CN101528942A - Methods and systems for detection of contaminants - Google Patents

Abstract

The present invention discloses an impedance biosensor for detecting a contaminant in a starting material, the biosensor comprising a housing, an input device supported by the housing, an output device supported by the housing, a microfluidic cell supported by the housing, the starting material being engagable with the microfluidic cell, and an impedance analyzer supported by the housing and operable to measure impedance of the starting material to detect the presence of a contaminant.

Description

The method and system that are used for detection of contamination

Related application

The application to submit on September 1st, 2006 60/841, No. 774 temporary patent application (Provisional Application No.60/841,774) reach in 60/876 of submission on December 22nd, 2006, No. 919 temporary patent application (Provisional Application No.60/876,919) priority request is proposed, the full content of described interim patent is merged in this patent at this by reference.

Foreword

Avian influenza virus H 5 N 1 was found for the end in nineteen ninety.According to report, surpassing 46 country's discovery animal cases, find human infections in 10 countries, there were 258 people infected from 2003, it is dead to have 153 examples.Recently, the draft report of the emergency preplan of United States Federal Government prophesy, if in U.S.'s burst bird flu, nearly 200,000,000 Americans may be infected, and 200,000 people may be dead in some months.U.S. CDC (CDC) estimates that unspecified flu outbreak can cause about 8.9 ten thousand to 20.7 ten thousand routine death, 31.4 ten thousand people to 73.4, ten thousand people prescription on individual diagnosis in hospital, 1,800 ten thousand to make an exception to forty-two million example outpatient service prescription on individual diagnosis and 2,000 ten thousand to 4,700 ten thousand in the U.S. and add illness.Directly financial loss may reach hundreds billion of dollars.Estimate that global flu outbreak will cause 2,000,000 to 7,400,000 examples human dead in the whole world.

In the U.S., the low pathogenicity bo bird flu (LPAI) of burst in 2001 and 2002 causes 4,500,000 chickens and turkey death, and estimates to have made poultry farming suffer about 1.25 hundred million dollars loss.According to the report of the World Bank, in 2005, it is dead or destroyed that bird flu has caused surpassing 1.40 hundred million poultry, and the estimated amount of damage that poultry farming is caused is above 10,000,000,000 dollars.

Summary of the invention

The key that spreads of control bird flu is promptly to detect this disease, then is the isolation of destroying infected animals, two mile radius scopes to prevent personnel and animal and move and to be that animal carries out vaccination outside isolated area.At present, it all is time-consuming, expensive or to the hypotype test out of true of bird flu (AI) virus being used to detect the technology of influenza-such as virus culture, real-time polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA)-often.Therefore, need be adapted at that wilderness or patient bedside are used, simple, rapidly, stand test and check reliably.

In one embodiment, provide impedance biosensor (impedance biosensor), be used for detecting the pollutent of starting material.Described biosensor comprises input unit, output equipment and micro-fluidic unit (microfluidic cell), and all these equipment are supported by seat board (housing).Described starting material can engage with described micro-fluidic unit and impedance analysis device, and this impedance analysis device is by described seat board support, and can operate the impedance of measuring described starting material, so that the existence of detection of contamination.

In another embodiment, be provided for detecting the method for the pollutent in the starting material.Described starting material partly contacts with the affinity that can be attached to described pollutent, to form target (target).Described affinity partly is coupled to magnetic nano-particle.Non-marked impedance biosensor described here is used to detect described target.The detection of described target can be indicated the existence of the pollutent in the described starting material.

In yet another embodiment, be provided for detecting the method for the virus in the starting material.Described starting material contacts with red corpuscle, and described virus can be attached to described red corpuscle to form mixture.Described mixture detects with biosensor.The detection of described mixture can be indicated the existence of the described virus in the described starting material.

In another embodiment, be provided for detecting the method for the pollutent in the starting material.Described starting material partly contacts with the affinity that can be attached to described pollutent, and to form target, wherein said affinity partly is coupled to magnetic nano-particle.Described target detects with impedance biosensor.The detection of described target can be indicated the existence of the described pollutent in the described starting material.

In another embodiment, be provided for detecting the method for the pollutent in the starting material.Described starting material partly contacts with the affinity that can be attached to described pollutent, and to form target, wherein said affinity partly is coupled to magnetic nano-particle.Described target separates from described starting material, and is delivered to micro-fluidic unit.Dispose interdigitated array microelectrode in the described micro-fluidic unit.Described target detects with described interdigitated array microelectrode biosensing device, and the detection of wherein said target can be indicated the existence of the described pollutent in the described starting material.

Description of drawings

Fig. 1 is the front elevation of portable biometric transmitter.

Fig. 2-4 is the different views of the biosensor shown in Fig. 1.

Fig. 5 is the interior views of the biosensor shown in Fig. 1, shows among the figure that the protecgulum of this biosensor is taken apart.

Fig. 6 is the structure iron of the biosensor shown in Fig. 1.

Fig. 7 is the electrical schematics of the biosensor shown in Fig. 1.

Fig. 8 is the structural map that is used for the box box assembly of biosensor.

Fig. 9 is micro-fluidic unitary top view, this micro-fluidic unit have the biosensor shown in Fig. 1, embed interdigitated array microelectrode (IDAM).

