CN101213453A - Method and apparatus for detecting analytes using an acoustic device - Google Patents

Abstract

Methods for detecting analytes are provided. A plurality of particles, each of which is coated with a capture agent having an affinity for the analyte is combined with the sample to form a plurality of analyte-particle complexes. The system also includes a transport arrangement for transporting the sample to the sensor surface, and optionally a magnetic field inducing structure constructed and arranged to establish a magnetic field at and adjacent to the sensor surface. The resonant sensor produces a signal corresponding to an amount of analyte-particle complexes that are bound to the sensor surface.

Description

Utilize the method and apparatus of acoustical device check and analysis thing

Technical field

The present invention relates to the method for one or more analytes in the test fluid sample.

Background of invention

() system for example, the chemistry and biology material, its remarkable difficult point comprises the concentration of analyte in the medium and analyte is transported to sensor surface for the analyte in the tracer liquid medium.For biological applications,, can produce concentration problems usually because the concentration of this analyte may be lower.In addition, biological analyte (for example, cell, cell fragment and big molecule are as protein and nucleic acid) may be bigger; So can produce transshipment problem very slowly in fluid solution because of these bigger analytes spread.

Except that cell, cell fragment with such as protein and the nucleic acid equimolecular, detect the useful mark that the micromolecule analyte can be used as the micromolecule library of the pharmacokinetics that diagnoses the illness, monitor patient's Chinese traditional medicine and screening potential drug target.Often need the many curative drugs in the monitoring patient body, comprise that small-molecule drug is with the beneficial effect that improves medicine as far as possible with avoid issuable detrimental effect.

For example, estradiol is the micromolecule that is used to assess many health status.For example, can adopt blood testing follicle-stimulating hormone (FSH) (FSH) and estradiol (E2) to check whether ovary deposit (ovarian reserves) and ovum function (eggfunction) be complete.In addition, can be with estradiol and metabolin thereof risk determinative as breast cancer.Estradiol level raises to be increased relevant with the risk that suffers from breast cancer.Yet estradiol level is low to be increased relevant with the spinal fracture risk.FDA Food and Drug Administration's approved uses the medicine Tamoxifen to prevent breast cancer in some patient.Yet Tamoxifen reduces the serum estradiol level can increase the spine fracture incidence.Therefore, need the doctor can often monitor patient's estradiol level, thereby guaranteeing that estradiol level maintains can reduce mammary cancer risk but can not increase in a certain scope of spine fracture risk.

Also need frequently to monitor the therapeutic agent in patient's body.Therapeutic agent comprises, for example immunosuppressive drug, for example cyclosporin, tacrolimus (FK-506), rapamycin and Mycophenolic Acid.The therapeutic index of immunodepressant is narrow, the difference degree height of bioavilability between the patient and in patient's body.The sample that uses immunodepressant to need to monitor the patient carefully, continually prevents the detrimental effect that the required immunosupress level of graft rejection avoids undue immunosupress to cause simultaneously with balance, and for example toxicity, infection and cancer stricken risk increase.

Detect micromolecule, for example estradiol is often difficult, be not only because its concentration may be low in the sample, and also be because of the suitable agent for capturing limited amount that can be used in conjunction with the micromolecule analyte.These limitation often cause and are difficult to design or improve existing test, for example to detecting the enough sensitive and special enzyme linked immunosorbent assay (ELISA) of low-level micromolecule analyte.

The analyte that detects in patient's sample need obtain sample doctor's office or clinic usually, and the sample that passes on performs an analysis.According to different analytes, analyze sustainable one day to several weeks.Analysis result is delivered to the doctor place, and it is adjusted therapeutic scheme with this information as required and gets in touch with the new therapeutic scheme of passing on the patient.The hysteresis of analytic sample makes the doctor be difficult to accurately formulate suitable therapeutic scheme.

Need to be used to detect the micromolecule analyte more easily, detect the improvement test of analytes in low concentration.In addition, need to improve, comprise the detection of micromolecule analyte, thereby the curative effect of can (by the patient) tailoring administration of drugs scheme keeping the individual patient Chinese traditional medicine reduces harmful side effect simultaneously analyte.In addition, need when nursing, the method and apparatus of detection of biological and/or clinical analyte of interest can be used for, thereby the hysteresis that obtains between sample and the acquisition test findings can be reduced.

The key metrics of competition experiments is the amount of analyte that is accumulated in the unit interval on the sensor.For obtaining good performance, cumulative speed (and the momentary signal (signal transient) that produces) need be faster than sensor drift speed.Another key performance metrics of analyte detection system is the degree of priority of the analytes of interest analytes on the systematic collection sensor surface.Because many biological samples contain external background component (for example, other protein, cell, nucleic acid, dust), need prevent the required detection of these background components interference.Therefore, analyte optionally can be attracted to sensor and make the conveyer method that interference background component passes through useful certainly.The selective binding of this method of coupling and analyte (for example, antibody, complementary dna chain etc.) and sensor surface can detect the analyte content in the sample that contains a large amount of external background components highly delicately.

Propose the method that various raising analytes are transported to sensor surface, comprised filtration, novel fluid geometry (flow geometries), sound field, electric field (time become and static) and magnetic field.

Used acoustically-driven (acoustic excitation) that cell is drawn to a node (field node), but single substance transportation is extremely surperficial with this technology difficulty.

Use electric field (electrophoresis and dielectrophoresis) to improve transhipment, but can not generally be applicable to all types of analytes and sample.They are more effective for bigger analyte (for example, cell) usually.In addition, given kind is different with microorganism electrical characteristics in the bacterial strain, thereby is difficult to the performance of prognoses system under all scheduled operation conditions.Sometimes the ionic strength that needs to change sample improves the performance of transhipment.This requirement conflicts mutually with the best combination or the wash conditions of test.Electric field is possibility consumed energy and electric conduction of heating fluid (for example, the 0.01M phosphate buffer solution) also, because heating can destroy biological analyte, this is disadvantageous.

Immunomagnetic isolation (IMS) method is the method for analyte in the sample separation known in the art.

Summary of the invention

The present invention relates to whether exist in the test sample method of one or more analytes.In one embodiment, this method comprises to be introduced a plurality of magnetic-particles that are coated with agent for capturing in the fluid chamber (fluid chamber), and wherein the acoustical device that combines first analyte is equipped with at least one surface of this fluid chamber.A plurality of magnetic-particles can contact with sample earlier, then these particles are introduced in this chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Thereby can near this acoustical device, produce magnetic flux and guide in described a plurality of magnetic-particles at least one into described surface.Thereby whether the signal output of monitoring described acoustical device generation can exist one or more analytes in the test sample.

In another embodiment, whether exist the method for one or more analytes to comprise that described particle is coated with first agent for capturing of energy bound analyte with the step of a plurality of particles and competition molecule introducing fluid chamber in the test sample.In some embodiments, these particles are magnetic-particles.Bag is equipped with by the acoustical device of energy in conjunction with second agent for capturing of competition molecule at least one surface of this fluid chamber.Thereby can near this acoustical device, produce magnetic flux and guide in described a plurality of magnetic-particles at least one into described surface, can whether have one or more analytes in the test sample thereby monitor the signal output that described acoustical device produces.A plurality of magnetic-particles can contact with the competition molecule with sample earlier, then these particles are introduced in this chamber, perhaps can earlier sample and competition molecule be introduced fluid chamber, introduce particle then or simultaneously.Thereby monitor the signal output energy check and analysis thing that described acoustical device produces.

The present invention also relates to determine whether to adjust the method for individual dosage.This method comprises the level of one or more analytes in the test sample.In one embodiment, the method for one or more analyte levels comprises a plurality of magnetic-particles that are coated with agent for capturing is introduced fluid chamber in the test sample, and wherein the above-mentioned acoustical device that combines first analyte is equipped with at least one surface of this fluid chamber.Thereby can near this acoustical device, produce magnetic flux and guide in described a plurality of magnetic-particles at least one into described surface.Thereby the signal of monitoring described acoustical device generation is exported the level of one or more analytes in the energy test sample.Whether the decision of the level of one or more analytes adjusts drug dose per sample.

In also having another embodiment, the method for the analyte that detection can combine with agent for capturing comprises introduces fluid chamber with a plurality of particles.Each magnetic-particle is coated with different analytes, and the acoustical device that combines agent for capturing is equipped with at least one surface of this fluid chamber.Thereby the signal output of monitoring above-mentioned acoustical device generation can detect the analyte that can combine with agent for capturing.A plurality of magnetic-particles can contact with sample earlier, then these particles are introduced in the fluid chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Thereby monitor the signal output energy check and analysis thing that described acoustical device produces.

The present invention relates to the method for the estradiol in the test sample.In one embodiment, this method comprises can be introduced the step of fluid chamber in conjunction with a plurality of magnetic-particles of the agent for capturing of estradiol with bag, and wherein the acoustical device that has connected estradiol is equipped with at least one surface of this fluid chamber.A plurality of magnetic-particles can contact with sample earlier, then these particles are introduced in the fluid chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Thereby the signal of monitoring described acoustical device generation is exported the estradiol in the energy test sample.

In another embodiment, this method comprises introduces the step of fluid chamber with a plurality of particles and competition molecule, and described particle is coated with can be in conjunction with first agent for capturing of estradiol.Bag is equipped with by the acoustical device of energy in conjunction with second agent for capturing of competition molecule at least one surface of this fluid chamber.A plurality of magnetic-particles can contact with sample earlier, then these particles are introduced in this chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Thereby the signal output of monitoring described acoustical device generation can detect estradiol.

The present invention relates to the method for one or more immunodepressant in the test sample.This method comprised with bag by can binding immunoassay a plurality of magnetic-particles of agent for capturing of inhibitor introduce the step of fluid chamber, wherein the acoustical device that combines described immunodepressant is equipped with at least one surface of this fluid chamber.A plurality of magnetic-particles can contact with sample earlier, then these particles are introduced in the chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Thereby the signal of monitoring described acoustical device generation is exported the immunodepressant in the energy test sample.

In another embodiment, the method that detects one or more immunodepressant comprises introduces fluid chamber with a plurality of particles and competition molecule, described particle is coated with first agent for capturing of energy binding immunoassay inhibitor, and wherein bag has been equipped with by the acoustical device of energy in conjunction with second agent for capturing of competition molecule at least one surface of this fluid chamber.A plurality of magnetic-particles can contact with sample earlier, then these particles are introduced in this chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Thereby the signal output of monitoring described acoustical device generation can detect immunodepressant.

The invention provides the improvement detection method of analyte.In some embodiments, described analyte can comprise micromolecule, includes but not limited to estradiol and curative drug.Result of the present invention is to be not difficult to measure the pharmacokinetics (parameter) of certain given medicine and to be easier to customization (for example, by doctor's customization) dosage regimen and keep drug effect and reduce adverse side effect simultaneously.In addition, can adopt detection of biological of the present invention and/or clinical analyte of interest and avoid delay (for example, sample being sent to the delay that causes away from the mechanism for testing at scene) in Division of Nursing, thereby can customize nursing (scheme) and improve the patient compliance level.Term used herein " Division of Nursing " comprises, for example doctor's office, clinic, emergency ward and the treatment mechanism's (for example, ambulance) of flowing.In one embodiment, the horizontal adjustment of medicine described in this method also comprises per sample gives individual curative drug dosage.The curative drug level can be different according to the known treatment scope of test curative drug.

The level of institute's check and analysis thing is utilized the present invention to diagnose the illness or is assessed ill risk in can be per sample.In one embodiment, described disease is a breast cancer.For example, but coupling detects method and other diagnostic test of breast cancer marker, detects individual breast cancer as mammogram.In another embodiment, can adopt the method that detects breast cancer marker to measure the possibility that individuality suffers from breast cancer.In addition, can utilize the situation of assess patient of the present invention, for example measure patient's fecundity according to estradiol level.Can utilize the present invention to monitor among the patient level of biological or clinical related substances.In addition, can adopt the present invention to measure this patient and whether be fit to accept medicine, or not be fit to include in drug research according to the level of certain given analyte among the patient.

System shown in Figure 1 100 is fit to operate individual sample in batches mode not because of relevant efficient and lower operational cost very much, and batch mode will obtain many samples before running test.In this way, the embodiments of the present invention are highly suitable for analytic sample on the individual primary.

In an embodiment of analyte detection system, analyte and magnetic-particle (for example, magnetic bead) thus in conjunction with forming analyte-particle composites.Analyte-particle composites is transported and is fixed to the senser element surface by applying magnetic field gradient.The magnetic material of magnetic field induction particle is arranged with the local magnetic line of force and is polarized.Particle is subjected to the clean power (effect) of gradient direction, thereby causes particle to the higher zone migration of field intensity.Change Distribution of Magnetic Field, analyte-particle composites inhaled from sample flow time, with them along the senser element surface distributed.Compare with magnetic-particle, the external background component in the sample (for example, cell, protein) usually magnetic susceptibility is very low, thus magnetic field can appreciable impact they.Therefore, have only this background objects mass-energy of few part and sensor surface to interact.

In one embodiment, senser element is (FPW) device of flexible Lamb wave (flexural plate wave), and it can have two with the reason that magnetic-particle well works especially.The first, the senser element surface exists magnetic-particle to cause the FPW signal to reply amplification.The combined volume and the density of analyte-particle composites are bigger, and the FPW signal of generation is replied and is higher than single analyte.The second, the sensor surface of FPW device is made of the film of normally counting micron thickness, thereby can produce bigger magnetic field and field gradient at sensor surface, because field source is placed more near sample flow.This analyte that causes catching from sample part is more.Because capture velocity and efficient are higher, may handle the sample of larger volume with less number of times.

On the one hand, the equipment of check and analysis thing comprises having the flexible board wave device that at least one fluid enters the fluid chamber of opening and defines at least a portion of at least one inside surface in this fluid chamber.This equipment also comprises the monitoring device of at least a signal output that monitoring flexible board wave device produces, be coated with to analyte have affinity agent for capturing a plurality of magnetic-particles and the magnetic-particle selectivity is attracted to first flux source of at least one inside surface of fluid chamber.

On the other hand, the post box of resonance device system comprises having the flexible board wave device that at least one fluid enters the fluid chamber of opening and defines at least one inside surface of this fluid chamber.This equipment also comprises first flux source that the magnetic-particle selectivity is attracted at least one inside surface of fluid chamber.

On the other hand, the method for check and analysis thing comprises mixes the fluid that contains analyte and is coated with a plurality of magnetic-particles that this analyte had the agent for capturing of affinity, thereby at least some magnetic-particles combine with at least some analytes.This method also comprises introduces the first fluid chamber with fluid-mixing, wherein the flexible board wave device at least one the surface with this first fluid chamber in fluid generation fluid contact (fluid communication).This method also is included in and produces first magnetic flux near this flexible board wave device, thereby with the magnetic-particle magnetic attraction of at least some combinations at least one surface to this flexible board wave device.

On the other hand, the method of check and analysis thing comprises with the first agent for capturing bag and is positioned at surperficial at least a portion of flexible board wave device in the fluid chamber, the fluid that will contain analyte is introduced this fluid chamber, thereby the agent for capturing on making some analytes and being positioned at the flexible board wave device combines.This method also comprises introduces this fluid chamber with the fluid that contains a plurality of magnetic-particles, described particle bag is by second agent for capturing, and near this flexible board wave device, produce magnetic flux, thereby with the surface of at least some magnetic-particle magnetic attraction to this flexible board wave device.

