of enzymatic reactions substrate; Enzyme reaction substrate to be detected joined on the substrate (also further contain coenzyme sometimes in the reaction system) in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot and enzyme in a plurality of microchambers, the variable quantity of the fluorescence intensity through water-soluble quantum dot realizes treating the detection by quantitative of the high selectivity of detection of enzymatic reactions concentration of substrate.
Detect in the time of described enzymatic activity and enzyme reaction substrate concentration; Be enzyme to be detected to be joined on the substrate (also further contain coenzyme sometimes in the reaction system) in the microchamber that is mounted with the reaction system that constitutes by water-soluble quantum dot and enzyme reaction substrate in a plurality of microchambers; Enzyme reaction substrate to be detected joined on the substrate (also further contain coenzyme sometimes in the reaction system) in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot and enzyme in a plurality of microchambers, the variable quantity of the fluorescence intensity through water-soluble quantum dot realizes respectively the detection by quantitative to the high selectivity of enzymatic activity to be detected and enzyme reaction substrate concentration to be detected.
Described substrate is quartz or glass sheet.
Described high molecular polymer is at least a in the group formed of gelatin, xanthans, cross-linked polyacrylamide, agar and carragheen.
The fluorescent emission wavelength of described water-soluble quantum dot is positioned at 400～700 nanometers, and quantum yield is greater than 20%.Described water-soluble quantum dot is CdTe quantum dot, CdTe/CdS quantum dot, CdTe/CdS/ZnS quantum dot, CdSe/ZnS quantum dot, ZnTe quantum dot or ZnSe quantum dot.
At least a in the group that described enzyme reaction substrate is selected from gamma-glutamyl-3-carboxyl-paranitroanilinum, glycylglycine, gamma-glutamyl-a naphthylamines, γ one glutamy paranitroanilinum, n-butyl lactate, L-lactic acid, pyruvic acid, phosphoric acid p-nitrophenyl, paranitrophenol disodium hydrogen phosphate, disodium phenyl phosphate, L-alanine, KG, acecoline, Choline Chloride, glucose, form nitro phenyl phosphate, sodium pyrophosphate and cholesterol.
Described coenzyme is NADH oxidation state (NAD
+) or its go back ortho states (NADH).
Described enzyme is gamma glutamyltransferase, lactic dehydrogenase, glutamic-pyruvic transaminase, cholinesterase, choline oxidase, glucose oxidase, cholesterol oxidase or alkaline phosphatase.
Single or the multiple physiology enzymatic activity that the enzyme chip that detects based on quantum dot fluorescence of the present invention can be used for being correlated with and the detection of physical signs, thus carry out angiocardiopathy, diabetes, the early detection of relevant diseases such as disease in the liver and gallbladder and cancer and diagnosis.That enzyme chip of the present invention has in use is simple to operate, detect fast, cost is low.
Enzyme chip that detects based on quantum dot fluorescence of the present invention and preparation method thereof, its detect principle and advantage following:
The water-soluble quantum dot that uses of the present invention be adopt a kind of fast, simple, environmental protection, the new method that can be continuously produced be synthetic.Compare with the organic phase quantum dot, this water-soluble quantum dot and enzyme molecule have better biocompatibility and compatibility, more are applicable to biochemistry detection.
2. the water-soluble quantum dot surface of the present invention's use has certain chemical active radical; When enzyme to be detected and enzyme reaction substrate or enzyme and enzyme reaction substrate to be detected reaction generation; The chemical valence link that produces changes and can produce disturbance to the photoluminescent property of water-soluble quantum dot; And this disturbance has certain corresponding relation with the amount of substance that reacts, thereby can realize the detection by quantitative of high selectivity.
3. the present invention detects the disease marker enzyme through the water-soluble quantum dot change in fluorescence, thereby realizes the early diagnosis of disease.Fluoroscopic examination has that information capacity is big, response speed is fast, highly sensitive, easy and simple to handle, with low cost, advantage such as the optics antijamming capability is strong; With respect to checkout equipments such as mass spectrum, microscope, electrochemical workstations, be easier to miniaturization, mechanization, the integrated and commercialization of the equipment of realizing.Advantages such as that enzyme reaction has is quick, single-minded, sensitivity.Therefore detect the enzyme amount through change in fluorescence, both can realize the qualitative detection that the enzyme biochemical indicator is quick, sensitive, also can carry out quantitative measurement.
4. method provided by the invention is that water-soluble quantum dot and enzyme detection architecture are structured on the substrate that adopts the making of mask lithography method.Because the fluorescent emission wavelength of water-soluble quantum dot can change through the change of water-soluble quantum dot particle diameter.Therefore combine with different enzyme reaction systems through water-soluble quantum dot different emission, when realizing plurality of enzymes, fast, detection easily.Through selecting suitable enzyme mark, carry out permutation and combination, can make up the enzyme chip that detects multiple disease simultaneously, thereby improve the detection efficiency of multiple disease.
Description of drawings
Fig. 1. the fluorescence spectrum figure of the enzyme chip detection lactic dehydrogenase of the embodiment of the invention 2." U " represents the active unit of enzyme among the figure.
Fig. 2. the linear fit figure of the fluoroscopic examination of the embodiment of the invention 2.
