CN100432235C - Quantitation of enzyme activity using planar waveguide - Google Patents
Quantitation of enzyme activity using planar waveguide Download PDFInfo
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- CN100432235C CN100432235C CNB2005101132371A CN200510113237A CN100432235C CN 100432235 C CN100432235 C CN 100432235C CN B2005101132371 A CNB2005101132371 A CN B2005101132371A CN 200510113237 A CN200510113237 A CN 200510113237A CN 100432235 C CN100432235 C CN 100432235C
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Abstract
Enzyme activity sensors comprising a planar waveguide can be used for sensitive and quantitative measurement of enzyme activity. Such enzyme activity sensors are useful in a variety of settings, including clinical, commercial, and public health settings.
Description
The sequence number that the application requires on September 8th, 2004 to submit to is the right of priority of the common unsettled provisional application of No.60/608043, and introduces it as a reference.
Invention field
The present invention relates to the method for quantitative enzymic activity.
The accompanying drawing summary
Fig. 1. adopt fluorescently-labeled slab guide Manifold technology diagram.
Fig. 2. be used for utilizing the slab guide Manifold technology diagram of " the hydrolysis lockout mode " of luciferase substrate.
Fig. 3. be used for utilizing the slab guide Manifold technology diagram of " the reaction transformation mode " of luciferase substrate.
Fig. 4. be used to utilize the slab guide Manifold technology diagram of fluorescently-labeled " coupling pattern ".
Fig. 5. be used to utilize the slab guide Manifold technology diagram of " the size exclusion pattern " of luciferase substrate.
Fig. 6. be used to utilize the slab guide Manifold technology diagram of " the cellular segregation pattern " of luciferase substrate.
Detailed Description Of The Invention
The invention provides and adopt the slab guide Manifold technology with sensitive and measure the enzymic activity transmitter of enzymic activity quantitatively.Enzymic activity transmitter of the present invention has many advantages, comprises small-scale operation, speed, and sensitivity, specificity, and be easy to produce.This transmitter can be used for any needs to be measured under the environment of enzymic activity, and can detect enzymic activity in the fluid system of complexity and without purification of samples.If desired, can carry out a plurality of enzymatic determinations concurrently.
Planar waveguide
Enzymic activity transmitter of the present invention contains planar waveguide, and it comprises one or more substrates that are used for one or more enzymes.Planar waveguide (for example: Ta typically comprises material that one deck has high refractive index
2O
5Perhaps TiO
2) film, be deposited on the transparent support of low-refraction (for example: glass or polymkeric substance).The method of making the planar waveguide layer is being known in the art, and can use any known method.Referring to, for example United States Patent (USP) 5959292; United States Patent (USP) 6078705; United States Patent (USP) 5846842; Rowe-Taitt etc., Biosensors ﹠amp; Bioelectronics 14,785-94,2000.
Enzyme substrates is fixing on planar waveguide
The substrate of tested enzyme is fixed on the planar waveguide.Method for immobilizing protein is being known in the art, and can enzyme substrates be fixed on the surveyed area with any these class methods.Referring to, for example, Scouten etc., Trends in Biotechnology 13,178-85,1995; Shriver-Lake etc., Biosensors and Bioelectronics 12,1101-06,1997; Rowe-Taitt etc., 2000.Method selected depends on the character of this certain enzyme substrate to a certain extent, and is as well known to those skilled in the art.Typically, substrate directly is fixed on the planar waveguide layer by covalent linkage or through chemically modified slab guide tube-surface, for example fixes through silanization or after applying polymer layer again.If necessary, wall that can one deck is thin (SiO for example
2) directly be added on the planar waveguide layer, thereby to help the fixing of on this waveguide enzyme substrates as short bonding coat.