Compare with conventional test, result of the present invention is can detection level low analyte far away.Many factors have improved the detection limit of analyte.In one embodiment, the particle that the every duplicate samples of the present invention is used is less, thereby makes that the analyte density of bag quilt is higher on the particle, even the concentration of analyte is low also like this in the sample.Higher each bag that causes of the contained analyte level of each particle is higher to the affinity of sensing surface by particle.

When test method is competitive trials, be that sensing surface is when being coated with analyte, can more effectively block combining of the particle that is coated with the higher density analyte and sensing surface because each particle contain can be free less in conjunction with the capture extender molecule of sensing surface analyte.In other words, the ratio of particle and analyte is low to cause the competition between the analyte of analyte and sensing surface in the sample fiercer, thus the lower analyte of concentration in the energy test sample.Therefore, the present invention has improved the accuracy and the sensitivity of system, and is because these particles are coated with more highdensity analyte, stronger to the combination of sensing surface.

In another embodiment, the device of making according to the principle of the invention and the method for execution are particularly useful for the particle of catching low concentration, because sensor surface thin (for example being the number micron orders in one embodiment).Embodiment coupling thin surface and magnetic field gradient and magnetic-particle.Because sensor surface is thin, can respond to big magnetic field gradient in a side of this sensor surface.Magnetic field gradient can accumulate in magnetic field greatly near the sensor surface and strengthen the sucking action of sensor sheet in the face of magnetic-particle.Magnetic field gradient can accumulate in magnetic field greatly near the sensor surface and strengthen the sucking action of sensor sheet in the face of magnetic-particle, and makes other flow of material mistake in the sample.The available in this way detection limit that can improve this system than the particle that hangs down quantity, because magnetic field gradient can make it particle aggregation and analyte that combines or agent for capturing interaction near sensing surface, this depends on test form.In various embodiments, can remove magnetic field gradient, thus the particle of any non-specific binding of energy flush away.In various embodiments, can remove magnetic field gradient, thus the particle of any non-specific binding of energy flush away.

The accompanying drawing summary

When reading in conjunction with the following drawings, can more fully understand the above and other purpose of the present invention, its various features and invention itself from following description, in these accompanying drawings:

Figure 1A has shown an embodiment of the analyte detection system that makes up according to the present invention.

Figure 1B has shown another embodiment of an analyte detection system part shown in Figure 1A.

Fig. 2 shows the FPW sensor shown in Figure 1A in greater detail.

Fig. 3 has shown the general detection scheme of coupling FPW sensor and analyte-particle composites detection of biological analyte.

Fig. 4 has shown a plurality of FPW sensor signals variations and the funtcional relationship of time in the exemplary detection scheme.

Fig. 5 shows that the final signal that is detected changes and the sketch plan of the funtcional relationship of original analysis substrate concentration.

Fig. 6 is similar to Fig. 4, shown the time progress figure (time evolution plot) of a detection scheme, but usefulness is the PSA analyte.

Fig. 7 is the synoptic diagram that certain analyte is attracted to three kinds of different modes of acoustical device sensing surface.

Fig. 8 is the synoptic diagram of an embodiment of the present invention, wherein competes molecule (for example, analyte) and is incorporated into sensing surface.

Fig. 9 is the synoptic diagram of an embodiment of the present invention, and wherein competing molecule is the analytes of interest analytes that links to each other with label, and the sensing surface of acoustical device is coated with can be in conjunction with the agent for capturing of this label.

Figure 10 is the synoptic diagram of an embodiment of the present invention, wherein compete molecule comprise carrier and with carrier-bound two or more interested analyte molecules, the sensing surface of acoustical device is coated with can be in conjunction with the agent for capturing of this analytes of interest analytes.

Figure 11 has shown that the signal of a plurality of FPW sensors changes and the funtcional relationship of PSA concentration.

Figure 12 shown the signal of a plurality of FPW sensors change with serum in the funtcional relationship of estradiol concentration.

Figure 13 is the dose-effect curve of FK-506 in the damping fluid.

Figure 14 shown the signal of a plurality of FPW sensors change with damping fluid in the funtcional relationship of FK-506 concentration.

Figure 15 is the dose-effect curve of FK-506 in the serum.

Figure 16 shown the signal of a plurality of FPW sensors change with serum in the funtcional relationship of FK-506 concentration.

Preferred implementation is described

The present invention relates to utilize acoustical device check and analysis thing, comprise the method for micromolecule analyte.Described micromolecule analyte comprises one or only comprise the discernible binding site of several agent for capturing usually.Because micromolecule has less several or only have a binding site, an embodiment of the invention adopt competitive in conjunction with detecting and/or the quantitative measurement analyte.When analyte contains the binding site that can be discerned by two or more agent for capturing, can adopt for example sandwich test, detect this analyte with two kinds of different agent for capturing.

Clinically, endogenous estradiol increases popular relevant with women breast cancer.For example, confirmed already that the women that Mammography breast density (clinical manifestation that breast tissue estrogen is excessively synthetic) increases had the high risk (Boyd, N.F. etc., N.Engl.J.Med.347:886-94 (2002)) that breast cancer takes place.In addition, confirmed already that the serum estradiol level was in the risk that the women of high quartile (upper quartile) suffers from breast cancer and is higher than the postmenopausal women (Cauley that the serum estradiol level is in normal lower quartile (lower quartile), J.A. etc., Ann.Intern.Med.130 (4 part 1): 270-7 (1999)).Therefore, can utilize the method that detects estradiol to detect breast cancer.In addition, the inventive method can with other diagnostic test, coupling detects individual breast cancer as mammogram.In another embodiment, can utilize the method that detects estradiol to measure the possibility that individuality suffers from breast cancer.

Can utilize the method assessment or the individual therapeutic scheme of exploitation that detect estradiol, or whether measure certain individuality suitable or qualified for certain concrete therapy, treatment or clinical research.For example, the individual candidate target that whether can be used as with the Tamoxifen treatment of available this method assessment.In another embodiment, available estradiol level monitoring patient's HRT comes the benefit of balance hormone replacement and the risk that estradiol level raises.

Generally, the check and analysis thing is based on the ability that the particle that is coated with agent for capturing (being also referred to as first agent for capturing at this paper) changes the acoustical device resonant frequency.This agent for capturing can bound analyte.In one embodiment, a plurality of particles that are coated with agent for capturing with the sensing surface of acoustical device are contacted with competition in the presence of the molecule at sample.Sensing surface can be the part of fluid chamber described herein or passage.In addition, bag can be contacted with sensing surface by static mode by particle, for example can be introduced in chamber and incubation regular hour by particle bag.In another embodiment, bag can be contacted with sensing surface by non-static mode by particle, for example makes bag be crossed fluid chamber or passage by grain flow.

As described herein, be surprised to find the analyte that the particle that uses low concentration can detect low concentration.This discovery is unexpected, because traditionally, relating to the test of catching by particle will be with a large amount of particles to improve capturing efficiency as far as possible.

In competitive combination of the present invention, can in conjunction with the consumption of the particle of acoustical device sensing surface usually and in the sample content of analytes of interest analytes be inversely proportional to.The level of analyte is high more in the sample, then the agent for capturing on the particle by analyte in the sample occupy many more, can be used on the particle in conjunction with the competition molecule agent for capturing few more, thereby have influence on and interactional number of particles of acoustical device sensing surface or quantity.As described below, the competition molecule can exist in solution, and maybe can be incorporated into the sensing surface of acoustical device.In a preferred embodiment, described particle is a magnetic, and magnetic flux puts near the acoustical device with at least one is drawn onto sensing surface in a plurality of magnetic-particles.

In one embodiment, the method that detects one or more analytes comprises introduces fluid chamber with a plurality of particles that are coated with the agent for capturing of energy bound analyte, and wherein the acoustical device that has connected the energy bound analyte is equipped with at least one surface of this fluid chamber.This mode is referred to herein as sandwich mode.A plurality of particles are contacted with sample, again these particles are introduced fluid chamber, perhaps can earlier sample be introduced fluid chamber, introduce particle then or simultaneously.Monitor the signal output of described acoustical device, thus one or more analytes in the energy test sample.

Test method

Although do not want to be confined to theory, be example with the micromolecule analyte, when test method is competitive trials, when promptly sensing surface is coated with analyte, utilize the lower particle of less particle or concentration can make the particle bag by more highdensity analyte.Can more effectively block be coated with density more the particle of high analyte thing combine with sensing surface because each particle contain can be free less in conjunction with the capture extender molecule of sensing surface analyte.In other words, the ratio of particle and analyte is low to cause the competition between the analyte of analyte and sensing surface in the sample better, thus the lower analyte of concentration in the energy test sample.The used concentration of particle can be about 1 * 10 ²-Yue 1 * 10 ⁷/ mL.In another embodiment, the concentration of particle is about 5 * 10 ³-Yue 5 * 10 ⁵/ mL.Fig. 7 has shown three kinds of embodiments of the sensing surface bag analyte of acoustical device.In figure B, analyte 10 directly combines with surface 12 by chemical joint.In figure A and C, analyte combines indirectly with the surface.In figure A, surface 12 is coated with in conjunction with the first right member 32.This that has connected one or more analyte molecules 10 can be combined first right member's combination by this of this surface with bag in conjunction with the second right member 22.In figure C, surface 12 is coated with in conjunction with the first right member.This can lip-deeply should use the agent for capturing 16 in conjunction with right first member, 32 marks can combine second right member's combination with this in conjunction with to first member's combination with being connected in conjunction with the second right member 22.The competition molecule 24 that contains the carrier that has connected one or more analyte molecules can combine with agent for capturing 16.

As shown in Figure 8, in one embodiment, the competition molecule comprises the analytes of interest analytes 10 (figure A) that combines with the sensing surface 12 of acoustical device.The particle 14 that is coated with agent for capturing 16 contacts (figure B) with the sample that contains analyte 18.The agent for capturing 20 that does not combine with the analyte of sample but combine with particle can freedom combine (figure C) with the analyte of acoustical device surface combination.As scheme shown in C and the D, the level of analyte is high more in the sample, and the particle that combines with the acoustical device sensing surface is few more, because more agent for capturing is occupied by the analyte of sample on the particle.The signal that the monitoring acoustical device produces is exported quantity and/or the number of measuring with the particle of surface combination, thereby can whether have one or more analytes in the test sample.In addition, but whether there are analyte or its quantity in the working sample, for example by comparison signal and contrast.As described herein, in another embodiment, described particle can be a magnetic-particle.After each particle introduced fluid chamber, near acoustical device, produce magnetic flux, thereby in a plurality of magnetic-particles at least one is drawn onto sensing surface (for example, being similar to Figure 1A, 1B and 2 described).

As shown in Figure 9, in another embodiment, the sensing surface 12 of acoustical device is coated with can be in conjunction with the agent for capturing 22 (being also referred to as second agent for capturing in this article) of competition molecule 24.The competition molecule can be that analytes of interest analytes 10, the second agent for capturing that for example are connected with label 26 can be in conjunction with this label.As shown in Figure 9, the level of analyte is high more in the sample, and the particle 14 that combines with the sensing surface of acoustical device is few more, because more agent for capturing 16 is occupied by the analyte of sample on the particle.Because second agent for capturing can have only when particle has combined competition and divide the period of the day from 11 p.m. to 1 a.m in conjunction with the label segment of competition molecule, bag is just combined with the sensing surface of acoustical device by particle.

As shown in figure 10, in another embodiment, competition molecule 24 can be that two or more analytes of interest analytes molecules that for example combine with carrier 26 or part 10, the second agent for capturing 40 can be in conjunction with this analytes.As shown in figure 10, second agent for capturing can be incorporated into the acoustical device sensing surface indirectly.Being incorporated into the surface can be identical agent for capturing with the agent for capturing that is incorporated into particle.The particle 14 that is coated with agent for capturing 16 with contain the sample of analyte 10 and contact (scheming B) with competition molecule 24.The agent for capturing that is incorporated into particle but do not combine with sample analytes can be free in conjunction with the competition molecule, this competition molecule and then combine (scheming C) with the agent for capturing that is incorporated into sensing surface.As scheme shown in the D, the analyte level that exists in the sample is high more, and the particle that combines with the sensing surface of acoustical device is few more, because the more agent for capturing on the particle are occupied by analyte in the sample.When analyte is when only containing the micromolecule of 1 certain given epi-position of copy or binding site, to have only particle to combine the competition molecule, wrap to be combined with the acoustical device sensing surface by particle.

Can utilize the present invention to screen one or more interested micromolecule.In this embodiment, a plurality of particles are introduced fluid chamber.Described a plurality of particle comprises the particle that is coated with different analytes.The acoustical device that combines agent for capturing is equipped with at least one surface of this fluid chamber, and wherein said agent for capturing can be in conjunction with interested analyte.Monitor the signal output that described acoustical device produces, thereby detect the analyte that can combine with agent for capturing.

Can adopt the concentration or the level that whether have analyte and/or determination and analysis thing in the inventive method test sample.The concentration that adopts the inventive method to detect can be absolute concentration or relative concentration.For example, concentration can be the relative value of the reference analyte concentration that exists in the same humoral sample.Can obtain this concentration to the similar data that obtain at different time by these data relatively, whether significant change take place thereby measure actual concentrations.In any embodiment as herein described, the reading that this method provides (read-out) can be positive reading or negative reading.In other embodiments, if detection is some threshold level of analyte, this method can provide positive reading.

For each embodiment as herein described, can make many variations and do not depart from the scope of the present invention following method.

Sample

Be applicable to that sample of the present invention comprises that suspection contains any material of analyte.Can directly obtain available sample from originating, perhaps can modify the gained sample according to one or more steps.Sample can be derived from any biological origin, for example physiology liquid (for example, blood, saliva, phlegm, blood plasma, serum, eye lens liquid (ocularlens fluid), cerebrospinal fluid, sweat, urine, milk, ascites, synovial membrane liquid, peritoneal fluid, amniotic fluid etc.) and ight soil.Available biology swab, for example nose or procto swab collected specimens.In addition, sample can be biological biopsy material.Sample can derive from people, primate, animal, bird or other suitable source.

Pretreatment sample for example prepares blood plasma, dilutes the liquid of thickness etc. from blood before use.Also can filter, distill, extraction or concentrating sample.For example, some small-molecule drugs can infiltrate haemocyte and with blood in protein bound (for example, FK506).Can extract micromolecule by adding protein precipitant and lysis agent.For example, the blood that zinc sulfate, polyglycol and methanol mixture can be added centrifugal back wash-out and solvent are mutually.Another kind of preparation method adds the potpourri that contains protein digestibility enzyme and washing agent destroying protein and cell, thereby discharges micromolecule.After centrifugal, the wash-out liquid phase is also analyzed to measure the micromolecule concentration in the primary sample.Also can handle sample some activity (material) with possibility interference analysis thing or testing process in deactivation or the modification sample.For example, can will separate compound antagonist (decomplexing antagonist) add sample so that analyte with possible in conjunction with agent for capturing and/or can disturb other molecular dissociation of agent for capturing bound analyte.This antagonist can be, for example the steroidal antagonist.With the estradiol detection is example, can add danazol and handle sample, and estradiol and sex hormone binding globulin matter are dissociated.

For carrying out environment or food test, can use other fluid sample except that physiology liquid, for example water, food etc.In addition, suspect that the solid matter that contains analyte can be used as specimen.Can modify solid test sample (for example, homogenate, extraction or curing) forms liquid medium or discharges analyte.