Figure 10 is the stereographic map of microchannel, and described microchannel is used for micro-fluidic unit.

Figure 11 is the top view of the microchannel shown in Figure 10.

Figure 12 is interdigitated array microelectrode (IDAM) chip, and this chip is used for the micro-fluidic unit shown in Fig. 9.

Figure 13-15 shows the making processes of interdigitated array microelectrode (IDAM) chip shown in Figure 12.

Figure 16 A is the synoptic diagram of non-marked bio-sensor system, and described non-marked bio-sensor system uses red corpuscle and magnetic nano-particle.

Figure 16 B shows the magnetic nano-particle separating device.

Figure 17 is Bode diagram (bode plot), and the Escherichia coli O 157 of magnetic nano-particle antibody complex (MNAC) is organized and had in its diagram contrast (control): the impedance magnitude of H7 group is to frequency curve.

Figure 18 is a graphic representation, when it is presented at the frequency of indicating among the figure, Escherichia coli O 157: H7MNAC and only have resistance difference (changing [NIC] expression with normalized impedance) between the control sample of magnetic nano-particle antibody complex (MNAC).

Figure 19 A-19D is a suite line chart, the impedance of the sample of indicating among their demonstration figure.Figure 19 A-19B shows the impedance measurement from the pure culture of bacterium.Figure 19 C-19D shows from the impedance measurement that is mixed with colibacillary hamburger sample.Figure 19 A and 19C show impedance spectrum, Figure 19 B and the 19D impedance when then display frequency is 16kHz.

Figure 20 is a graphic representation, and it shows based on coating anti-hemocyte cohesion plain (anti-hemagglutinin, HA) antibody immobilization impedance biosensor and impedance measurement pre-incubated with red corpuscle, avian influenza virus (bird flu H 5 N 1).

Figure 21 is a graphic representation, and it shows based on coating the impedance measurement that anti-hemocyte condenses plain (anti-HA) antibody, adds the avian influenza virus of erythrocytic immobilization impedance biosensor after the bird flu combination.

Figure 22 is a graphic representation, and it is presented at after the magnetic nano-particle contact avian influenza virus of coating plain (anti-HA) antibody of anti-hemocyte cohesion, based on the impedance measurement non-marked impedance biosensor, avian influenza virus.

Figure 23 A and 23B are a suite line chart, and they are presented at the impedance measurement of carrying out when avian influenza virus and new city eqpidemic disease and infectious bronchitis virus exist jointly.The curve display of Figure 23 A is based on the impedance measurement of coating anti-plain (anti-HA) antibody immobilization of hemocyte cohesion avian influenza virus impedance biosensor, in having new city eqpidemic disease and infectious bronchitis virus and erythrocytic mixture.The curve display of Figure 23 B is based on the impedance measurement of coating anti-plain (anti-HA) antibody immobilization of hemocyte cohesion avian influenza virus impedance biosensor, in the mixture that has new city eqpidemic disease and infectious bronchitis virus, and wherein red corpuscle adds described biosensor to after adding described antibody.

Figure 24 is a graphic representation, and it is presented at after the magnetic nano-particle contact avian influenza virus of coating plain (anti-HA) antibody of anti-hemocyte cohesion, based on the impedance measurement of the avian influenza virus non-marked impedance biosensor, in the mixture that has new city eqpidemic disease and infectious bronchitis virus.

Figure 25 A and 25B are a suite line chart, their show based on coat plain (anti-HA) antibody immobilization impedance biosensor of anti-hemocyte cohesion, after pre-the cultivation with red corpuscle, contrast tracheae swab (Figure 25 A) and be mixed with the impedance measurement of the tracheae swab (Figure 25 B) of avian influenza virus.

Figure 26 is a graphic representation, and it is presented at after the magnetic nano-particle contact avian influenza virus of coating plain (anti-HA) antibody of anti-hemocyte cohesion, based on the impedance measurement non-marked impedance biosensor, that contrast the tracheae swab and be mixed with the tracheae swab of avian influenza virus.

Figure 27 A and 27B are a suite line chart, their show based on coat plain (anti-HA) antibody immobilization impedance biosensor of anti-hemocyte cohesion, after pre-the cultivation with red corpuscle, contrast cloaca swab (Figure 27 A) and be mixed with the impedance measurement of the cloaca swab (Figure 27 B) of avian influenza virus.

Figure 28 A and 28B are a suite line chart, their show based on coat plain (anti-HA) antibody immobilization impedance biosensor of anti-hemocyte cohesion, after pre-the cultivation with red corpuscle, contrast cloaca swab and be mixed with the impedance measurement of cloaca swab of the avian influenza virus of ten times of dilutions.Figure 28 A display frequency is 10, the impedance during 400Hz, and Figure 28 B then shows impedance spectrum.

Figure 29 A and 29B are a suite line chart, they be presented at after the magnetic nano-particle contact avian influenza virus of coating plain (anti-HA) antibody of anti-hemocyte cohesion, based on the non-marked impedance biosensor, after pre-the cultivation with red corpuscle, contrast cloaca swab and be mixed with the impedance measurement of cloaca swab of the avian influenza virus of ten times of dilutions.Figure 29 A display frequency is 4, the impedance during 150Hz, and Figure 29 B then shows impedance spectrum.