In other embodiment, the planar waveguide layer is coated with avidin, and enzyme substrates can be coupled to the biotin moiety that the antibiont fibroin has very strong avidity.Referring to Rowe-Taitt etc., 2000.In other embodiments, waveguide is coated with the aquagel membrane that is made of the polyisobutene hydrazides, wherein the treated generation free of polyisobutene hydrazides dimaleoyl imino; With some enzyme substrates oxidation formation reaction thiol groups, afterwards with the maleimide radical reaction.Also have in other embodiments, the waveguide surface-coated of silanization has by quadrol base deutero-polyoxyethylene glycol.These groups also can react with the enzyme substrates of oxidation.
Non-covalent interaction also can be used for substrate is fixed on the planar waveguide.This interaction includes, but not limited to hydrophobic absorption, model ylid bloom action, hydrogen bonding and electrostatic force.
If the use coating, its preferably be added to can obtain on the planar waveguide reproducible, the constant layer thickness.The example that typically can be used for applying the method for planar waveguide comprises spraying, scraper coating, spin coating, perhaps dip coated.Can carry out quality control to coating by any appropriate means, comprise microscopy, interferometric method, oval method of masurement and contact angle method of masurement.
Different enzyme substratess can be fixed on the difference on the planar waveguide and the zone of mutual exclusion.Substrate preferably is arranged to the space addressable array.For example, can have a plurality of plate holes or passage on the waveguide surface so that can the detectable label of contrast and sample solution be compared simultaneously.Randomly, can strengthen the brightness of evanescent field like this at the most coated reflectance coating of waveguide surrounding edge to prevent light from ovfl.Can also prepare the space addressable array as the zone or the point in order to detect the plurality of enzymes activity in the single fluid sample.
Detectable label
In some embodiments that are described below, enzyme substrates contains detectable mark.In other embodiments, thus enzyme activity change substrate comprise detectable mark.Be used for detectable label of the present invention and be typically the photoresponse mark, for example fluorescent mark, phosphorescence mark and ultraviolet radiation absorption mark.Fluorescent mark is especially suitable, does not penetrate because the field needs a large amount of fluids to see through the width of cloth to exciting of fluorophore, thereby has limited background signal, has improved sensitivity.The method that is connected detectable label on polypeptide and other enzyme substrates is being known in the art.
Suitable mark comprises rhodamine, fluorescein derivative, coumarin derivatives, distyryl biphenyl class, stilbene derivatives, phthalocyanine pigment, naphthalocyanines, polypyridine base-ruthenium complexe are as three (2,2 '-bipyridyl)-ruthenium chloride, three (1, the 10-phenanthroline) ruthenium chloride, three (4,7-phenylbenzene-1,10-phenanthroline) ruthenium chloride and polypyridine base-azophenlyene-ruthenium mixture, platinum-metalloporphyrin complex such as octaethyl-platinum-porphyrin, long lifetime europium and terbium coordination compound or cyanine dyes.Absorption and the dyestuff of emission wavelength in the scope of 600-900nm especially are suitable for analyzing blood or serum.
Especially the detectable label of Shi Yonging is a dyestuff, for example contains the fluorescein derivative of functional group, by this functional group its can be covalently bound to substrate, fluorescein isothiocyanate for example.Equally very suitable also have functional fluorescence dye, can be available from Biological Detection Systerms Inc., and for example single and bifunctional CY5.5
TMDyestuff, Clinical Chemistry 40,1819-22,1994.If necessary, the substrate that is used for different enzymes can contain different detectable labels.
The example of enzymic activity transmitter
Enzymic activity transmitter of the present invention can be used in some different " patterns ".According to employed pattern, enzyme substrates can comprise or not comprise above-mentioned detectable label.According to employed pattern, enzymic activity detects the minimizing or the increase that can relate to the amount that detects detectable label simultaneously.
For example, the enzymic activity transmitter that contains the substrate after can detecting ground mark can be used for " hydrolysis lockout mode ".Under this pattern, enzyme downcuts substrate from the slab guide tube-surface, and the signal that is weakened just reflects the activity of enzyme.Referring to Fig. 2.