Sample volume may be as few as 10 μ L or as many as 250mL.In one embodiment, sample volume may be as few as 50 μ L or as many as 5mL.In one embodiment, sample volume is the about 5mL of about 1-.

In one embodiment, an available following post box moves a duplicate samples.In addition, for test contains a group objects of one or more analytes, a duplicate samples can be divided into two equal portions or many equal portions.Can test the different analytes in each sample aliquot, for example want test analyte to utilize different post boxes for every kind.In this way, can individuality be basis when the patient obtains (that is, from) rather than with batch mode, for example, thereby the accumulation multiple sample is simultaneously or with an instrument analytic sample in service.

Agent for capturing

The used suitable agent for capturing of the present invention comprises can be in conjunction with any molecule of analytes of interest analytes.Term " agent for capturing " comprises can be in conjunction with the molecule or the polymolecular compound of analytes of interest analytes.Agent for capturing preferably in specific mode basically in conjunction with their binding partners.Preferred dissociation constant (KD) is less than about 10 ^-6Agent for capturing.Agent for capturing also can be, for example polypeptide, nucleic acid, carbohydrates, nucleoprotein, glycoprotein, glycolipid and lipoprotein.Antibody or antibody fragment extremely are suitable as agent for capturing.Can commercialization buy the standard method preparation that maybe can adopt generation antibody in conjunction with the antibody of selected analyte.

Antigen also can be used as agent for capturing, because they can binding antibody.The acceptor of binding partner is another example of possible agent for capturing.Should know, protein catch agent be not limited to by noncovalent interaction only with the interactional material of their binding partners.Agent for capturing also can be chosen the protein that is covalently attached to its combination wantonly.For example, agent for capturing can be crosslinked in its binding partners with optical excitation in conjunction with the back.

Term " antibody " comprises any immunoglobulin (Ig) that produces no matter natural generation or synthetic wholly or in part.This term also comprises the antibody derivatives that keeps with the analytes of interest analytes binding ability.This term also comprises any protein that contains with the binding structural domain of certain immune globulin binding structural domain homology or most of homology.These eggs can derive from natural origin, synthetic generation wholly or in part from matter.Antibody can be monoclonal or polyclonal.Antibody can be any immunoglobulin like protein, comprises IgG, IgM, IgA, IgD and IgE.When known analyte can be when certain carrier protein combines, described antibody can be special to the carrier combining form of the free form of this analyte or this analyte.Can commercialization buy or adopt the known method preparation that produces antibody in conjunction with the antibody of selected analyte.

Term antibody also comprises antibody fragment.Term " antibody fragment " refers to any derivant of any antibody less than total length.Antibody fragment has preferably kept the ability in conjunction with analytes of interest analytes at least.The example of antibody fragment includes but not limited to: Fab, Fab ', F (ab ') ₂, scFv, Fv, dsFv double antibody (diabody) and Fd fragment.Can prepare antibody fragment in any way.For example, can prepare antibody fragment, or produce antibody fragment with the genetic recombination of coded portion antibody sequence by enzymatic or the complete antibody of chemical method fragmentation.Perhaps, can synthesize the generation antibody fragment wholly or in part.It is single chain antibody fragments that antibody fragment can be chosen wantonly.Perhaps, this fragment can comprise many chains that link together by (for example) disulfide bond.It is the polymolecular compound that this fragment also can be chosen wantonly.The functional antibodies fragment contains usually at least about 50 amino acid, more common containing at least about 200 amino acid.

Strand Fv (scFv) only contains by the peptide linker light chain (V that links to each other of covalency each other _L) variable region and heavy chain (V _H) recombinant antibody fragment that constitutes of variable region.V _LOr V _HCan be NH ₂-end structure territory.The length of peptide linker can be different with composition, as long as described two variable regions do not have serious spatial interference through bridging.Joint is usually by the glycocoll that is inserted with some glutamic acid or lysine residue with the serine residue extension constitutes and easily dissolving." double antibody " is the scFv of dimerization.The peptide linker that contains in the composition of double antibody is shorter than most of scFv usually, and their demonstrations preferentially are combined into dimer." Fv " fragment is only to contain a V who links together by noncovalent interaction _HDomain and a V _LThe antibody fragment that domain constitutes.Term used herein " dsFv " refers to contain engineered intermolecular disulfide bond and stablizes V _H-V _LThe Fv of pairing." F (ab ') ₂" fragment is when being equal to pH 4.0-4.5 basically, the antibody fragment that obtains with pepsin digestion immunoglobulin (Ig) (generally being IgG).Can recombinate and produce this fragment." Fab " fragment is to be equal to basically by reduction F (ab ') ₂The antibody fragment that connects the one or more disulfide bond acquisition of two heavy chains in the fragment.Can recombinate and produce Fab ' fragment." Fab " fragment is to be equal to the antibody fragment that obtains with pepsin digestion immunoglobulin (Ig) (generally being IgG) basically.Can recombinate and produce the Fab fragment.The heavy chain section of Fab fragment is Fd.

Suitable polypeptide agent for capturing in fact also comprises can be in conjunction with analytes of interest analytes or micromolecule, any peptide, polypeptide or the protein of for example little organic molecule.In one embodiment, agent for capturing is can be in conjunction with the antibody of analytes of interest analytes.In preferred embodiment, agent for capturing can be in conjunction with micromolecule.For example, can commercialization buy, adopt recombination method, adopt synthesis preparation method or obtain suitable polypeptide agent for capturing from the natural origin purifying.Polypeptide comprises, for example cell surface and soluble receptor body protein, for example protein that lymphocytic cell surface acceptor, steroid receptor, nucleoprotein, signal transducers, transcription factor, allosteric enzymes inhibitor, clotting factor, enzyme (as proteinase and thymidylate synthetase, serine/threonine kinase, threonine kinase, phosphatase, bacterial enzyme, fungal enzyme and viral enzyme), DNA and/or RNA are synthetic or degraded is relevant etc.As will be detailed later, when using multiple agent for capturing, agent for capturing can be, for example isotype each other.

Agent for capturing also can be nucleic acid, for example RNA or DNA or peptide nucleic acid.In one embodiment, nucleic acid or peptide nucleic acid can be hybridized with nucleic acid or peptide nucleic acid analyte.In addition, agent for capturing can be fit, a kind of can be in conjunction with the nucleic acid of non-nucleotide analyte (for example, protein, little organic molecule or inorganic molecule)." fit " used herein can be RNA or the DNA chain that the nucleotide by natural generation or modification constitutes.

Suitable agent for capturing also comprises in conjunction with right member.Suitable combination is to comprising the derivant (for example, Streptavidin and neutral Avidin (neutravidin)) of biological example element and Avidin or biotin and Avidin.

Agent for capturing can be incorporated on following surface or the pearl, or adopts standard techniques such as connecting polypeptide, nucleic acid to be incorporated into the surface.

Analyte

Term used herein " analyte " refers to, for example the molecular structure discerned of agent for capturing.For example, term " analyte " refers to the epi-position that antibody is discerned, perhaps can comprise part and receptor binding moiety.Term " analyte " also comprises the big molecule that contains molecular structure that agent for capturing is discerned.Analyte can be the part of cell, for example cell surface protein.(for example, selecting in advance) analytes of interest analytes that analyte can the person of being to use be selected.Can select with the ability that agent for capturing interested combines according to analyte, for example in the micromolecule library, screen.

As described herein, can utilize the present invention to detect one or more analytes in the set of analytes.This set of analytes can comprise one or more analytes that available competitive form described herein detects.This set of analytes can comprise one or more analytes that available sandwich test method described herein detects.In one embodiment, detect each analyte with following post box respectively.For testing a group objects of one or more analytes, a duplicate samples can be divided into two equal portions or many sample aliquot.Can test the different analytes of each sample aliquot, for example utilize different post boxes for every kind of analyte being tested.In this way, can test many different analytes of group and need not to obtain multiple sample and/or detect different analytes with dissimilar equipment.

In one embodiment, interested analyte is a micromolecule.Micromolecule comprises that molecular weight grades is about 1000g/mol or lower organic or inorganic molecule.The micromolecule analyte contains one or have only several binding sites usually.Because micromolecule has several or has only a binding site, the present invention adopts competitive in conjunction with detecting and/or quantitative measurement micromolecule analyte.

Yet, should know that available sandwich test form detects some micromolecule.For example, can detect rapamycin with being fixed on a kind of agent for capturing of sensor surface or the another kind of agent for capturing on protein interaction companion and the magnetic head or protein companion by sandwich immunoassay.Be responsible for two kinds of protein in conjunction with rapamycin and be 12-kD FK506 in conjunction with albumen (FKBP) be called the 100-amino acid structure territory of the rapamycin mammal target (mTOR) of FKBP-rapamycin binding structural domain (FRB).Having confirmed is not already having in the presence of the rapamycin, and FKBP and FRB do not have obvious affinity each other.

Micromolecule can comprise, for example steroids, lipid, carbohydrates, peptide and heterogeneous ring compound (for example, alkali comprises co-factor, as FAD and NADH).Analyte (for example, micromolecule) can be the part in little organic molecule library, comprise aldehyde, ketone, oxime, hydrazone, semicarbazones, carbazone, primary amine, secondary amine, tertiary amine, the hydrazine that N-replaces, hydrazides, alcohol, ether, mercaptan, thioether, thioesters, disulfide, carboxylic acid, ester, acid amides, urea, carbamate, carbonic ester, ketal, thioketones (thioketal), acetal, thioacetal (thioacetal), virtue halogen, the sulfonic acid aromatic ester, alkane halogen, sulfonic acid alkane ester, aromatic compounds, heterogeneous ring compound, aniline, alkene, alkynes, glycol, amino alcohol,  azoles alkane,  azoles quinoline, thiazolidine, thiazoline, enamine, sulfanilamide (SN), epoxide, aziridine, isocyanates, sulfonic acid chloride, diazo-compounds and/or acyl chlorides, preferred aldehydes, ketone, primary amine, secondary amine, alcohol, thioesters, disulfide, carboxylic acid, acetal, aniline, glycol, amino alcohol and/or epoxide, most preferably aldehyde, ketone, primary amine, secondary amine and/or disulfide and their combination.

In an embodiment, analyte is an estradiol.Term " estradiol " comprises estradiol and all detectable estradiol metabolites.Therefore, term " estradiol " can comprise oestrone-3-glucosiduronic acid (E3G), estradiol-3-glucosiduronic acid, estradiol-17-glucosiduronic acid, estriol-3-glucosiduronic acid, estriol-16-glucosiduronic acid and oestrone-3-sulfuric ester.

In one embodiment, can utilize estradiol level to measure patient's ovary deposit state and/or prediction patient's fecundity.Available the present invention detects a part or a set of analytes is measured ovary deposit state or fertility state.The analyte group of objects can comprise, for example estradiol and FSH.Forecasting Methodology known in the art, Buyalos etc. for example, Fertil Steril 68:272-277, described in 1997, its content is included this paper in as a reference in full.

Analyte of the present invention also can be biological analyte, for example polypeptide, nucleic acid, carbohydrates, nucleoprotein, glycopeptide or glycolipid.Useful analyte comprises, for example enzyme, steroids, hormone, transcription factor, growth factor, immunoglobulin (Ig), steroid receptor, nucleoprotein, signal transduction component, allosteric enzyme correctives etc.For example, can commercialization buy, recombinate, synthetic or obtain interested analyte from the natural origin purifying.In preferred embodiment, interested analyte is relevant with concrete human disease or illness.Suitable growth factor comprises: cell factor, hematopoietin/EPO for example, granular leukocyte colony costimulatory receptor, the granular leukocyte macrophage colony costimulatory receptor, TPO (TPO), IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12, growth hormone, prolactin, human placental lactogen (LPL), CNTF and octostatin.Suitable steroids includes but not limited to: estradiol, progesterone, testosterone and their derivant.Other suitable analyte comprises, for example insulin, type-1 insulin like growth factor (IGF-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), placenta growth factor (PLGF), TGF-α and TGF-β, other hormone and acceptor, for example bone morphogenetic factor, follicle-stimulating hormone (FSH) (FSH) and corpus luteum hormone (LH), the necrosis factor (TNF), antiapoptotic factors-1 and-2 (AP-1 and AP-2) and mdm2.Biological analyte also comprises the cell analysis thing, and it comprises that for example suitable detectable label is as surface receptor.

In one embodiment, analyte is a breast cancer marker.Breast cancer marker comprises, for example above-mentioned estradiol and metabolin thereof.In addition, breast cancer marker includes but not limited to authorize the US6 of Watkins etc., the protein of describing in 936,424, and the content of this piece patent is included this paper in as a reference in full.

Interested analyte can also be a curative drug, and it can be used for detecting the levels of drugs in patient's sample, for example is the drug therapy purpose.Suitable curative drug includes but not limited to: protease inhibitors and immunodepressant.Suitable protease inhibitors comprises An Ruinawei (ageneraser), Rui Ta taste (reyataz), lexiva, Fu Shanawei (telzir), indinavir, kaletra, viracept see nelfinaivr (viracep), norvi, inverase, aortovase, tipranavir (aptivus) etc.Suitable immunodepressant comprises cyclosporin, tacrolimus (FK-506), rapamycin, Mycophenolic Acid etc.In one embodiment, adopt competitive trials to detect interested curative drug.

In another embodiment, detect curative drug with sandwich test form.As mentioned above, curative drug can or combine protein combination with carrier, and available energy detects described curative drug in conjunction with the agent for capturing of carrier by sandwich test.In another embodiment, curative drug is that biology is treated agent.The example that biology is treated agent comprises: monoclonal antibody, enzyme, hormone and other protein.Because these molecules are very big, they have a plurality of epi-positions usually and available sandwich test detects.

Usually the prescription of opening to the transplant patient is the coupling immunosuppressive drug.For example, the neurocalcin inhibitor, for example cyclosporin and tacrolimus are often prescribed with Mycophenolic Acid.In addition, can singly use rapamycin (sirolimus) or with tacrolimus and cyclosporin coupling.Therefore, a patient being carried out a series of detections assists the doctor to determine that their levels of drugs of improvement is useful.One set of analytes can comprise micromolecule and biological substance (being also referred to as biomarker in this article).One set of analytes can comprise, for example one or more prescribed treatment medicines and optional immune response mark, for example one or more inflammatory cytokines.In addition, but joint-detection curative drug and biomarker, thus can monitor infection or rejection.Can adopt similar method for other patient's illness and treatment agent.

Interested analyte can be pathogen or microorganism, for example bacterium or microbial spores, virus, parasite, prion or other pathogen or their cell membrane or surface component, for example Grain-positive peptide glycan, lipoteichoicacid and LTA and Grain-negative endotoxin, for example lipopolysaccharides.The bacterial analysis thing comprises, Shigella (Shigella sp.) for example is as Shigella dysenteriae (Shigella dysenteriae); Campylobacter (Campylobacter sp.) is as campylobacter jejuni (Campylobacter jejuni); Enterococcus spp (Enterococcus sp.) is as enterococcus faecalis (Enterococcus faecalis); Bacillus anthracis (Bacillusanthracis); Yersinia pestis (Yersinia pestis); Pertussis is won special bacterium (Bordetellapertussis); Streptococcus (Streptococcal species); Staphylococcus aureus (Staphylococcusaureus); Much's bacillus (Mycobacterium tuberculosis); Clostridium difficile (Clostridiumdifficile); Clostridium tetani (Clostridium tetani); Clostridium botulinum (Clostridium botulinum); Escherichia coli (Escherichia coli); Salmonella typhimurtum (Salmonella thyphimurim); Intestines detection of Salmonella (Salmonella enterica); Chlamydia (Chlamydia species); Spirochaeta pallida (Treponemapallidum); NEISSERIA GONORRHOEAE (Neisseria gonorrhoeae); B. burgdorferi (Borreliaburgdorferi); Comma bacillus (Vibrio cholerae); Bacterium diphtheriae (Corynebacteriumdiphtheriae) and helicobacter pylori (Helicobacter pylori).Parasite comprises, for example giardia lamblia, plasmodium and Cryptosporidium.The virus analysis thing comprises, for example rhinovirus, yellow fever virus, B group's Coxsackie virus (CB1, CB2, CB3, CB4, CB5 and CB6), canine parvovirus (CPV), herpes simplex types 1 virus (HSV1), vaccinia virus, T4-sample virus, adenovirus, influenza B virus, influenza A virus, avian influenza virus, rhinovirus, coronavirus (as SARS), human immunodeficiency virus (HIV), hepatitis virus, herpesviral, west nile virus and Ebola virus.