Fig. 3. the enzyme chip of the embodiment of the invention 3 detects the fluorescence spectrum figure of lactic dehydrogenase and glucose simultaneously;
Wherein: curve (1) is the fluorescence spectrum figure that the enzyme chip of QD-545 and QD-600 is housed respectively in two microchambers;
Curve (2) is in two microchambers QD-545+NAD to be housed respectively
+The fluorescence spectrum figure of the enzyme chip of+n-butyl lactate and QD-600+ glucose oxidase;
Curve (3) is the fluorescence spectrum figure that adds the enzyme chip of 0.03U lactic dehydrogenase and 4.4mM glucose in two microchambers in curve (2) respectively;
Curve (4) is the fluorescence spectrum figure that adds the enzyme chip of 3U lactic dehydrogenase and 8.9mM glucose in two microchambers in curve (2) respectively;
Curve (5) is the fluorescence spectrum figure that adds the enzyme chip of 1.5U lactic dehydrogenase and 2.2mM glucose in two microchambers in curve (2) respectively;
Wherein " U " represents the active unit of enzyme.
Embodiment
Embodiment 1
1, the preparation of cadmium telluride quantum dot (with reference to CN 200810101429.4 preparations),
(1) in deionized water, adds cadmium nitrate, TGA successively; The mol ratio that makes cadmium nitrate and TGA is 1: 50, after complex reaction is accomplished, adds and the equimolar sodium hydrogen telluride of cadmium nitrate; Obtain the precursor solution of water-soluble quantum dot, the concentration of final cadmium ion is 10
-5M;
(2) precursor solution that step (1) is obtained is added in the ultrasonic atomizer, and through behind the ultrasonic atomizatio, precursor solution exists with the form of sub-micron droplet, obtains the droplet of the precursor solution of water-soluble quantum dot;
(3) feed nitrogen; Nitrogen flow is 5L/min; The droplet of the precursor solution that step (2) is obtained is brought in the reaction unit of temperature stabilization, and the droplet of precursor solution is through heating (temperature is 450 ℃) reaction, and reaction product is absorbed by water and obtains water miscible cadmium telluride quantum dot; The fluorescent emission wavelength is 569nm, and quantum yield is 50%.
2, adopt the substrate (with reference to CN 200710177651.8 preparations) of mask lithography fabrication techniques enzyme chip
With piezoid base material 1 is in the sodium hydrate aqueous solution, acetone or alcohol of 1wt% in concentration successively, stirs 5 minutes with 3000rpm respectively under the room temperature, washes drying 3 times;
In the electric paper micro-cup of anti-solvent glue material 2: oligomer: EM7666 (sour modification cresol-novolak epoxy acrylate) (Changxing, Taiwan chemical industry company limited), concentration is 10wt%, SB401 (aromatic acid methacrylic acid half ester) (U.S. Sartomer company), concentration is 40wt%, CN988 (amino ester/acrylic ester) (U.S. Sartomer company); Concentration is 10wt%, monomer: 1, and 6-hexanediol double methacrylate; Concentration is 10wt%, two trimethylolpropene tetraacrylate, and concentration is 10wt%, light trigger: 2-hydroxyl-aminomethyl phenyl propane-1-ketone; Concentration is 1wt%, 2,4,6-trimethylbenzoyl-diphenyl phosphine oxide; Concentration is 1wt%; Modifier: maleic anhydride, concentration are 10wt%, surfactant: BYK306 (polyether-modified dimethyl silicone polymer interpolymer solution) (German Bi Ke chemical company); Concentration is 1wt%, BYK109 (HMW alkane hydroxyl amino acid amides) (German Bi Ke chemical company); Concentration is 1wt%, CD9050 (U.S. Sartomer company), and concentration is 6wt%, and 30 ℃ of lucifuges stirred 15 minutes;
Steel wire scraper with 250 μ m applies one deck electric paper micro-cup glue material 2 on piezoid base material 1; Baked 10 minutes in advance for 80 ℃; Add polyethylene terephthalate (PET) mask 3 that has rectangular patterns,, remove mask then in 4 times exposures of 1000W high-pressure mercury light 30 seconds; The sodium carbonate that with concentration is 2wt% is to develop 50 seconds in the developer solution of main body, and with deionized water rinsing 3 times, 80 ℃ were toasted 5 minutes, and promptly got the high microchamber pattern of identical 0.2mm with mask, microchamber length of side 20mm.