Figure 30 is a bar graph, and it is shown as the impedance measurement that detects the avian influenza virus H 5 N 1 in the poultry swab sample and use the non-marked bio-sensor system to carry out.

Figure 31 A-31E is a suite line chart, and they show impedance measurement, and these impedance measurements use non-commercial antibody that avian influenza virus H 5 N 1 is carried out.

Embodiment

Before at length explaining at least one structure of the present invention, should understand the restriction that diagrammatic structure detail and assembly are arranged in that the present invention is not subjected to display in the following description or the accompanying drawing.The present invention can implement with other structures, and can carry out or realization with multitude of different ways.

The ability of the existence of the small amounts of contamination of detection such as bacterium and virus, the especially ability of the existence of detection small amounts of contamination under complex background are to the prevention particularly important of biotechnology, medical diagnosis and pandemic disease.Detect and differentiate infected curee on one's body food or supply water in pollutent, necessary to safeguarding health and safety.In addition, the rapid detection of small amounts of contamination will make the clinical diagnosis of disease accelerate, and can obtain better prognosis.During a small amount of pollutent of (in food or soil), it detects very difficulty in must detecting a large amount of samples or in the complicated sample.That this area needs is other, highly sensitive, accurately and rapidly method detects and quantitative contamination.In addition, also need to use in the wilderness hand-held or handheld device detects and quantitative contamination.

The contriver is used for detecting and the method and system of the pollutent of quantitative starting material in this description.Describe based on impedance biosensor micro-fluidic and interdigitated array microelectrode at this.Described biosensor can be implemented on handheld device, and it can carry out highly sensitive, accurately and rapidly detection to pollutent, and minimizes the time between the sample.The present invention also provides the several different methods that detects the pollutent in the starting material.Described method comprises that the affinity can be attached to described pollutent partly contacts described starting material, to form target.Described affinity part can be coupled to magnetic nano-particle, to form magnetic target.Then, detect described magnetic target with biosensor suitably, such as detecting described magnetic target with impedance biosensor described here.In addition, the invention provides the influenza that detects in the starting material and the method for Avian pneumo-encephalitis virus, described method contacts described starting material forming mixture with red corpuscle, and suitably with biosensor, be impedance biosensor described here, detect described mixture.

Described biosensor technology comprises based on one or more notions: (1) uses magnetic nano-particle, and the target virus in the poultry swab sample is carried out high-level efficiency and separation rapidly; (2) use the microflow controlled biochip that has embedding interdigitated array microelectrode, to realize accurately sending and the high sensitive measurement of described target virus; And the mixture of (3) formation red corpuscle and nanoparticle, amplify to obtain bigger impedance signal.Finished the experiment of carrying out based on chicken swab sample laboratory scale, to preparing with inactivation avian influenza virus H 5 N 1 and other virus.Experimental result shows that described biosensor can detect avian influenza virus H 5 N 1 clearly, and its detection is limited to: be less than in 30 minutes, and in poultry cloaca or the tracheae swab sample, 100EID at least ₅₀/ ml.Described biosensor can use in the wilderness, and its cost estimation is less than 10 dollars of every samples.Biosensor described here has proposed the notion of the real-time detection of bird flu.

Impedance biosensor

Referring to Fig. 1, Fig. 1 diagram impedance biosensor 20, this impedance biosensor 20 can be operated the existence that detects the pollutent in the solution that is introduced in this impedance biosensor 20, and the quantity of quantitative described pollutent.Can comprise by biosensor 20 detections and quantitative pollutent, for example bacterium, virus, eukaryotic cell, polypeptide or other biological or chemical pollutant.Biosensor 20 is a separate equipment, and it can be operated and measure resistance value, with the pollutent in detection and the volumetric soiutions.Biosensor 20 also can be carried easily by the user.

Continuation is referring to Fig. 1 and in addition referring to Fig. 2-5, and biosensor 20 comprises seat board 24, and its seat board 24 comprises front portion 28 and rear portion 32, and front portion 28 and rear portion 32 can connect movably with the fastening piece (not shown).Biosensor 20 also comprises liquid-crystal display (LCD) 36 and input unit or keypad 40, and keypad 40 is supported by anterior 28.Liquid-crystal display (LCD) 36 and keypad 40 are respectively the user input and output resource are provided.Supply socket 44 supported by rear portion 32, and receives and support power supply 48, and for example a battery or a plurality of battery are so that provide electric energy for biosensor 20.In some embodiments, power supply 48 is a pair of AA 1.5V batteries, and it provides direct current energy.In other embodiment, power supply 48 is cord and electrical connector receptacle, and it provides AC energy.Opening 52 is arranged on rear portion 32, so that power supply 48 is inserted socket 44 or removes power supply 48 from socket 44.The position that movably be connected to rear portion 32 of lid 56 on opening 52 is so that selectively cover or open socket 44.

Referring now to Fig. 6 and 7,, biosensor 20 comprises microcontroller 60, data-carrier store 64, power converter 68, power strainer 72, warning howler 76, communication interface 80, impedance detector 84 and box box assembly 88.Should be appreciated that embodiments of the invention comprise hardware, software and electric component or module, but for the ease of discussing, these embodiment may or describe to such an extent that be embodied as hardware and/or software individually as some assembly by diagram.Yet those of ordinary skill in the art reaches and it will be understood that at least one embodiment that based on the reading to this detailed description the aspect based on electric component and software of the present invention may be embodied as different hardware and software arrangements.Therefore, should be appreciated that, can use a plurality of equipment and a plurality of different structure assemblies to implement the present invention based on hardware and software.In addition, as described in following paragraph, the purpose of diagrammatic specific mechanical configurations is illustration one embodiment of the present of invention among Fig. 6 and 7, but also might adopt other substituting structures.

