CN101529250A - Methods of detection using acousto-mechanical detection systems - Google Patents
Methods of detection using acousto-mechanical detection systems Download PDFInfo
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Abstract
Methods for detecting target biological analytes within sample material using acousto-mechanical energy generated by a sensor are disclosed. The acousto-mechanical energy may be provided using an acousto-mechanical sensor, e.g., a surface acoustic wave sensor such as, e.g., a shear horizontal surface acoustic wave sensor (e.g., a LSH-SAW sensor). The detection of the target biological analytes in sample material are enhanced by coupling of the target biological analyte (e.g., through the use of magnetic particles), application of a magnetic field to draw the target analyte to the sensor surface, and subsequent removal of the magnetic field before measuring detection.
Description
Government rights
According to the DAAD 13-03-C-0047 that Ministry of National Defence is authorized, U.S. government can enjoy some right to the present invention.
Background technology
With regard to sound-mechanical pick-up device, many biological analyte are together introduced in this sensor with liquid-carrier.Liquid-carrier is the sensitivity of reduction sound-mechanical detection systems unfriendly.In addition, this Selectivity of Sensor may depend on can not fast detecting characteristic, for example, specimen may need to hatch for a long time or grow.
In order to address this problem, can obtain selectivity by making the target organism analyte be bonded to (for example) detection surface.Yet, when employed sensor depends on sound-mechanical energy and detects the target organism analyte, the known target biological analyte optionally is bonded to detect the surface and may has problems.
Sonic sensor is because their testing mechanism is mechanical wave or sound wave and named.When sound wave passed the material surface propagation or propagates thereon, any variation of propagation path characteristic all can influence wave propagation velocity and/or amplitude.The variation of speed can be monitored by the frequency or the phase propetry of survey sensor, this variation can be associated with the corresponding physical quantity of measuring then.
Pass piezoelectric substrate or the mode propagated is described acoustic wave device by ripple on piezoelectric substrate.Upload sowing time when sound wave at substrate surface, it is called as surface wave.Show that sonic sensor (SAW) and shearing horizontal surface acoustic wave (SH-SAW) sensor are the surface wave device that is most widely used.A key character of SH-SAW sensor is that it allows sensing in liquid.
Shearing horizontal surface acoustic wave sensor is designed to the plane propagation sound-mechanical energy ripple along the sensor surface.In some systems, waveguide can be arranged on the detection surface so that the sensitivity (comparing) that sound-mechanical wave is positioned at the surface and increases sensor with the sensor of no waveguide.This improved shearing horizontal surface acoustic wave often is called as the shearing horizontal surface acoustic wave sensor of Love ripple (" LSH-SAW sensor ").
Use has combined the detection of relative less chemical substance with the size of target analytes wherein of this sensor and other materials.Therefore, most of target analytes are positioned at effective wave field (for example, for being about 60 nanometers (nm) in the water for the sensor of the frequencies operations of (for example) 103 megahertzes (MHz)) of sensor to a great extent.
For these sensors, analyte absorption can be disturbed the propagation of sound wave on whole sensor from the teeth outwards, and making can the check and analysis thing.These interference can be used as the phase place of equipment and the measure of the change of decay.In typical sensing experiment, sensor stablize a period of time, with the also variation of Measurement Phase and decay to the sensor of the analyte injection paid close attention to.The variation of estimating the variation of phase place and/or decay is relevant with the existence of target analytes and may be correlated with its concentration.
Sensor may be restricted in detection, when especially the concentration of target analytes is low in sample.For this result, there are several reasons, comprise the analyte ratio of actual acquisition on the sensor surface of existence; The quality and/or the size of institute's captured object analyte; And the intrinsic sensitivity of SAW equipment.Therefore, still need to improve to detecting target analytes with sound-mechanical detection systems.
Summary of the invention
The invention provides the method for enhancing to the detection of the target organism analyte in the specimen material, this detects and uses the sound-mechanical energy that is produced by sensor.This method comprises makes target analytes combine with magnetic-particle, utilizes the SAW sensor surface to catch the target analytes that is attached to magnetic-particle then.Sound-mechanical energy preferably use sound-mechanical pick-up device provides, for example, such as shearing horizontal surface acoustic wave sensor (as the LSH-SAW sensor), show sonic sensor, but other sound-mechanical pick-up device technology can be used in some cases in conjunction with method of the present invention.Use method as herein described can increase the improvement of detection limit aspect, this detection limit can realize 50 times more than increase.
In one embodiment, the method that detects the target organism analyte is provided, this method comprises makes specimen material contact with magnetic-particle, and wherein target organism analyte in the specimen material and magnetic-particle interact and make the target organism analyte be attached on the magnetic-particle in specimen material; Provide to comprise the system with the sound-plant equipment that detects surface (trapping agent is positioned on this detection surface), wherein trapping agent can optionally be attached to the target organism analyte and detect on the surface; The detection surface of sound-plant equipment is contacted with specimen material; Magnetic field generator is provided, and this magnetic field generator can provide the magnetic field on proximity detection surface, and this magnetic field is attracted to sensor surface with target analytes and the magnetic-particle that adheres to; Target organism analyte and the magnetic-particle that adheres to optionally are attached to the detection surface; Cut off magnetic field generator significantly to weaken the magnetic field on proximity detection surface; And when immersing in the liquid, the surface moves the target organism analyte that this-plant equipment is adhered to detection when detecting.
In another embodiment, provide the method for detection of biological analyte, this method comprises that the target organism analyte to being positioned at specimen material separates; The target organism analyte of separation is contacted with magnetic-particle, and wherein the target organism analyte that separates in the specimen material interacts with magnetic-particle and makes the target organism analyte that separates be attached on the magnetic-particle in specimen material; Provide to comprise the system with the surface acoustic wave device that detects surface (trapping agent is positioned on this detection surface), wherein trapping agent can optionally be attached to the target organism analyte and detect the surface; The detection surface of surface acoustic wave device is contacted with specimen material; Magnetic field generator is provided, and this magnetic field generator can provide the magnetic field on proximity detection surface, and the target analytes that this magnetic field will be attached to magnetic-particle is attracted to sensor surface; The target organism analyte optionally is attached to detects the surface; Magnetic field generator is moved to apart from detecting surface distance enough far away significantly to weaken the magnetic field on proximity detection surface; And show the separate targets biological analyte that sonic sensor adheres to detection when detecting to move when the surface is immersed in the liquid.
As used in this paper and the appended claim, singulative " ", " a kind of " and " being somebody's turn to do " comprise that plural number refers to, unless context indicates other implications clearly.Therefore, for example mentioned " a kind of target organism analyte " comprises the plurality of target biological analyte, and mentioned " this sensing chamber " comprises one or more sensing chamber of finger and equivalent well-known to those skilled in the art thereof.
When term " comprises " and variations does not have the implication of restriction when appearing in embodiment or the claim.
These and other features of detection system of the present invention and method and advantage can be described in conjunction with a plurality of exemplary embodiment of the present invention hereinafter.
Description of drawings
Fig. 1 is the diagram of sonic transducer.
Fig. 2 is an exemplary detection schematic representation of apparatus that comprises biology sensor.
Fig. 3 is the synoptic diagram that comprises the pick-up unit of biology sensor.
Fig. 4 is the synoptic diagram of sonic transducer detection system.
Embodiment
In the following specific descriptions of exemplary embodiment of the present invention, will be with reference to the accompanying drawings, these accompanying drawings constitute its part, and in these accompanying drawings, show in illustrational mode and can implement specific embodiments of the invention.Should be appreciated that, can utilize other embodiment and can carry out structural change and do not depart from the scope of the present invention.
Method of the present invention relates on the surface of magnetic-particle the acquisition target molecule and catch this magnetic-particle on sensor surface.The use of magnetic-particle has increased the amount that can be delivered to the target analytes of sensor surface.In addition, magnetic-particle (passing through target analytes) is coupled to sensor surface and can strengthens the sensor response.
The problem that may cause when as mentioned above, whether the target organism analyte exists in use sound-mechanical energy test sample material be the target organism analyte effectively be coupled to detect the surface so that from the sound-mechanical energy of sensor with the affected ability of detectable mode.As used herein, " target organism analyte " can comprise (for example) microorganism (as bacterium, virus, endospore, fungi, protozoan etc.), protein, peptide, amino acid, fatty acid, nucleic acid, carbohydrates, hormone, steroids, lipoid, vitamin etc.
In certain embodiments, detection method of the present invention can provide the target organism analyte that multiple technologies are used for the test sample material.This method comprises: choose wantonly the target organism analyte in the specimen material is separated or decomposes (as cracking target organism analyte), magnetic-particle is bonded to the target organism analyte and the target analytes that is attached to magnetic-particle is contacted with the SAW sensor surface.Use the trapping agent that target analytes is had affinity to apply the SAW sensor.Apply magnetic field on the surface of sensor and be attracted to sensor surface with the target analytes that will be attached to magnetic-particle.In case be attracted to sensor surface, just remove the response of magnetic field and survey sensor.