3, adopt the activity of enzyme chip detection gamma glutamyltransferase:
(1) preparation of gelatin solution: with the gelatin gelatin solution that obtains soluble in water, the weight percent concentration of gelatin is 2%, 60 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject gelatin solution, cool to room temperature forms one deck gelatin gel in the microchamber bottom;
(2) preparation quantum dot solution: cadmium telluride quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains cadmium telluride quantum dot solution, the volumetric molar concentration of the cadmium telluride quantum dot in the cadmium telluride quantum dot solution is 6.9 * 10
-4Mol/L;
(3) preparation enzyme reaction substrate solution: gamma-glutamyl-3-carboxyl-paranitroanilinum and glycylglycine are dissolved in respectively in the phosphate buffer (the pH value is 6.8) of 0.01 mol; Obtain gamma-glutamyl-3-carboxyl-paranitroanilinum and glycylglycine solution respectively, the volumetric molar concentration of the gamma-glutamyl-3-carboxyl-paranitroanilinum in gamma-glutamyl-3-carboxyl-p-nitrophenyl amine aqueous solution is 9.8 * 10
-3Mol/L, the volumetric molar concentration of the glycylglycine in the glycylglycine solution is 3.8 * 10
-3Mol/L;
(4) solution with step (2) and step (3) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the cadmium telluride quantum dot in the mixed liquor after the dilution is 3.6 * 10
-7Mol/L, the volumetric molar concentration of gamma-glutamyl-3-carboxyl-paranitroanilinum is 6.5 * 10
-3Mol/L, the volumetric molar concentration of glycylglycine is 2.5 * 10
-3Mol/L;
(5) mixed solution of step (4) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains detecting the active enzyme chip of gamma glutamyltransferase based on quantum dot fluorescence;
(6) gamma glutamyltransferase solution to be detected is joined in the microchamber of enzyme chip of step (5) assembling the reaction 10 minutes down of system normal temperature; Adopt fluorospectrophotometer to detect the enzyme chip, obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the active data of gamma glutamyltransferase.
Embodiment 2
1, the preparation of cadmium telluride quantum dot is with embodiment 1.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 30mm.
3, adopt the activity of enzyme chip detection lactic dehydrogenase:
(1) preparation of agar solution: with the agar agar solution that obtains soluble in water, the weight percent concentration of agar is 5%, 70 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject agar solution, cool to room temperature forms one deck agar gel in the microchamber bottom;
(2) preparation quantum dot solution: cadmium telluride quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains cadmium telluride quantum dot solution, the volumetric molar concentration of the cadmium telluride quantum dot in the cadmium telluride quantum dot solution is 8.9 * 10
-6Mol/L;
(3) preparation enzyme reaction substrate solution: n-butyl lactate is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes n-butyl lactate is 5.2 * 10
-2Mol/L;
(4) preparation coenzyme solution: the NADH oxidation state is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes the NADH oxidation state is 4.5 * 10
-1Mol/L;
(5) with step (2), the solution of step (3) and step (4) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the cadmium telluride quantum dot in the mixed liquor after the dilution is 6.7 * 10
-7Mol/L, the volumetric molar concentration of n-butyl lactate is 2.2 * 10
-3Mol/L, the volumetric molar concentration of NADH oxidation state is 4.8 * 10
-3Mol/L;
(6) mixed solution of step (5) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains detecting based on quantum dot fluorescence the enzyme chip of lactic acid dehydrogenase activity;
(7) lactic dehydrogenase enzyme solutions to be detected is joined in the microchamber of enzyme chip of step (6) assembling the reaction 5 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the lactic acid dehydrogenase activity data.The fluorescence spectrum figure of the enzyme chip detection lactic dehydrogenase that obtains sees Fig. 1, and the linear fit figure of fluoroscopic examination sees Fig. 2.
1, the preparation of cadmium telluride quantum dot (with reference to CN 200810101429.4 preparations)
(1) in deionized water, adds cadmium nitrate, TGA successively; The mol ratio that makes cadmium nitrate and TGA is 1: 3, after complex reaction is accomplished, adds and the equimolar sodium hydrogen telluride of cadmium nitrate; Obtain the precursor solution of water-soluble quantum dot, the concentration of final cadmium ion is 5 * 10
-2M;
(2) precursor solution that step (1) is obtained is added in the ultrasonic atomizer, and through behind the ultrasonic atomizatio, precursor solution exists with the form of sub-micron droplet, obtains the droplet of the precursor solution of water-soluble quantum dot;
(3) feed nitrogen; Nitrogen flow is 3L/min; The droplet of the precursor solution that step (2) is obtained is brought in the reaction unit of temperature stabilization, and the droplet of precursor solution is through heating (temperature is respectively 220 ℃ and 280 ℃) reaction, and reaction product is obtained the water soluble cadmium telluride quantum point of two kinds of fluorescent emission wavelength by the water absorption; Wherein the fluorescent emission wavelength is that 545nm is expressed as QD-545, and fluorescent emission wavelength 600nm is for being expressed as QD-600.The quantum yield of QD-545 is 30%, and the quantum yield of QD-600 is 40%.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 35mm.