Such as described further below, microcontroller 60 is carried out the multiple operation and the function of biosensor 20, and communicates with the different assemblies of biosensor 20.In one embodiment, the AT89C55WD type microchip unicircuit of microcontroller 60 for providing by Atmel Corporation (ATMEL Corporation).Yet those of ordinary skill in the art it will be understood that and can use other assemblies and combination of components, replaces microcontroller 60 such as microprocessor, digital signal processor, application specific integrated circuit (ASICs) or the like.With Fig. 7 is example, and microcontroller 60 is connected to the pierce circuit that comprises vibrator X2 and electrical condenser C12 and C13 and comprises inductor block L3, electrical condenser C7 and the VCC/ reset circuit of C14, diode D2 and resistor R 34.

Keypad 40 and liquid-crystal display (LCD) 36 is respectively the user input and output resource is provided, but also can use the input and the output equipment of other classifications.Such output equipment belongs to legacy equipment in the art, so will not be further described at this.With Fig. 7 is example, the LCD1604B type liquid-crystal display of liquid-crystal display (LCD) 36 for being provided by OPTREX company.

Data-carrier store 64 devices are in biosensor 20, and it stores data on biosensor 20.Data-carrier store 64 can comprise multiple conventional memory device, for example the AT24LC256 type memory integrated circuit that is provided by Atmel Corporation (ATMEL Corporation).Should be appreciated that though display data memory 64 is independent storer among the figure, in other embodiments, described data-carrier store can make up with other elements (for example microcontroller 60) of biosensor 20.

Communication interface 80 is used for communicating by letter between biosensor 20 and the external electrical equipment (such as Personal Computer, printer or the like).With Fig. 7 is example, and communication interface 80 comprises electrical condenser C22, C23, C24, C25 and C26 and equipment U9, and equipment U9 can be the MAX232 type communication integrated circuit that is provided by Maximum company.

Impedance detector 84 can be operated the impedance of responding to the sample of introducing box box assembly 88, and provides the relevant value of impedance with institute's sensing to microcontroller 60.With Fig. 7 is example, and impedance detector 84 comprises inductor block L2; Electrical condenser C15, C27 and C28; Resistor R 2, R3 and R32; Vibrator X1; And impedance unicircuit U4, impedance unicircuit U4 can be by Analog Devices Inc (Analog Devices, the AD5934 type impedance unicircuit that Inc.) provides.Impedance detector 84 itself or be combined to form impedance analysis device 85 with other circuit (for example microcontroller 60) is so that determine according to institute's induced impedance in other things.For example, impedance analysis device 85 can be used for the existence of detection of contamination.Other that to discuss below that impedance analysis device 85 carries out are determined.

Power converter 68 receives the electric energy from power supply, and the appropriate voltage that the circuit shown in Fig. 7 uses is changed/be adjusted to described electric energy.With Fig. 7 is example, and described power converter comprises inductor block L1; Electrical condenser C6, C10 and C11; Diode D1; Voltage unicircuit U2, voltage unicircuit U2 can be the LT 1302 type voltage unicircuit that provided by Linear Techn Inc. (Linear Technology Corporation); And voltage unicircuit U3, voltage unicircuit U3 can be the LX8117-33CDD type voltage unicircuit that is provided by Microsemi company.

Warning howler 76 provides visual and/or can listen warning.With Fig. 7 is example, and warning howler 76 comprises resistor R 35 and R36, light emitting diode D3, transistor Q1 and calling set SP1.

Power strainer 72 filters VCC voltage and comprises electrical condenser (is example with Fig. 7) C2, C3, C4, C5, C9, C17, C18, C19, C20, C31, C32, C33 and C34.

Referring to Fig. 5 and 8, below box box assembly 88 will be described in more detail.Box box assembly 88 is placed in the top of seat board 24, and it is divided into part of detecting 92 and waste material storing chamber part 96.Sample is introduced into part of detecting 92 for test, and after test, described sample is applied to waste material storing chamber part 96 then, and described sample or a plurality of sample are in 96 accumulations of waste material storing chamber part, till disposing.Described sample is introduced into biosensor 20, the outside that injection port 100 can be led to seat board 24 by injection port 100.Inlet flexible pipe 104 is connected between injection port 100 and the micro-fluidic unit 108 (see following more go through), and outlet hose 112 is connected between micro-fluidic unit 108 and the waste material storing chamber 96.Discharge outlet 116 fluidly is connected to waste material storing chamber 96, so that from the described sample of waste material storing chamber 96 dischargings.