The target organism analyte can be by any suitable method from being or comprising that the specimen material of test sample obtains, and may depend primarily on the type of target biological agent to be detected.For example, test sample can obtain from individual (people, animal etc.) or other sources by (for example) collection of biological tissue and/or fluid sample (as blood, urine, ight soil, saliva, seminal fluid, bile, eye lens liquid, synovial membrane liquid, celiolymph, fester, sweat, exudate, mucus, lactation, skin, hair, nail etc.).In other examples, specimen can be used as acquisitions such as environmental samples, product sample, food sample.The test sample that obtains can be liquid, gas, solid or their combination.
Before being delivered to system and method for the present invention, can allow specimen material and/or test sample accept early stage and handle, for example, the dilution viscous fluid, concentrate, filter, distillation, dialysis, add reagent, chemical treatment etc.
It is by use the magnetic-particle that target analytes has the trapping agent coating of affinity to be finished that target analytes is captured magnetic-particle.Trapping agent can combine with target analytes by specificity combination or non-specific binding.For example, can be with such as by deriving from (Invitrogen (the Carlsbad of Carlsbad, California city hero life technology company limited, and (Berlin, DE) particle that applies of the Streptavidin of Chemicell GmbH company is used to catch albumin A-biotin in the Berlin, Germany CA)).Similarly, can catch other target analytes to the biotinylated protein (as the magnetic-particle of Streptavidin coating) that target analytes has specific biotinylated antibody coating by use.In one embodiment, catch target analytes (for example staphylococcus aureus) and comprise that the magnetic-particle that specimen and Streptavidin are applied contacts, be attached with the biotinylation derivant of monoclonal antibody (for example being filed in the U.S. Patent application sequence No.11/562 of the title on November 22nd, 2006, Mab 107 monoclonal antibodies described in 747) on this magnetic-particle for " ANTIBODY WITH PROTEIN A SELECTIVITY ".Then magnetic-particle is expelled on the sensor surface and by the magnetic field generator on proximity sense surface and catches.
The magnetic-particle that is attached to target analytes captured be subjected to a plurality of factor affecting on the sensor surface, comprise magnetic field intensity (by the distance decision of magnet strength and magnet), magnetic field generator with respect to the granularity of the orientation of the position of the sensor surface in the X-Y plane, magnetic field generator, magnetic-particle with form apart from sensor, and during magnetic field generator is caught and is moved the flow velocity of specimen on sensor.For example, can be close to sensor surface (for example distance is 0 millimeter) places magnetic field generator and catches all particles for given flow velocity in the forward position of sensor.Can remove magnetic field generator then bunch makes it flow on sensor with release particles.Alternatively, can still keep attracting the magnetic field of magnetic-particle arranging magnetic field generator, and use suitable flow velocity uniform particles to be coated on the sensor single step away from a certain distance of sensor.
Although utilizing the magnetic-particle of band coating to catch the target organism analyte is known in the prior art, also do not recognize up to now to produce to be used to handle the magnetic particle detectability to the SAW sensor has adverse influence to the magnetic field on surface.After magnetic-particle was captured to sensor surface, the magnetic field (as by removing or perhaps cutting off) of significantly weakening the proximity sense surface was so that the sensor response.When magnetic field generator is in place, have sensor response, yet this has nothing to do with analyte concentration.On the other hand, after cutting off magnetic field generator, observe the big signal relevant with analyte concentration.Therefore, remove the sensitivity that magnetic field generator has improved check and analysis after catching.Even it also is like this all uniform particles being layered under the situation on the sensor at the position by optimizing magnetic field generator and flow velocity.
In a preferred embodiment, during catching magnetic-particle, magnet is remained on the fixed position on proximity sense surface, it is moved hardly.Mobile or the sensor surface of magnet can cause unsettled sensor response with respect to moving of magnet.Although do not expect bound by theoryly, magnet can be arrived by sensor with respect to moving of sensor surface, so the disturb sensor response.
In a preferred embodiment, when small part is covered by fluid, remove (or cut off) magnetic field and carry out follow-up sensor measurement when sensor surface.In most of embodiment, sensor surface is immersed in the fluid, as is immersed in the fluid sample material that comprises the magnetic-particle that is attached with target analytes.
In one embodiment, utilize method as herein described, sensor can increase nearly 50 times to the detection limit or the sensitivity of given target organism analyte (as albumin A).As further illustrational in the example hereinafter; when the target organism analyte is attached to magnetic-particle or does not apply/cut off when moving sensor under the situation of magnetic field generator of not as described herein or this paper prescription protection, sensor only can the test sample material in 50ng/500mL or higher target organism analyte.By contrast, use the method for described herein or this paper prescription protection, sensor is low to moderate the target organism analyte of 1ng/500mL in can the test sample material.
In addition, when using the SH-SAW sensor, preferably use granularity less than 1 micron magnetic-particle.Surpass 1 micron as fruit granule, using SH-SAW to detect can affect adversely, even because be the coating of dense accumulation, the quantity of some contact also can reduce, and this ripple that has influenced sensor surface is propagated.In a preferred embodiment, magnetic-particle is of a size of 250nm, and 100nm more preferably.
Shown that traditional Love wave type sensor has phase response.For check and analysis method of the present invention, we find not have significant phase response.After magnetic field generator was removed from sensor, response was on the contrary but in decay.
The data that generate in the experiment are normally collected in frequency domain.This data-switching can be become time domain and can carry out time threshold (time gating) algorithm.In typical algorithm, use the time signal that thresholding (gate) filtering is not expected, data-switching can be returned frequency domain then.Have the situation of reference channel for sensor, the reference channel signal can be deducted the noise of not expecting to filter out from the active channel signal.Response is that part of data computation after magnetic field generator is almost completely weakened from the proximity sense surface normally.Decaying offset is measured as the just in time decay before magnetic field generator weakens and magnetic field generator and weakens difference between the decay of back in a few minutes.If before magnetic field generator weakens, have drift, can before removing magnetic field generator, on data point, use linear regression to come the estimated sensor drift.At this drift the skew of being surveyed is proofreaied and correct then.
Use is bonded to magnetic-particle on the target analytes and has improved on the sensor surface capture rate to target analytes.Significantly more the target analytes of a large amount can be delivered to sensor surface.For example, single dependence diffusion causes about 0.1% to 1% available targets analyte arrival sensor surface.Yet to being bonded to the magnetic-particle applying a magnetic field generator of target analytes, the target analytes up to 100% is delivered to and/or remains on the sensor surface is possible.Because target analytes is attached on the magnetic-particle that is attracted to sensor surface, so target analytes moves to the surface with the speed that is higher than other components of sample.Thereby, realize preferentially target analytes being attached to sensor surface.
Particle adheres to
The target organism analyte is attached to the target organism analyte is had on the magnetic-particle of selective affinity.Can allow particle adhere in conjunction with separation/decomposition technique (wherein, for example, particle may be attached on the fragment of cell membrane or the like).In certain embodiments, the target organism analyte is separated or perhaps resolves into less fragment or particle, make increase that the target organism analyte that is bonded to magnetic-particle of number percent can remain in effective wave field of sound-mechanical pick-up device and/or can with the effectively coupling of detection surface of sound-mechanical pick-up device.
Chemical method, mechanical means, method for electrically, by the use of thermal means be can pass through, or separation or decomposition finished by the combination of two or more these technology.The example of the chemical separation technology that some may be fit to can relate to (as) use one or more enzymes, hypertonic solution, hypotonic solution, scaling agent etc.The example of the mechanical separation technology that some may be fit to can comprise that (for example) is exposed to acoustic energy, mechanical raking (as decomposing to strengthen), alkaline bleach liquor cleavage etc. under the situation that has microballon or other particles.Thermal release can be undertaken by (for example) heating target biological agent.Other separation/decomposition techniques also can use in conjunction with the present invention.Be filed among on Dec 17th, 2004, the U.S. Patent Application Publication No.2005-0153370-A1 of title and described cytolytic use for " Methodof Enhancing Signal Detection of Cell-Wall Components of Cells " (method of the input of the cell-wall components of enhancing pair cell), as a kind of method that the target organism analyte is separated, this method can be used in conjunction with the present invention with cytolysis.
In other cases, can specially not use magnetic-particle under the situation of separation/decomposition goal biological analyte.For example, magnetic-particle can be used to catch the whole cell of bacterium, as be filed on November 22nd, 2006, title U.S. Patent application 60/867 for " Method of Capturing Bacterial WholeCells " (catching the method for the whole cell of bacterium), described in 016, and the document is incorporated this paper into way of reference in full.Particle can optionally be attached on the target organism analyte or they can non-selectively be attached on the material in the specimen.