3, adopt the enzyme chip to detect the activity of glucose and lactic dehydrogenase simultaneously:
(1) preparation of gelatin solution: with the gelatin gelatin solution that obtains soluble in water, the weight percent concentration of gelatin is 3%, 50 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject gelatin solution, cool to room temperature forms one deck gelatin gel in the microchamber bottom;
(2) preparation quantum dot solution: the cadmium telluride quantum dot that above-mentioned two kinds of fluorescent emission wavelength are respectively QD-545 and QD-600 is dispersed in respectively in the phosphate buffer (the pH value is 6.8) of 0.01 mol; Obtain two kinds of cadmium telluride quantum dot solution respectively, the volumetric molar concentration of the cadmium telluride quantum dot in two kinds of cadmium telluride quantum dot solution is 1.3 * 10
-7Mol/L;
(3) preparation enzyme reaction substrate solution: n-butyl lactate is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes n-butyl lactate is 6.2 * 10
-3Mol/L;
(4) preparation enzyme solutions: glucose oxidase is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the concentration that makes glucose oxidase is 4.9 * 10
5Individual unit of activity/L;
(5) preparation coenzyme solution: the NADH oxidation state is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes the NADH oxidation state is 5.8 * 10
-3Mol/L;
(6) solution of QD-545, step (3) and the step (5) of step (2) preparation being prepared mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein, the volumetric molar concentration of the cadmium telluride quantum dot QD-545 in the mixed liquor after the dilution is 2.1 * 10
-8Mol/L, the volumetric molar concentration of n-butyl lactate is 8.2 * 10
-4Mol/L, the volumetric molar concentration of NADH oxidation state is 7.4 * 10
- 4Mol/L;
(7) QD-600 of step (2) preparation and the solution of step (4) preparation are mixed, add phosphate buffer (the pH value is 6.8) dilution, wherein the volumetric molar concentration of the cadmium telluride quantum dot QD-600 in the mixed liquor after the dilution is 3.1 * 10
-8Mol/L, the concentration of glucose oxidase is 8.6 * 10
3Individual unit of activity/L;
(8) two kinds of mixed solutions of step (6) and step (7) preparation are joined step (1) respectively and make in two different microchambers of substrate, assembling obtains detecting simultaneously based on quantum dot fluorescence the enzyme chip of lactic acid dehydrogenase activity and concentration of glucose;
(9) lactic dehydrogenase to be detected and glucose mixed solution are joined in two microchambers of enzyme chip of step (8) assembling the reaction 12 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the data of lactic acid dehydrogenase activity and concentration of glucose to be detected.The enzyme chip detection lactic dehydrogenase that obtains and the fluorescence spectrum figure of glucose see Fig. 3; Wherein:
Curve (1) is the fluorescence spectrum figure that the enzyme chip of QD-545 and QD-600 is housed respectively in two microchambers;
Curve (2) is in two microchambers QD-545+NAD to be housed respectively
+The fluorescence spectrum figure of the enzyme chip of+n-butyl lactate and QD-600+ glucose oxidase;
Curve (3) is the fluorescence spectrum figure that adds the enzyme chip of 0.03U lactic dehydrogenase and 4.4mM glucose mixed solution in two microchambers in curve (2) respectively;
Curve (4) is the fluorescence spectrum figure that adds the enzyme chip of 3U lactic dehydrogenase and 8.9mM glucose mixed solution in two microchambers in curve (2) respectively;
Curve (5) is the fluorescence spectrum figure that adds the enzyme chip of 1.5U lactic dehydrogenase and 2.2mM glucose mixed solution in two microchambers in curve (2) respectively;
Wherein " U " represents the active unit of enzyme.
1, the preparation of zinc telluride quantum dot (with reference to CN 200810101429.4 preparations)
(1) in deionized water, adds zinc acetate, TGA successively; The mol ratio that makes zinc acetate and TGA is 1: 20, after complex reaction is accomplished, adds and the equimolar sodium hydrogen telluride of zinc acetate; Obtain the precursor solution of water-soluble quantum dot, the concentration of final zinc ion is 3 * 10
-2M;
(2) precursor solution that step (1) is obtained is added in the ultrasonic atomizer, and through behind the ultrasonic atomizatio, precursor solution exists with the form of sub-micron droplet, obtains the droplet of the precursor solution of water-soluble quantum dot;
(3) feed nitrogen; Nitrogen flow is 1L/min; The droplet that drives liquid solution that step (2) is obtained is brought in the reaction unit of temperature stabilization, and the droplet of precursor solution is through heating (temperature is 110 ℃) reaction, and reaction product is absorbed by water and obtains water miscible zinc telluride quantum dot; The fluorescent emission wavelength is 465nm, and quantum yield is 25%.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 8mm.
3, adopt enzyme chip detection alkaline phosphatase activity:
(1) preparation of carragheen and cross-linked polyacrylamide solution: with carragheen and cross-linked polyacrylamide carragheen and the cross-linked polyacrylamide solution of obtaining soluble in water; The weight percent concentration of carragheen is 7.5%; The weight percent concentration of cross-linked polyacrylamide is 2.5%, 80 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject carragheen and cross-linked polyacrylamide solution, cool to room temperature forms one deck carragheen and crosslinked polyacrylamide gels in the microchamber bottom;
(2) preparation quantum dot solution: zinc telluride quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains zinc telluride quantum dot solution, the volumetric molar concentration of the zinc telluride quantum dot in the zinc telluride quantum dot solution is 2.5 * 10
-3Mol/L;
(3) preparation enzyme reaction substrate solution: disodium phenyl phosphate is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes disodium phenyl phosphate is 3.0 * 10
-1Mol/L;
(4) solution with step (2) and step (3) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the zinc telluride quantum dot in the mixed liquor after the dilution is 6.5 * 10
-4Mol/L, the volumetric molar concentration of disodium phenyl phosphate is 6.0 * 10
-2Mol/L;
(5) mixed solution of step (4) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains detecting based on quantum dot fluorescence the enzyme chip of alkaline phosphate ester enzymatic activity;
(6) alkaline phosphatase enzyme solutions to be detected is joined in the microchamber of enzyme chip of step (5) assembling the reaction 30 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the alkaline phosphatase activities data.
