CN100504286C - Fiber-optic assay apparatus based on phase-shift interferometry - Google Patents
Fiber-optic assay apparatus based on phase-shift interferometry Download PDFInfo
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Abstract
Apparatus and method for detecting the presence or amount or rate of binding of an analyte in a sample solution is disclosed. The apparatus includes an optical assembly having first and second reflecting surfaces separated by a distance 'd' greater than 50 nm, where the first surface is formed by a layer of analyte-binding molecules, and a light source for directing a beam of light onto said first and second reflecting surface. A detector in the apparatus operates to detect a change in the thickness of the first reflecting layer resulting from binding of analyte to the analyte-binding molecules, when the assembly is placed in the solution of analyte, by detecting a shift in phase of light waves reflected from the first and second surfaces.
Description
The related application cross reference
Cross reference the application case of related application is advocated the right of following application case: the people's such as Hong Tan that on November 4th, 2004 filed an application name be called " based on the fiber-optic assay apparatus of phase-shift interferometry " act on behalf of the of file number 24377-09611US _ _ _ _ _ _ _ _ number U.S.'s non-provisional application case; The 60/518th, No. 068 U.S. Provisional Application case that on November 6th, 2003 filed an application; Reach the 60/558th, No. 381 U.S. Provisional Application case of filing an application on March 31st, 2004, for various purposes, whole disclosures of above-mentioned application case all are incorporated herein in full with way of reference.
About the research of federal government's subsidy or the statement of exploitation
Inapplicable.
Technical field
The present invention relates to a kind of device and method that is used for detecting in existence, quantity or the association rate of the one or more analytes of a sample, and in particular to the device and method based on the optical fibre interference method.
Background technology
Diagnostic test in conjunction with the binding events between the right member has been widely used in medical treatment, animal doctor, agricultural and the research application based on an analyte-anti--analyte.Usually, these class methods are used to detect the existence of the analyte in the sample or quantity and/or this analyte association rate of anti--analyte therewith.Typical analyte-anti--analyte is to comprising complementary nucleic acid chain, Ag-Ab to, and acceptor-receptor-binding agents, and wherein analyte can be arbitrary member of described centering, and anti-analyte molecule is its opposition member.
The diagnostic method of this type usually adopts one to have fixing anti-analyte molecule that sample analysis thing molecule will combine with its specificity and the solid surface that has high-affinity on a detection zone that defines.In this alanysis that is called solid phase assays, impelling analyte to be bonded under the condition of fixing resisting-analyte molecule, solid surface is exposed to sample.Can be directly can indicate the feature of a binding events to detect binding events by (for example) quality, reflectivity, thickness, color or other.When (for example) uses chromophore or fluorescence or radioactive label to come preliminary making analyte position, can detect binding events according to the existence and/or the quantity of detectable label on detection zone.Perhaps, can at analyte after combination on the described detection zone, come this analyte of mark with (for example) auxiliary, fluorescently-labeled resisting-analyte antibody.
A kind of fibre optic interferometer analytical equipment that is bonded to the analyte of a fiber end face in order to detection that designs of the 5th, 804, No. 453 (＇ 453 patents) U.S. Patent Publications owning together, this United States Patent (USP) is incorporated herein with way of reference.Detection of analytes is based on one because of the variation in thickness of analyte molecule to the fiber end face that causes of combination on surface, and analyte quantity is many more, and the interference signal relevant with thickness that is produced changes with regard to more greatly.Specify as institute in Fig. 7 a of ＇ 453 patents and 7b, it is to be caused by the light that reflects from the end of optical fiber and the phase shift between the light that the binding layer that places this optical fiber end reflected that interference signal changes.This device easy operating also provides a kind of rapid analysis for detection of analytes.
In theory, the interferometer analytical equipment will produce the spectrum peak and paddy (extreme value) change in location that can be easy to observe, described change in location is in the scope of traditional visible light light-splitting meter (or rather, in the visible-range between about 450-700nm), thus the marked change that relatively little optical thickness change all can be used as the spectral position of interfering wavelength peak and paddy on the optical fiber end detects.A restriction of observing with device described in ＇ 453 patents is not to be easy to the wave spectrum extreme value discerned in this spectral range.
Design the present invention limits to overcome this, keeps the speed of the disclosed device in front and the advantage of simplicity, but significantly improves sensitivity and degree of accuracy.The present invention also provides a kind of replaceable more easily capitiform formula and a kind of multiple analyte array format of using with what (for example) genetic chip and protein-chip.
Summary of the invention
One aspect of the present invention comprises a kind of device that is used for detecting a sample analyte, and it comprises the existence of check and analysis thing, the quantity of analyte or the association and/or the dissociation rate of analyte and analyte binding molecule.This device comprises an optical element, and this optical element has be separated by a near-end reflecting surface and a far-end reflecting surface of 50nm at least.Be incident upon these two reflecting surfaces and from these two reflecting surfaces reflections from the light beam of an optical fiber.So far optical fiber and interference are mutually returned in institute's beam reflected coupling.This optical element also comprises an analyte molecular binding layer through the location, with the interference between the toilet folded light beam along with analyte is bonded to described analyte molecular binding layer and changing.
Interfere and change and to cause by different physical phenomenons.For example, analyte is in conjunction with causing the optical path length between these two reflecting surfaces or the change of physical distance.Perhaps, analyte is in conjunction with the optical absorption or the reflectivity change that can cause the material between these reflecting surfaces.Analyte changes thereby cause interfering in conjunction with causing that also the analyte molecular binding layer expands.
In a specific design, the far-end reflecting surface comprises the analyte molecular binding layer.For example, when analyte was bonded to the analyte molecular binding layer, optical path length or physical distance between these two reflecting surfaces may increase.In the present invention on the other hand, a kind of transparent solid material between reflecting surface and, optionally, the near-end reflecting surface comprises that one has the material greater than the reflectivity of this transparent solid material.Perhaps, an air-gap can be between reflecting surface.In another design, this far-end reflecting surface is between near-end reflecting surface and analyte molecular binding layer.For example, analyte moves near the near-end reflecting surface far-end reflecting surface in conjunction with the analyte molecular binding layer is expanded.In another design, the analyte molecular binding layer is between these two reflecting surfaces.The analyte combination can make this layer expansion or change its reflectivity, and then changes two interference between the folded light beam.