Micro-fluidic unit

Miniflow many advantages that learned a skill,, precise control little comprising S/V rate height, sample quantities and cost are low.Designed and made microfluidic channel (cross sections of dark 40 μ m, wide 50 μ m).Please ConsultLi Shi and Su Shi are in 2006 writing in " rapid method in the microbiology and automatization periodical " 14 phase 96-109 pages or leaves (Journal of Rapid Methods andAutomation in Microbiology 14:96-109) (full content of described book is merged in this patent at this by reference).Designed and made interdigitated array microelectrode (electrode that the 3 pairs of width are 25 μ m refers to) in described microfluidic channel, to carry out impedance measurement to the biological target sensitivity in the sample.(people such as Young writes (Impedance biosensors to detectSalmonella typhimurium and E.coli O157:H7 cells captured onto thesurface of an interdigitated array microelectrode modified with specificantibody) to " be used to detect the Salmonella typhimurtum and the Escherichia coli O 157 that capture with the surface of the interdigitated array microelectrode of specific antibody modification: the impedance biosensor of H7 cell ", 2004, " analytical chemistry " is 76 phase 1107-1113 pages or leaves (AnalyticalChemistry), the full content of described book at this by reference, be merged in this patent) or " by interdigitated array electrode " (Passing through aninterdigitated array electrode) (people such as Varshney, 2007, " biosensor and biological electronics " (Biosens Bioelectron) 22 (11) phase 2408-2424 pages or leaves) be described (full content of above book is merged in this patent at this by reference).

Also further referring to Fig. 9-12, micro-fluidic unit 108 is placed in the part of detecting 92 referring to Fig. 5 and 8 in continuation, and with a plurality of stages making.The purpose of making processes described here is only in illustration.Therefore, can use other making processes make can carry out desired operation and also belong to micro-fluidic unit in spirit of the present invention and the claim.

In the stage one, on the chip glass 124 that is coated with the gold layer, make interdigitated array microelectrode (IDAM) chip 120.In this stage, with

Gold and with

Chromium as bonding coat sputtering glass wafer 124.Follow AZ 4330 photosensitive and corrosion material spin-coating glass wafers 124 with 4 μ m bed thickness.Use the photolithography described photosensitive and corrosion material of finalizing the design, described photosensitive and corrosion material then develops.Photosensitive and corrosion material with typing comes Jin Jige on the etching glass wafer 124 as shielding layer, to obtain described electrode.In some embodiments, interdigitated array microelectrode (IDAM) chip 120 comprises 50 pairs of gold electrodes and refers to, every finger beam 25 μ m, and Interdigital Space is 25 μ m.

In the stage two, prepare mould 128, so that make microchannel 132.In this stage, with SU-8140 spin coating silicon (Si) wafer 136, soft then roasting (soft bake) silicon wafer 136.Then, cover silicon wafer 136 and SU-8 coating 140, be exposed to ultraviolet ray then with development SU-8 structure 140.Then hard roasting (hard bake) mould 128.

Stage three, microchannel 132 form and with interdigitated array microelectrode (IDAM) chip 120 bondings.In this stage, (polydimethylsiloxane PDMS) 144 is introduced into mould 128 to polydimethylsiloxane, solidifies (cured) 1 hour on 60 ℃ ground, temperature condition lower section then.Then, polydimethylsiloxane (PDMS) passage 144 is pulled out from mould 128.For polydimethylsiloxane (PDMS) passage 144 is bonded on interdigitated array microelectrode (IDAM) chip 120, polydimethylsiloxane (PDMS) passage 144 is alignd with microelectrode 148, hot key is incorporated into described chip under 80 ℃ temperature condition then, for the time 8 hours.Synthetic microchannel 132 comprises inlet 152, outlet 156 and along the sensing chamber 160 of between the two direction.In some embodiments, microchannel 132 dark 15 μ m, wide 500 μ m, and the size of sensing chamber 160 is 0.5 * 0.5 * 0.02mm, its volume is 5nl.Micro-fluidic unit 108 also comprises electrical cnnector 164, these electrical cnnectors 164 are connected to described interdigitated array microelectrode (IDAM), so that provide electric energy and transmission information between described interdigitated array microelectrode (IDAM) and microprocessor 60 to it.

The operation of impedance biosensor

Since described biosensor 20, the purposes and the exemplary operation of biosensor 20 will be described below.By impedance measurement, biosensor 20 can be used to detect and quantitatively be introduced into the existence of the pollutent of sample.

The following operation of making relevant biosensor 20 is described, and is a mode of the existence of the pollutent in detection and the quantitative sample, and its purpose is not to be to limit the scope of the invention.Therefore, also can use the mode of other detections and quantitative contamination, for example use the other biological transmitter, also belong within spirit of the present invention and the claim.

In some embodiments, described interdigitated array microelectrode operation as following.The flow through electric current of described interdigitated array microelectrode, the spatial electric current between the described electrode of particularly flowing through refers to is stoped by the existence of the target in the described passage.Described impedance is directly proportional with the target quantity of existence, and is the function of the frequency of the voltage signal that is applied in.

Partly contact with affinity and after affinity part-pollutent " target " formed, described target separated from other samples, and uses biosensor 20 subsequently quantitatively at described sample.If described affinity partly is coupled to magnetic nano-particle, then can use magnetic separator to remove described " particle adds target " mixture (beads-plus-target complexes) (seeing the further discussion of following relevant magnetic nano-particle).

An aspect of native system is to contact with described interdigitated array microelectrode the mechanism of described target, particularly is the use of continuous-flow system.By using continuous-flow system, a plurality of samples can pass through biosensor 20 continuously, reach inferior limit the stoppage time between the sample.