As mentioned above, in some embodiments, detected multiple analytes.In some embodiments, analyte has identical molecular structure.Analyte can be for example identical molecules of interest.In another embodiment, different analytes (being also referred to as first and second analytes in this article) are more macromolecular parts.Therefore, analyte can be and the big particular combination site (for example, epi-position) that molecule links to each other or it is contained.Therefore, the different analytes of detection can be the parts of different molecular.

As described below, analyte can be incorporated into surface or pearl or adopt and be incorporated into each surface in conjunction with standard techniques such as micromolecule, polypeptide, nucleic acid.In one embodiment, analyte is incorporated into the surface indirectly.For example, analyte can be by being incorporated into the surface in conjunction with first member being wrapped by the surface indirectly.Analyte and this combination to second member in conjunction with or link to each other, analyte is incorporated into this surface by this in conjunction with the interaction between the first and second right members then.Suitable combination is to comprising the derivant of biological example element and Avidin or biotin and Avidin, for example Streptavidin and neutral Avidin.

According to an embodiment of the inventive method, can adopt variously multi-form described a plurality of particle to be contacted with sample.For example, in one embodiment, a plurality of particles contact the back and introduce fluid chamber with sample.Can first concentrating sample, the particle that will contact with sample is again introduced fluid chamber.For example, can come concentrating sample by taking out the pearl in the solution and being resuspended in the liquid of smaller size smaller.

In another embodiment, earlier sample is introduced fluid chamber, introduce a plurality of particles again.In also having another embodiment, earlier a plurality of particles are introduced fluid chamber, again sample is added this fluid chamber.

As mentioned above, in another embodiment, a plurality of particles are contacted with the competition molecule with sample.Can adopt multitude of different ways that a plurality of particles are contacted with the competition molecule with sample.For example, in one embodiment, a plurality of particles are contacted with the competition molecule with sample, introduce fluid chamber again.In another embodiment, earlier sample and competition molecule are introduced fluid chamber, introduce a plurality of particles again.In also having another embodiment, earlier a plurality of particles are introduced fluid chamber, in this fluid chamber, add sample and/or competition molecule again.

As mentioned above, in an embodiment of the invention, a plurality of magnetic-particles and competition molecule are introduced fluid chamber.By magnetic-particle, acoustical device is equipped with at least one surface of fluid chamber with the first agent for capturing bag that can bound analyte, and this acoustical device is coated with can be in conjunction with second agent for capturing of competing molecule.In one embodiment, described competition molecule comprises and links to each other with label or the analyte of combination, and described second agent for capturing can be in conjunction with this label.This label can be any part of agent identification of being captured.In one embodiment, this label is in conjunction with to one of member, and this agent for capturing is that this is in conjunction with another right member.For example, label can be a biotin, and agent for capturing can be Avidin, Streptavidin or neutral Avidin.In this embodiment, adopt the connection molecule to be connected biotin and analyte, thereby form the competition molecule with the known method of biotin.Can adopt any appropriate method to use in conjunction with right second member bag by described surface.For example, can be as described below with the biotin bag by this surface, this biotinylated surface is contacted with the derivant of Avidin or Avidin.

In another embodiment, the competition molecule comprises two or more analyte molecules that link to each other with carrier, and second agent for capturing can be in conjunction with this analyte.Carrier can be can link to each other with two or more analyte molecules or any molecule of combination.Carrier can be protein, nucleic acid or other polymkeric substance.In one embodiment, carrier is an albumin, for example bovine serum albumin(BSA).In another embodiment, carrier is a horseradish peroxidase.In this embodiment, can as described below two or more analyte molecules be linked to each other with carrier, perhaps can adopt known interconnection technique to form the competition molecule.As follows, available agent for capturing bag is by the surface.

As mentioned above, available the present invention screens one or more interested micromolecule in the micromolecule library.Can form the micromolecule library type that can be used for the inventive method by technology well known in the art, perhaps can commercialization buy (being that the chemical bridge company (ChemBridge) of chembridge.com buys for example) from network address.The micromolecule library comprises combinatorial libraries.Combinatorial libraries can be to contain in a large number (generally 10 ³-10 ⁶Between) different sequence oligomers (its feature is the sequence difference of subunit usually), or the molecular library of not homotactic side chain and connecting key or the combination of different substituents compound.The various solid phases or the liquid-phase synthesis process in known preparation micromolecule library.In a method, alternately mix and the pearl that separates the continuous precursor that contains the target compound that forms the library, each step to each component from pearl in one of the plurality of reagents that brings Selection In (Furka etc., Int.J.Pept.Protein Res.37:487-493 (1991); Chen etc., J.Am.Chem.Soc.116:2661-2662 (1994); Pham etc., WO 9513538 (1995); Dillard etc., WO 9408051 (1994)).In this embodiment, each pearl only contains a kind of chemical substance.Can adopt above-mentioned known method synthesis analysis thing on each particle surface to prepare a plurality of particles.An embodiment synthetic different analytes on each particle.

In one embodiment, this method comprises that preparation is combined with a plurality of particles of different analytes.In one embodiment, a plurality of particles contain two or more sets particles, wherein respectively organize particle and are coated with different analytes.Can with not on the same group particle contact with different analytes and prepare particle, thereby make the particle in each group be coated with different analytes.Each can be organized particle then and be used for the inventive method, or earlier they be mixed, be used further to the inventive method.

Control signal/standardization

In another embodiment, acoustical device reacted to the contact sample and the output of the signal that produces and control signal relatively, or according to the control signal standardization.Can provide or obtain control signal by the user from the user.For example, control signal can the person of being to use according to concrete analysis thing, agent for capturing, concrete model or the model of used device or the numerical value that form provides.In one embodiment, obtain control signal according to many principles.For example, control signal can be to represent the signal of many concrete analysis things of user's acquisition.Representative signal can, for example before the sample test, in the test or obtain by experiment afterwards.In some embodiments, control signal be by, for example analyze the analyte of known quantity and the typical curve that obtains with the acoustical device of specific agent for capturing and specific model.

In another embodiment, obtain control signal according to purposes.For example, can obtain unique control signal during with certain acoustic device detection specific analyte and/or agent for capturing when each.Yet, not having in the presence of the sample, an embodiment can obtain control signal with identical analyte and/or agent for capturing.Can obtain control signal by second part of a plurality of particle introduced the fluid chamber that is coated with agent for capturing.The acoustical device that combines second analyte is equipped with at least one surface of this fluid chamber.Monitoring signal that described acoustical device produces exports and measures the control signal that is used for testing subsequently.

Acoustical device

Acoustical device mainly interact by the acoustics between this device and the fluid and with the fluid coupling.Typical acoustical device comprises surface acoustic wave device, flexible board wave device, lamb wave devic and cantilever device (cantilever device).Acoustical device also interact by some viscosity between this device and the fluid and with the fluid coupling, yet described coupling mainly is the acoustics coupling.Viscosity interaction device mainly interact by the viscosity between this device and the fluid and with the fluid coupling.Typical viscosity interaction device comprises quartz crystal microbalance (QCM) device, shears harmonic wave surface acoustic wave device (shear harmonic surfaceacoustic wave devices) and acoustics plate mode device (acoustic plate mode device).Term " surface acoustic wave " refers to carry in the device architecture mode of energy, rather than device how with the mode of fluid coupling.Acoustical device is fluid interactional device on the actual area of device plane.Acoustical device is to reacting outside the plane in fact and with near this device plane the motion of fluid (that is, kinetic energy, potential energy and loss mainly lie in the fluid) acoustics coupling.Viscosity interaction device mainly in the plane but not with this device plane near the motion of hydroacoustics coupling react.

Overall consideration

For relating to, the for example application of biological or chemical material in detection and the quantitative measurement fluid, coupling between acoustical device and the fluid common (occurring in) is with respect between the about 10 μ m of the about 100nm-of the planar thick of this device, and the coupling between viscosity interaction device and the fluid common (occurring in) is with respect between the about 100nm of the about 10nm-of the planar thick of this device.

Surface acoustic wave device and shearing harmonic wave surface acoustic wave device can carry energy in a similar manner in their each self-structures.Obvious and the hydroacoustics coupling of surface acoustic wave device mainly interacts and the fluid coupling by viscosity and shear the harmonic wave surface acoustic wave device.

Figure 1A shows an embodiment of analyte detection system constructed according to the invention 100.This system 100 comprises channel network 102, is used for by FPW device 104 transmission various test solutions (being also referred to as " test solution " or " fluid ").Include this paper following United States Patent (USP) and patented claim as a reference in and described the example that is suitable for use in various types of FPW devices of the present invention: United States Patent (USP) 5,129,262, United States Patent (USP) 5,189,914, United States Patent (USP) 6,688,158 B2, U.S. Patent application 10/324,685, United States Patent (USP) 5,668,303, United States Patent (USP) 5,836,203, U.S. Patent application 20040038195.

For example, United States Patent (USP) 5,129,262 have described a kind of ultrasonic sensor, and it has the thin plate material piece that is used to form blue nurse (Lamb) ripple propagation medium.Lamb wave is also referred to as plate-mode wave, only can the limited material of penetration thickness.It is the hundred times of the wavelength of the SAW that just propagating that surface acoustic wave (SAW) requires the thickness of propagation medium, compare with surface acoustic wave, it at most only is that several wavelength are so thick that Lamb wave requires propagation medium, and only is the part of the wavelength of the Lamb wave just propagated usually.The thickness of above-mentioned material sheet is not more than about 20 microns.The Lamb wave generator produces Lamb wave in the planar materials sheet, and output device produces an electric signal that is used to represent the propagation characteristic of Lamb wave when material piece is propagated.Measuring equipment is measured the characteristic of selected output electric signal.The certain physical characteristics of this planar materials sheet depends on the value of the measurand that acts on this material piece, and result, those physical characteristicss have determined along the propagation characteristic of the Lamb wave of this material piece propagation.Because the electric signal from output device has been represented propagation characteristic, so this electric signal has also been represented the value that acts on the measurand on this material piece.

United States Patent (USP) 5,129,262 described Lamb wave equipment can be used for biological sensing.Above-mentioned planar materials sheet is the coated antibody molecule in advance, makes to contain in the liquid of corresponding antigens or change with the frequency of this equipment after this liquid phase contacts in immersion.The Ag-Ab of propagation medium surface is in conjunction with making the velocity of wave of the Lamb wave in the material piece change.Velocity of wave changes makes oscillation frequency change by the delay line oscillator form of this equipment.In addition, in order to quicken the Ag-Ab combination, this material piece can be made by porous and transparent material, thereby allows the bigger surf zone of this material piece of antibody sandwich, also allows the flow of liquid that contains antigen cross this film.Other biology interacts and also can sensedly arrive, and other application can comprise that immunity test, clinical labororatory are detected, interior biomedical the monitoring and biomedical research of body.

Test solution (such as lock solution 106, sample 108 and damping fluid 110) used in the above-mentioned embodiment all is derived from holding tank container 112.Each bar channel path of holding tank 112 all has a valve 114, flows to point 116 with control fc-specific test FC solution, and this point 116 leads to the inlet 118 of FPW device 104.Test solution flows through FPW device 104 and flows out by outlet 120, and pump 122 is led in this outlet 120.Pump 122 extracts the test solution that passes through channel network 102 and pass through FPW device 104, and test solution is guided to waste canister 124.

Figure 1B shows another embodiment of analyte detection system 100.Present embodiment is packaged into a post box 103 with FPW device 104 and associated fluid chamber 160 thereof, i.e. detachable and a replace consumable components.Some embodiments can comprise fluid control spare 101 (such as stopper, obturator or baffle plate), are used to change the fluid that passes this device 104.In one embodiment, compare with the situation that does not have fluid control spare 101, fluid control spare 101 can make fluid more close sensor surface 143 when flowing through device 104.In addition, the source of input test solution is shown as an input fluid chamber 105, and this input fluid chamber 105 has an outlet 107 that is used for test solution is guided to the inlet 109 of post box 103.In some embodiments, magnetic-particle is arranged in input fluid chamber 105 at first, and the fluid that contains analyte with import fluid chamber 105 in magnetic-particle mix, be directed to FPW device 104 then and be positioned wherein post box 103.Can magnetic-particle be mixed in input fluid chamber 105 with the fluid that contains analyte with a device (such as by pump or magnetic stirrer).Figure 1B also shows the output fluid chamber 111 that has an inlet 113, is used to receive the fluid from the outlet 115 of post box 103.This output fluid chamber 111 can comprise above-mentioned one or more fluid control spares, and it can comprise one or more mechanisms of storing and/or disposing waste liquid of being used to.

In at least one embodiment, the outlet 107 of input fluid chamber 105 is constructed and arranged to allow repeatably to be connected and to disconnect with inlet 109 junctions of post box 103.Similar is that the outlet 115 of post box 103 is constructed and arranged to allow repeatably to be connected and to disconnect with inlet 113 junctions of output fluid chamber 111.In some embodiments, these junctions are constructed and arranged to be used to the instrument that connects and disconnect, need spanner or other instrument to realize connecting and disconnecting such as being threaded.In other embodiments, these junctions are constructed and arranged to allow rapidly and are easy to manually connect and disconnect, and without any need for extra instrument or annex.These need instrument all is known in the art with not needing being connected of instrument.In some embodiments, a plurality of input fluid chamber and output fluid chamber are arranged.In some embodiments, one or more inputs and/or output fluid chamber are the parts of post box 103.In addition, in some embodiments, the source of one or more magnetic flux is parts of this post box.

Fig. 2 illustrates in greater detail FPW device 104.In FPW device 104, strain energy and tension force in this device, have been produced when crooked.In some embodiments, the thickness-wavelength ratio of expectation FPW device 104 is less than 1, and in some cases much smaller than 1.Usually, the wavelength of FPW device 104 " λ " approximates the spacing of the electrode of above-mentioned staggered arrangement greatly.In one embodiment, the thickness-wavelength ratio of FPW device 104 is 2 μ m/38 μ m.In other embodiments, FPW device 104 is designed to isolate the bandwidth of specific pattern (such as from the zeroth order pattern to any pattern of higher order mode more) or a plurality of patterns relevant with this device.For example, its thickness/wavelength ratio is the 80th pattern that the FPW device 104 of 2 μ m/38 μ m will be isolated FPW device 104.Thereby FPW device 104 can be designed to by selecting specific pattern to realize above-mentioned effect at the electrode of the staggered arrangement that is deposited on this device.In one embodiment, the shape of FPW device 104 is rectangles.FPW device 104 can be circular or oval-shaped or certain other flat shape.