The particle that is attached to target organism analyte (or their fragment) is a magnetic, makes that they can work by the magnetic field that is applied to sensor before measuring detection.In this system, proximity detection surface in magnetic field is provided with, and makes the target organism analyte be attracted and be attached to and detects the surface, can detect them by sound-mechanical pick-up device there.
Magnetic-particle can strengthen the detection of target organism analyte in many ways.Magnetic-particle is used in the target organism analyte that will adhere under the influence in magnetic field and is driven to the detection surface, thereby quickens to catch and/or improve capture rate.When being coupled to sensor surface by target analytes, self can also provide the detection of enhancing the magnetic-particle that adheres to.
The method of using the conventional method of magnetic-particle and preparing magnetic-particle is at (for example) U.S. Patent No. 3,970,518 (Giaever) U.S. Patent No. 4,001,197 people such as () Mitchell; And describe to some extent among the open No.0016552 of European patent people such as () Widder.These methods can be suitable for using in conjunction with the present invention.
Can also add and make the reagent that changes with the viscosity, elasticity and/or the viscoelastic property that detect the surperficial specimen material that contacts.The example of the material sex change technology (mass-modificationtechniques) that some are fit to can be that (for example) is used in combination fibrinogen and staphylococcus species, be filed in the U.S. Patent application sequence No.60/533 on Dec 30th, 2003 as (for example), 171 and U.S. Patent Publication No.2006-0019330-A1 described in.
Selectivity is adhered to
Detection system of the present invention and method can preferably be provided for the target organism analyte optionally is attached to magnetic-particle and detection surface or is attached to the another kind of component that can be coupled to the detection surface.Can realize that selectivity adheres to by how sharp technology.Some examples can comprise the combination of (for example) Ag-Ab; Affine combination (as avidin-biotin, nickel sequestrant, glutathione-glutathione s-transferase (GST)); Covalently bound (as azlactone, three chlorotriazines, phosphonitrilic chloride trimer or N-sulfonyl amino carbonyl-acid amides chemistry); Or the like.
Can realize directly that the selectivity of target organism analyte adheres to, promptly target organism analyte self optionally is attached to and detects the surface.The example of the direct selectivity attachment techniques that some are such can comprise the use trapping agent, and for example, Ag-Ab is caught in conjunction with (wherein target organism analyte self comprises the antigen that detects lip-deep antibody in conjunction with being fixed on), DNA etc.
It also can be indirect that selectivity is adhered to, and promptly wherein the target organism analyte optionally is attached to optionally to be adhered to or be attracted to detect surperficial magnetic-particle.Indirect selectivity attachment techniques comprises that magnetic-particle optionally is bonded to the target organism analyte makes that the target organism analyte can be by magnetic attraction to detecting the surface and being retained on this detection surface.
Adhere in conjunction with selectivity, preferred system and method for the present invention can be provided for other biological analyte or low (for example) detection surface that is bonded to of material non-specificly.Non-specific binding can have a negative impact to the result's that obtains with detection system of the present invention and method correctness.Non-specific binding can be permitted different ways and be solved.For example, can before specimen is introduced the detection surface, the known biological analyte of non-specific binding problem and the material of may causing be removed from this specimen.In another method, can use interrupter technique to reduce and detect lip-deep non-specific binding.
Usually depend on the coating that is arranged on the sound-mechanical detection surface, layer etc. because selectivity is adhered to, therefore must be noted that these materials be relative thin and can not make sound-mechanical energy decay to the degree of the effective detection of inhibition.
Adhere to another relevant problem with selectivity and be to use the technology that is commonly called " fixing " technology, this technology can be used for trapping agent being kept or being fixed on the surface of (for example) sound-mechanical pick-up device self.Detection system of the present invention and method can relate to the use of various fixed technology.
As described herein, conventional method is the surperficial trapping agent of detection that applies or offer sound-mechanical pick-up device equipment, for example (as) antibody, peptide, fit or any other trapping agent of the target organism analyte being had affinity.The surface coverage of lip-deep trapping agent and compactedness can determine the sensitivity of sensor.The fixedly chemistry that trapping agent is connected to the sensor surface can filled trapping agent, keep working in the trapping agent activity (if it is a biomolecule), and also can help the repeatability and the life-span of sensor.
If trapping agent is protein or antibody, then they may be adsorbed onto the surface non-specificly and be lost the ability (activity) that they catch the target organism analyte.Various fixed method and sound-mechanical pick-up device can be united use to realize the target of high yield, activity, life-span and stability.These trapping agents preferably adopt glutaraldehyde cross-linking chemistry, acrylamide to catch or the general coupling chemistry as carbodiimide, epoxide, cyanogen bromide etc. applies.Depend on employed trapping agent, the concentration of the trapping agent on the sensor surface becomes very important in optimizing the sensor response.
Except in conjunction with trapping agent and still keeping its active chemical property, all there is other character of surface in any fixedly chemistry that uses in conjunction with the present invention, and these characteristics may need to consider and may become relevant in sound-mechanical pick-up device is used.For example, fixedly chemistry can cause preferably that the limited decay of sound-mechanical energy makes immobilization can not learn to stop sensor to produce available data.Fixedly chemistry can also determine how antibody or protein are attached to the surface, thus the orientation of decision trapping agent avtive spot.Fixedly chemistry can preferably provide repeatably characteristic to obtain repeatably data and sensitivity from sound-mechanical pick-up device of the present invention.
Can in (for example) following patent, description: U.S. Patent Application Publication No.2005-01070615-A1 be arranged in conjunction with some technique for fixing that system and method for the present invention uses, be filed in the 2005-0112672-A1 and the United States Patent (USP) sequence No.60/533 on Dec 30th, 2003,162, be filed in 60/533 of on Dec 30th, 2003,178, U.S. Patent Application Publication No.2005-0142296-A1,2005-0106709-A1,2005-0227076-A1,2006-0135718-A1,2006-0135783-A1, and the title that is filed on Dec 17th, 2004 is the open No.WO2005/066092 of the PCT of " Acoustic Sensors and Methods " (sonic transducer and method).
Fixing means can be included in waveguide on the sound-mechanical base and the tack coat between the fixed bed.An exemplary tack coat can be (for example) diamond like carbon glassy layer, for example described in open No.WO 01/66820 A1 in world people such as () David.
Sound-mechanical pick-up device
System and method of the present invention preferably detects the existence of target organism analyte in the specimen by use sound-mechanical energy, sound-mechanical energy can be measured or sensing to determine wave attenuation, phase change, frequency change and/or variation of resonant frequency.
Can use (for example) to produce sound-mechanical energy based on surface acoustic wave (SAW) equipment of piezoelectricity.As (for example) U.S. Patent No. 5,814, described in 525 people such as () Renschler, this class can further be subdivided into surface acoustic wave (SAW), sound Lamb wave (APM) or QCM (Quartz Crystal Microbalance) (QCM) equipment according to their detection wave mode based on the sound plant equipment of piezoelectricity.
Method as herein described adopts sonic transducer, and is sound mechanical biological sensor more specifically, but the signal of the variation of this at least one physical characteristics of sensor and generation response change detected.Preferably, the sound mechanical biological sensor of this paper employing is surface acoustic wave (SAW) biology sensor.Sound wave produces from the comb transducer (IDT) on the piezoelectric substrate as surface acoustic wave or bulk acoustic wave in these equipment.The 2nd IDT can change the telegram in reply signal with sound wave so that measure.This is referred to as lag line.Alternatively, this equipment can be used as resonator work.Can change two intervals between the IDT with the coating that comprises the bioactive molecule that is used for chemistry or biological sensing application.
With reference to Fig. 1, the sound mechanical biological sensor surface 100 between the IDT 15 preferably includes two lag lines in certain embodiments.Provide first passage (i.e. " activity " passage 20) to be used for the acceptance test sample.Provide second channel (i.e. " reference " passage 30) as baseline or contrast.Therefore, physical characteristics is changed to difference between active channel and the reference channel.When needs, acoustic duct 10 (its edge only is shown in Fig. 1) covers area and the IDT self between IDT usually.Available mathematical algorithm translation data is to improve sensitivity.The optional structure of an exemplary sound mechanical pick-up device be included in the title that is filed on Dec 17th, 2004 for the PCT of " Acousto-mechanical Detection Systems and Methods of Use " (sound-mechanical detection systems and using method) disclose among the No.WO2005/075973 disclosed those.
Usually detect minute changing capability of quality or viscosity based on their and work based on the SAW biology sensor of piezoelectricity.As U.S. Patent No. 5,814, described in 525, this class can further be subdivided into surface acoustic wave (SAW), sound Lamb wave (APM) or QCM (Quartz Crystal Microbalance) (QCM) equipment according to the wave mode of their mass change based on the sound mechanical biological sensor of piezoelectricity.