In another aspect, this device comprises an optical module, and it has first and second reflecting surface of the above distance ' ' d ' ' of 50nm of being separated by.This optical module is made up of a transparent optical element, and this optical element can have a near-end and the 50nm at least between the distal face, preferable between 400 to 1 that is defined in this element, the thickness between the 000nm.First reflecting surface places on the distal face of optical element, and is formed by one analyte-molecular binding layer.Second reflecting surface is formed by the transparent material coating of a kind of refractive index greater than this optical element refractive index.This coating can by a preferred thickness between 5 and 50nm between Ta
2O
5Layer forms.Optical element can be SiO2, and has and between about 100 to 5, between the 000nm, preferable 400 to 1, and the thickness of 000nm.
Also comprise a light source, it is used for a light beam is mapped to described first and second reflecting surface; And a detector cell, when this assembly was placed in the analyte solution, its operation was bonded to the optical thickness change in first reflection horizon that the analyte binding molecule causes to detect one because of analyte.The first reflection horizon optical thickness change is with relevant by the phase propetry displacement from two formed interference waves of light wave of described first and second surface reflection.Described phase propetry can be the displacement of the spectral position of one or more peaks of interference wave and paddy, or the variation in this ripple whole circulation cycle.
Light source can comprise that its far-end is suitable for adjoining the optical fiber that second reflecting surface in this assembly is placed, and this device further comprises a photo-coupler that is used for reflecting light is reflexed to from assembly detecting device.
In first embodiment, this optical module is fixedly installed on the described optical fiber, and the far-end of optical fiber contacts with second reflecting surface simultaneously.In a second embodiment, this optical module further comprises one second transparent optical element, and it has less than the refractive index of second coating and greater than the thickness of about 100nm, and wherein the coating of this high-index material is sandwiched between these two transparent optical elements.In one embodiment of back, this assembly is the remote area that movably is attached to optical fiber, has one in the far-end of optical fiber and the assembly between the opposite face of second transparent optical element less than 100nm or greater than the interval of 2 μ m.
In order to detect multiple analytes (for example multiple nucleic acid substances), the analyte molecular binding layer can be by forming such as discrete analysis thing calmodulin binding domain CaM arrays such as single-chain nucleic acids.These zones can be effectively in conjunction with different analytes.This optical fiber comprises the optical fiber that complex root is independent, aims at one of in each optical fiber and these zones, and this detecting device comprises a plurality of detection zones, and optically-coupled be used for each root of complex root optical fiber therewith each in the zone be coupled.
Analyte binding molecule in this assembly can be (for example) and (i) resists material (anti-species) antibody molecule, it is used to screen the hybrid cell storehouse and hides the existing of antibody, (ii) antigen molecule to seek, and it is used for the existence of detection specificity at the antibody of this antigen; (iii) protein molecule, it is used to detect this combination of proteins partner's existence; (iv) protein molecule, it is used to detect the existence that protein therewith forms the multiple bound substances of polyprotein matter complex compound; Or (v) single stranded nucleic acid molecule, it is used to detect the existence of nucleic acid binding molecule.
This detecting device can be the spectrometer that is used to measure the intensity of reflected light in the selected wavelength coverage.Perhaps except that it, this light source can comprise a plurality of light emitting diodes, and each light emitting diode all has a spectrum of properties frequency, and this detecting device is used for writing down the catoptrical light intensity under each frequency of different LED frequency.In an embodiment again, this light source comprise a white light source and this detecting device through design to write down the catoptrical light intensity under each wavelength in a plurality of different wave lengths.
On the other hand, the present invention includes a kind of be used for the detecting existence of a sample solution analyte or the method for quantity.The method comprises makes this sample solution and the reaction of first reflecting surface, this first reflecting surface by thickness on the distal surface that places transparent optical element at least the analyte molecular binding layer of 50nm formed, thereby by analyte therewith the combination of analyte binding molecule in the layer increase the thickness in first reflection horizon.Measure the variation in thickness in first reflection horizon by the phase propetry displacement that detects interference wave, this interference wave is by forming from ground floor and two light waves reflecting from second reflection horizon, and this second reflection horizon is formed on the opposed proximal end face of this optical element and it has the refractive index big than optical element.
Detecting step can comprise the light from optical fiber is guided on these two reflecting surfaces and by optically-coupled and will be mapped on the detecting device from the reflected light on these two surfaces.This detecting device can be a spectrometer, wherein detects to comprise the spectral position displacement of measurement by these two one or more interference extreme values that reflecting light produced.
If the method is used for the association rate of Measurement and analysis thing to the second layer, then can implement this reactions steps till observing first reflector thickness and increasing near maximal value.If the method is used for the dissociation rate of Measurement and analysis thing and the second layer, then reactions steps can comprise the second layer is dipped in the disassociation damping fluid a period of time till observing the reduction of first reflector thickness.If the method is used for measuring the quantity of the analyte that sample exists, then this is detected the period that is enough to measure first reflector thickness on a plurality of different time points of implementing.
If the method is used for measuring one or more in a plurality of analytes of sample, then first reflection horizon is made up of a discrete analysis thing calmodulin binding domain CaM array, zones of different can be effectively in conjunction with different analytes, and this measures and detects effectively because of each regional variation in thickness in analyte and these zones that combining of analyte binding molecule causes.
In conjunction with the accompanying drawings, by reading hereinafter detailed description of the invention, these and other purpose of the present invention and feature will add very clear.
Description of drawings
With reference to hereinafter explanation and accompanying drawing, will understand these and other feature of the present invention, aspect and advantage better, wherein:
Fig. 1 shows the ultimate system setting of bioprobe and device thereof;
Fig. 2 shows an optical module according to one embodiment of the invention formation;
Fig. 3 A and 3B show along 7 peak valley order (3A) and are in the part of an interference wave of this spectrum visible part (3B);
Fig. 4 shows an optical module of constructing according to another embodiment of the present invention;
Fig. 5 show one have analyte in conjunction with array and according to another embodiment of the present invention the structure once division analyse the thing optical module;
Fig. 6 shows the order combination of three kinds of molecules;
Fig. 7 shows association and the dissociation curve that association and the disassociation because of antibody produces;
Fig. 8 shows the curve of the antibodies of two kinds of variable concentrations to its antigen;
Fig. 9 demonstration comes fixedly diamine base PEG (MW3300) of specificity by forming amido link.PEG (MW8000) is as the non-specific binding of a negative control with supervision PEG polymkeric substance; And
Figure 10 shows micromolecular negative control and the base measurement that combines with big molecule.