Another aspect of native system is, described affinity part does not need to be integrated into described interdigitated array microelectrode (being called " non-marked " (label-free) biosensor), but in the embodiment that some has changed, described affinity partly can be used as the part that flows through material.Because this is specific, biosensor 20 can partly be reused with many different affinities, therefore can change under the situation of microelectrode not needing, and is used to detect multiple different pollutent.In other system, what form contrast is, described affinity part (being generally antibody) is coupled to described microelectrode, and the result to be described specific microelectrode be merely able to be used to detect a kind of pollutent.In addition, use " non-marked " (label-free) system's analytical sample more promptly, if this is owing to use " non-marked " (label-free) system, just need between reading, not peel off or remove resistates from described biosensor, and use " immobilization " (immobilized) biosensor, that is: be attached with affinity biosensor partly, but need like that.

After described target is introduced biosensor 20, can apply the oscillating voltage signal of a range of frequency or a single-frequency, and the record resultant impedance.When not having positive reaction (positive reaction) between pollutent in the described sample and the described affinity part, described affinity part will can not form the target mixture, and therefore described affinity part will have measurable different profile on impedance spectrum.On the contrary, when having positive reaction, the described target mixture that the affinity of formation partly adds pollutent will cause the measurable increase of impedance of some part of frequency spectrum, cause to produce special mark.

In typical a measurement, sample solution is introduced into described injection port 100, and by the inlet flexible pipe 104 of microchannel 132 (Fig. 8) and the micro-fluidic unit 108 of 152 inflows that enters the mouth.Comprise that interdigitated array microelectrode (IDAM) chip 120 of made and the micro-fluidic unit 108 of microchannel 132 are used for gathering the pollutent of the active coating on the described interdigitated array microelectrode (IDAM), and when impedance measurement is carried out, minimize the interference of the particulate in the sample.Described active coating is positioned at the surperficial several microns places of described interdigitated array microelectrode (IDAM), and the maximum part of strength of electric field just here.When having the 0.1M mannitol solution, can in the range of frequency of 10Hz to 1MHz, measure the size and the phase angle of impedance.Selectively, when having other solution and concentration, can measure described size and phase angle at different frequency.After microchannel 132, sample leaves micro-fluidic unit 108 by the outlet 156 and the outlet hose 112 of microchannel 132 in sample stream.Then, sample flows to waste material storing chamber 96, flows out biosensor 20 by discharge outlet 116 at last.

The impedance measurement of described solution can be carried out with above-mentioned impedance biosensor system, or carry out with traditional impedance detector or analyzer, for example with IM-6 impedance analysis device (Lafayette city, west, U.S. Indiana State BioAnalytical Systems Inc. company, West Lafayette, Indiana) carry out, or the use traditional software, for example IM-6/THALES software carries out.For all impedance measurements, described interdigitated array microelectrode (IDAM) has been applied the Sine Modulated alternating-current of 100mV voltage, and be size and phase angle that 10Hz to 1MHz range of frequency is measured impedance.Use the magnetic nano-particle antibody complex (MNAC) tested, from the detection total time that is sampled to measurement be 35 minutes.An electrode of described interdigitated array microelectrode (IDAM) chip is connected to test probe and pickup probe, and another electrode then is connected to the reference electrode and the counter electrode (counter electrode) of described impedance analysis device.

Frequency when adhering to the sample solution of magnetic nano-particle antibody complex (MNAC) and the maximum of the impedance measurement difference between the control group in order to determine to be with has been drawn the graphic representation of normalized impedance variation (NIC) to frequency.Normalized impedance changes the value of (NIC) and obtains according to following formula (1):

$\frac{Z_{\mathrm{sample}}-Z_{\mathrm{control}}}{Z_{\mathrm{control}}}\×100---\left(1\right)$

Wherein, Z _ControlBe the impedance magnitude of control group, and Z _SampleImpedance magnitude for sample solution.

Method described here can be used for detecting the existence at the multiple pollutent that comprises influenza virus of the different multiple starting material of the complexity aspect antigen diversity, density and the volume.Except the starting material that is used for following examples, can reasonably understand the pollutent that can detect in numerous food, animal and environment and the clinical sample, and starting material can comprise liquid, solid or comprise liquid and the material of solid mixture.Described starting material can comprise vegetables, fruit, meat mincing, beef, poultry, marine products, milk preparation, water, air, soil, blood, urine, ight soil; Take from skin surface, organ, tracheae or cloacal swab; Or tissue sample.The food sample can be to give birth to sample or ripe sample.For example, poultry prod can comprise carcass, the wash water from carcass, the poultry of boning, broken poultry or the poultry pie after butchering.Described method also is suitable for food or environmental inspection or clinical diagnosis or monitoring.For example, described method can be in food-processing, store, sell or or even during market is sold, be used to monitor food.Before being used for described method, solid or semi-solid starting material may stand to homogenize.