In certain embodiments, sound mechanical biological sensor comprises second trapping agent or the reactant (as antibody) that target analytes is attached to piezoelectricity sound mechanical biological sensor surface.The surface velocity of wave propagation is to detect the sensitive probe of the variation such as quality, elasticity, viscoelasticity, conduction and specific inductive capacity.Therefore, any one variation in these characteristics will cause detectable surface acoustic wave to change.That is to say, when material contacts, adsorbs or causes the surface coating that is attached to SAW equipment, will produce corresponding response.
APM also can work when equipment contacts with liquid.Similarly, the alternating voltage that is applied on two comparative electrodes of QCM (be generally AT-and cut quartz) equipment comprises the thickness shear wave mode, and the mass change in the variation of its resonant frequency and the coating material is proportional.
The direction of sonic propagation (as in the plane parallel with waveguide or vertical with planar waveguide) by quartz-Qie decision with the piezoelectric of its structure sound mechanical biological sensor.Since with liquid that the surface contact in have too many acoustic attenuation, so SAW biology sensor (its most of sound wave (being R wave (Rayleigh wave), most of Lamb wave (Lamb-wave)) is propagated the turnover plane) use in the liquid sensing is used usually.
For the fluid sample medium, shearing horizontal surface acoustic wave biology sensor (SH-SAW) is preferably with such piezoelectric structure: this piezoelectric has quartz-Qie and makes wave propagation rotate to the orientation on horizontal shear wave (promptly in the plane of biology sensor waveguide), cause with liquid that biosensor surface contact in acoustic attenuation loss reduction.The horizontal shear sound wave comprises (for example) sound Lamb wave (APM), plunder face bulk wave (SSBW), Love wave, leakage shows sound wave (LSAW) and Bleustein-Gulyaev wave (Bleustein-Gulyaev) (BG).
Specifically, love wave sensor is by supporting SH wave mode (for example SSBW of ST quartz or 36 ° of YXLiTaO
3Leaky wave) substrate form.By using slim sound guide layer or waveguide these wave modes are changed into the Love wave.These ripples be frequency dependence and the prerequisite of velocity of wave that can be lower than piezoelectric substrate at the shear wave velocity of ducting layer under generate.SiO
2Be used as the sound wave conducting shell on the quartz.Other thermoplastic and crosslinked polymer waveguide materials such as polymethylmethacrylate, phenol formaldehyde resin (as trade name NOVALAC), polyimide and polystyrene also are used.
Alternatively, QCM equipment also can use with the fluid sample medium, but is to use these equipment sound waves to cause becoming the lower equipment of common sensitivity because of the serious decay of liquid medium.
The biology sensor of employing sound mechanical hook-up and the parts of this biology sensor are known.Referring to (for example) U.S. Patent No. 5,076,094,5,117,146,5,235,235,5,151,110,5,763,283,5,814,525,5,836,203,6,232,139.SH-SAW equipment can (obtain as the Sandia National Laboratory in Albuquerque, New Mexico city (SandiaNational Laboratories, Albuquerque, NM)) from a plurality of manufacturers.Some SH-SAW biology sensor is also at " 36 ° of YXLiTaO of Low-level detection of a Bacillus anthracis stimulant usingLove-wave biosensors of
3" (with 36 ° of YXLiTaO
3The Love wave biology sensor is to the low level detection of Bacillus anthracis analogies) describe to some extent in (Biosensors andBioelectronics, 19,849-859 (2004)).SAW biology sensor and detection of biological compositions and methods be also at the U.S. Patent application sequence No.60/533 that is filed on Dec 30th, 2003, describes to some extent in 169.
In certain embodiments, the surface of biology sensor comprises second trapping agent or the reactant (as antibody) that covers on the ducting layer.In this embodiment, biology sensor usually detects the viscosity and/or the mass change of being adhered to by second trapping agent or reactant.For this embodiment, biology sensor preferably includes fixed bed (covering on the ducting layer) and optional tack coat.
Can provide fixed bed to be used to making second trapping agent or reactant (as antibody) be bonded to the surface.The material that can be used for fixed bed comprise above-described those.
Detection system and detection tube
As described herein, material of the present invention and method can be used for sensor so that waveguide, fixed bed, capture material or their combination to be provided.Following discussion provides some possible systems and has detected the example of tube, wherein can utilize the sensor that uses material of the present invention.
Fig. 2 is a synoptic diagram that comprises the pick-up unit of biology sensor.Device is shown can be chosen wantonly and comprise reagent 322, specimen 324, washing buffer 326 and magnetic-particle 327.Component that can these are different is introduced in (for example) stand chamber (staging chamber) 328, and here they can mix and/or react to each other.Alternatively, can allow one or more these components before one or more other components are introduced into stand chamber 328, be present in this stand chamber.
For example, expectation introduces in the stand chamber 328 reagent 322 and specimen 324 so that reagent 322 acts on and/or be attached to the target organism analyte in the specimen 324.Subsequently, magnetic-particle 327 can be introduced in the stand chamber 328.Magnetic-particle 327 can optionally be attached to the target organism analyte material in the stand chamber 328, although they not necessarily need to do like this.
After the target organism analyte in the specimen 324 is attached to magnetic-particle 327, specimen 324 (and associate magnetic-particle) can be moved to the sensing chamber 330 from stand chamber 328, but the detection surface 332 of the target organism analyte feeler in the specimen material here.Detect surface 332 and be preferably the type that comprises position trapping agent thereon so that the target organism analyte in the specimen material can optionally be attached to detection surface 332.
If target organism analyte and magnetic-particle associate, then expectation comprises and can detect the magnetic device 333 that surperficial 332 places produce magnetic field, so that the target organism analyte that associates with magnetic-particle can be used to use detection by the sensor 334 of controller 335 manipulations to detecting the surface by magnetic attraction.The use of the magnetic-particle that combines with the target organism analyte can to make the target organism analyte when not having (for example) magnetic attraction desired moves to quickly and detect surface 332 and improve detection by (for example).
322 pairs of target organism analytes of preferred reagent have selectivity, and promptly the other biological analyte in the specimen 324 can be by reagent 322 modifications.Alternatively, reagent 322 can be nonselective, and promptly it can act on the multiple biological analyte in the specimen 324, no matter whether this biological analyte is the target organism analyte.In certain embodiments, reagent 322 can be preferably Chemical Decomposition agent (chemical fractionating agent), for example (as) one or more enzymes, hypertonic solution, hypotonic solution, washing agent etc.
The target organism analyte is to the description to some extent in the open No.WO02/090565 (Ritterband) in the world and WO 00/70040 people such as () Bitner of adhering to roughly of (for example) magnetic-particle, and this patent has been described in the kit use in order to the magnetic bead and the magnetic response particle of concentrating cells.Biological agent to the selectivity of magnetic-particle (as paramagnetic microsphere) adhere to also " Impedance characterization of a piezoelectric immunosensor partII:Salmonella typhimurium detection using magnetic enhancement " people such as (for example) Kim (
Biosensors and Bioelectronics18 (2003) 91-99) describe to some extent in.
After the target organism analyte in the specimen 324 is attached to magnetic-particle 327, specimen material (specimen 324 and the magnetic-particle that associates) can be moved to sensing chamber 330 from stand chamber 328, the target organism analyte in sensing chamber in the specimen material can contact with detection surface 332.Because target organism analyte and magnetic-particle associate, can detect the magnetic field generator 333 that surperficial 332 places produce magnetic field so expectation comprises, detect the subsequent detection that is used to use the sensor of handling by controller 335 334 on the surface so that the target organism analyte that associates with magnetic-particle can be retained in.In other words, the magnetic force that is provided by the magnetic field on proximity detection surface 332 can be attracted to magnetic-particle (and the target organism analyte that adheres to) and detect surface 332.Magnetic field generator 333 can be that any suitable equipment that is arranged to magnetic-particle is attracted to the magnetic field of detecting the surface can be provided, for example, and permanent magnet, electromagnet etc.
The use of the target organism analyte that combines with magnetic-particle can to make the target organism analyte when not having (for example) magnetic attraction effectively desired and/or move to quickly and detect surface 332 and improve detection by (for example).
Before detecting target analytes, magnetic field generator 333 moves distance enough far away (perhaps cutting off (for example cutting out) magnetic field generator 333) significantly to weaken the magnetic field of proximity sense 334 from sensor 334 with sensor 334.Keep magnetic field if (produce and detect acoustic energy in this) during detecting processing by sensor 334, then magnetic field will influence unfriendly and very likely can stop accurate detection to the target organism analyte time.