Usually, by using micro-fabrication technology known in the art, from silicon wafer 130, construct FPW device 104.In the above-described embodiment, chamber 132 is etched in the wafer 130 to produce film 134 thin, that poise, and this film 134 about 1.6mm are long, 0.3mm is wide and 2 μ m are thick.The thickness of entire wafer 130 is approximately 500 μ m, so the degree of depth in chamber 132 just is slightly less than the thickness of wafer 130.Shown in Fig. 2 enlarged drawing, go up the thick layer 136 of 0.5 μ m that deposit aluminum nitride (AlN) constitutes at the outside surface (promptly with chamber 132 opposite surfaces) of film 134.Two groups of staggered metal electrodes 138 of settling on the AlN layer, have been deposited.Go up the thin layer 140 (about 500 dusts) that deposited gold constitutes at the inside surface of film 134 surface of chamber 132 (promptly facing to), fix (hereinafter can describe in more detail) to promote agent for capturing.

In operating process, instrument/control electronic equipment 126 (with reference to Figure 1A) power transformation signal when at least one group of electrode 138 applies is to produce vibration in the film 134 that poises.Instrument/control electronic equipment 126 is also by receiving the vibration characteristics of monitoring film 134 at least from the sensor signal of the second group electrode 138.When liquid contacted with the chamber face 132 of film 134, the peak response of plate structure was about 15-25MHz.Instrument/control electronic equipment 126 will compare from the sensor signal and the reference signal of second group of electrode, to determine as the relative amplitude of the sensor signal of the function of frequency and the variation of phase angle.These change instrument/control electronic equipment 126 translations to detect the existence of target analyte.In some embodiments, instrument/control electronic equipment is also determined the concentration of the target analyte on the inside surface of film 134.

As mentioned above, the agent for capturing with the target analytes of interest analytes is fixed on the thin gold layer 140 of coverlay 134 inside surfaces.This surface can be wrapped by suitable connection compound.Suitable connection compound is but that commercialization is buied.In one embodiment, connect compound and comprise hereinafter described biotin PEG disulfide.In another embodiment, make the alkyl chain of mercaptan end-blocking be connected to above-mentioned gold surface, thereby but form the individual layer (SAM) that self assembles.The part of SAM chain reaction active groups (such as carboxyl) end-blocking, thus can use biochemical treatment step known in the art that agent for capturing is covalently bound to the SAM chain.The remainder of SAM chain is with reactionless reactive group (preferably having water-wet behavior) end-blocking, to stop non-specific binding (such as, the oligomer of ethylene glycol).Other finishing chemistry (method) all has description in the literature and can be used to produce catch surface.

FPW device 104 is packaged into the electrode 138 on the outside surface that allows to be electrically connected to film 134.In addition, passage block 142 is mechanically supporting FPW device 104, with the inside surface engaged test solution of permission film 134, and provides an interface so that sensor surface 143 contacts with fluid sample.Passage block 142 produces a paths (fluid chamber 160), so that allow test solution flow into, pass the inside surface of film 134, to come out from exporting 120 from input port 118.Formed sealing 144 between FPW device 104 and passage block 142, spilt from path 10 2 to prevent test solution, this path 10 2 is to form within the combination of FPW device 104 and passage block 142.Thus, passage block 142 forms fluid chamber, and wherein FPW device 104 comprises one of a plurality of inwalls.

Path 10 2 diameters that combination by FPW device 104 and passage block 142 constitutes are approximately 0.5mm.Passage block 142 can be made of various materials, comprising plastics, metal or pottery and other material.

System 100 comprises one or more fluid control spares, is used at least a fluid behaviour in the change system 100, such as flow velocity, pressure or track etc.Pump 122 shown in Figure 1A and valve 114 all are the examples of fluid control spare, and they are used for guiding and control various test solutions flowing through this device and arriving sensor surface 143 (as the execution method of testing is desired).Usually, at least a fluid behaviour of at least one near surface in the fluid chamber 160 of fluid control spare change device 104.Usually, realize this point, so that magnetic-particle is along at least a portion distribution of sensor surface 143.As mentioned above, in some embodiments, fluid control spare be pump (such as, peristaltic pump, centrifugal pump, rotary pump, electroosmotic pump).In some embodiments, this pump is positioned the entrance side of this fluid chamber, and in other embodiments, pump is positioned at the exit of fluid chamber.In some embodiments, this device is the shunt (such as stopper, obturator or baffle plate) that is provided with respect to fluid chamber, with near the stream of the fluid at least one inside surface that changes fluid chamber.

With reference to Figure 1A, single pump 122 is placed in refuse one side of FPW device 104.The suction that pump 122 produces is drawn from damping fluid 110 in each holding tank 112 of supply one side of FPW device 104 or the analyte in the sample 108.Valve 114 is placed in supply one side of device 104, and which kind of test solution any moment will guide on the sensor surface 143 in the method for testing so that be controlled at.The flow velocity of pump 122 these tests of control.

The device (such as thermoelectric (al) cooler) that is used to regulate temperature can be associated with FPW device 104 and passage block 142.Thereby this has reduced the influence of variable environmental baseline to 104 outputs of FPW device by device 104 is remained under constant relatively known temperature.In the alternative, temperature sensor is included within the system 100, for example, and as the part of FPW device 104.Based on the output of temperature sensor, in the sensor signal of adjustment of specific moment (or in a period of time), so that produce the signal that not influenced by temperature variation effects from FPW device 104.Certain hybrid combining that this adjustment can be based on mathematical model or analytical model or mathematics and analytical model realizes.

In some embodiments of system 100, in the path of test solution, comprise a filtrator with the particle of filtering specific dimensions (such as magnetic-particle and biomaterial) optionally thus prevent that them from entering fluid chamber.As example, specific method of testing can be included in the step of change filter between detection period.This will allow during the different piece of this detection the analyte and the magnetic-particle of dissimilar (being size) are guided in the fluid chamber, thereby can be detected them by system 100.

In one embodiment, its pan coating has the magnetic-particle (such as paramagnetism or superparamagnetism pearl or microballoon) of agent for capturing to mix mutually with the sample that contains analyte.After the incorporation time of regulation, obtain analyte-particle composites 146, obtain equally in conjunction with the particle 147 of non-specific material and less than particle 148 in conjunction with any material.Particle 146,147 and 148 is positioned within the sample storage groove 112.

System 100 also comprises magnetic field induction structure 150, is used for producing near film 134 magnetic flux.In Figure 1A, the source of magnetic flux is a contractile magnet 150, be disposed in usually FPW device 104 film 134 near.When magnet 150 was near film 134, magnet 150 produced significant gradient magnetic near film 134.Under the control of instrument/control electronic equipment 126, contractile magnet 150 can be from film 134 toward having bounced back segment distances, and this distance is enough near the magnetic field that reduces on sizable degree the film 134.In one embodiment, when near film 134, magnet 150 is positioned at the sensor surface 143 about 200 μ m from film 134.In another embodiment, when near this film, magnet 150 is positioned at sensor surface 143 about 50 μ m from film 134 to 100 μ m.

When magnet 150 during near film 134, magnet 150 provides the source of a magnetic flux so that magnetic-particle is drawn onto sensor surface 143 from sample.Analyte-particle composites 146 and contain the particle 147 of non-specific binding material and do not shift out from fluid sample in conjunction with the particle 148 of any material runs into sensor surface 143 up to them.Analyte combines with agent for capturing on the sensor surface 143.Thus, analyte has formed being connected between magnetic-particle and the sensor surface.Magnetic field will be contained the particle 147 of non-specific binding material and not remain on sensor surface 143 places in conjunction with the particle 148 of any material.In addition, weak adhesion can act between particle 146,147,148 and the sensor surface 143.In the washing step (hereinafter can describe in more detail) of said method, shrink the magnetic force of magnet 150 to reduce to be experienced at the particle that sensor surface 143 places have been accumulated.Increase the washing flow velocity, to remove those particles 147 and 148 that are not attached to above-mentioned surface by analyte.Because compare with analyte-particle composites 146 particle 147 that contains the non-specific binding material and with not ing in conjunction with the particle 148 of any material and sensor surface 143 combine a little less than, so make them from sensor surface 143 disengagings with lower washing flow velocity (with corresponding hydrodynamic force).Therefore, use moving magnet 150 (promptly significantly reducing the particle 146,147 and 148 magnetic force of being experienced at sensor surface 143 places), distinguish the particle 146 that contains analyte and do not contain the particle (147 and 148) of analyte with those.A kind of technology that is used to engage and shrinks magnet 150 is that it is installed in the pylon (not shown) that is activated by the camming (not shown).

Therefore the material of magnet 150, geometric configuration and determined magnetic field shape and magnetic field gradient jointly from the distance of sensor surface 143, also determined the power that analyte-particle composites 146 is experienced.High-strength permanent magnets as collapsible magnet 150 is commercially available.For example, 1mm diameter circle cylindricality NdFeB magnet can have been bought from some suppliers (such as Dexter magnetic technology company).In one embodiment, when being engaged with each other, diameter is that 1mm and length are that the NdFeB magnet 150 of 5mm is positioned within the 0.1mm of sensor surface 143.When shrinking, magnet 150 has 0.5mm at least from sensor surface 143.Because the film of FPW device 104 134 extremely thin (2 μ m) and be made of nonmagnetic substance (such as silicon, aluminium nitride or gold) is not so film 134 is significantly upset the magnetic field of sensor surface 143 these sides of device 104.As a result, as high collection efficiency is necessary, can realize high-intensity magnetic field and very big magnetic field gradient.

Sample flow rate by path 10 2 is (such as being specified by operating personnel) that is determined by the necessary residence time of good collection efficiency.Regulate sample flow rate, make average velocity on the sensor surface 143 between 1 to 5mm/s.Be about the iron oxide paramagnetic particle of 3 μ m for diameter, can realize collection efficiency near 50%.

Can use other configuration in the source 150 (being magnet) of magnetic flux.For example, can use electromagnet to substitute permanent magnet.Electromagnet comprises the pole piece of extension, magnetic fluxes can be focused near the sensor surface 143 of device 104.

Perhaps, (within the 0.1mm) can form and settle magnetisable material near sensor surface 143, and independent magnet combines so that induce magnetic field in magnetisable material with the free face of magnetisable material.The magnetic field that is induced in the above-mentioned material is used to make desired field gradient to be positioned near the sensor surface 143.Like this, according to the formation of above-mentioned material, can use bigger low-cost magnet, and can use single magnet to solve a plurality of sensors.Spendable for this purpose examples material is pure iron, high mu-metal (such as alloy 49, high nickel content iron), sna silicon steel (common siliceous 1-2%).The benefit of using this magnetisable material that combines with magnet is to simplify sensor configuration, thereby allows more low-cost manufacturing.Can use the low accuracy actuator to engage and shrink above-mentioned magnet, because this magnet only needs the catalytic oxidation ferromagnetic core or only need to shrink fully.In the above-described embodiment, magnet 150 is positioned near the sensor surface 143, needs higher levels of degree of accuracy to realize good test repeatability.Although use this method can lose some field intensity, still also may design total system and realize good capture rate (such as＞10%).

The pointed shape of field induced junction structure (such as magnet or ferromagnetic material) can be adjusted to the field gradient that strengthens and/or concentrate above-mentioned surface.(, make the one or more positions of magnetic field concentration to the sensor surface 143 because the size of FPW device 104 so can be adjusted near the part of the field induced junction structure of film 134 such as 0.3mm * 1.6mm) usually less than the magnet that forms according to a conventional method or the inductor of machining.Adjust this tip and can increase local field strength and local field gradient.For example, wedge shaped tip is well suited for current FPW device geometries.

An embodiment of system 100 comprises the second optional flux source 150a, and it is relative or partly relative with first flux source 150.The second flux source 150a expels some to adhere to the magnetic-particle of sensor surface 143.For example, its expulsion does not have the magnetic-particle 148 in conjunction with any analyte; They can not adhere to sensor surface 143 consumingly as those particles 146 of bound analyte.In some embodiments, first flux source 150 is closed or removes from sensor surface 143, then, the second flux source 150a with respect to fluid chamber above-mentioned at least one the surface and the location optionally to remove magnetic-particle.For example, the realization this point can be removed those not in conjunction with the magnetic-particle of any analyte, and therefore, they can not be attached to sensor surface 143 consumingly.This will realize and increase the similar effect of rate of flow of fluid, thereby remove the magnetic-particle 148 that does not have in conjunction with any analyte.

The distribution situation of control analysis thing-particle composites 146 on the surface 143 of device 104 just can be improved the performance of this device, because device 104 has the film 134 that poises and be not that all parts of film 134 all contribute to detectable resonance motion quality equably.For example, system 100 can construct and be arranged to make analyte-particle composites 146 along the centre 2/3rds of film 134 longitudinal axis center lines distribute and be in FPW device 104 width 1/3rd within.Consider the fluid field effect, the shape at the tip of field induced junction structure (such as magnet 150) can be so that an amplitude and field gradient constantly increase on the flow direction on the sensor film 134.That is, compare higher field and the field gradient of analyte-particle composites 146 experience in the downstream area (wherein marginal layer part degree has exhausted analyte) with the analyte-particle composites 146 in the upstream region.

Usually, system 100 can be constructed and arranged to make magnetic-particle to focus in one or more specific regions of sensor surface 143.On sensor surface 143, the response of device 104 may be uneven, and this may be that the feature of manufactured materials or the details of sensor design cause.Thus, the high sensitivity zone of device 104 can be uneven and asymmetric about the major axis and the minor axis center line of device 104.Thus, the tip of field induced junction structure can be shaped as magnetic-particle is focused in the zone of maximum sensitivity.

For given Distribution of Magnetic Field, change the more even covering that the flow velocity that passes device 104 also can be used to realize analyte-particle composites 146.For given field, magnetic-particle may fall under the situation that gravity exists the spitting image of the object that is emission by determined such and sensor surface 143 interactions of a large amount of rate of flow of fluids.Yet in this case, magnetic strength stress accounts for leading.By changing flow velocity, analyte-particle composites 146 is interacted at the diverse location place and the sensor surface 143 of streamwise.In addition,, can make flow inversion and forward more next,, transmit more particles with sensor surface 143 thus so that this accumulation is pulled back along with magnetic-particle is piled up (if they will feeler surface 143, then not expecting to occur this situation).In an embodiment of system 100, by in detection method, optionally changing flux source and, just having realized making magnetic-particle optionally along sensor surface 143 location along in the fluid mobility matter of sensor surface 143 one or more.

An embodiment of system 100 comprise a kind of device that is used to portray at least a character that is attached to or is attracted to the magnetic-particle on the sensor surface 143 (such as optics or magnetics).This device can be FPW device 104 part of the whole, and perhaps it can be the part of magnet 150, and perhaps it can be the discrete assembly that separates mutually with other assembly of system 100.This device can be used to detect the existence of above-mentioned particle, also is used for determining the parameter (for example, be attracted to size, quantity, concentration or the density of the particle of sensor surface 143) relevant with these particles.

An embodiment of system 100 comprises the sign device, thereby allows the specific components of operating personnel or computer identity system 100 or this system so that follow the tracks of the operating position of this system or assembly.The sign device can comprise symbol or image (such as bar code), identification number or other identity marking.This sign device can comprise that the passive or active block (such as RFID label, integrated circuit or other assembly known in the art) of a reality is so that provide identification information.Many this devices all are known in the art, although any sign device that is contemplated to all is operable.