1, the preparation of zinc telluride quantum dot is with embodiment 4.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 0.5mm.
3, adopt the activity of enzyme chip detection cholinesterase:
(1) preparation of xanthan gum solution: with the xanthans xanthan gum solution that obtains soluble in water, the weight percent concentration of xanthans is 0.6%, 65 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject xanthan gum solution, cool to room temperature forms one deck xanthans gel in the microchamber bottom;
(2) preparation quantum dot solution: zinc telluride quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains zinc telluride quantum dot solution, the volumetric molar concentration of the zinc telluride quantum dot in the zinc telluride quantum dot solution is 4.2 * 10
-5Mol/L;
(3) preparation enzyme reaction substrate solution: acecoline is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration of acecoline is 3.6 * 10
-3Mol/L;
(4) solution with step (2) and step (3) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the zinc telluride quantum dot in the mixed liquor after the dilution is 2.3 * 10
-6Mol/L, the volumetric molar concentration of acecoline is 7.3 * 10
-4Mol/L;
(5) mixed solution of step (4) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains detecting based on quantum dot fluorescence the enzyme chip of cholinesterase activity;
(6) cholinesterase solution to be detected is joined in the microchamber of enzyme chip of step (5) assembling the reaction 6 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the cholinesterase activity data.
Embodiment 6
1, the preparation of zinc selenide quantum dot (with reference to CN 200810101429.4 preparations)
(1) in deionized water, adds caddy, mercaptopropionic acid successively; The mol ratio that makes caddy and mercaptopropionic acid is 1: 5, after complex reaction is accomplished, adds and the equimolar sodium hydrogen selenide of caddy; Obtain the precursor solution of water-soluble quantum dot, the concentration of final cadmium ion is 7 * 10
-3M;
(2) precursor solution that step (1) is obtained is added in the ultrasonic atomizer, and through behind the ultrasonic atomizatio, precursor solution exists with the form of sub-micron droplet, obtains the droplet of the precursor solution of water-soluble quantum dot;
(3) feed nitrogen; Nitrogen flow is 4L/min; The droplet of the precursor solution that step (2) is obtained is brought in the reaction unit of temperature stabilization, and the droplet of precursor solution is through heating (temperature is 250 ℃) reaction, and reaction product is directly absorbed by water and obtains water miscible zinc selenide fluorescence nano particle; The fluorescent emission wavelength is 652nm, and quantum yield is 35%.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 50mm.
3, adopt the activity of enzyme chip detection lactic dehydrogenase:
(1) preparation of carrageenan solutions: with the carragheen carrageenan solutions that obtains soluble in water, the weight percent concentration of carragheen is 1%, 40 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject carrageenan solutions, cool to room temperature forms one deck carragheen gel in the microchamber bottom;
(2) preparation quantum dot solution: zinc selenide quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains zinc selenide quantum dot solution, the volumetric molar concentration of the zinc selenide quantum dot in the zinc selenide quantum dot solution is 1.8 * 10
-4Mol/L;
(3) preparation enzyme reaction substrate solution: pyruvic acid is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes pyruvic acid is 8.7 * 10
-4Mol/L;
(4) preparation coenzyme solution: with NADH also ortho states be dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, make NADH also the volumetric molar concentration of ortho states be 6.5 * 10
-4Mol/L;
(5) solution with step (2), step (3) and step (4) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the zinc selenide in the mixed liquor after the dilution is 4.8 * 10
-6Mol/L, the volumetric molar concentration of pyruvic acid is 9.2 * 10
-5Mol/L, the NADH also volumetric molar concentration of ortho states are 2.3 * 10
-4Mol/L;
(6) mixed solution of step (5) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains detecting based on quantum dot fluorescence the enzyme chip of lactic acid dehydrogenase activity;
(7) lactic dehydrogenase enzyme solutions to be detected is joined in the microchamber of enzyme chip of step (5) assembling the reaction 7 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the data of lactic acid dehydrogenase activity.
Embodiment 7
1, the preparation of cadmium telluride quantum dot is with embodiment 3.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 40mm.