Embodiment
Definition
Except that because of hereinafter having in addition clearly the definition, employed term all should be considered as its implication commonly used that the those skilled in the art understands in these claims and the instructions.Numerical range described in these claims and the instructions should be considered as comprising the extreme value that defines described scope.
Term " in vivo " is meant the process that takes place in live organism.
" analyte in conjunction with " molecule is meant arbitrary molecule that can participate in the specificity association reaction of analyte molecule.Example includes, but is not limited to for example antibody-antigen association reaction and nucleic acid hybridization reaction.
" specificity association reaction " be meant saturable, usually reversible and can with the association reaction of excessive a kind of competition in these reactants.The specificity association reaction thus in the specificity association reaction shape between the participant, electric charge and other complementarity in conjunction with determinative characterize.
" antibody " is meant the immunoglobulin molecules with two heavy chains and two light chains of the method preparation known or exploitation afterwards by field under arbitrary and comprises for example by giving such as former polyclonal antibody that produces of mammal injecting immune and the monoclonal antibodies by using known Kohler Milstein hybrid cell integration technology to be produced such as goat, mouse, rabbits.This term comprises the use antibody that gene engineering method produced (for example those uses (for example) are through the SCID mouse manufacturing person of human immunoglobulin gene reorganization) and has used the known humanized antibody of surface replacement technology in affiliated field.
" antibody fragment " is meant the fragment of the antibody molecule that produces by chemical cracking or technique for gene engineering, also refers to use combination gene storehouse and the exhibition of antibiotic body to show single chain variable fragment (SCR such as survivor that technology is produced such as those
VS).Usually keep in conjunction with the ability of its isogeneic and therefore comprise variable sequence and antigen-binding site according to antibody fragment used in the present invention.
" micromolecule " is meant the organic compound that has less than about 500 daltonian molecular weight.Micromolecule is useful initial screening material, and it can be determined lead compound and then can be optimized to generate new medicine by conventional medicament chemistry, structure-activity relationship research.But the small-molecule drug compound has the advantage of common oral bioavailability.Micromolecular example comprises compound cited in the following database:
MDL/ACD (http://www.mdli.com/), MDL/MDDR (http://www.mdli.com/), SPECS (http://www.specs.net/), CNPD (CNPD) (http://www.neotrident.com/), and national drug screening centralization compound sample data storehouse (http://www.screen.org.cn/).Used abbreviation comprises as follows in the application's case: " ss " is meant strand; " SNP " is meant single nucleotide polymorphism; " PBS " be meant phosphate buffered solution (0.01M phosphate buffer, 0.0027M kali and 0.137M sodium oxide molybdena, pH7.4); " NHS " is meant N-hydroxy-succinamide; " MW " is meant molecular weight; " Sulfo-SMCC " is meant carboxylic acid 4-[N-maleimide ylmethyl] cyclohexane-1-sulfosuccinimide base ester.Must be noted that, as used in this instructions and the claims of enclosing, unless context has clearly indication in addition, otherwise singulative " ", and " being somebody's turn to do " comprise a plurality of objects.
Advantage and effectiveness
With reference to the graphic and embodiment that hereinafter more elaborates advantage of the present invention and effectiveness are described.These comprise and need not the ability that usage flag can real time monitoring analyte association reaction, thereby reduce cost and possible toxicity.Another advantage comprises the ability of using the visible wavelength light source to implement the method.The optical properties at detecting device tip can provide other advantage, and this most advanced and sophisticated permission monitors association reaction and makes fibre bundle can carry out the analysis of high complexity together to association reaction in minimum volume of sample (comprising " external " space).
Fig. 1 shows an interferometric measuring means 20 constructed according to the invention with synoptic diagram.When adopting primary element, this device comprises light source 22, optical module 26 and detector cell 28, optical module 26 plays sensing element or detecting device tip and hereinafter is described in further detail with reference to Fig. 2,4 and 5, and detector cell 28 is used to detect the interference signal that is produced by the interference light wave from optical module 26 reflections.
To be incident upon on the optical module 26 from the light in source 22, and by one by the optically-coupled assembly reflected back of dotted line 30 places indications detecting device so far.In a preferred embodiment, this coupling assembly comprises from light source and extends to first optical catheter of optical module or second optical catheter or an optical fiber 34 and the optical coupler with optical mode coupled fiber 32,34 that optical fiber 32, one will be loaded onto detecting device from the reflected light of optical module.In ＇ 453 patents that optical fiber that is fit to and coupling unit are specified in above to be quoted.A kind of exemplary coupling mechanism can comprise Ocean Optics certainly, and (Dunedin is Florida) interior many seller persons of buying.
Perhaps, this coupling assembly can comprise a lens combination, its through structure with so that focus on the upper surface of a light beam at optical module and will guide to detecting device from the reflection interference light of optical module.Back one system does not need optical fiber, but this will propose strict relatively requirement to the location of the lens element that is used for optically-coupled.
Light source in the device can be such as light emitting diode white light sources such as (LED), its (for example) 400nm or with down to 700nm or above, usually produce light on the wide spectrum in the spectral range at 100nm at least.Perhaps, this light source can be the plurality of sources that has the different characteristic wavelength separately, for example is designed in visible-range the LED with different selected wavelength emission light.Identical functions can be incident upon the light of the selected wavelength of difference on the optical module by the single source with suitable wave filter (for example white light source) and realize.
This detecting device is preferably spectrometer, for example can write down the charge-coupled device (CCD) from the reflection interference light spectrum of optical module.Perhaps, when light source operation when being incident upon different selected wavelength on the optical module, detecting device can be a simple photodetector that is used to write down variant irradiation wavelength place light intensity.In an embodiment again, detecting device can comprise a plurality of wave filters, and this allows in a plurality of selected wavelength of interfering reflection wave each wavelength place to detect light intensity from (for example) white light source.Exemplary light source and detector configurations are set forth in above-mentioned ＇ 453 patents, especially with reference to Fig. 8 and 10 of described patent, and should be appreciated that these structures also are applicable to the present invention.