Thereby Fig. 3 shows the universal test method 200 of coupling FPW device 104 and analyte-particle composites 146 detection of biological analytes.

The first step 202 of detection method 200 is to obtain and prepare analyte sample.According to the particular type of test analyte, need before test, carry out various set-up procedures earlier.For example, in order to detect the microorganism (such as the Escherichia coli of twisting in the thin beef) in the food, earlier sample is mixed with the meat soup that concentrates, again through digestion (stomach), cultivation and filtration.For the protein that detects in the blood, sample will be at first after filtration or is centrifugal and isolate serum.This paper has described the specific example of the method for testing that comprises the sample preparation process.

Next step of testing process 204 is that the paramagnetic particle of compatibility bag quilt (being pearl) is mixed mutually with off-the-shelf analyte sample.Paramagnetism or supperparamagnetic particles can have been bought from many suppliers (such as the Dynal biotech company of Norway Oslo).Typical diameter be from 50nm to 10 μ m, and this particle can obtain in advance, has been coated with the affinity reagent (such as antibody, protein and nucleic acid probe) of the various analytes of target (such as cell, protein and nucleic acid) on it.Perhaps, can buy and have various reactive chemistry groups the particle of (epoxy, carboxyl and amine), so that in conjunction with selected agent for capturing.The standard biochemical method can be used for this purpose.

The paramagnetic particle of stirred sample and interpolation (206), the mixing time amount is determined by concrete analysis thing and agent for capturing.In this process, these particles combine with analyte, make analyte be captured on these particles.In some cases, can directly test this sample this moment.But in other cases, preferably carry out separating step 208, so that in conjunction with all the other component separating of the analyte and the primary sample of particle.These separating steps 208 have reduced the interference of other biomaterial in the test.Being used to carry out the manual of this separating step or equipping automatically is (such as Dexter magnetic technology company, the Dynal biotech company) that can buy.Above-mentioned basic process uses magnet that paramagnetic particle is positioned on the surface, makes the major part of sample liquids to be sucked out.Then, remove magnet (such as the magnet 150 of Figure 1A), and add cleaning damping fluid so that particle suspend again.

In one embodiment, before testing treated analyte sample, carry out baseline step 210 earlier with FPW device 104.In baseline step 210, reference solution 106 flows through this system with flushing/sealing sensor 104, and instrument/control electronic equipment 126 starters 104 and record are from the initial baseline signal of device 104.

After baseline step 210, be sample transmitting step 212.Under the situation that engages magnet 150, the sample 108 that contains analyte-particle composites 146 flows on sensor surface 143.Analyte-particle composites 146 is collected on the sensor surface 143.After the sample 108 of prescribed volume has flow through this device 104, shrink magnet 150 so that do not have the particle 147 and 148 of bound analyte to peel off, and fluid is switched to wash solution (such as buffer solution 110) from sensor surface 143.Increase the flow velocity of wash solution, with the particle 147 that helps to remove loose combination and 148 and sample in may be attached to other material of sensor surface 143.

After sample transmitting step 212, be obtaining step 214.Reference solution 106 passes device 104 once more, and instrument/control electronic equipment 126 starters 104 are to obtain and to write down the final background signal from device 104.

By relatively initial baseline signal and final background signal (they correspond respectively to before the obtaining step 214 and the vibration performance of FPW device 104 in reference solution) afterwards, system 100 has determined the amount of analyte of being accumulated on sensor surface 143 during the transmitting step 212.Analyte-the particle composites 146 that is attached to sensor surface 143 has changed the vibration performance of FPW device 104, and the variation of vibration performance amount is corresponding to the amount of the analyte-particle composites that is attached to sensor surface 143.

Fig. 4 shows for typical detection method from the signal of a plurality of FPW devices 104 over time.Square symbol is corresponding to the device 104 of contact analysis thing; Triangle and star are corresponding to negative control (wherein not having analyte on pearl).Data represented typical detection method shown in Fig. 4 (with described Fig. 5 hereinafter), the analyte that this method is suitable for are Escherichia coli or cell analysis thing normally.

In specific test, the frequency of harmonic peak is tracked.Fig. 4 shows along with magnetic-particle 146,147 and 148 is accumulated on above-mentioned surface, and the resonance frequency of FPW device 104 is reducing.In case remove magnet 150 and some magnetic-particles are washed out, then the frequency of device 104 can increase.Finally, this system sets up final baseline, and initial baseline in the time of this final baseline can being begun with detection or contrast compares.

Fig. 5 shows the synoptic diagram of the final signal variation of the initial analyte concentration function of detected conduct.

State in the use under the situation of system 100, can use other detection method.According to the various requirement of application-specific or analyte, can cancel or add some other steps.For example, Fig. 6 shows the time-evolution figure that is used for detection method, to shown in Figure 4 similar.Yet Fig. 6 has described to be used for the detection method of PSA test, and it has proved that system 100 can detect protein.In addition, in the method, can make flow direction reverse.For example, make flow direction oppositely can be used for washing away the material of non-specific binding, perhaps more effectively use resulting sample from this device.

Another variant of this detection method comprises hocket washing step and integrating step.This can allow to use better the dynamic range of this device, and in some cases, a big chunk in the above-mentioned particle does not have bound analyte, especially when analyte concentration is low.By combination repeatedly and washing granule, also may accumulate more analysis thing-particle composites 146, improve the sensitivity of this measurement thus.

The Distribution of Magnetic Field at 143 places, change or control pick-up surface can increase the probability that the specific particle that is combined with analyte runs into sensor surface 143 during transmitting step 212.For example, if during combination, alternately change the space distribution in magnetic field, the paramagnetic particle at sensor surface 143 places is rolled.In some embodiments, by the space distribution of controlling magnetic field, operating personnel or instrument/control electronic equipment 126 can be used to control the rolling of paramagnetic particle along sensor surface 143.

As mentioned above, with the specificity that just can improve in the drawing-in system 100 of second magnetic field (i.e. second flux source) the control and the increase test of test condition.For example, in the combination or washing step of aforesaid method, apply second magnetic field to sensor surface 143 magnetic-particle of weak combination is broken away from.The intensity in second magnetic field can be adjusted on the analyte-particle composites 146 at sensor surface 143 places and produce acting force, and this acting force is lower than the adhesion of specificity bound analyte but is higher than the typical combination power of non-specific binding material.Can make in test above-mentioned steps repeat repeatedly specificity in proper order with further this detection of increase.

By in washing step, increasing (continuously or discretely) this magnetic force, monitor the response of FPW device 104 simultaneously, just can determine the relative bond strength of various analyte-particle composites 146 on sensor surface 143.Information about relative bond strength can be used for distinguishing between the different analytes of sample 108.

In other embodiments, sample can be different with device 104 interactional ad hoc fashions.In the above-described embodiment, system 100 makes sample flow cross passage so that set up contact between analyte-particle composites 146 and sensor surface 143.In the alternative variant of system 100, FPW device 104 is installed on the probe and immerses test solution at least in part, and this test solution comprises the magnetic-particle that is attached to analyte.For present embodiment, the immersion process is enough to the magnetic-particle through combination is placed near the sensor surface 143, makes to attract these particles and next detect them towards sensor surface 143.In order to obtain background signal, device 104 (or post box 103) is dipped in the reference test solution, and in some embodiments, the part of device 104 (such as film 134) is installed on the probe.In addition, have only the part of the sensor surface 143 of film 134 just to contact with the above-mentioned solution that contains the magnetic-particle that is attached to analyte.In these embodiments, immersion or controlled move of above-mentioned probe in fluid has been enough to the magnetic-particle through combination is placed near the sensor surface 143, makes to attract these particles and next detect them towards sensor surface 143.

Another alternate embodiment of system 100 relates to above-mentioned device 104 is installed to a tube interior, this pipe parts can be immersed one and hold in the hole of sample 108 and next shrink.When immersing sample, pump applies suction sample sucked in the above-mentioned pipe and to be drawn onto on the sensor surface 143 (or post box 103).Then, by pump being reversed or making the pipe emptying simply, the sample re-injection is gone in this hole.The circulation of this absorption and release sample can repeat, thereby can increase collection efficiency and therefore improve the performance of testing.

For an embodiment of said system 100, following examples have illustrated preparation and have utilized system 100 to come many steps of check and analysis thing.

Embodiment

Example I: the universal method on agent for capturing functionalization flexible board wave device surface

1. with the surface (for example, sensor surface 143) of deposition of gold, clean this gold surface 143 with for example oxygen plasma to flexible board wave device 104.

2. the ideal surfaced chemistry (characteristic) of gold surface 143 should be able to provide 1) prevent non-specific binding and 2) be positioned at the reaction active groups that is used for the covalent bond agent for capturing on this surface.The exemplary table surface chemistry (characteristic) of gold surface 143 is self-assembly individual layers (SAM) of alkanethiol.The potpourri of available two kinds of alkanethiols forms SAM; An end contains reaction active groups and is used for linking to each other with the agent for capturing covalency subsequently, and an end contains the nonreactive activity group.For example, can use the terminal C that is for this purpose ₁₁The EG of-alkanethiol ₆-OCH ₂COOH (EG6) and EG ₃The potpourri of-OH (EG3).In one embodiment, flexible board wave device 104 (being the surface 143 of device 104 specifically) contacts with alkanethiol solution, and room temperature is cultivated, for example about 16 hours.Clean the surface 143 of this flexible board wave device 104 then with ethanol, dry up with nitrogen.

3. next step comprises the surface 143 that agent for capturing or analyte is covalently attached to this flexible board wave device 104.Can adopt covalently bound agent for capturing of many methods or analyte.A kind of exemplary method comprises the biotin shank is covalently attached to SAM, makes biotinylated antibody or analyte be incorporated into flexible board wave device surface 143 by the Streptavidin articulamentum then.

Example II: adopt, method for example shown in Figure 3 detects the Escherichia coli O 157 in the thin beef of strand: H7 (Fig. 5 comprises the colibacillary data of the various concentration of representative)

1. preparation contains the Escherichia coli O 157 that concentration is higher than about 100cfu/ml: the H7 analyte sample.

2. carry out immunomagnetic isolation and concentrate this analyte sample solution.Can utilize extensive stock instrument (for example, the Bead Retriever of the Pathatrix of model micrology company (Matrix Microsciences) and Dynal biotech company (Dynal Biotech)) or manual method to carry out this immunomagnetic isolation.Exemplary manual method comprises:

A. the resuspended magnetic bead (for example, the Dynabeads company available from the Dynal biotechnology resists-Escherichia coli O 157) that is coated with Escherichia coli antibody disperses until the magnetic bead that is deposited in the test tube bottom.Microcentrifugal tube is placed (for example, Dynal MPC-S) on the magnetic sheet frame.1-20 μ L magnetic bead storing solution is sucked this test tube (selecting magnetic bead storing solution volume according to required final pearl concentration).

B. the 1mL analyte sample is added the test tube in the magnetic sheet frame, close this test tube.

C. the shelf that reverses for several times.Room temperature is cultivated solution 10-60 minute in the test tube, softly stirs in case the magnetic bead sedimentation continuously simultaneously.

Thereby the shelf that d. reverses makes magnetic bead accumulate in test tube one side for several times and forms sediment.Suitably reclaimed about 3 minutes.

E. open test tube, carefully draw and discard the liquid in sample supernatant and the test tube lid.

F. remove magnetic sheet.

G. in test tube, add 1mL lavation buffer solution (PBS-tween).Close the test tube lid, the reversing shelf makes pearl resuspended for several times.

H. repeating step d-g is twice.

I. the inclusions of utilizing vortex mixer (vortex mixer) simply to mix test tube.

3. detection Escherichia coli O 157: H7

A. use Escherichia coli O 157: the surface 143 of H7 antibody functionization (to A.3 described similar) flexible board wave device 104 with step A.4.

B. the first inlet flexible pipe is placed and contain the standard wash damping fluid (1 * PBS) the test tube that contains 0.05% polysorbas20 places the second inlet flexible pipe to contain to combine Escherichia coli O 157: the test tube of the magnetic bead of H7 (B.2 preparation).Connect the first and second inlet flexible pipes with the t-joint, thereby this two flexible pipes fluid is linked to each other, the fluid in the first inlet flexible pipe or the second inlet flexible pipe can be introduced the inlet of fluid chamber 160.Two flexible pipes are equipped with respectively that valve allows or limit fluid flows through flexible pipe separately.

First outlet hose is linked to each other with the outlet fluid of fluid chamber 160.The fluid collection of first outlet hose is gone into the waste collection bottle.

D. utilize the flexible board wave device to obtain the baseline output signal.The standard wash damping fluid is flowed into and effluent fluid chamber 160 was detected background signal in about 5 minutes with standard pump speed (for example, 200 μ L/ minutes).

E. engage first source of magnetic flux 150, the fluid that will contain then in the test tube of analyte is introduced the inlet of fluid chamber 160.Fluid is introduced the inlet of fluid chamber 160, thereby can at least one surface 143 of flexible board wave device 160, accumulate the magnetic bead that is combined with Escherichia coli O 157: H7, on this at least one surface 143, combine requirement.In one embodiment, the output frequency drift (frequency shift) when for example flexible board wave device 104 when being about 4000ppm, reaches requirement.

F. the fluid of ending to contain the analyte test tube then flows.Then the fluid of lavation buffer solution test tube is introduced material (that is, except that the 1) magnetic bead and 2 of fluid chamber 160 with the flush away non-specific binding) be combined with the material the magnetic bead of analyte).

G. remove first source of magnetic flux 150 then.

H. start automatic washing scheme to remove any magnetic bead or matrix components.

I. detect the final signal output of flexible board wave device 104 then.Background signal is compared the concentration of determining Escherichia coli O 157: H7 in the analyte sample with final signal.

EXAMPLE III: adopt, method step for example shown in Figure 3 detects the prostate specific antigen (PSA) in the human serum

1. preparation contains the human serum analyte sample by what centrifugal human blood sample obtained.

2. carry out immunomagnetic isolation and concentrate this analyte sample solution.Can utilize extensive stock instrument (for example, the Bead Retriever of the Pathatrix of model micrology company and Dynal biotech company) or manual method to carry out this immunomagnetic isolation.Exemplary manual method comprises:

A. the resuspended magnetic bead that is coated with PSA antibody (for example, available from the Dynabeads of power biotech company anti--PSA) disperse until the magnetic bead that is deposited in the test tube bottom.Microcentrifugal tube is placed (for example, Dynal MPC-S) on the magnetic sheet frame.1-20 μ L magnetic bead storing solution is sucked this test tube (selecting magnetic bead storing solution volume according to required final pearl concentration).

B. the 1mL analyte sample is added the test tube in the magnetic sheet frame, close this test tube.

C. the shelf that reverses for several times.Room temperature is cultivated solution 10-60 minute in the test tube, softly stirs in case the magnetic bead sedimentation continuously simultaneously.

Thereby the shelf that d. reverses accumulates in magnetic bead test tube one side for several times and forms sediment.Suitably reclaimed about 3 minutes.

E. open test tube, carefully draw and discard the residual liquid in sample supernatant and the test tube lid.

F. remove magnetic sheet.

G. in test tube, add 1mL lavation buffer solution (PBS-tween).Close the test tube lid, the reversing shelf makes pearl resuspended for several times.

H. repeating step d-g is twice.

I. utilize vortex mixer simply to mix the inclusions of test tube.

3. detect PSA

A. use the surface 143 of PSA antibody functionization (to A.3 described similar) flexible board wave device 104 with step A.4.