Comprise that position selectivity trapping agent thereon makes the target organism analyte can optionally be attached to and detects surface 332 if detect surface 332 when removing magnetic field, then can remove magnetic field the magnetic-particle that does not carry any target organism analyte (or not entrained by any target organism analyte) is removed from detecting surface 332 by (for example).In the SH-SAW sensor, because the local detection district of SH-SAW, it is not very important to remove the magnetic-particle that does not carry any target organism analyte (or not entrained by any target organism analyte) that washing detects surface 332.At the magnetic-particle that introduce to associate (promptly carry the target organism analyte or by the entrained magnetic-particle of target organism analyte) before, the magnetic-particle that can use additive method to remove not-associate, the i.e. magnetic-particle that does not associate with any target organism analyte.
The preferred sensor of being handled by optional control module 335 334 that uses carries out selectivity is attached to the detection of any target organism analyte that detects the surface.Control module 335 is the control pick-up 334 feasible sound-mechanical energy that are fit to that produce preferably.But control module 335 also can be chosen the mobile and monitoring sensor 334 that suitable flow velocity, controlling magnetic field generator 333 are set wantonly so that can have or not the target organism analyte to determine to detecting on the surface 332.
Drive and the example of the technology of the sound-mechanical pick-up device of monitoring such as those the-mechanical pick-up device that can use in conjunction with the present invention can find in (for example) following patent: U.S. Patent No. 5,076,094 (people such as Frye), 5,117,146 (people such as Martin), 5,235,235 (people such as Martin), 5,151,110 (people such as Bein), 5,763,283 (people such as Cernosek), 5,814,525 (people such as Renschler), 5,836,203 (people such as Martin), and 6,232,139 (people such as Casalnuovo) etc.Other example can be described in (for example) following document: " 36 ° of YX LiTaO of Low-level detection of a Bacillus anthracis simulant using Love-wavebiosensors on of people such as Branch
3" (with 36 ° of YX LiTaO
3On the Love wave biology sensor to the low level detection of Bacillus anthracis analogies) (Biosensors andBioelectronics, 19,849-859 (2004)), and the U.S. Patent application No.60/533 that is filed on Dec 30th, 2003,177 and be filed in Dec 17 in 2004 purpose titles and be the open No.WO2005/066622 of PCT of " Estimating Propagation Velocity Through A Surface Acoustic WaveSensor " (by showing sonic sensor assessment velocity of propagation).
Can be although above discussed in conjunction with the exemplary detection device of the present invention's use in conjunction with Fig. 2, those devices can be contained in and can be described as in the integrated unit that detects tube.Exemplary detection tube has further description in following patent: the title that is filed on Dec 17th, 2004 is that open No.WO2005/075973 of the PCT of " Acousto-mechanical Detection Systems and Methods of Use " (sound-mechanical detection systems and using method) and the title that is filed on Dec 17th, 2004 are " Detection Cartridges; Modules; Systems andMethods " (detection filter cylinder, module, system and method) the open No.WO2005/064349 of PCT, it has been described can be in conjunction with the detection tube of the present invention's use and/or the additional features of module.
Fig. 3 shows an exemplary embodiment of the detection tube 610 that comprises stand chamber 620, sensing chamber 630 and waste chamber 640.Detect tube 610 and comprise sensor 650 and magnetic field generator 656 with the detection surface 652 that is exposed in the sensing chamber 630.
In preferred embodiment shown in Figure 3, preferably magnetic field generator 656 is shifted near sensor 650 by the opening in the tube 610.In alternate embodiment, magnetic field generator 656 proximity senses 650 be provided with and do not have an opening, this depend on magnetic field generator 656 intensity, be used to construct the material of tube 610 etc.
Some examples that sound-mechanical pick-up device are attached at the method that may be fit in the tube that can use in conjunction with the present invention can find in (for example) following patent: be filed in the U.S. Patent application sequence No.60/533 on Dec 30th, 2003,176 and be filed in the title on Dec 17th, 2004 and be the open No.WO 2005/066621 of the PCT of " Surface Acoustic Wave Sensor Assemblies " (showing the sonic sensor assembly).
In some cases, the method of using in the above-mentioned appointment document can be used with sonic transducer, this sonic transducer comprises the contact mat outside the ducting layer edge that is exposed on the sensor, uses to be arranged on the carrier of sensor contact mat and attached sensors or the Z-axle adhesive agent between the track on the supporting mass element.Yet alternatively, the method described in those documents can be used for producing electrical connection by ducting layer, wherein the characteristic of Z-axle adhesive agent and waveguide material is (as glass transition point (T
g) with fusing point) be similar.In this method, do not need waveguide material is removed from the contact mat on the sensor, the conducting particles in the Z-axle adhesive agent produces electrical connection by the waveguide material on the sensor contact mat.
The embodiment of Fig. 3 comprises the ventilating opening 678 in the waste chamber 640, and it can be arranged in waste chamber 640 and the inner space that ambient atmosphere is communicated with.Thereby the fluid stream that enters waste chamber 640 and pass tube 610 is controlled in the opening and closing that can utilize ventilating opening 678.In addition, ventilating opening 678 can be used for this ventilating opening 678 being vacuumized and wait the pressure that reduces in the waste chamber 640 by (for example).
Although be illustrated as and carry out direct fluid with waste chamber 640 and be communicated with, one or more ventilating openings can be set and make them directly be connected any correct position place of leading to the inner space (as stand chamber 620, sensing chamber 630 etc.) of detecting tube 610.Ventilating opening 678 can adopt any suitable form, for example one or more spaces, pipe, accessory etc.
Refer again to tube shown in Figure 3, stand chamber 620 can be set at the upstream of sensing chamber 630.Stand chamber 620 can provide a space, various ingredients can be introduced wherein before entering sensing chamber 630.Although not shown, be to be understood that stand chamber 620 can comprise multiple structure example as (as) be positioned at wherein one or more reagent (as be dried or be received be used for carrying out selectivity and discharge), coating (as hydrophilic coating, hydrophobic coating etc.), configuration/shape (can the formation of (for example) minimizings/inhibition bubble, improve/cause the configuration/shape of mixing etc.) in the suitable time.
In addition, the fluid passage between stand chamber 620 and the sensing chamber 630 can be opened, as shown in Figure 3.Alternatively, fluid passage between stand chamber 620 and the sensing chamber 630 can comprise the multiple structure that can carry out one or more functions, for example (as) filtration (using (for example) porous membrane, volume exclusion structure, pearl etc.), fluid containment structure
(using (for example) one or more valves, porous membrane, capillary vessel or passage, fluid restrictor etc.), coating (as hydrophilic coating, hydrophobic coating etc.), configuration/shape (formation that can (for example) minimizing/inhibition bubble and/or the configuration/shape of transfer, raising mixing etc.).
The optional structure of other of sensor tube (for example fluid monitor 627 and the module 680 of sending multiple material) further describes in the open No.WO2005/075973 of PCT.
Although exemplary embodiment as herein described can comprise single-sensor, detection tube of the present invention can comprise two or more sensors, and these two or more sensors are similar substantially or different each other.In addition, each sensor that detects in the tube according to the present invention can comprise two or more passages (as sense channel and reference channel).Alternatively, can use different sensors that sense channel and the reference channel that detects in the tube is provided.If a plurality of sensors are provided, then they can be arranged in the same sensing chamber of detection tube or different sensing chamber.
The other argumentation that relates to multiple detection system and parts (the detection tube that for example comprises biology sensor) can be found in (for example) following patent: the U.S. Patent application No.60/533 that is filed on Dec 30th, 2003,169, the title that is filed on Dec 17th, 2004 is the open No.WO2005/075973 of the PCT of " Acousto-mechanical Detection Systems and Methods of Use " (sound-mechanical detection systems and using method), and the title that is filed on Dec 17th, 2004 (detects tube for " Detection Cartridges; Modules, Systems andMethods ", module, system and method) the open No.WO2005/064349 of PCT.
System design
Expect the unit that tube can be docked to or be connected to can (for example) provides multiple function (for example data that power supply, receiving sensor produce are provided, ability that makes the user import control fluid stream and/or sensor operation or the like is provided for sensor or other equipment in the tube of detecting) that detects of the present invention.
Fig. 4 schematically shows a kind of such system 500, and it preferably includes power supply 501 and user interface 502 (as button, keyboard, touch-screen, microphone etc.).System 500 also can comprise identification module 503 in addition, and this module is suitable for using the concrete detection tubes 510 of identification such as (for example) bar code, radio frequency identification equipment, physical construction.
The input that processor 505 receives from biosensor analysis instrument 504, this analyser can comprise (for example) and pass the relevant measurement of sound-mechanical pick-up device or the ripple on sound-mechanical pick-up device propagation.Processor 505 can determine whether there is the target organism analyte in the specimen material then.Although the present invention is not limited to this aspect, the sensor that detects in the tube 510 can be electrically coupled to biosensor analysis instrument 504 by detecting tube 510 insertion systems 500 slit or other docking structures interior or on it.Processor 505 can be contained in the unit identical with biosensor analysis instrument 504 or can be the part of independent unit or independent computing machine.