Fig. 2 shows the remote area adjoining segment of the optical fiber 32 that fixedly is attached to it according to the optical module 26 of one embodiment of the invention structure and this optical module.As can be seen, assembly 26 comprises a transparent optical element 38, and optical element 38 has respectively at (far-end) under it and goes up first and second reflecting surface 42,44 that forms on (near-end) end face.According to a key character of the present invention, the thickness " d " of (promptly between these two reflecting surfaces) is at least 50nm between its far-end of optical element and the proximal end face, and the preferable 100nm that is at least.One example thicknesses between the 000nm, is preferably 400 to 1,000nm between about 100 to 5.First reflecting surface 42 is formed by specificity analyte binding molecule 46 effectively and layer with analyte binding molecule (for example molecule 44) of high-affinity.In other words, analyte and anti-analyte molecule be the above-mentioned type in conjunction with right opposition member, it is right to, complementary nucleic acid and acceptor-bond that it can include, but is not limited to Ag-Ab.
The formed layer of free analyte binding molecule so that mainly take place but not interface between optical element and analyte binding molecule from the reflection of far-end under the optical module in the preferable refractive index that is similar to first reflecting surface of the refractive index of this optical element.Equally, when analyte molecule is bonded to the lower floor of optical module, mainly comes across by the formed layer of the analyte of analyte binding molecule and institute's combination from the light of assembly lower end reflection but not come across this interface zone.A kind of Exemplary materials that forms optical element is SiO
2, for example have the high quality glass of about 1.4 to 1.5 refractive indexes.Optical element also can be formed by transparent polymer, for example have preferable in 1.3 to 1.8 scopes the polystyrene or the tygon of refractive index.
Second reflecting surface that forms in the optical module is the transparent material layer of high refractive index than optical element roughly as having one, so this layer can be used for the light that antireflection part is incident upon optical module.Preferably, the second layer has the refractive index greater than 1.8.A kind of Exemplary materials that is used for the second layer is the Ta that reflectivity equals 2.1
2O
5This layer usually by a traditional vapor deposition coating or hierarchical process form on the optical element thickness less than 50nm, usually between 5 and 30nm between layer.
The thickness of first (analyte in conjunction with) layer through design with according to particular hardware and optics optimization overall sensitivity.Use the conventional fixed chemical substance to come the analyte molecular binding layer to be connected to the lower surface of optical element with chemical mode (for example with covalent manner).For example, multiple bifunctional reagent, it comprises one and is used for chemistry and is connected to SiO
2Siloxane group and hydroxyl, amine, carboxyl or other reactive group that is used for connecting such as the biomolecule of protein (for example antigen, antibody) or nucleic acid.But also conventional etch or otherwise handle the density of a glass surface with the hydroxyl that increases the bound analyte binding molecule.When this optical element is when forming such as polymkeric substance such as polystyrene, then can use several different methods to be used for exposing such as available chemically reactive surface groups such as amine, hydroxyl and carboxyls by a kind of.
The analyte binding layer is preferable to be formed under following condition: wherein the distal surface of optical element is through densification coating, thus analyte molecule therewith the combination of layer force this layer thickness to change, rather than be filled in this layer.This analyte binding layer can be a single or multiple lift matrix.
By can measure existence, concentration and/or the association rate of analyte on the optical module from the interference of the folded light beam of two reflecting surfaces in the optical module.Particularly, when analyte molecule connects so far the surface or when it dissociates, the average thickness respective change in first reflection horizon.Because the thickness of every other layer remains unchanged, so by reflecting from the phase shift of the formed interference wave of the light wave on these two surfaces with this variation in thickness.
Suppose two folded light beams are arranged: from first light beam of first surface reflection, it is the far-end interface between analyte binding molecule and institute's bound analyte and the surrounding medium; Reach second light beam from the second surface reflection, it is the near-end interface between optical element (ground floor) and the high refractive index layer (second layer).The interference wave intensity that depends on total wavelength is:
Wherein I is an intensity, I
1And I
2Be the intensity of two interfering beams, Δ is an optical path difference, and λ is a wavelength.
When (during 2 π Δs/λ)=N π, if N be an integer 0,1,2 ..., then curve is at its peak or the paddy place.
Thickness d=the Δ of ground floor/2n.Therefore, at the peak or paddy (extreme value) locate λ=4nd/N.
For the first few value of N (that is, and 0,1,2 ... .7), and supposition d is 700nm, and then equation is:
N=0: λ=∞ (paddy)
N=1: λ=4nd=4,496.80nm (peak)
N=2: λ=2nd=2,248.40nm (paddy)
N=3: λ=4nd/3=1,498.9nm (peak)
N=4: λ=nd=1,124.20nm (paddy)
N=5: λ=4nd/5=899.36nm (peak)
N=6: λ=2nd/3=749.47nm (paddy)
N=7: λ=4nd/7=642nm (peak)
N=8: λ=nd/2=562nm (paddy)
N=9: λ=4nd/9=499.64nm (peak)
N=10: λ=4nd/10=449.6nm (paddy)
This shows, and in Fig. 3 A and 3B, further specify, at least three peak/paddy (N=7-9) in limit of visible spectrum, occur.
Change if use the 7th paddy to calculate molecular layers thick, then when the molecular layer that is connected to ground floor when 0nm increases to 10nm, the 7th time paddy will be moved to 650.74nm.Therefore, the actual phase of the 7th paddy move and variation in thickness between ratio equal (650.74-642.40)/10=0.834.
On the contrary, if the initial separation between these two reflection horizon is made of the analyte binding molecule on the optical fiber end fully, suppose that this layer thickness is 25nm, then first secondary peak will come across the 146nm place, obviously outside limit of visible spectrum, therefore device will only be seen 0 paddy and the part in peak-to-peak zone for the first time, but will can't see any peak, thereby be difficult to accurately measure the spectral signature displacement of interference wave.
Gross thickness up to the reflection horizon just first secondary peak can occur near about 100nm in visible spectrum.Suppose that total thickness variations is up to 50nm, the thickness of optical element just can be little of 50nm, but be preferably hundreds of approximately nanometers, so as can be easily spectral position by more times peak or paddy (for example wherein N=3-10) move the periodicity phase shift or the variation of measuring interference wave.