B. the first inlet flexible pipe is placed and contain the standard wash damping fluid (1 * PBS) the test tube that contains 0.05% polysorbas20 places the second inlet flexible pipe in the test tube that contains the magnetic bead (B.2 preparation) that combines PSA.Connect the first and second inlet flexible pipes with the t-joint, thereby these two flexible pipe fluids are linked to each other, the fluid in the first inlet flexible pipe or the second inlet flexible pipe can be introduced the inlet of fluid chamber 160.Two flexible pipes are equipped with respectively that valve allows or limit fluid flows through flexible pipe separately.

First outlet hose is linked to each other with the outlet fluid of fluid chamber 160.The fluid collection of first outlet hose is gone into the waste collection bottle.

D. utilize flexible board wave device 104 to obtain the baseline output signal.The standard wash damping fluid is flowed into and effluent fluid chamber 160 was detected background signal in about 5 minutes with standard pump speed (for example, 200 μ L/ minutes).

E. engage first source of magnetic flux 150, the fluid that will contain then in the analyte test tube is introduced the inlet of fluid chamber 160.Fluid is introduced the inlet of fluid chamber 160, thereby can at least one surface 143 of flexible board wave device 104, accumulate the magnetic bead that combines PSA, on this at least one surface 143, combine requirement.In one embodiment, when the output frequency drift of for example flexible board wave device 104 is about 4000ppm, reach requirement.

F. the fluid of ending to contain the analyte test tube then flows.Then the fluid of lavation buffer solution test tube is introduced material (that is, except that the 1) magnetic bead and 2 of fluid chamber 160 with the flush away non-specific binding) be combined with the material the magnetic bead of analyte).

G. remove first source of magnetic flux 150 then.

H. start automatic washing scheme to remove any magnetic bead or matrix components.

I. detect the final signal output of flexible board wave device 104 then.Background signal is compared the concentration of determining PSA in the analyte sample with final signal.

EXAMPLE IV: the PSA among the verification liquid I (PSA in Calibrator I)

In order to illustrate, utilize system shown in Figure 1A 100 to experimentize to obtain data according to the principle of the invention.Will with anti--(PN 90205 for prostate specific antigen (PSA) capture antibodies; San Diego agency of company of Scripps Laboratories (Scripps Laboratories Inc.), California) the super paramagnetic beads of the Dynal tosyl-activation of functionalization contacts with sample.Sample contains 1 * PBS (phosphate-buffered saline) and 1% bovine serum albumin(BSA) (BSA), admixture about 0pg/mL, 10pg/mL, 100pg/mL and 500pg/mL free PS A[Fitzgerald industry (the Fitzgerald Industries International of international corporation, Inc.), Concord agency, the Massachusetts].In this experiment, the pearl concentration used for sample is about 2 * 10 ⁴Pearl/mL.The soft continuous stirring of admixture sample of cultivating with pearl 1 hour.

Earlier with the 1 * PBS[sigma-Aldrich company (Sigma-Aldrich Co.) that contains 0.05% polysorbas20 (polyglycol Arlacel-20), St. Louis agency, MO] preliminary sensitization is contained in the post box (not shown) 8 flexible Lamb waves shown in Figure 2 (FPW) device 104 on the chip, (PN 90197 to use complementary anti-PSA antibody again, company of Scripps Laboratories, San Diego agency, the California) functionalization.

As acquisition as described in the U.S. Patent application (attorney piece number BIO-008) of " method and apparatus that test detects " by name submitted to by Masters etc. on May 2nd, 2006 with analyze data.Make about 17800 seconds 8 kinds of unifrequent baseline detected values (it is above-mentioned to be similar to this paper), each is corresponding to the sensor phase of following the trail of, and is relevant with reference signal separately.For each device, at first select the tracking phase place (tracking phase) in this device resonance wave band, this wave band is positioned at response amplitude to have for frequency change near the frequency of the obvious range of linearity near peak value and phase response (phase response).When following the trail of the sensor phase of each time point, find each device track frequency by following means: 1) scan the frequency range of each device and write down the phase place of each stimulating frequency with respect to the response of reference signal, 2) match stimulating frequency related function detects the phase place and 3 of each device) utilize this function to calculate track frequency corresponding to the tracking phase place of measuring in the past.

This embodiment is being operated these devices near 20MHz, sweep limit is about 20kHz.The phase characteristic of this scope is linear basically, thereby the function of match is linear.The reference signal of each device comprises the output valve that is made of network the passive electric assembly that excites (frequency) to drive simultaneously, resistor and capacitor.But select this reference network matching attenuation effect, for each device provides near the preferred phase drift that resonates.Bareline heart rate is with reference to track frequency and according to the track frequency standardization, with parts per million (ppm) (ppm) expression of selected time point.

Sensing surface 143 with each device 104 of agent for capturing functionalization.With the chip of oxygen plasma source clean Jin Bao quilt, its typical treatment conditions are about 50W, about 2 minutes.Subsequently chip was immersed in the straight alcohol 30 minutes.Then chip is moved to the ethanolic solution (catalog number (Cat.No.) 41151-0895, the pure company of polymerization (Polypure AS), Olso agency, Norway) of 0.5mM biotin PEG disulfide, cultivation is spent the night.Chip is shifted back straight alcohol solution, 30 minutes.Simply clean chip with ethanol at last, nitrogen dries up.Can change preparation condition and still can obtain analog result.

Make the neutral Avidin flow of solution of 10 μ g/ml cross the biotinylation surface 1 hour that device 104 produced, thereby with neutral Avidin (PN 31000, Pierre Si biotech company (Pierce Biotechnology, Inc.), Rockford agency, IL) bag is by this biotinylation surface.Operation instructions biotinylated antibody (PN F-6347 according to the manufacturer, hero Life Technologies, Inc. (Invitrogen Corporation), Carlsbad agency, the California), make the 5 μ g/ml solution (with 1 * PBS 0.1%BSA damping fluid dilution) of biotinylated antibody flow through neutral Avidin bag then by surface 1 hour, thus with the surperficial coupling of neutral Avidinization.

Introduce the PSA sample, produce the about 18200 seconds magnetic field of about 17900-at 143 places, proximity transducer surface simultaneously.Each sample of about 0pg/mL, 10pg/mL, 100pg/mL and 500pg/mL free PS A is provided to two kinds of different devices 104 (8 devices altogether).Sample is with about 100 μ L/ minute flows through sensor, and the gross sample running/volume of flows through sensor is 500 μ L.To wrap in this way by the super paramagnetic beads of Dynal tosyl-activation of free PS A be incorporated into the essence surface (surperficial 143) of device 104.Each pearl combines with the surface 143 of device 104 by many keys.The adhesion difference that each root of many keys produces, each and the surface combination of this device.For every duplicate samples, the association of pearl system and the feature of dissociating have been determined the concentration of analyte in this sample.

Substituted sample at about 18200 seconds with sensitization damping fluid (1 * PBS, 0.05% polysorbas20 (polyglycol Arlacel-20)).As shown in figure 11, observed each signal from 18200 seconds-18400 seconds change (seeing position 30), this variation has been represented with sample fluid and has been changed back the relevant fluid overall permanence variation of damping fluid fluid.Removed magnetic field at about 18400 seconds.(1 * PBS) flow velocity that contains 0.05% polysorbas20 is about 50 μ L/ minutes (corresponding to the sensor surface about 1.5mm/ in the 143 places flow velocity of second) to damping fluid fluid between about 18200 seconds-18400 seconds.

In subsequently 450 seconds (about 18400 seconds-18850 seconds), flow velocity increased to about 300 μ L/ minutes (flow velocity increased about 17 μ L/ minutes in per 30 seconds, totally 450 seconds, was reduced to then about 50 μ L/ minutes) with linear incremental from about 50 μ L/ minutes.In this way, by the flow velocity that improves between about 18850 seconds of about 18400-each pearl is applied controlled external action (that is the flow velocity in the present embodiment).The quantity of the pearl (with other non-specific material) that the signal indication of the part representative of these curves between about 18850 seconds of about 18400-combined with sensor surface 143 in this time changes.

Figure 11 is data and the time relation figure that obtains.The relative amplitude of the Y-axle of this figure signal output that to be device 104 produce at the tracing sensor phase place place near device 104 resonance changes (in 1,000,000/).The X-axle of this figure is the time in second.The track frequency of this figure changes with reference to the track frequency of selected time point and according to this track frequency standardization.

According to this curve about 18350 seconds numerical value and with reference to introduce the further standardization of bareline heart rate that detects before the sample fluid in 1,000,000 of track frequency/ each curve data.Therefore, compare when introducing sample, the numerical value with each data and curves during by about 18350 seconds is 100% proportional convergent-divergent.Remove the related data of (from about 18400 seconds-Yue 18850 seconds) each standardized curve during 450 seconds behind the magnetic field when being incorporated into about 18400 seconds then.

Integrate divided by the time that obtains summation by the signal level (each interval level multiply by each interval time) of each device that adds up and with final summation.This can provide the quantity of the material element (pearl) that combines with device surface 143 after time standardization, and these numerical value show the detected value of the correlation analysis substrate concentration that is each sample in this article.In this way, can change the concentration of coming the determination and analysis thing according to the material number of elements that during about 18400 seconds-Yue 18850 seconds, combines with the surface 143 of each device 104.As shown in figure 11, behind the sample drawing-in system, in about 9 minutes, detect the free PSA of about 10pg/mL.

In some embodiments, save some data points, for example observed normal deviate data point in addition in the curve.For example, compare with adjoining data point, the pseudo-data point of numerical value change above an order of magnitude left out in analysis subsequently.For example, can remove these data points in the data by operator or computer program.

EXAMPLE V: the competitive trials of serum estradiol

Adopt the standard method estradiol specificity sheep monoclonal antibody of will dissociating to be coupled to Dynal M280-tosyl activated beads.

As mentioned above, by with disulfide-PEG-biotin and golden sensor surface coupling, streptavidin (Pierce PN 31000) and biotinylated antibody make up sensor surface in the coupling then.

Make the estradiol of 10 μ g/mL BSA couplings of 1 * PBS damping fluid dilution flow through antibody surface 1 hour, thereby with the estradiol of BSA coupling, E2-6-CMO-BSA (Sigma PN E5630) is coupled to the antibody surface.Because estradiol is coupled to BSA and goes up a plurality of amidos site (30 moles of estradiol of every mole of BSA coupling), said method can provide the micromolecule surface of intermediate density in solution.

Preparation contains (charcoal striped) human serum (Texas biological products company (Texas Biologicals)), the 30%1 * PBS of 70% activated carbon treatment, the sample solution of 0.05% polysorbas20 (polyglycol Arlacel-20), wherein admixture the free estradiol (sigma) of 0pg/ml, 10pg/ml, 30pg/ml, 200pg/ml.With concentration about 2 ⁴The pearl of/ml and sample were cultivated 2 hours.Adopt conventional scheme to make each pearl flow through FPW then, obtained the result at about 0.5 hour.

In some samples, also added danazol as separating compound antagonist to separate any interference molecule (for example sex hormone binding globulin) and estradiol.The level that adds about 10-100ng/ml.

Make sample flow through FPW with 70 μ L/ minutes when engaging magnetic field, each pearl is accumulated in sensor surface.Is 50 μ L/ minutes with the pearl of combination when sensor washes that used washing scheme begins, and is 500 μ L/ minutes during end, increases by 50 μ L/ minutes in per 30 seconds.According to these signals during exposure level (exposure level) standardization FPW signal that obtains standard signal and the integration washing.As shown in figure 12, in the presence of antagonism adjuvant 36, can detect and be lower than 10pg/mL, be lower than 50pg/mL not having to detect in the presence of the antagonism adjuvant 34.

As shown in figure 12, detect 1, the estradiol of 000pg/mL-10pg/mL concentration.

The competitive trials of example VI: FK506

1. FK-506 being added 2ml damping fluid (0.05%1 * PBS polysorbas20 contains 1%BSA), to produce FK506 concentration be 0,0.5,2.0 and the analyte sample of 20ng/ml.

2. with 8 μ l 1 * 10 of above-mentioned preparation ⁷/ ml is anti--and FK-506 IgM bag added in each sample by pearl, and room temperature was cultivated 30-60 minute.Operation instructions (hero Life Technologies, Inc.) according to the manufacturer will resist-FK506IgM (Fitzgerald industrial group) and paramagnetic beads coupling to prepare each pearl.Concentrating analysis sample solution as mentioned above.

3. the 500 μ l solution that will contain the carrier (HRP) of useful FK-506 (Dai Suolin company (Diasorin Inc.)) mark add in the sample, and room temperature was cultivated 60 minutes.

4. with these samples of FPW device analysis, the sensor of this device has been used agent for capturing (anti--KF-506 IgM) functionalization.With reference to Figure 10, as mentioned above,, adopt standard biological elementization method biotinylation to resist-FK-506 32 with neutral Avidin 30 functionalized sensing devices 12.Shown in the B figure among Figure 10, analyte 10 (being FK-506 in this example) is mixed with competition molecule 24 with the particle 16 of agent for capturing 14 (being anti--FK-506 antibody in this example) mark.For present embodiment, competition molecule 24 comprises the carrier HRP with analyte FK-506 mark.Shown in C and D figure, estimate that FK-506 concentration makes particle combine with the competition molecule than low energy in the sample, thereby combine with sensor surface.The FK-506 that estimates higher level can cause the particle that combines with sensing surface less, because more particles can combine with the FK-506 in the sample.Shown in Figure 13 and 14, behind the sample drawing-in system, the minimum FK-506 that can in 15 minutes, detect the 0.5ng/ml-20ng/ml concentration range.

Example VII A: the competitive trials of FK-506 in treated people's whole blood matrix

1. give four part of 100 μ l blood sample admixture 0,3,10 or 30ng/ml FK-506.Implement the protein digestibility method according to manufacturer's operation instructions (Dai Suolin company) and from blood matrix, extract medicine.

2. in each sample, add 600 microlitre protein digestibility reagent.

3. revolve (vortex) biased sample that shakes, room temperature (about 23 ℃) was cultivated 15 minutes, obtained translucent potpourri.

4.75 ℃ cultivation sample obtained dark brown mixture to stop digestion reaction in 35 minutes.

5. revolve the biased sample that shakes, with 1800g centrifugal 10 minutes, obtain 500-600 μ l supernatant.

6. 500 microlitre supernatants are transferred in the new test tube, four duplicate samples of (containing) same concentrations FK-560 are incorporated in the test tube, obtain the 2ml supernatant of various concentration FK-506.

7. analytic sample as mentioned above.Shown in Figure 15 and 16, sample introduced device after, the fastest FK-506 that can in 8 minutes, detect the 0.5ng/ml-20ng/ml concentration range.

The present invention can be presented as other concrete form and not break away from its design or essential characteristic.Therefore, should think that these embodiments are illustratives and nonrestrictive, scope of the present invention is embodied rather than above narration by additional claims, so these claims should comprise all changes that connotation and scope with these claim equivalences accompany.

Claims (110)

1. whether there is the method for one or more analytes in the test sample, comprises:

A) a plurality of magnetic-particles that are coated with agent for capturing are introduced fluid chamber, wherein the acoustical device that combines first analyte is equipped with at least one surface of this fluid chamber;

B) near this acoustical device, produce magnetic flux to guide at least one of a plurality of magnetic-particles into described surface; With

C) the signal output of the described acoustical device generation of monitoring, thus detected whether there are one or more analytes in the sample.

2. the method for claim 1 is characterized in that, described acoustical device is the flexible board wave device.