For instance, processor 505 can be the general purpose microprocessor of saved software in the execute store 506.In this case, processor 505 can be loaded in custom-designed computing machine, general purpose personal computer, workstation, handheld computer, the laptop computer etc.Alternatively, processor 505 can be special IC (ASIC) or other custom-designed processors.Under any circumstance, any required data analysis technique of processor 505 preferred execution is to determine whether there is the target organism analyte in specimen.
Other available data analysis techniques can be described in the document of the use that relates to sonic transducer as herein described.Use the system and method that shows sonic sensor although wherein described to relate to, be to be understood that the use of these system and methods also can be used for other sound-mechanical pick-up devices.
Preparation sound-mechanical pick-up device
As described herein, the present invention depends on use sound-mechanical pick-up device detects the target organism analyte in the specimen that flows on detecting the surface existence.Available several different methods and technology implementation apply or provide required multiple material to think that sound-mechanical pick-up device provides the selection attachment characteristic of expectation.
When being used for sonic transducer, employed waveguide material, immobilization material, trapping agent etc. can deposit by any suitable technology or method on the sensor.Usually, preferably with the substrate of these material delivery to the carrier fluid, carrier fluid and material form (for example) solution or dispersion.When as above sending, some be suitable for from the teeth outwards the example of the deposition technique of deposition materials can include, but is not limited to irritate be coated with, spin coating, printing, noncontact deposition (as ink-jet, spraying etc.), mould are coated with, blade coating etc.In certain embodiments, preferred deposition technique has the ability that mould is coated with the surface, that is, and and deposition materials on the selected part on surface only.Be filed in the U.S. Patent application sequence No.10/607 on June 27th, 2003,698 have described the mould coating method that is applicable in conjunction with sensor use according to the present invention.
In certain embodiments (title that for example is filed on Dec 17th, 2004 for describe among the open No.WO2005/066092 of the PCT of " Acoustic Sensors and Methods " (sonic transducer and method) those or the like), some material can be simultaneously as the immobilization material of the following suprabasil waveguide material and second trapping agent.In other embodiments, same material can be used as waveguide material, immobilization material and capture material.In these two kinds of versions, but in the material preferred deposition of the present invention substrate below, this substrate itself can be insoluble to carrier fluid effectively makes carrier fluid can not have a negative impact to following substrate.
Yet,, preferably use the noncontact techniques of deposition material such as (for example) ink-jet, spraying etc. if surface of waveguide material to be deposited, immobilization material and/or trapping agent shows to have certain solubility at the carrier fluid that is used for delivery materials on it.For example, to use (for example) butyl acetate to deposit if following substrate is the waveguide that is formed by (for example) polyimide, acrylate etc. on the sensor base and the material of fixed bed as carrier fluid, then preferably use the noncontact deposition process with the formation of restriction waveguide and preferred leaving exposed in the functional characteristic of the immobilization material of gained coating surface.Same Consideration is applicable to the coating of trapping agent on the surface.
In spray application technology, there are some controllable variablees, comprise percentage of solids, nozzle diameter, carrier fluid in rate of sedimentation, substrate velocity (with respect to fog-spray nozzle), sheath gas velocity, sheath gas, grating space, raster pattern, port number, the spray liquid/dispersion, wait to deposit the composition etc. of the lower surface of material of the present invention on it.Can deposit material of the present invention can rule of thumb determine with the actual conditions that produces suitable coating.
Method of the present invention can be in conjunction with uses such as the multiple material described in (for example) a plurality of U.S. Patent applications hereinafter described, method, system, devices, and all patents all are incorporated herein with way of reference with their full text separately.These patents comprise: the U.S. Patent application sequence No.60/533 that is filed on Dec 30th, 2003,162, be filed in 60/533 of on Dec 30th, 2003,178, U.S. Patent Application Publication No.2005-0142296-A1,2005-0107615-A1,2005-0112672-A1,2005-0106709-A1,2005-0227076-A1, be filed in the U.S. Patent application sequence No.60/533 on Dec 30th, 2003,171, U.S. Patent Application Publication No.2006-0019330-A1, be filed in the U.S. Patent application sequence No.60/533 on Dec 30th, 2003,177, be filed in 60/533 of on Dec 30th, 2003,176, U.S. Patent Application Publication No.2005-0153370-A1,2006-0135718-A1,2006-0135783-A1, be filed in the PCT open No.WO2005/066622 of the title on Dec 17th, 2004 for " Estimating Propagation Velocity Through A Surface Acoustic WaveSensor " (by showing sonic sensor assessment velocity of propagation), the title that is filed on Dec 17th, 2004 is the open No.WO2005/066621 of the PCT of " Surface AcousticWave Sensor Assemblies " (showing the sonic sensor assembly), the title that is filed on Dec 17th, 2004 is the open No.WO2005/075973 of the PCT of " Acousto-mechanicalDetection Systems and Methods of Use " (sound-mechanical detection systems and using method), the title that is filed on Dec 17th, 2004 (detects tube for " Detection Cartridges; Modules; Systems and Methods ", module, system and method) the open No.WO2005/064349 of PCT, and be filed in the title on Dec 17th, 2004 and be the open No.WO2005/066092 of the PCT of " Acoustic Sensors and Methods " (sonic transducer and method).
Example
These examples only are in order schematically to illustrate, and have no intention to limit the scope of subsidiary claim.Except as otherwise noted, otherwise all umbers in example and the instructions remainder, percentage, ratio etc. all by weight.
Example 1
The method of preparing sensor and carrying out test experience
This experiment uses spin coating shearing horizontal surface acoustic wave (SH-SAW) sensor of waveguide (as be filed in be 50: 50 methyl methacrylates preparing described in the example W1 of the PCT patent disclosure No.WO2005/066092 of " Acoustic Sensors and Methods " (sonic transducer and method) and the multipolymer of isobornyl methacrylate on Dec 17th, 2004, title) to be arranged (by Com Dev (Cambridge, Ontario, Canada) or by Sandia NationalLaboratory (Albuquerque, New Mexico) provide).Utilize fixedly chemicals spray application sensor, this fixedly chemicals comprise as being filed in the isobornyl methacrylate/methyl methacrylate/asccharin-methacrylate/terpolymer of acryloxy benzophenone (35/35/30/0.5) in butyl acetate/acetonitrile (50/50) of preparation as described in the example MP26 that on Dec 17th, 2004, title discloses No.WO2005/066092 for the PCT of " Acoustic Sensors and Methods " (sonic transducer and method).In some cases, will to albumin A have that specific monoclonal antibody (Mab107) manually applies or spray application to two (use and with reference to) sensor passage on.In other cases, Mab107 antibody or rabbit are resisted-staphylococcus aureus (RaSa) (Accurate Chemical ﹠amp; Scientific Corporation, Westbury, NY) manually apply or spray application to a sensor passage (active channel) is gone up and with non-specific chicken IgY (Jackson ImnunoResearch LaboratoriesInc, West Grove PA) manually applies or spray application arrives on other passages (reference channel).By electroconductive binder (anisotropic conducting film bonding agent 7313 (Anisotropicconductive film adhesive 7313), 3M company, Sao Paulo, Minnesota State city) sensor of band coating is thermally bonded to flexible circuit.Bonding sensor is attached to the flow device (flowpod) of Controllable Temperature by the double-sided adhesive film.Then sensor is connected to electrical measurement template (passing through flexible circuit), this electrical measurement template is driven by the software program that is written in the LABVIEW software by network analyzer.LabVIEW software can derive from Texas Austin city American National instrument company (National Instruments, Austin, TX).In whole experiment, collect decay and phase propetry in the required frequency range.
In order to begin experiment, with PBSL running buffer (as described below) via syringe pump with the mean flow rate flows through sensor of 0.1mL/min and be adjusted to required flow velocity subsequently.Begin experiment with software program then.To rise to the position below the sensor by the rare-earth magnet that neodymium-iron-boron (NdFeB) forms.When flow enough stable after, by time of software appointment by introduction valve injection sample and allow its flows through sensor.
After sample had arrived and accumulated on the sensor surface, (in the time by the software appointment) removed magnet (" decline ") distance enough far away significantly to weaken the magnetic field intensity of sensor surface.Usually magnet is moved＞65mm.Yet, littler significantly weakening apart from magnetic field.We are visually observed, when magnet from sensor surface be removed to＞5mm apart from the time magnetic-particle can not keep (because flow of block bulk liquid flow power) at sensor surface.
Flowing, it is stable until phase place and deamplification to continue time enough.Usually, this can determine by the phase place and the decay original signal that show on the visual observation computer screen.When original signal over time with magnet descend the desired signal in back change compare relative than hour, think that then signal is stable.