Other enzyme substratess contain by enzyme modification and become detectable label or part (" reaction transformation mode ") that no longer can detected mark.If this part of enzyme modification makes it produce detectable label, then the enhanced signal reflects enzymic activity.On the other hand, if this part of enzyme modification no longer can detect it, the signal that then weakens reflects enzymic activity.Referring to Fig. 3.
And in other embodiments, enzyme is coupled to (" coupling pattern ") on the enzyme substrates with detectable label.Referring to Fig. 4.The enhanced signal reflects enzymic activity in these embodiments.
In some embodiments, by spatially separating different enzyme substratess, for example, can strengthen the active selectivity of target enzyme with substrate connection or spacer structure by the mechanism (" size exclusion pattern ") of locus, ion or hydrophobic interaction.Referring to Fig. 5.These mechanism are particularly useful for enzymic activity transmitter of the present invention, but because the feasible sensitivity that can control testing process of the ability of the mark part of spatial positioning detection molecules, because the brightness of evanescent field and decaying to the planar waveguide sensor surface apart from the exponentially formula.Shriver-Lake etc., 1997.
For example, use the spacer of different lengths can the balance multiple testing in the fluorescent signal of different loci.Similarly, because the site of enzyme reaction also can be controlled by the identical factor, therefore under the situation that has multiple potential reaction molecule, can realize enhancing or elimination to some specific reactions.
Any above-mentioned embodiment all can be used in the original position cell cultures, to detect the substratum (" cellular segregation pattern ") of emiocytosis enzyme.Referring to Fig. 6.In this case, by the macroporous polymer layer, hydrophilic polymer (for example polyhydroxylated methacrylic ester or polysaccharide) for example, enzymic activity transmitter can be from cell masses, or separate in the individual cells.The cellular segregation pattern allows under physiological conditions, do not need cell and substrate or possible reaction product to contact, and detection is by viable cell excretory enzyme.Many enzymic activity detection systems are not compatible with viable cell.Thereby this embodiment is improvements over the prior art, detects the activity that there is the enzyme of disadvantageous effect in those deleterious or pair cells because this reaction system can be used in.The cellular segregation pattern can also not measured under the condition of the biological property of potential change cell owing to radiation.
The application of enzymic activity transmitter
Enzyme sensor disclosed herein can be used in any device that need qualitatively or quantitatively determine enzymic activity.Can detect the activity of the enzyme of any kind, include but not limited to, proteolytic ferment (for example, aminopeptidase, aspartyl protease, serine protease, metalloprotease, cysteinyl proteolytic enzyme, stomach en-, trypsinase, zymoplasm, N,O-Diacetylmuramidase, Factor IX: C), Glycosylase, esterase, lytic enzyme, nuclease, synthetic enzyme, isomerase, polysaccharase, kinases, Phosphoric acid esterase, reductase enzyme comprises oxydo-reductase, transferring enzyme, ligase enzyme, restriction enzyme, Ntn hydrolase, the ATP enzyme, carbohydrase, lipase, cellulase, desaturase, and oxydase.The substrate of these enzymes is being known in the art and can buying in many suppliers.
Enzymic activity transmitter of the present invention is particularly suited for as diagnostic tool, because it can in situ, the height multiplicity study complicated biological enzyme system in real time.Especially, these transmitters can be monitored enzymic activity in real time, even in muddy fluid, for example without the serum or the blood of specimen preparation.Therefore this transmitter is easy to be used as the quick diagnosis instrument under the nurse environment.
In clinical instrumentation, with the contacted fluid of planar waveguide be biogenetic derivation, include but not limited to, blood, blood plasma, urine, cerebrospinal fluid, saliva, bronchial perfusate, aqueous humor, ascites, EVLW, folliculi liquor, labyrinthine fluid, endolymph, perilymph, lymph, chyle (chyle), nose irrigating solution, and synovia.These fluids can also be the substratum of wherein having cultivated the cell that comes from the patient.