As following for embodiment described in, the pollutent of many classifications can detect or quantitatively with method described here.In the following example, Escherichia coli O 157: H7 and H 5 N 1 avian influenza have been detected.Except these pollutents, can reasonably understand those of ordinary skill in the art can use described method to multiple potential pollutant, includes but not limited to such as listeria monocytogenes (Listeria monocytogenes), campylobacter jejuni (Campylobacter jejuni), false unit cell Bacillus proteus (Pseudomonas mirabilis), Salmonellas (Salmonella) species and faecalis (Enterococcus) species; Eukaryotic cell; Polypeptide comprises PrPC (prions), toxin and blood or urine albumen; Virus is such as influenza virus; Or other chemical pollutants, such as sterilant or weedicide.What be significant is that viable cell and dead cell can detect with method described here.Yet, can the pre-treatment starting material, remove any active pollutant that may threaten so that kill to the technician's that carries out method described here health.

Magnetic nano-particle

Nanoparticle was described in the past.The treatise of delivering at 2005 " food protection periodical " (Journal of Food Protection), 68 phase 1804-1811 referring to people such as Varshney; Fritzsche and Taton are at 2003 " nanotechnology " (Nanotechnology) treatise delivered of 14 phase R63-R73 pages or leaves; People such as the treatise that people such as Tan deliver at " medical research comment " (MedicalResearch Review) 24 phase 621-638 pages or leaves in 2004, Zhao are in treatise, the U.S. 6 that 2004 " institute of NAS periodical " (Proceedings of the National Academy of Sciences), 101 volume 15027-15032 pages or leaves are delivered, 623, No. 982 patents and the U.S. 6,645, No. 731 patents, the full content of all these treatises and patent is merged in this patent at this by reference.The diameter of nanoparticle is between 1-300nm, suitably between the 50-150nm diameter.The nanoparticle nuclear that can be magnetic, its magnetic core can comprise different metal, and the same with microparticle, described magnetic core may be paramagnetic or super paramagnetic.The nanoparticle that is suitable for method described here comprises can be from " molecular probe company " (Molecular Probes, Inc.) nanoparticle of Gou Maiing in Ore. (Oregon) Eugene city (Eugene).

The nanoparticle exhibit magnetic properties fluidic characteristic of 50-150nm, and remain stable colloid.These nanoparticles are gathered in the magnetic individual layer, and this allows to wash off unnecessary background.With other isolation technique, such as centrifuging, filtration, and the use of microparticle is relatively, and magnetic nano-particle provides the higher capture efficiency to avian influenza virus in the poultry sample, this so that improve the susceptibility of described biosensor.In case described nanoparticle contacts with described starting material and described magnetic target forms, described magnetic target can be separated from described starting material with multiple mode.Described target can separate by filtration or centrifuging or by producing magnetic field.The magnetic resolution equipment that is suitable for described method comprises " Dai Nuo the company " (Dynal by New York, United States (New York) Sa Kesesi Hu Shi (Lake Success), Inc.) the magnetic particle concentrator that provides (Magnetic Particle Concentrator, MPC) and at title be the U.S. 11/328 of " using the separation system of magnetic nano-particle and effectively capturing of pollutent " (Separation System and EfficientCapture of Contaminants Using Magnetic Nanoparticles), No. 808 patent applications (U.S.Patent Application No.11/328,808) separation system of describing in, the full content of described United States Patent (USP) is merged in this patent (seeing Figure 16 B) at this by reference.

Described nanoparticle can be directly or is coupled to the affinity part that pollutent is had affinity indirectly.Be coupled to the nanoparticle that the market of affinity part can get by number of chemical reaction and prepare, but nanoparticle also can be prepared by the final user.Nanoparticle can be coupled to the affinity part by several different methods, and these methods include but not limited to be attached in advance streptavidin (streptavidin), avidin (avidin), Protein G or albumin A; The kit utility that uses market to get, nanoparticle can be directly by being covalently bound to antibody; Surface exposed functionality carboxyl or amino to be used for the nanoparticle of coupling multiple polypeptides; Or be linked to can binding antibody the Fc district, such as Fc acceptor or anti--Fc antibody, or can be in conjunction with the nanoparticle of the polypeptide in the non-Fc district of described antibody.The method biotinylation that polypeptide can be known with those of ordinary skill in the art, and described vitamin H can form the bridge joint mixture, this bridge joint mixture is linked to the affinity part by being attached to streptavidin or avidin with nanoparticle.Can reasonably understand, those of ordinary skill in the art can use multiple different chemical method to make described antibody directly or indirectly be coupled to described nanoparticle.

Described magnetic nano-particle can be dependent on and be coupled to affinity part and be attached to described pollutent.The affinity part has the polypeptide of affinity suitably to described pollutent, and will be attached to described pollutent when being with near described pollutent.Affinity partly comprises the acceptor that described pollutent is had specific antibody, can make the ligand on the described pollutent of receptors bind and be attached to described pollutent.Except following illustrative to useful those affinities part of method described here, it will be also suitable partly that the different pollutent that can reasonably understand other has the affinity of affinity.Suitable antibody can use antibody source guide to identify, for example uses " Linscott immunity and biological reagent catalogue " (Linscott ' s Directory ofImmunological and Biological Reagents) or " manufacturers's specification and with reference to outline-main antibody catalogue " (MSRS Catalog of Primary Antibodies) to identify.The method that is used to produce mono-clonal and polyclonal antibody is known by those of ordinary skill in the art.

The step of described method can be finished with several different order.For example, described antibody can be coupled to described magnetic nano-particle before the described starting material of contact, or described antibody can add after described pollutent-antibody target forms.Selectively, described antibody and described magnetic nano-particle can the described starting materials that adds to of while.