(8753ET/ES network analyzer user manual 3-35 and 3-36 page or leaf, Agilent Technologies (Agilent Technologies)) is used for handling original phase and the attenuation data that experiment produces with the time threshold algorithm.Except as otherwise noted, otherwise the time interval unit that is used for data aggregation be 13 seconds and when the software startup data aggregation, pick up counting (as, time point 100 occurs in back 1300 seconds of experiment beginning).Can specify the suitable thresholding of this algorithm based on the concrete sensor design of using.Can directly use this algorithm by network analyzer and make that the data that obtain from experiment are to have passed through the time threshold processing.Alternatively, can collect raw data and can using is written on Matlab (software program in Massachusetts (Natick, MA)) carries out the time threshold processing for Mathworks company, Nei Dike town.
Can further analyze the data handled through time threshold to determine phase place and decaying offset.Use Matlab software to finish all this data processing.There is not the situation of reference channel for those, by calculating in two passages about phase place and both signal bias of decaying in the magnet value of the back value of signal when stablize when deducting magnet and just having descended that descend.
For the sensor with active channel and reference channel, the decay and the phase signal that deduct reference channel respectively by decay and phase signal with active channel come the calculated difference signal.Deduct the skew of the data computation difference signal before magnet has just descended by the stabilization signal of back acquisition that magnet is descended.
Example 2
Albumin A-biotin-conjugated is to magnetic-particle
The albumin A of biotin-conjugated derives from sigma chemical company (the Sigma Chemical Company (St.Louis, MO)) of St. Louis, Missouri.To derive from Invitrogen company (Carlsbad city, the California) or Chemicell Gmbh (Berlin, the magnetic-particle of Streptavidin-coating Germany) is in 1mL phosphate buffered saline (PBS) (PBS, 137mMNaCl, 2.7mM KCl and 10mM phosphate, pH7.40) middle pre-wash.This magnetic-particle and albumin A-biotin specimen are mixed in 1mL PBS with required concentration.This suspending liquid is hatched 〉=30 minutes (stirring simultaneously) under 37 ℃.Then sample is washed three times to remove any unconjugated target substance in 1mLPBS.Washing process comprises: place magnet so that magnetic-particle is fixed on the tube wall against sample hose, remove supernatant, add isopyknic fresh PBS and suspended particle again.For final washing, particle is suspended in 1mL PBSL64 damping fluid (the PBS damping fluid contains 0.2%w/v PLURONIC L64 (BASF AG in Fu Lunhan park, New Jersey city (BASF, Florham Park))) again.
Example 3
By the Mab107-biotin albumin A is conjugated to magnetic-particle
According to the instructions of manufacturer, (Illinois Rockford city (Rockford, Pierce company IL)) carries out biotinylation to Mab107 with EZ-Link NHS-PEO4-Biotin kit.Obtain albumin A and it is diluted to required test concentrations among PBS from Invitrogen.The magnetic-particle and the biotinylation-Mab107 antibody of Streptavidin coating are mixed with required concentration and hatches in the PBSL damping fluid in 37 ℃ 〉=1 hour.Then sample is washed three times to remove any unconjugated antibody in the PBSL damping fluid.At last washing step end, will suspend again in the albumin A specimen of particle in 1mL PBSL damping fluid, and under 37 ℃, hatch 〉=30 minutes.
Example 4
In the SAW sensor, detect the albumin A of no particle combination
This examples show do not use the detection of the albumin A that magnetic-particle carries out.Described in example 1, prepare sensor.Prepare binary channels, 103MHz, interdigital (two-way IDT) shearing horizontal surface acoustic wave (SH-SAW) sensor (Sandia National Laboratory), making has RaSa antibody on the active channel, and chicken IgY is arranged on the reference channel.Binary channels, 113MHz, SpudTA (unidirectional IDT) shearing horizontal surface acoustic wave (SH-SAW) sensor (be coated with waveguide, provided by Com Dev) are provided, and making has Mab107 antibody on the active channel, and chicken IgY is arranged on the reference channel.
Carry out independent experiment at 125ng/mL to the albumin A between the 1000ng/mL with concentration range.At time point 150 specimen is expelled on the SAW sensor, and in the skew of time point 300 Measurement Phase with the decay difference signal.Data are shown in the table 1.
Table 1: when not using magnetic-particle to the phase place and the convergent response of albumin A sample
| Sensor | The antibody activity passage | Concentration (ng/mL) | Phase response | Convergent response | |
| Sandia | RaSa | 1000 | 0.50 | 0.03 | |
| | RaSa | 500 | 0.42 | 0.03 | |
| Sandia | RaSa | 250 | 0.33 | 0.02 | |
| Sandia | RaSa | 125 | 0.13 | 0.01 | |
| Com Dev | Mab107 | 1000 | 0.55 | 0.03 |
Data show, produce less relatively phase place and decaying offset under the employed albumin A concentration range in these tests.The least concentration (125ng/mL) of the albumin A of test can detect by the relative less variation with convergent response of phase place (on noise level) of SAW sensor in these experiments.
Example 5
In SAW sensor (fixing magnet), detect combination with next-door neighbour's sensor surface
Albumin A-biotin to 1 μ m magnetic-particle
This examples show magnet is being risen to proximity sense surface and when experimental session remains on this position, is utilizing determination method to detect albumin A-biotin based on magnetophoresis.The magnetic-particle that uses in this example is
MyOne
TMStrepto-is affine 1 μ m microballon (Invitrogen).Described in example 1, prepare sensor.Binary channels, 113MHz, SpudTA (unidirectional IDT) shearing horizontal surface acoustic wave (SH-SAW) sensor (be coated with waveguide, provided by ComDev) are provided, and making has Mab107 antibody on the active channel, and chicken IgY is arranged on the reference channel.
The microballon that the Streptavidin of 1mg/mL applies is puted together with the albumin A-biotin of different amounts, and as described in example 2, washed then.Washed microballon further is diluted to 1: 10 in PBS (v: v) and with the aliquot of 500 μ L specimen as the SAW sensor.In whole experiment, magnet (size and configuration are shown in the synoptic diagram 1) is fixed in sensor surface under (promptly＜0.5mm).
The size and the configuration of the magnet that uses in the synoptic diagram 1.SAW sensor device.Note the direction of magnet with respect to the sample flows through sensor.
(100nm diameter; Chemicell GmbH)
When the magnetic-particle in the sample deposits to after sensor surface (analyte capture) finishes, measure the skew of convergent response.The result is shown in the table 2.Use this detection method, the decaying offset that causes in conjunction with albumin A-biotin composite of 5000ng/mL is insignificant.The combination of this magnetic-particle that shows 1 μ m size and the magnet of next-door neighbour's sensor do not allow to albumin A in the sample carry out sensitivity (≤detect 5000ng/mL).These data show that also this detection method (use the magnetic-particle of 1 μ m and keep magnet next-door neighbour sensor) is poorer than method (example 4) sensitivity of nonmagnetic particle.
Table 2 uses the method that does not remove magnet 1 μ m to be attached to the magnetic of albumin A-biotin
The sensor response of particle.Decaying offset has shown the scope from the result of 4 repeated experiments
| Magnetic-particle concentration (mg/mL) | Albumin A-biotin concentration (ng/mL) | Flow velocity (mL/min) | Decaying offset (dB) |
| 0.1 | 5000 | 0.03 | 0 to 0.06* |
* the scope of representing the result of 4 repeated experiments
Example 6
Magnet positions is bonded to the albumin A of sub-micron magnetic-particle in the SAW sensor for detection
Influence
As preparation sensor as described in the example 1.Binary channels, 113MHz, SpudTA (unidirectional IDT) shearing horizontal surface acoustic wave (SH-SAW) sensor (be coated with waveguide, provided by Com Dev) are provided, and making has Mab107 antibody on the active channel, and chicken IgY is arranged on the reference channel.Will be with Streptavidin (100nm diameter; Chemicell GmbH) magnetic-particle of Tu Fuing is used for the albumin A-biotin of experiments of measuring sample.The 100nm particle of 1mg/mL is puted together with the albumin A-biotin of different amounts, and as described in example 2, washed.Then these particles are carried out 10 times of specimen of diluting and 500 μ L volumes being used as the SAW sensor in PBSL.For these experiments, with magnet (as described in synoptic diagram 1, constructing) be fixed in sensor just below (＜ejection testing sample 0.5mm) and when the time point 150.
When magnet remains on original position, do not observe significant response in phase place and the decay difference signal.When time point 400, magnet is removed＞65mm from sensor.In containing the sample of the albumin A of 200ng/mL at least,, will observe big (＞0.8dB) convergent response (table 3) in case remove magnet.