The enzyme of concern comprises in clinical, for example, and amylase and lipase (pancreatic disease); Creatinine kinases, serum lactic dehydrogenase, AHB, serum glutamic oxalacetic transaminase and creatine phosphokinase (heart attack, muscle disease and apoplexy); Amino alanine transaminase, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transminase and serum lactic dehydrogenase (hepatopathy); Alkaline phosphatase (liver or ephrosis); Angiotensin-converting enzyme (active sarcoidosis, atypical mycobacteria, primary biliary cirrhosis, high Xue Shi disease or leprosy); Factor IX: C (hemophilia); Feritin (renovascular hypertension); Galactose-1-phosphate uridyltransferase (galactosemia); Glucocerebrosidase (high Xue Shi disease); And biotinylation enzyme (biotinylation enzyme deficiency disease).
Enzymic activity transmitter of the present invention also can be used in commerce or the research equipment, in order to monitor the enzymic activity in big or the small-scale enzyme purification process.It can be used for monitoring the enzymic activity in the fermention medium.It can be used for detecting public health harm, for example is used to detect the enzyme of diagnosis malignant bacteria, virus and fungi.Enzymic activity transmitter of the present invention also can be used in the high flux screening mensuration that can reduce the active therapeutical agent of certain enzyme.
The generation of detectable label and detection
Enzymic activity transmitter of the present invention can be got considerably less liquid level sample and be used for detectable mark, has wherein adopted the evanescent field that is produced by the reflection of planar waveguide inner laser bundle.This evanescent field extends into about 100nm in the liquid, and intensity is exponential decay; Therefore, can not detect this detectable label of portion outside the venue.Referring to Fig. 1.
The fluid that will contain determined enzyme is arranged to contact with described planar waveguide.Contact can be static, also can make fluid pass through the top of planar waveguide.With polarized light, modal is the polarized light that comes from LASER Light Source such as mninidiode laser apparatus, is directed to by total reflection on the interface of ducting layer.The ripple that at the interface produce fadout or the field of internal reflection between the different refractivity material of waveguide boundary by light.The intensity of evanescent field depends on the ratio of the specific refractory power of the thickness of ducting layer itself and ducting layer and surrounding medium.Under the situation of thin-film guide pipe, when promptly its layer thickness is equal to or less than the wavelength that is conducted, can distinguish by the discrete mode of light conducting.
On the optics of planar waveguide in the thinner medium, but only be directly to be adjacent to by the place of conduction light wave, evanescent field stimulated luminescence.The luminous method and apparatus that is used to detect this evanescent excitation is well known in the art, and for example is described in United States Patent (USP) 4582809, United States Patent (USP) 5081012, WO 90/06503 and Biosensors ﹠amp; Bioelectronics 6 (1991), among the 595-607.Can adopt photorectifier, photoelectric cell, photomultiplier, CCD photographic camera and detector array, for example the CCD battery.The light that is sent can for example mirror, prism, lens, Fresnel lens and gradient index lens be come imaging with optical element.Known element is spectral filter, prism, monochromatic filter, dichroic mirror for example, and diffraction grating can be used for selecting suitable emission wavelength.
All patents, the patent application of being quoted in this disclosure, and reference is all specially introduced in full as a reference at this.
Claims (23)
1. enzymic activity transmitter that contains planar waveguide, comprising first kind of substrate of first kind of enzyme and second kind of substrate of second kind of enzyme, wherein:
(1) described first kind of substrate comprises first kind of detectable mark, and described second kind of substrate comprises second kind of detectable mark; Perhaps
(2) thus first kind of substrate of the activity change of described first kind of enzyme comprises first kind of detectable mark, thereby and second kind of substrate of the activity change of second kind of enzyme comprise second kind of detectable mark; And wherein first kind of substrate contains first kind of spacer structure between planar waveguide and first kind of substrate, and wherein second kind of substrate contains second kind of spacer structure between planar waveguide and second kind of substrate, and wherein first kind of spacer structure is different with the length of second kind of spacer structure.