3, adopt the enzyme chip to detect lactic dehydrogenase and alkaline phosphatase activity simultaneously:
(1) preparation of gelatin solution: with the gelatin gelatin solution that obtains soluble in water, the weight percent concentration of gelatin is 6%, 45 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject gelatin solution, cool to room temperature forms one deck gelatin gel in the microchamber bottom;
(2) preparation quantum dot solution: two kinds of cadmium telluride quantum dots that above-mentioned two kinds of fluorescent emission wavelength are respectively QD-545 and QD-600 are dispersed in respectively in the phosphate buffer (the pH value is 6.8) of 0.01 mol; Obtain two kinds of cadmium telluride quantum dot solution respectively, the volumetric molar concentration of the cadmium telluride quantum dot in two kinds of cadmium telluride quantum dot solution all is 7.6 * 10
-5Mol/L;
(3) preparation enzyme reaction substrate solution: n-butyl lactate is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes n-butyl lactate is 6.0 * 10
-4Mol/L.To be dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol the nitro phenyl phosphate, making the volumetric molar concentration to the nitro phenyl phosphate is 4.3 * 10
-3Mol/L;
(4) preparation coenzyme solution: the NADH oxidation state is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the volumetric molar concentration that makes the NADH oxidation state is 9.6 * 10
-5Mol/L;
(5) QD-545 of step (2) preparation, the n-butyl lactate solution of step (3) preparation and the coenzyme solution of step (4) preparation are mixed; Add phosphate buffer (the pH value is 6.8) dilution, wherein the volumetric molar concentration of the cadmium telluride quantum dot QD-545 in the mixed liquor after the dilution is 1.6 * 10
- 5Mol/L, the volumetric molar concentration of n-butyl lactate is 8.0 * 10
-5Mol/L, the volumetric molar concentration of NADH oxidation state is 3.6 * 10
-5Mol/L;
(6) with the QD-600 of step (2) preparation and step (3) preparation nitro phenyl phosphate solution is mixed, add phosphate buffer (the pH value is 6.8) dilution, wherein the volumetric molar concentration of the cadmium telluride quantum dot QD-600 in the mixed liquor after the dilution is 1.6 * 10
-5Mol/L is 2.4 * 10 to the volumetric molar concentration of nitro phenyl phosphate
-4Mol/L;
(7) two kinds of mixed solutions of step (5) and step (6) preparation are joined step (1) respectively and make in two different microchambers of substrate, assembling obtains detecting simultaneously based on quantum dot fluorescence the enzyme chip of lactic acid dehydrogenase activity and alkaline phosphatase activities;
(8) lactic dehydrogenase to be detected and alkaline phosphatase mixed solution are joined in two microchambers of enzyme chip of step (7) assembling the reaction 20 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the data of lactic dehydrogenase and alkaline phosphatase activities.
Embodiment 8
1, the preparation of zinc selenide quantum dot is with embodiment 6.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 45mm.
3, adopt the concentration of enzyme chip detection cholesterol:
(1) preparation of agar solution: with the agar agar solution that obtains soluble in water, the weight percent concentration of agar is 4%, 55 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject agar solution, cool to room temperature forms one deck agar gel in the microchamber bottom;
(2) preparation quantum dot solution: zinc selenide quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains zinc selenide quantum dot solution, the volumetric molar concentration of the zinc selenide quantum dot in the zinc selenide quantum dot solution is 5.7 * 10
-4Mol/L;
(3) preparation enzyme solutions: cholesterol oxidase is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the concentration that makes cholesterol oxidase is 2.8 * 10
6Individual unit of activity/L;
(4) solution with step (2) and step (3) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the zinc selenide in the mixed liquor after the dilution is 4.3 * 10
- 5Mol/L, the concentration of cholesterol oxidase is 7.5 * 10
4Individual unit of activity/L;
(5) mixed solution of step (4) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains the enzyme chip based on quantum dot fluorescence cholesterol detection concentration;
(6) cholesterol solution to be detected is joined in the microchamber of enzyme chip of step (5) assembling the reaction 15 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the data of cholesterol concentration.
Embodiment 9
1, the preparation of cadmium telluride quantum dot is with embodiment 1.
2, the substrate of mask lithography fabrication techniques enzyme chip is with embodiment 1, and the substrate microchamber length of side that makes is 37mm.
3, adopt the concentration of enzyme chip detection pyruvic acid:
(1) preparation of carrageenan solutions: with the carragheen carrageenan solutions that obtains soluble in water, the weight percent concentration of carragheen is 10%, 75 ℃ of water temperatures.In on-chip each microchamber that step 2 is made, inject carrageenan solutions, cool to room temperature forms one deck carragheen gel in the microchamber bottom;
(2) preparation quantum dot solution: cadmium telluride quantum dot is dispersed in the phosphate buffer (the pH value is 6.8) of 0.01 mol and obtains cadmium telluride quantum dot solution, the volumetric molar concentration of the cadmium telluride quantum dot in the cadmium telluride quantum dot solution is 3.5 * 10
-4Mol/L;
(3) preparation enzyme solutions: lactic dehydrogenase is dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, the concentration that makes lactic dehydrogenase is 3.6 * 10
4Individual unit of activity/L;
(4) preparation coenzyme solution: with NADH also ortho states be dissolved in the phosphate buffer (the pH value is 6.8) of 0.01 mol, make NADH also the volumetric molar concentration of ortho states be 7.2 * 10
-4Mol/L;
(5) solution with step (2), step (3) and step (4) preparation mixes, and adds phosphate buffer (the pH value is 6.8) dilution, and wherein the volumetric molar concentration of the cadmium telluride in the mixed liquor after the dilution is 7.4 * 10
-6Mol/L, the concentration of lactic dehydrogenase is 1.2 * 10
3Individual unit of activity/L, the NADH also volumetric molar concentration of ortho states are 8.5 * 10
-5Mol/L;
(6) mixed solution of step (5) preparation is joined in the substrate microchamber that step (1) makes, assembling obtains detecting based on quantum dot fluorescence the enzyme chip of pyruvic acid concentration;
(7) pyruvic acid solution to be detected is joined in the microchamber of enzyme chip of step (6) assembling the reaction 25 minutes down of system normal temperature.Adopt fluorospectrophotometer to obtain the fluorescence spectrum of system, the variation of the fluorescence intensity through analysis of fluorescence spectrum records the data of pyruvic acid concentration.