It is believed that the ratio between actual (real) thickness and the measured phase shift is to measure the key factor of sensitivity.Be appreciated that adjustable lay the grain why learns the thickness and the refractive index thereof of element and improve and optimize sensitivity to adapt to electronics and optical design.
Fig. 4 is presented at the optical module 50 that movably places optical fiber 52 far-ends in the analytical equipment.As shown in the figure, this optical element comprises a plurality of such as flexible clamp arms such as arms 54, and these clamp arm designs are formed in the optical fiber end slip and come grip optical fibers by annular rim on the optical fiber or detent 56 with the interlock that is formed at the complementary connected in star in the arm.This annex be used for optical module be positioned on the optical fiber with the far-end of optical fiber and assembly relative (on) form between the face less than 100nm or greater than the air-gap of 2 μ m.If air-gap is greater than about 100nm but less than 2 μ m, then can significantly facilitates and not expect interference what accuracy of detection caused adverse effect from the internal reflection of optical module upper surface.
Continuation is referring to Fig. 4, and this optical module comprises that one is similar to first optical element 60 of above-mentioned optical element 38, and it has first and second reflection horizon 62,64 that is respectively that corresponds respectively to above-mentioned reflection horizon 40,42.This assembly comprises that further the thickness of second optical element, 66, the second optical elements 66 is preferable greater than 100nm, usually 200nm and its refractive index are similar to the refractive index of first optical element 60 at least.Preferably, these two optical elements constitute by identical glass or polymeric material with the refractive index between 1.4 and 1.6.Form and have preferable layer by high-index material and 64 be sandwiched in as shown in the figure between 2 optical elements less than about 30nm thickness.
During operation, this optical module is positioned on the distal fiber end and on this optical fiber clicks into place.Then, helping under sample analysis thing and the condition that the analyte binding molecule that forms reflection horizon 62 combines, the lower surface of this assembly is being exposed to an analyte sample.Along with analyte molecule layer combination therewith, the thickness of this layer increases, thereby the distance ' ' d ' ' between reflecting surface 62 and 64 increases.Described with reference to Fig. 3 A and 3B as mentioned, this produces by the extreme value displacement from this two-layer interference wave that reflection produced.The variation in thickness on (distal-most end) reflection horizon is down determined in the change of this extreme value or wavelength or wavelength period and then be used for.After using up, can remove and abandon this optical module, and replace with original element and be used for new analysis (identical or different analyte is analyzed).
Fig. 5 has illustrated optical module and the fibre bundle in one embodiment of the invention, and it detects in the sample one or more in a plurality of analytes (for example not homotactic nucleic acid analyte) through being designed for.Fibre bundle 72 waits the array (for example annular array) of each optical fiber to form by for example optical fiber 74.Optical module (indicating with 70 usually) is made up of basic optical element described above with reference to Fig. 4 but that be array format.Particularly, first optical element 80 in this element its down distal surface provide one such as regional 84 arrays such as conversion zone such as analyte such as grade, each array all comprises the analyte molecular binding layer that one of effectively is bonded in the sample in the different analytes.Each zone all forms first reflection horizon in this optical module.One preferable sensing can provide not homotactic nucleic acid (for example cDNA or oligonucleotides) array, and it is hybridized with different sequencing nucleic acid amalyzing substances in specific and the sample through design.In other words, this array surface formation one is used for detecting " genetic chip " of a plurality of each sequence of different genes sequence.
Comprise also in this optical module that one second optical element 78 and is sandwiched between these two optical elements and the high-index material layer 79 of second reflecting surface is provided in optical module.This assembly places on the fibre bundle 72 such as annular rim on flexible sway braces such as arm 76 and the fibre bundle or the interlock between the detent 86 by a pair of.Because assembly is positioned on the fibre bundle, the following far-end of optical fiber and the opposite of optical element 78 are separated by air-gap 85, and the spacing of air-gap 85 is preferable less than 100nm or greater than 2 μ m.In addition, each root in the optical fiber all with the correspondence analysis regional alignment of optical module so that each optical fiber all is mapped to light on the surveyed area of its aligning, and receive the reflected light in zone since then.Equally, be used in this device the photo-coupler that multifiber is coupled to detecting device is kept aiming between the relevant position on array region and the fluorescence detector (for example two-dimensional CCD).The material of various optical module parts and gauge all are similar to they above with reference to the exponent of Fig. 4 institute.
More particularly, the device described in the present invention can be used for following application:
(i) screen hybridoma expression system to obtain to have the clone of high antibody expression with the opposing material antibody that is carried on the tip;
(ii) come the qualitative antibody that this antigen is had high-affinity with the antigen that is carried on the tip;
(iii) come qualitative this combination of proteins of discriminated union partner (DNA, RNA, protein, carbohydrates, organic molecule) with the protein that is carried on the tip;
(iv) with being carried on the binding partner (for example DNA, RNA, protein, carbohydrates, organic molecule) that carbohydrates on the tip or glycosyl part come qualitative this carbohydrates of discriminated union;
(v) think on the tip that with being carried on the protein that participates in polyprotein matter complex compound is next qualitative in conjunction with component and/or complex compound formation dynamics;
(vi) with being carried on the protein bound agent that the small protein binding molecule on the tip comes qualitative this molecule of discriminated union;
(vii) with being carried on the antibody on the tip, the calibration curve that one group of standard analysis thing of use is drawn this analyte.Use this calibration curve, people can determine the analyte concentration in the unknown solution (cell culture supernatant, Biosample, processing potpourri etc.) thus.
(viii) differentiate the molecule of nucleic acid specificity combination therewith with the single-chain nucleic acid (for example ssDNA or RNA) that is carried on the tip.
Use a temperature controll block, this device and method also can be used for monitoring through the combination of fixing ssDNA oligonucleotides to the solution and to this in conjunction with characterizing to implement snp analysis.
The following example illustrates the whole bag of tricks of the present invention and application, but is intended to limit the scope of the invention by no means.