2. the described enzymic activity transmitter of claim 1 wherein further comprises measuring element.
3. the described enzymic activity transmitter of claim 1, wherein said first kind of substrate comprises first kind of detectable mark, and wherein said second kind of substrate comprises second kind of detectable mark.
4. the described enzymic activity transmitter of claim 1, thereby first kind of substrate of the activity change of wherein said first kind of enzyme comprises first kind of detectable mark, thereby and second kind of substrate of the activity change of wherein said second kind of enzyme comprise second kind of detectable mark.
5. the described enzymic activity transmitter of claim 3, wherein first kind and second kind of detectable mark are selected from down group: fluorescent mark, phosphorescence mark and ultraviolet radiation absorption mark.
6. the described enzymic activity transmitter of claim 4, wherein said first kind of detectable fluorescence part that is labeled as.
7. the described enzymic activity transmitter of claim 1, wherein said first kind of enzyme is selected from down group: Ntn hydrolase, esterase, oxydase, reductase enzyme, polysaccharase, transferring enzyme, ligase enzyme, restriction enzyme and proteolytic enzyme.
8. the described enzymic activity transmitter of claim 1, wherein first kind of substrate and the second kind of substrate substrate that is different enzymes.
9. the described enzymic activity transmitter of claim 1, wherein first kind of substrate and second kind of substrate are arranged to the form of space addressable array.
10. method that detects enzymic activity comprises step:
(a) under the condition that allows the enzyme-to-substrate effect, the planar waveguide of the described enzymic activity transmitter of claim 1 is exposed to sample;
(b) the described planar waveguide of irradiation is to produce evanescent field;
(c) existence of detectable label in the described evanescent field of detection.
11. the described method of claim 10, wherein said first kind of substrate comprises first kind of detectable mark.
12. the described method of claim 11, the activity of wherein said first kind of enzyme make described first kind of detectable mark discharge from first kind of substrate.
13. the described method of claim 11, the activity of wherein said first kind of enzyme has reduced the detectability of described first kind of detectable mark.
14. the described method of claim 10, thus the activity change of wherein said first kind of enzyme described first kind of substrate comprise first kind of detectable mark.
15. the described method of claim 14, wherein said first kind of enzyme is coupled to described first kind of detectable mark on described first kind of substrate.
16. the described method of claim 14, wherein said first kind of detectable fluorescence part that is labeled as.
17. the described method of claim 11, wherein said first kind of detectable photoresponse mark that is labeled as.
18. the described method of claim 17, wherein said photoresponse mark is selected from down group: fluorescent mark, phosphorescence mark and ultraviolet radiation absorption mark.
19. the described method of claim 18, wherein first kind of substrate and the second kind of substrate substrate that is two kinds of different enzymes.
20. the described method of claim 10, wherein said first kind and second kind of substrate are arranged to the form of space addressable array.
21. the described method of claim 10, wherein said sample comprises the fluid that is selected from down group: blood, blood plasma, urine, cerebrospinal fluid, saliva, bronchial perfusate, aqueous humor, ascites, EVLW, folliculi liquor, labyrinthine fluid, endolymph, perilymph, lymph, chyle, nose irrigating solution, synovia, fermention medium, and cell culture medium.
22. the described method of claim 10, wherein step (c) comprises detection by quantitative.
23. the described method of claim 10, wherein step (c) comprises qualitative detection.
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| Application Number | Priority Date | Filing Date | Title |
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| US60804304P | 2004-09-08 | 2004-09-08 | |
| US60/608043 | 2004-09-08 | ||
| US11/099462 | 2005-04-06 |
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| WO2004042377A1 (en) * | 2002-11-07 | 2004-05-21 | C-Cit Ag | Sensor for determining analysed substances and method for determining enzymatic activities |
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