3. the method for claim 1 is characterized in that, removes described magnetic flux before step c).

4. the method for claim 1 is characterized in that, also comprises c) signal output make comparisons with control signal.

5. the method for claim 1 is characterized in that, a plurality of magnetic-particles are contacted with sample, introduces described fluid chamber again.

6. the method for claim 1 is characterized in that, earlier sample is introduced described fluid chamber, introduces a plurality of particles again.

7. the method for claim 1 is characterized in that, described sample is a biological sample.

8. method as claimed in claim 6 is characterized in that, described biological sample is selected from the group that following material constitutes: saliva, phlegm, cerebrospinal fluid, blood, serum, blood plasma, urine and biopsy material.

9. method as claimed in claim 4 is characterized in that described control signal is a typical curve.

10. method as claimed in claim 4 is characterized in that, is not having to obtain described control signal in the presence of the sample.

11. method as claimed in claim 4 is characterized in that, obtains described control signal by following steps:

A) second part of a plurality of magnetic-particle that are coated with agent for capturing introduced fluid chamber, wherein the acoustical device that combines second analyte is equipped with at least one surface of this fluid chamber;

B) near this acoustical device, produce magnetic flux to guide at least one of second part of a plurality of magnetic-particle into described surface; With

C) the signal output of the described acoustical device generation of monitoring.

12. method as claimed in claim 11 is characterized in that, described first and second analytes have identical chemical constitution.

13. method as claimed in claim 11 is characterized in that, described the first/the second analyte is a more macromolecular part.

14. the method for claim 1 is characterized in that, described analyte is incorporated into described surface indirectly.

15. method as claimed in claim 14 is characterized in that, described pan coating has in conjunction with the first right member, and described analyte combines second right member's combination with this.

16. method as claimed in claim 15 is characterized in that, described combination is to being selected from biotin/avidin, biotin/Streptavidin and biotin/neutral Avidin.

17. the method for claim 1 is characterized in that, described agent for capturing is an antibody.

18. whether have the method for one or more analytes in the test sample, comprising:

A) a plurality of magnetic-particles and competition molecule are introduced fluid chamber, described magnetic-particle be coated with can bound analyte agent for capturing, wherein bag has been equipped with by can be in conjunction with the acoustical device of second agent for capturing of competing molecule at least one surface of this fluid chamber;

B) near this acoustical device, produce magnetic flux to guide at least one of described a plurality of magnetic-particles into described surface; With

C) the signal output of the described acoustical device generation of monitoring, thus whether there are one or more analytes in the test sample.

19. method as claimed in claim 18 is characterized in that, described a plurality of magnetic bead is contacted with the competition molecule with sample, introduces described fluid chamber again.

20. method as claimed in claim 19 is characterized in that, described competition molecule comprises the analyte that combines with label, and described second agent for capturing can be in conjunction with this label.

21. method as claimed in claim 20 is characterized in that, described label is a biotin.

22. method as claimed in claim 20 is characterized in that, described competition molecule comprises and carrier-bound two or more analyte molecules, and described second agent for capturing can be in conjunction with this analyte.

23. method as claimed in claim 22 is characterized in that, described carrier is selected from horseradish peroxidase and albumin.

24. method as claimed in claim 18 is characterized in that, earlier sample and competition molecule is introduced fluid chamber, introduces a plurality of particles again.

25. method as claimed in claim 18 is characterized in that, described sample is a biological sample.

26. method as claimed in claim 25 is characterized in that, described biological sample is selected from: saliva, phlegm, cerebrospinal fluid, blood, serum, blood plasma, urine and biopsy material.

27. method as claimed in claim 18 is characterized in that, also comprises c) signal output make comparisons with control signal.

28. method as claimed in claim 27 is characterized in that, described control signal is a typical curve.

29. method as claimed in claim 27 is characterized in that, is not having to obtain described control signal in the presence of the sample.

30. method as claimed in claim 27 is characterized in that, described agent for capturing is incorporated into described surface indirectly.

31. method as claimed in claim 30 is characterized in that, described pan coating has in conjunction with the first right member, and described agent for capturing combines second right member's combination with this.

32. method as claimed in claim 31 is characterized in that, described combination is to being selected from biotin/avidin, biotin/Streptavidin and biotin/neutral Avidin.

33. method as claimed in claim 18 is characterized in that, described agent for capturing is an antibody.

34. a method that determines whether to adjust individual drugs dosage comprises:

A) level of one or more analytes in the test sample comprises:

I) sample and a plurality of magnetic-particle that is coated with agent for capturing are introduced fluid chamber, wherein the acoustical device that combines first analyte is equipped with at least one surface of this fluid chamber;

Ii) near this acoustical device, produce magnetic flux to guide at least one of a plurality of magnetic-particles into described surface; With

Iii) monitor the signal output that described acoustical device produces, thus in the test sample level of one or more analytes and

B) per sample in the level of one or more analytes determine whether to adjust drug dose.

35. a method that detects the analyte of energy integrating capture agent comprises:

A) a plurality of particles are introduced fluid chamber, wherein said a plurality of particles are coated with different analytes, and wherein the acoustical device that combines agent for capturing is equipped with at least one surface of this fluid chamber; With

B) the signal output of the described acoustical device generation of monitoring.

36. method as claimed in claim 35 is characterized in that, described acoustical device is the flexible board wave device.

37. method as claimed in claim 35 is characterized in that, described a plurality of particles are magnetic, and described method also is included in and produces magnetic flux near the described acoustical device to guide at least one of a plurality of particles into described surface.

38. method as claimed in claim 35 is characterized in that, also comprise the preparation described a plurality of particles, comprise particle is contacted with a group analyte, thus with different analyte bags by described a plurality of particles.

39. method as claimed in claim 35 is characterized in that, also comprises the described a plurality of particles of preparation, is included in synthesis analysis thing on the surface of described particle.

40. the method for estradiol in the test sample, this method may further comprise the steps:

A) will be coated with and can introduce fluid chamber in conjunction with a plurality of particles of the agent for capturing of estradiol, wherein the acoustical device that combines estradiol is equipped with at least one surface of this fluid chamber; With

B) the signal output of the described acoustical device generation of monitoring, thus the estradiol in the sample detected.

41. method as claimed in claim 40 is characterized in that, described acoustical device is the flexible board wave device.

42. method as claimed in claim 40 is characterized in that, compares with reference, the estradiol level increase shows that the possibility that individuality suffers from breast cancer increases.

43. method as claimed in claim 40 is characterized in that, described a plurality of particle is contacted with sample, introduces fluid chamber again.

44. method as claimed in claim 40 is characterized in that, earlier described sample is introduced fluid chamber, introduces a plurality of particles again.

45. method as claimed in claim 40 is characterized in that, described particle is a magnetic, and described method also is included in and produces magnetic flux near the described acoustical device to guide at least one of a plurality of magnetic-particles into described at least one surface.

46. method as claimed in claim 45 is characterized in that, removes magnetic flux before step b).

47. method as claimed in claim 40 is characterized in that, the concentration of estradiol is about 10pg/ml in the described sample.

48. method as claimed in claim 40 is characterized in that, estradiol is selected from free estradiol, the estradiol that combines with sex hormone binding globulin (SHBG), oestrone-3-glucosiduronic acid and estradiol-3-glucosiduronic acid.

49. method as claimed in claim 40 is characterized in that, described sample is selected from: blood, serum, blood plasma, urine, saliva and biopsy material.

50. method as claimed in claim 40 is characterized in that, also comprises comparison b) signal output and control signal.

51. method as claimed in claim 50 is characterized in that, described control signal is a typical curve.

52. method as claimed in claim 50 is characterized in that, is not having to obtain described control signal in the presence of the sample.

53. method as claimed in claim 40 is characterized in that, described estradiol is incorporated into described surface indirectly.

54. method as claimed in claim 53 is characterized in that, described pan coating has in conjunction with the first right member, and described estradiol combines second right member's combination with this.

55. method as claimed in claim 54 is characterized in that, described combination pairing is selected from biotin/avidin, biotin/Streptavidin and biotin/neutral Avidin.

56. the method for claim 1 is characterized in that, described agent for capturing is an antibody.

57. the method for estradiol in the test sample, this method may further comprise the steps:

A) a plurality of particles and competition molecule are introduced fluid chamber, described particle is coated with can be in conjunction with the agent for capturing of estradiol, and wherein bag has been equipped with by the acoustical device of energy in conjunction with second agent for capturing of competition molecule at least one surface of this fluid chamber; With

B) the signal output of the described acoustical device generation of monitoring, thus estradiol detected.

58. method as claimed in claim 57 is characterized in that, described acoustical device is the flexible board wave device.

59. method as claimed in claim 57 is characterized in that, compares with reference, the estradiol level increase shows that the possibility that individuality suffers from breast cancer increases.

60. method as claimed in claim 57 is characterized in that, described a plurality of particle is contacted with the competition molecule with sample, introduces fluid chamber again.

61. method as claimed in claim 60 is characterized in that, described competition molecule comprises the estradiol that combines with label, and described second agent for capturing can be in conjunction with this label.

62. method as claimed in claim 61 is characterized in that, described label is a biotin.

63. method as claimed in claim 58 is characterized in that, described competition molecule comprises and carrier-bound two or more estradiol molecules, and described second agent for capturing can be in conjunction with estradiol.

64., it is characterized in that described carrier is selected from horseradish peroxidase and albumin as the described method of claim 63.

65. method as claimed in claim 57 is characterized in that, described particle is a magnetic, and described method also is included in and produces magnetic flux near the described acoustical device to guide at least one of a plurality of magnetic-particles into described at least one surface.

66. as the described method of claim 65, it is characterized in that, before step b), remove magnetic flux.

67. method as claimed in claim 57 is characterized in that, the concentration of estradiol is about 10pg/ml in the described sample.

68. method as claimed in claim 57 is characterized in that, estradiol is selected from free estradiol, the estradiol that combines with sex hormone binding globulin (SHBG), oestrone-3-glucosiduronic acid and estradiol-3-glucosiduronic acid.

69. method as claimed in claim 57 is characterized in that, described sample is selected from: blood, serum, blood plasma, urine, saliva and biopsy material.

70. method as claimed in claim 57 is characterized in that, also comprises comparison b) signal output and control signal.

71., it is characterized in that not having to produce described control signal in the presence of the sample as the described method of claim 70.

72., it is characterized in that described control signal is a typical curve as the described method of claim 70.

73. method as claimed in claim 57 is characterized in that, described agent for capturing is incorporated at least one surface of fluid chamber indirectly.

74., it is characterized in that described at least one pan coating has in conjunction with the first right member as the described method of claim 73, described agent for capturing links to each other in conjunction with the second right member with this.

75., it is characterized in that described combination is to being selected from biotin/avidin, biotin/Streptavidin and biotin/neutral Avidin as the described method of claim 74.

76. method as claimed in claim 57 is characterized in that, described agent for capturing is an antibody.

77. the method for one or more immunodepressant in the test sample, this method comprises:

A) a plurality of particles that will be coated with the agent for capturing of energy binding immunoassay inhibitor are introduced fluid chamber, and wherein the acoustical device that combines described immunodepressant is equipped with at least one surface of this fluid chamber; With

B) the signal output of the described acoustical device generation of monitoring, thereby the immunodepressant in the test sample.

78. as the described method of claim 77, it is characterized in that, also comprise per sample described in the horizontal adjustment of medicine give the dosage of individual described immunodepressant.

79., it is characterized in that as the described method of claim 77, described a plurality of particle is contacted with sample, introduce fluid chamber again.

80., it is characterized in that as the described method of claim 77, earlier described sample is introduced fluid chamber, introduce described a plurality of particle again.

81., it is characterized in that described particle is a magnetic as the described method of claim 77, described method also is included in and produces magnetic flux near the described acoustical device to guide at least one of a plurality of magnetic-particles into described at least one surface.

82. as the described method of claim 81, it is characterized in that, before step b), remove magnetic flux.

83., it is characterized in that described sample is selected from as the described method of claim 77: blood, serum, blood plasma, cerebrospinal fluid, urine, saliva and biopsy material.

84., it is characterized in that described immunodepressant is selected from as the described method of claim 77: cyclosporin, tacrolimus, rapamycin and Mycophenolic Acid.

85. as the described method of claim 77, it is characterized in that, also comprise comparison b) signal output and control signal.

86., it is characterized in that not having to produce described control signal in the presence of the sample as the described method of claim 85.

87., it is characterized in that described control signal is a typical curve as the described method of claim 85.

88., it is characterized in that described immunodepressant is incorporated into described surface indirectly as the described method of claim 77.

89., it is characterized in that described pan coating has in conjunction with the first right member as the described method of claim 88, described immunodepressant combines second right member's combination with this.

90., it is characterized in that described combination is to being selected from biotin/avidin, biotin/Streptavidin and biotin/neutral Avidin as the described method of claim 88.

91., it is characterized in that described agent for capturing is an antibody as the described method of claim 77.

92. the method for one or more immunodepressant in the test sample, this method comprises:

A) a plurality of particles and competition molecule are introduced fluid chamber, described particle be coated with can the binding immunoassay inhibitor first agent for capturing, wherein at least one surface of this fluid chamber is equipped with bag and can be competed the acoustical device of second agent for capturing of molecule in conjunction with this; With

B) the signal output of the described acoustical device generation of monitoring, thereby the immunodepressant in the test sample.

93., it is characterized in that described acoustical device is the flexible board wave device as the described method of claim 92.

94. as the described method of claim 92, it is characterized in that, also comprise per sample described in the horizontal adjustment of medicine give the dosage of individual described immunodepressant.

95., it is characterized in that as the described method of claim 92, described a plurality of particle is contacted with the competition molecule with sample, introduce fluid chamber again.

96., it is characterized in that described competition molecule comprises the immunodepressant that combines with label as the described method of claim 95, described second agent for capturing can be in conjunction with this label.

97., it is characterized in that described label is a biotin as the described method of claim 96.

98., it is characterized in that described competition molecule comprises and carrier-bound two or more immunosupress agent molecules as the described method of claim 95, described second agent for capturing can be in conjunction with this immunodepressant.

99., it is characterized in that described carrier is selected from horseradish peroxidase and albumin as the described method of claim 98.

100., it is characterized in that described particle is a magnetic as the described method of claim 92, described method also is included in and produces magnetic flux near the described acoustical device to guide at least one of a plurality of magnetic-particles into described at least one surface.

101. as the described method of claim 100, it is characterized in that, before step b), remove magnetic flux.

102., it is characterized in that described sample is selected from as the described method of claim 92: blood, serum, blood plasma, cerebrospinal fluid, urine, saliva and biopsy material.

103., it is characterized in that described immunodepressant is selected from as the described method of claim 92: cyclosporin, tacrolimus, rapamycin and Mycophenolic Acid.

104. as the described method of claim 92, it is characterized in that, also comprise comparison b) signal output and contrast.

105., it is characterized in that not having to produce described control signal in the presence of the sample as the described method of claim 104.

106., it is characterized in that described control signal is a typical curve as the described method of claim 104.

107., it is characterized in that described agent for capturing is incorporated into this surface indirectly as the described method of claim 92.

108., it is characterized in that described pan coating has in conjunction with the first right member as the described method of claim 107, described agent for capturing combines second right member's combination with this.

109., it is characterized in that described combination is to being selected from biotin/avidin, biotin/Streptavidin and biotin/neutral Avidin as the described method of claim 108.

110., it is characterized in that described agent for capturing is an antibody as the described method of claim 92.

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