Claims (10)
1. enzyme chip that detects based on quantum dot fluorescence; It is characterized in that: the described enzyme chip that detects based on quantum dot fluorescence be one have a plurality of microchambers on-chip each microchamber that is used for detecting be mounted with the reaction system that constitutes by water-soluble quantum dot, enzyme reaction substrate and coenzyme; Or be mounted with the reaction system that constitutes by water-soluble quantum dot, enzyme and coenzyme, or be mounted with reaction system that constitutes by water-soluble quantum dot, enzyme reaction substrate and coenzyme and the reaction system that constitutes by water-soluble quantum dot, enzyme and coenzyme respectively at the said on-chip different microchamber that is used for detecting.
2. the enzyme chip that detects based on quantum dot fluorescence according to claim 1 is characterized in that: in the described reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme, the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10
-8～6.5 * 10
-4Mol/L, the volumetric molar concentration of enzyme reaction substrate is 8 * 10
-5～6 * 10
-2Mol/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L;
In the described reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme, the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10
-8～6.5 * 10
-4Mol/L, the concentration of enzyme is 1.2 * 10
3～7.5 * 10
4Individual unit of activity/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L.
3. the enzyme chip that detects based on quantum dot fluorescence according to claim 1 is characterized in that: one deck high molecular polymer is arranged at described microchamber bottom;
Described high molecular polymer is at least a in the group formed of gelatin, xanthans, cross-linked polyacrylamide, agar and carragheen.
4. the enzyme chip that detects based on quantum dot fluorescence according to claim 1 and 2, it is characterized in that: the fluorescent emission wavelength of described water-soluble quantum dot is positioned at 400～700 nanometers, and quantum yield is greater than 20%.
5. the enzyme chip that detects based on quantum dot fluorescence according to claim 4, it is characterized in that: described water-soluble quantum dot is CdTe quantum dot, CdTe/CdS quantum dot, CdTe/CdS/ZnS quantum dot, CdSe/ZnS quantum dot, ZnTe quantum dot or ZnSe quantum dot.
6. the enzyme chip that detects based on quantum dot fluorescence according to claim 1 and 2 is characterized in that: at least a in the group that described enzyme reaction substrate is selected from gamma-glutamyl-3-carboxyl-paranitroanilinum, glycylglycine, gamma-glutamyl-a naphthylamines, γ one glutamy paranitroanilinum, n-butyl lactate, L-lactic acid, pyruvic acid, phosphoric acid p-nitrophenyl, paranitrophenol disodium hydrogen phosphate, disodium phenyl phosphate, L-alanine, KG, acecoline, Choline Chloride, glucose, form nitro phenyl phosphate, sodium pyrophosphate and cholesterol;
Described coenzyme is that NADH oxidation state or its are gone back ortho states.
7. the enzyme chip that detects based on quantum dot fluorescence according to claim 1 and 2, it is characterized in that: described enzyme is gamma glutamyltransferase, lactic dehydrogenase, glutamic-pyruvic transaminase, cholinesterase, choline oxidase, glucose oxidase, cholesterol oxidase or alkaline phosphatase.
8. one kind according to any described enzyme chip production method that detects based on quantum dot fluorescence of claim 1～7, it is characterized in that this method may further comprise the steps:
(1) prepare quantum dot solution: water-soluble quantum dot is dispersed in obtains quantum dot solution in the phosphate buffer, the volumetric molar concentration of the water-soluble quantum dot in the quantum dot solution is 1.3 * 10
-7～2.5 * 10
- 3Mol/L;
(2) prepare enzyme reaction substrate solution: enzyme reaction substrate is dissolved in obtains enzyme reaction substrate solution in the phosphate buffer, the volumetric molar concentration of the enzyme reaction substrate in the enzyme reaction substrate solution is 6 * 10
-4～3 * 10
-1Mol/L;
(3) prepare enzyme solutions: enzyme is dissolved in obtains enzyme solutions in the phosphate buffer, the concentration of the enzyme in the enzyme solutions is 3.6 * 10
4～2.8 * 10
6Individual unit of activity/L;
(4) prepare coenzyme solution: coenzyme is dissolved in obtains coenzyme solution in the phosphate buffer, the volumetric molar concentration of the coenzyme in the coenzyme solution is 0～5 * 10
-1Mol/L;
(5) solution with step (1), step (2) and step (4) preparation mixes; Add the phosphate buffer dilution and obtain the reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme, wherein: the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10 in the reaction system
-8～6.5 * 10
-4Mol/L, the volumetric molar concentration of enzyme reaction substrate is 8 * 10
-5～6 * 10
-2Mol/L, the volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L; Or
The solution of step (1), step (3) and step (4) preparation is mixed, add the phosphate buffer dilution and obtain the reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme, wherein: the volumetric molar concentration of water-soluble quantum dot is 2.1 * 10 in the reaction system
-8～6.5 * 10
-4Mol/L, the concentration of enzyme is 1.2 * 10
3～7.5 * 10
4Individual unit of activity/L; The volumetric molar concentration of coenzyme is 0～8 * 10
-2Mol/L;
(6) reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme that step (5) is prepared joins the on-chip microchamber that is used for detecting, and assembling obtains being used to detect the enzyme chip based on the quantum dot fluorescence detection of enzymatic activity; Or
The reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme that step (5) is prepared joins the on-chip microchamber that is used for detecting, and assembling obtains being used for the enzyme chip based on the quantum dot fluorescence detection of detection of enzymatic reactions concentration of substrate; Or
The reaction system that constitutes by water-soluble quantum dot, enzyme reaction substrate and coenzyme that step (5) is prepared; With join the on-chip different microchamber that is used for detecting respectively by water-soluble quantum dot, enzyme and reaction system that coenzyme constitutes; Assembling obtains being used to detect the enzyme chip based on quantum dot fluorescence multienzyme joint-detection of enzymatic activity and detection of enzymatic reactions concentration of substrate, the enzyme chip that promptly detects based on quantum dot fluorescence.