Example
It below is the example that is used to implement specific embodiment of the present invention.These given examples only are used for illustration purpose, and do not desire to limit the scope of the invention in any form.Although make every effort to make used variable (for example quantity, temperature etc.) to reach accurately, should allow some experimental error and deviation certainly.Except as otherwise noted, otherwise should use protein chemistry, biological chemistry, recombinant DNA technology and pharmacological common method in this technology to put into practice the present invention.These technology are explained fully in the document.Proteins:Structures and MolecularProperties (W.H.Freeman and Company, 1993) referring to (for example) T.E.Creighton; The Biochemistry of A.L.Lehninger (WorthPublishers company limited, current [); People's such as Sambrook Molecular Cloning:A LaboratoryManual (the 2nd edition, 1989); Methods In Enzymology (S.Colowick and N.Kaplan edit, AcademicPress company limited); Remington ' s Pharmaceutical Sciences, the 18th edition (Easton, Pennsylvania:MackPublishing company, 1990); The 3rd edition (Plenum Press) A of Carey and Sundberg Advanced Organic Chemistry and B volume (1992).
Example 1: micromolecule-protein bound reaction.
This example is used for verifying protein bound also is bonded to a plurality of antibody subsequently to the micromolecule that is fixed in the sensor tip the ability that detects.The double-decker of fiber optic tip is used in this test.The one Ta
2O
5The thickness of layer is 25nm and the 2nd SiO
2The thickness of layer is 770nm.Optical fiber is from Ocean Optics (Dunedin, Florida) person of buying.Manually be cut into the long section of 40mm.The two ends of these sections all are finished to standard minute surface quality.Finishing method used herein and those methods with what optical lens and catoptron are identical.The optical coating chamber is supplied with coating Ta in a surface of these fiber segments
2O
5Layer and SiO
2Layer.This supplier uses ion beam-assisted physical vapour deposition (PVD) (IAPVD) coating machine of being made by Leybold.IAPVD is the coating technique that is generally used for antireflection and light filter.These experimental procedures are included in hereinafter (except as otherwise noted, otherwise institute all at room temperature implement in steps):
With the polymer monolayers painting optic fibre tip that derives through biotin.Use biotinylated lipid (using always) preparation polymer monolayers.Use this lipid to form a lipid monolayer on the aqueous solution surface.Use UV light to make this individual layer crosslinked 15 minutes.Make the optical fiber of clean drying contact this unsteady film and the biotin polymkeric substance is absorbed in this fiber optic tip then.Then that these optical fiber are following dry 1 hour in 60 ℃.Under environmental baseline, store this optical fiber then.
This biosensor tips is dipped in 50 μ g/ml in PBS (Invitrogen, Carlsbad, CA; Catalog number 14190078) also simply washed with PBS then in 9 minutes in the streptavidin in (PierceBiotechnology, Rockford IL, catalog number 21122).
Same tip is dipped in rabbit-anti--streptavidin solution (AbCam, Cambridge, the MA of 10 μ g/ml in PBS; Catalog number ab6676-1000) also simply washed with PBS then in 36 minutes in.
At last, this tip is dipped in donkey-anti--rabbit antibody-solutions (JacksonImmunoResearch, West Grove, the PA of 50 μ g/mL in PBS; Catalog number 711-005-152) in 25 minute.In PBS solution, washed 10 minutes at last.
Fig. 6 shows the real-time response curve of this order in conjunction with experiment.The longitudinal axis is 7 paddy phase shifts representing with nanometer.It is clearly showed: the tip is fixed in streptavidin to the combination of biotin earlier, and is that anti-streptavidin antibody is to streptavidin and the second antibody combination of first antibody so far subsequently.In the time of 900 seconds, can find out the most advanced and sophisticated disassociation (minimizing that optical thickness is little) since then of streptavidin layer.
Example 2: the biomolecular interaction analysis of bio-molecular interaction dynamics and compatibility.
This example is set forth dynamics and the compatibility that the invention process biomolecular interaction analysis (BIA) is used to measure bio-molecular interaction.Identical pointed tip configuration described in the use-case 1.That this experimental procedure comprises is following (except as otherwise noted, otherwise used step all at room temperature implement):
Use following program to prepare the tip of hydrosulphonyl silane coating.With the optical fiber of clean drying at toluene: caproic acid: under room temperature, cultivated 24 hours in sulfydryl propyl group three TMOSs (10:2:1 volume ratio).These optical fiber with 10ml toluene flushing 2 times, were washed 5 minutes at every turn.These optical fiber are with 10mL alcohol flushing 1 time and in dry under the argon gas stream and store under environmental baseline then.
At first pass through with solution (Jackson ImmunoResearch, WestGrove, the PA of 10 μ g/ml rabbit-IgG in PBS; Catalog number 309-005-003) dipping obtained biosensor tips in 1 hour.
This tip through coating is dipped in 10 μ g/ml goats-anti--rabbit antibody (Jackson ImmunoResearch, WestGrove, PA; Catalog number 111-005-003) solution in PBS also kept 15 minutes therein.
It is most advanced and sophisticated and wash in PBS to take out this.For impelling the first antibody disassociation since then of this second antibody, manually rocked this PBS20 minute.
Then, this tip is dipped in once more in identical goat-anti--rabbit solution to show of the reappeared association of goat-anti--rabbit to rabbit-IgG.
Fig. 7 shows the association and the association and the dissociation curve that dissociate and produced by rabbit-IgG and goat-anti--rabbit.Once more, the longitudinal axis is the 7th paddy phase shift.This phase shift is with 0.834 ratio and average thickness positive correlation.Can detect association and dissociation curve reliably be crucial to measuring interaction dynamics and compatibility.
Example 3: calculate affinity costant by antibody-antigen combination and release profiles.
This experiment demonstration is calculated affinity costant by association and the dissociation curve of measuring two kinds of antibody and its antigen.With these exclusive antibody labelings is Ab-1 and Ab-2.The molecular weight of this antigen is about 30 kilodaltons.Use with example 1 in identical pointed tip configuration.Use with example 2 in identical hydrosulphonyl silane optical fiber preparation thing.These experimental procedures (except as otherwise noted, otherwise used step all under room temperature, implement) as described below:
Activate this fiber optic tip to be used for the covalently bound of described antigen.The optical fiber of hydrosulphonyl silane coating is by being dipped in the sensor tip 50mg/mL sulfo-SMCC (Pierce Biotechnology, the Rockford EL of 50 μ L; Catalog number 22322) in DMF (Sigma-Aldrich Chemical Company, St Louis, MO; Catalog number 494488) activated in 2 hours in the solution in.With the simple flushing and dry in DMF of this sensor tip.