Use the magnetic-particle of same concentrations to carry out control experiment, the no albumin A-biotin of this experiment is attached on this particle.Convergent response in this experiment is less than 0.2dB.After removing, sensor do not having significant phase response after the injection or at magnet.Thereby, in the sample of 200ng/mL and 5000ng/mL, to observe big decaying offset and show, the magnetic-particle of 100nm can be used for the amplifying signal decay, and precondition is that magnet is descended.
Be attached to the 100nm magnetic-particle of albumin A-biotin when removing magnet after table 3. is caught
The sensor response
| The concentration of biotinylated protein A (ng/mL) | The distance of magnet and sensor (mm) | Flow velocity (mL/min) | Decaying offset (dB) |
| 0 | 0 | 0.03 | 0.14 |
| 200 | 0 | 0.03 | 0.84 |
| 200 | 0 | 0.03 | 1.21 |
| 5000 | 0 | 0.03 | 1.55 |
Digital proof removes the sensor response that magnet produces to be increased from sensor.
Example 7
Be attached to the SAW sensor response of albumin A of the various concentration of sub-micron magnetic-particle
This examples show use determination method to detect albumin A based on magnetophoresis.Binary channels, 113MHz, SpudTA (unidirectional IDT) shearing horizontal surface acoustic wave (SH-SAW) sensor (be coated with waveguide, provided by Com Dev) are provided, and making all has Mab 107 antibody on active channel and the reference channel.
Will be with Streptavidin (250nm diameter; Chemicell GmbH) magnetic-particle of Tu Fuing is used for the albumin A of experiments of measuring sample.With the 250nm particle of 0.1mg/mL as puting together with the albumin A sample of variable concentrations (0,2,20 and 200ng/mL) as described in the example 3.In each experiment, use biotinylation-Mab 107 antibody (shown in following table) of multiple amount.With the specimen of 500 μ l Ma volumes as the SAW sensor.For these experiments, magnet (size of magnet and configuration are shown in the synoptic diagram 2) is arranged on below the sensor 3mm place and specimen is injected on the SAW sensor at time point 50 or 150 places.As illustrate shown in Figure 2ly, the direction of fluid passage is for from left to right on the top surface.With time interval collecting sensor data as shown in table 4.At time point 200 or 300 with magnet away from sensor and measure the skew of convergent response.
Synoptic diagram 2. is used for the synoptic diagram of the magnet of example 7 described experiments.
As shown in table 4, the convergent response after magnet removes be with specimen in the concentration of albumin A proportional.In this experiment, detected the concentration of 2ng/mL.This expression is compared with the determination method of not using magnetic-particle (example 4), and there has been remarkable improvement the detection limit aspect of albumin A.In these experiments, use minimum antibody concentration (0.01 μ g/mL) to realize for the viewed maximum attenuation response of 20ng/mL albumin A.
Table 4: when removing magnet after catching to the biography of the 250nm magnetic-particle that is attached to albumin A
The sensor response
| Albumin A (ng/mL) | Data Ab is concentration (sec) (μ g/mL) at interval | Sample is injected magnet and is put time point fall time | The distance of magnet and sensor (mm) | Flow velocity (mL/min) | Decaying offset (dB) |
| 2 | 8 0.01 | 150 300 | 3 | 0.08 | 0.29 |
| 20 | 8 0.01 | 150 300 | 3 | 0.08 | 0.96 |
| 20 | 8 0.1 | 50 200 | 3 | 0.08 | 0.5 |
| 0 | 13 1 | 150 300 | 3 | 0.08 | -0.06 |
| 20 | 13 1 | 150 300 | 3 | 0.08 | 0.14 |
| 200 | 13 1 | 150 300 | 3 | 0.08 | 2.02 |
All lists of references as herein described and patent disclosure all are incorporated herein with way of reference clearly in full.This paper has discussed exemplary embodiment of the present invention and has mentioned the possible variant that is in the scope of the invention.Under the prerequisite that does not depart from the scope of the present invention, these and other variant and modification will be conspicuous to those skilled in the art, and should be appreciated that the present invention is not limited to the exemplary embodiment that this paper proposes.Therefore, the present invention only is subjected to the qualification of following appending claims and equivalent thereof.
Claims (18)
1. method that detects the target organism analyte, described method comprises:
Specimen material is contacted with magnetic-particle, and target organism analyte in the wherein said specimen material and described magnetic-particle interact, and make described target organism analyte be bonded to the described magnetic-particle in the described specimen material;
The system that comprises sound-plant equipment is provided, and described sound-plant equipment comprises the detection surface, and trapping agent is positioned on the described detection surface, and wherein said trapping agent can optionally be attached to described target organism analyte described detection surface;
The described detection surface of described sound-plant equipment is contacted with described specimen material;
Magnetic field generator is provided, and this magnetic field generator can provide the contiguous described magnetic field of detecting the surface, and the described target analytes that this magnetic field will have the magnetic-particle that adheres to is attracted to described sensor surface;
The described target organism analyte that will have the magnetic-particle that adheres to optionally is attached to described detection surface;
Cut off described magnetic field generator significantly to weaken the contiguous described described magnetic field of detecting the surface; And
When immersing in the liquid, the surface operates the described target organism analyte that described sound-plant equipment is adhered to detection described the detection.
2. method according to claim 1, wherein said sound-plant equipment comprises surface acoustic wave device.
3. method according to claim 2, wherein said surface acoustic wave device comprise shearing horizontal surface acoustic wave equipment.
4. method according to claim 1, wherein said magnetic-particle has the particle mean size less than 1 micron.
5. method according to claim 1 also comprises described target analytes is contacted with separating agent.
6. method according to claim 1 is wherein cut off described magnetic field generator and is comprised described magnetic field generator is removed to and be enough to significantly to weaken the contiguous described distance that detects the described magnetic field on surface.
7. method that detects the target organism analyte, described method comprises:
Separate the target organism analyte that is positioned at specimen material;
The target organism analyte of separation is contacted with magnetic-particle, the target organism analyte of the described separation in the wherein said specimen material and described magnetic-particle interact, and make the target organism analyte of described separation be bonded to the interior described magnetic-particle of described specimen material;
The system that comprises saw sensor is provided, and described saw sensor comprises the detection surface, and trapping agent is positioned on the described detection surface, and wherein said trapping agent can optionally be attached to described target organism analyte described detection surface;
The described detection surface of described surface acoustic wave device is contacted with described specimen material;
Magnetic field generator is provided, and this magnetic field generator can provide the contiguous described magnetic field of detecting the surface, and the described target analytes that this magnetic field will be attached to described magnetic-particle is attracted to described sensor surface;
Make described target organism analyte optionally be attached to described detection surface;
Described magnetic field generator is moved to apart from the described surface distance enough far away that detects significantly to weaken the contiguous described magnetic field of detecting the surface; And
When immersing in the liquid, described detection surface operates described saw sensor to detect the target organism analyte of accompanying separation.
8. method according to claim 7, wherein said separation comprise the described target organism analyte in the described specimen material of Chemical Decomposition.
9. method according to claim 7, wherein said separation comprise that machinery separates the described target organism analyte in the described specimen material.
10. method according to claim 7, wherein said separation comprise the described target organism analyte in the described specimen material of thermal release.
11. method according to claim 7, wherein said separation comprise that electricity separates the described target organism analyte in the described specimen material.
12. method according to claim 7, wherein said saw sensor comprise the shearing horizontal surface acoustic wave sensor of Love wave type.
13. according to claim 1 or 7 described methods, wherein said target organism analyte comprises bacterial cell.
14. according to claim 13 described methods, wherein said bacterial cell comprises staphylococcus aureus.
15. according to claim 1 or 7 described methods, wherein said trapping agent is an antibody.
16. according to claim 1 or 7 described methods, wherein said trapping agent is monoclonal antibody Mab-107.
17. according to claim 1 or 7 described methods, the concentration of wherein said target organism analyte in described specimen material is 1ng/500mL or can be more detected when higher.
18. method according to claim 1, wherein said target organism analyte is whole bacterial cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111148572A (en) * | 2017-06-06 | 2020-05-12 | 西北大学 | Cross-interface magnetic separation |
| CN113865906A (en) * | 2021-09-28 | 2021-12-31 | 重庆交通大学 | Integrated test system for micro-channel flow and on-way loss of aerosol collector |
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2007
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111148572A (en) * | 2017-06-06 | 2020-05-12 | 西北大学 | Cross-interface magnetic separation |
| CN111148572B (en) * | 2017-06-06 | 2022-05-06 | 西北大学 | Trans-interface magnetic separation |
| CN113865906A (en) * | 2021-09-28 | 2021-12-31 | 重庆交通大学 | Integrated test system for micro-channel flow and on-way loss of aerosol collector |
| CN113865906B (en) * | 2021-09-28 | 2024-05-17 | 重庆交通大学 | Micro-channel flow and along-path loss integrated test system of aerosol collector |
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