9. preparation method according to claim 8 is characterized in that: the concentration of described phosphate buffer is 0.01 mol, and the pH value is 6.8.
10. application according to any described enzyme chip that detects based on quantum dot fluorescence of claim 1～7 is characterized in that: enzyme to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme in a plurality of microchambers; Or
Enzyme reaction substrate to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme in a plurality of microchambers; Or
Enzyme to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme reaction substrate and coenzyme in a plurality of microchambers, enzyme reaction substrate to be detected is joined on the substrate in the microchamber that is mounted with the reaction system that is made up of water-soluble quantum dot, enzyme and coenzyme in a plurality of microchambers;
Reaction at normal temperatures; Adopt fluorospectrophotometer to detect the enzyme chip, the variable quantity of the fluorescence intensity through water-soluble quantum dot obtains the fluorescence spectrum of enzyme chip, and the variation of the fluorescence intensity through analysis of fluorescence spectrum records enzymatic activity to be detected and/or enzyme reaction substrate concentration to be detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397077A (en) * | 2013-08-02 | 2013-11-20 | 中国人民解放军63975部队 | Method for determining activity of acetylcholin esterase by using bifunctional quantum dot sensor system |
| CN103645166A (en) * | 2013-12-02 | 2014-03-19 | 兰州理工大学 | Method for detecting Hg<2+> by fluorescence quenching |
| CN104597005A (en) * | 2014-07-24 | 2015-05-06 | 南京工业大学 | Lipase enzyme activity detection fluorescence analysis method |
| CN108414482A (en) * | 2018-02-28 | 2018-08-17 | 复旦大学 | Utilize the method for molybdenum disulfide quantum dot inner filtering effect fluoroscopic examination alkaline phosphatase activities |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101077976A (en) * | 2007-06-28 | 2007-11-28 | 复旦大学 | Method of preparing CdTe/CdS/ZnS core-shell-core structure quantum points |
| CN101281131A (en) * | 2008-05-26 | 2008-10-08 | 南开大学 | Method for detecting enoxacin in biological body fluid with Mn doping ZnS quantum point room temperature phosphorescent |
| CN101299025A (en) * | 2008-06-20 | 2008-11-05 | 桂林工学院 | CdTe quantum point fluorimetric determination method of chlorine dioxide in water |
| JP2009008603A (en) * | 2007-06-29 | 2009-01-15 | Canon Inc | Fluorescence detection device and biochemical reaction analyzing apparatus and fluorescence detection method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101077976A (en) * | 2007-06-28 | 2007-11-28 | 复旦大学 | Method of preparing CdTe/CdS/ZnS core-shell-core structure quantum points |
| JP2009008603A (en) * | 2007-06-29 | 2009-01-15 | Canon Inc | Fluorescence detection device and biochemical reaction analyzing apparatus and fluorescence detection method |
| CN101281131A (en) * | 2008-05-26 | 2008-10-08 | 南开大学 | Method for detecting enoxacin in biological body fluid with Mn doping ZnS quantum point room temperature phosphorescent |
| CN101299025A (en) * | 2008-06-20 | 2008-11-05 | 桂林工学院 | CdTe quantum point fluorimetric determination method of chlorine dioxide in water |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397077A (en) * | 2013-08-02 | 2013-11-20 | 中国人民解放军63975部队 | Method for determining activity of acetylcholin esterase by using bifunctional quantum dot sensor system |
| CN103397077B (en) * | 2013-08-02 | 2016-01-20 | 中国人民解放军63975部队 | A kind of method of bifunctional quantum dot sensing system of determination acetylcholine esterase active |
| CN103645166A (en) * | 2013-12-02 | 2014-03-19 | 兰州理工大学 | Method for detecting Hg<2+> by fluorescence quenching |
| CN104597005A (en) * | 2014-07-24 | 2015-05-06 | 南京工业大学 | Lipase enzyme activity detection fluorescence analysis method |
| CN104597005B (en) * | 2014-07-24 | 2017-09-26 | 南京工业大学 | A kind of XRF for detecting lipase activity |
| CN108414482A (en) * | 2018-02-28 | 2018-08-17 | 复旦大学 | Utilize the method for molybdenum disulfide quantum dot inner filtering effect fluoroscopic examination alkaline phosphatase activities |
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