By being flooded in the PBS of the antigen of 20 μ g/ml solution, this activated tip made this antigen in 20 minutes with the so far activated fiber optic tip of covalent manner combination.This tip was washed 2 minutes with PBS.After the PBS flushing, this is most advanced and sophisticated with 100 μ M monoethanolamine pH, 8.5 (Sigma-Aldrich Chemical Company, St Louis, MO; Catalog number E9508) aqueous solution quenching 5 minutes was also washed in PBS 2 minutes subsequently again.
This same tip is dipped in the antibody of the experiment that is used for associating and writes down 9 to 15 minutes real-time binding data (deciding according to antibody character and concentration).In case write down these data, this tip is dipped among the PBS once more and stirs with measure 9 to 15 minutes leave curve (off curve) (that is the disassociation between immobilized antigen and the institute's binding antibody).The antibody (25nM, 150nM and 430nM) of use variable concentrations also repeats this in conjunction with (association curve) and disassociation (dissociation curve) measurement with two kinds of different antibodies that are labeled as Ab-1 and Ab-2.
Fig. 8 shows association and the dissociation curve under the variable concentrations.Because it is extremely slow to associate, therefore do not finish the Ab-2 test of this concentration under the concentration of 25nM.These shown curves are raw-data maps.
By obtaining K from these curves with single order exponential function match raw data
On, K
OffAnd K
DBy two groups of data are average, obtain following dynamics and compatibility coefficient:
| Ab-1 | Ab-2 |
| K on＝1.35?x?10 5(M -1S -1) | K on＝2.01?x?10 5(M- 1S -1) |
| K off＝5.55?x?10 -５(S -1) | K off＝8.15?x?10 -５(S -1) |
| K D＝K off/K on＝3.99?x?10 -9(M) | K D＝K off/K on＝4.45?x?10 -9(M) |
The tip of example 4:NHS-ester activation.
Adopt the identical pointed tip configuration described in the example 1.Adopt identical hydrosulphonyl silane optical fiber preparation thing described in the example 2.By the sensor tip being dipped in 50uL50mg/mL sulfo-SMCC (Pierce Biotechnology, RockfordEL; Catalog number 22322) in DMF (Sigma-Aldrich Chemical Company, St Louis, MO; Catalog number 494488) activated the optical fiber of hydrosulphonyl silane coating in the solution in 2 hours.Simple this sensor of flushing is most advanced and sophisticated and dry in DMF.
The molecule that contains amine can be so far surperficial with the covalent manner combination by forming stable amide linkage.The molecule that does not contain unhindered amina can not pass through the NHS partial fixing, but this equimolecular still can be incorporated into this surface by non-specific binding.This non-specific binding can be multilayer and validity that should be by the NHS ester in individual layer and accessibility are controlled the Covalent Immobilization by the NHS ester.
In this group experiment, (Shearwater Polymers, San Carlos CA) is bonded to activating surface as test compounds with covalent manner to use diamine base PEG (MW 3300).The PEG (MW8000) (Sigma-Aldrich Chemical Company, St Louis, the MO that do not comprise free amino; Catalog number 04162) as negative control.This negative control is used for seeking any non-specific or multilayer combination that the PEG polymkeric substance may be intrinsic on this surface.
Fig. 9 displaying is handled the time course at activated hydrosulphonyl silane tip with test molecule.When this activation is most advanced and sophisticated when being exposed to the diamine base PEG (MW 3300) of 0.1mg/mL in PBS, its optical thickness shows that one obviously increases.When diamine base PEG solution replaces with the PBS damping fluid, stop to increase.Be exposed to 0.1mg/mL PEG (MW8000) and be stored in that the activation of the potpourri (it does not comprise amine) among the PBS is most advanced and sophisticated to be showed that less optical thickness is initial and increase but this vestige becomes smooth very soon.Can reach a conclusion since then: the PEG polymkeric substance do not have intrinsic non-specific binding and the diamine base PEG that seen in conjunction with specificity covalency solid owing to amido.
Example 5: the antibody derivatization tip of using NHS-esterification material to obtain.
This example has been set forth low-molecular-weight molecule and has been bonded to through the fixedly combination of high molecular weight molecules.Identical pointed tip configuration described in the surface of the identical NHS ester termination described in the employing example 4 and the example 1 is fixed in 3 optical fiber with an anti--biotin antibody.Be dipped in 20 μ g/mL mouse anti-biotin antibody (Biodesign, Saco MN by optical fiber under room temperature that this is activated; Catalog number H61504M) finished the fixing of antibody in the solution in PBS in 1 hour.This tip was washed 2 minutes with PBS.After the PBS flushing, this is most advanced and sophisticated with 100 μ M monoethanolamine pH, 8.5 (Sigma-AldrichChemicalCompany, St Louis, MO; Catalog number E9508) aqueous solution quenching 5 minutes was also washed in PBS 2 minutes then again.
First optical fiber is exposed to 200 μ g/mL biotin (Pierce Biotechnology, Rockford IL; Catalog number 29129) solution in PBS.Use sucrose (Sigma-Aldrich Chemical Company, St Louis, MO; Catalog number S8501) (2mg/mL) on second and third optical fiber, implements contrast to determine baseline noise with the solution of PBS.The data displays that obtain from these tests are in Figure 10.Biotin is increased in conjunction with being considered as optical thickness, do not show and be higher than the perceptible increase of baseline (PBS) and be exposed to sucrose.(from Calbiochem, what San Diego CA buied resists-Lewis Y antibody to use the irrelevant antibody that is fixed in the same manner on above-mentioned resisting-biotin antibody; Catalog number 434636) implements another negative control.This is exposed to 200 μ g/mL biotin solution through fixing antibody.Do not have to show that in conjunction with the biotin of antibody so far the biotin combination of anti--biotin antibody so far is because specificity interacts rather than because non-specific binding.
Although above show and set forth the present invention according to a preferred embodiment and various alternate embodiment are concrete,, those who familiarize themselves with the technology should be appreciated that, spirit and the category that can make various modifications and not deviate from the present invention wherein form and details.
Go out all purposes of what, all lists of references, publication and the patent application case quoted in this instructions main body all are incorporated herein in the mode that its integral body is quoted.
Claims (32)
1, a kind of being used for based on the assembly of interfering the analyte that detects a sample, it comprises:
Optical fiber with an end;
Be attached to the optical element of described optical fiber end, described optical element conforms to the size of described optical fiber end, and is coupled to light source by described optical fiber,
Described optical element comprises transparent material, first reflecting surface and second reflecting surface, separate by described second reflecting surface of described transparent material and described first reflecting surface, described first and second reflecting surface 50nm at least of being separated by, wherein said first reflecting surface comprises the analyte molecular binding layer, interference between the light that is reflected into described optical fiber from described first and second reflecting surfaces changes and changes along with the analyte the sample that is attached to described analyte binding molecule, and wherein said second reflecting surface comprises the material layer of refractive index greater than described refractive index of transparent materials.
2, assembly as claimed in claim 1, the spacing between wherein said first and second reflecting surface is between 100nm and 5, between the 000nm.
3, assembly as claimed in claim 2, the spacing between wherein said first and second reflecting surface is between 400nm and 1, between the 000nm.
4, assembly as claimed in claim 1, the refractive index of wherein said optical element transparent material is less than 1.8.
5, assembly as claimed in claim 4, wherein said optical element transparent material is for being selected from by SiO
2The material of the group that forms with transparent polymer.
6, assembly as claimed in claim 5, wherein said transparent polymer comprises polystyrene or tygon.
7, assembly as claimed in claim 1, the described material layer of wherein said second reflecting surface has the refractive index greater than 1.8.
8, assembly as claimed in claim 7, the described material layer of wherein said second reflecting surface comprises Ta
2O
5
9, assembly as claimed in claim 8, the thickness of the described material layer of wherein said second reflecting surface is between 5nm and 50nm.
10, assembly as claimed in claim 1, wherein said analyte molecular binding layer comprises the molecule that is selected from the group that is made up of protein, micromolecule, nucleic acid and carbohydrates.
11, assembly as claimed in claim 10, wherein said protein is selected from the group that is made up of avidin, streptavidin, antibody and antibody fragment.
12, assembly as claimed in claim 1, its further comprise between described optical element and described optical fiber and with second optical element of described optical fiber interlock.
13, assembly as claimed in claim 12, the thickness of wherein said second optical element is greater than 100nm.
14, assembly as claimed in claim 13, the thickness of wherein said second optical element is greater than 200nm.
15, assembly as claimed in claim 12, it further comprises:
Make the mechanical couplings of described second optical element and described optical fiber interlock.
16, assembly as claimed in claim 15, wherein said mechanical couplings provides air-gap between described second optical element and described optical fiber.
17, assembly as claimed in claim 16, wherein said air-gap is less than 100nm.
18, assembly as claimed in claim 16, wherein said air-gap is greater than 2 μ m.
19, a kind of two-dimensional array of forming by the described assembly of claim 1.
20, a kind of two-dimensional array of forming by the described assembly of claim 12.
21, a kind of device that is used for the check and analysis thing, it comprises:
The described assembly of claim 1;
Be used for light be incident upon described first with described second reflecting surface on light source; With
Detecting device, it receives from described first and is exposed to described analyte with the light of described second reflecting surface and described first reflecting surface so that the optical thickness change of described first reflecting surface is detected.
22, a kind of device that is used for the check and analysis thing, it comprises:
The described assembly of claim 12;
Be used for light be incident upon described first with the light source of described second reflecting surface; With
Detecting device, it receives from described first and is exposed to described analyte with the light of described second reflecting surface and described first reflecting surface so that the optical thickness change of described first reflecting surface is detected.
23, a kind of device that is used for the check and analysis thing, it comprises:
Two-dimensional array as claimed in claim 19;
Be used for light be incident upon described first with described second reflecting surface on light source; With
Detecting device, it receives from described first and is exposed to described analyte with the light of described second reflecting surface and described first reflecting surface so that the optical thickness change of described first reflecting surface is detected.
24, a kind of device that is used for the check and analysis thing, it comprises:
Two-dimensional array as claimed in claim 20;
Be used for light be incident upon described first with described second reflecting surface on light source; With
Detecting device, it receives from described first and is exposed to described analyte with the light of described second reflecting surface and described first reflecting surface so that the optical thickness change of described first reflecting surface is detected.
25, a kind of cover group, it comprises:
One assembly, described assembly comprise that one is coupled to the optical element of light source by optical fiber, and described optical element comprises transparent material, first surface and second reflecting surface, described first surface and described second reflecting surface 50nm at least of being separated by;
Wherein said first surface comprises the material layer of a refractive index greater than described optical element refractive index of transparent materials in conjunction with an analyte molecular binding layer and described second reflecting surface; With
Be used to make described analyte molecular binding layer to be bonded to the instructions of described first surface.
26, cover group as claimed in claim 25, it further comprises and is used for modifying the reagent of described first surface and the operation instructions of described reagent with chemical mode.
27, cover group as claimed in claim 25, wherein said assembly further comprise one between described optical element and described optical fiber and with second optical element of described optical fiber interlock, described second optical element is coupled to described light source by a mechanical couplings, described mechanical couplings makes described second optical element and described optical fiber interlock, and provides air-gap between described second optical element and described optical fiber.
28, cover group as claimed in claim 26, wherein said assembly further comprise one between described optical element and described optical fiber and with second optical element of described optical fiber interlock, described second optical element is coupled to described light source by a mechanical couplings, described mechanical couplings makes described second optical element and described optical fiber interlock, and provides air-gap between described second optical element and described optical fiber.
29, a kind of method that is used for the test samples analyte, it comprises:
Device as claimed in claim 21 and sample are provided;
With described first reflecting surface be exposed to described sample and
Determine whether described exposure causes the optical thickness change of described first reflecting surface.
30, a kind of method that is used for the test samples analyte, it comprises:
Device as claimed in claim 22 and sample are provided;
With described first reflecting surface be exposed to described sample and
Determine whether described exposure causes the optical thickness change of described first reflecting surface.
31, a kind of method that is used for the test samples analyte, it comprises:
Device as claimed in claim 23 and sample are provided;
With described first reflecting surface be exposed to described sample and
Determine whether described exposure causes the optical thickness change of described first reflecting surface.
32, a kind of method that is used for the test samples analyte, it comprises:
Described device of claim 24 and sample are provided;
With described first reflecting surface be exposed to described sample and
Determine whether described exposure causes the optical thickness change of described first reflecting surface.
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| CN101639447A (en) | 2010-02-03 |
| CN1875243A (en) | 2006-12-06 |
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