Biosensor can be used for detecting and the described pollutent of quantitative described starting material.Suitable biosensor includes but not limited to impedance biosensor, QCM (Quartz Crystal Microbalance) biosensor and visco-elasticity biosensor, such as the U.S. 11/329 that at title is " method of the concentration of the types of unknown pollutants in the detection material " (Method for Detecting an Unknown ContaminantConcentration in a Substance), No. 009 patent application (U.S.PatentApplication No.11/329,009) description person in, the full content of described patent is merged in this patent at this by reference.Suitably, biosensor described here is used for detecting and the described pollutent of quantitative described starting material.

Influenza and Avian pneumo-encephalitis virus can the aggegation red corpuscle, to form virus-red corpuscle mixture.The contriver describes by detect the method for the virus in the described starting material with red corpuscle contact starting material.Described virus in the described starting material can be attached to described red corpuscle, and described red corpuscle-viral mixture can then detect with biosensor.Suitably, impedance biosensor is used to detect described virus-red corpuscle mixture, and more suitably, impedance biosensor described here is used to detect described virus-red corpuscle mixture.Described virus-red corpuscle mixture can further contact with antibody, so that provide specificity to described biosensor.When being used for biosensor described here, described antibody coupling is to magnetic nano-particle.As previously discussed, the step of described method can be carried out with different order.For example, described virus can be incorporated into described affinity part-nano-particle compound, cultivates described virus with red corpuscle then or with described virus culture red corpuscle.In immobilized impedance biosensor based on antibody, described virus can be compound with red corpuscle before adding described biosensor to, or compound with red corpuscle after described virus is partly captured by described affinity in described biosensor.

In the specific embodiment of Figure 16 A, magnetic nano-particle is coated with special viral antibody, and is used for target virus is separated from poultry swab sample.Red corpuscle as biomarker can form described nanoparticle-virus-red corpuscle mixture with viral mixing of the target of being captured.Microflow controlled biochip design and be made into the equipment of flowing through so that described mixture is delivered to the interdigitated array microelectrode of embedding, is made for impedance measurement.The impedance variations of described nanoparticle-virus-red corpuscle mixture and the concentration of the avian influenza virus H 5 N 1 in the original sample are interrelated.

The usefulness of following examples is intended to illustration the present invention, rather than is intended to limit patent right requirement of the present invention.

Embodiment

Embodiment 1-material and method

In all following embodiment, following material and method have been used, except as otherwise noted.

Cultivation of bacterium and surface seeding (Culture and plating of bacteria)

Refrigeration stock Escherichia coli O 157: H7 (ATCC 43888) under-70 ℃ of temperature condition brain-heart soak the collection liquid substratum (brain-heart of Kan. Le Nasa city Remel Inc. soak the collection liquid substratum-BHI, Remel Inc., Lenexa keeps in Kansas).Brain-the heart of described culture after maintaining 37 ℃ of temperature 18 to 22 hours soaks collection in the liquid substratum of collection (BHI).In order to enumerate, pure growth is at 0.01M, serial dilution in the phosphate buffered saline buffer of pH 7.4 (PBS), and surface seeding is at sorbitol-MacConkey agar (the sorbitol MacConkey that cultivates 20 to 22 hours under 37 ℃ of temperature condition, SMAC agar) (U.S. Remel Inc. company, Lenexa, Kansas) on.

Avian influenza virus

Avian influenza virus (H5N1) gets by cultivate and gather allantoic fluid in Embryo Gallus domesticus, and is slaughtered by the American National veterinary service laboratory that is positioned at A Meisi city, U.S. Iowa (Iowa) (Ames) (National Veterinary Services Laboratory).Described viral stock solution contains about 1 * 10 ⁷Every milliliter of egg infective dose (EID) (EID/ml).

Chemical and reagent

(St. Louis city-St.Louis Missouri) obtains phosphate buffered saline buffer (PBS) (0.01M, pH 7.4) from Sigma-Aldrich company.1.0%wt vol ^-1Bovine serum albumin (BSA) (the EM Science company-Gibbstown in N.J. gibbs soup city, New Jersey) in phosphate buffered saline buffer (PBS), be prepared into the blocking-up damping fluid (PBS BSA).(St. Louis city-St.Louis Missouri) obtains albumin A (deriving from streptococcus aureus Cowan strain cell wall) from Sigma-Aldrich company.(St. Louis city Sigma-Aldrich company-St.Louis Missouri) is used to washing and suspension bacterium and virus, and is used to wash electrode the mannitol solution that places de-ionized water of 1/10 moles (0.1M).All solution is all so that (Massachusetts, United States Bedford city-Bedford, de-ionized water MA) (Milli-Q, 18.2 M Ω cm) prepares from Millipore company.

Nanoparticle and antibody

(mean diameter is 145nm, 0.5mg Fe ml with streptavidin bonded magnetic nano-particle ^-1) (Molecular Probes, Inc.) (Eugene, OR, USA city-Eugene Oregon) obtains from " molecular probe company ".Magnetic nano-particle contains and surpasses 85% oxide compound such as Z 250 (Fe ₃O ₄), about 80%wt wt ^-1Magnetite, and about 4 * 10 ¹¹Every milligram of (particles mg of particle ^-1) iron.

(maine state of u.s.a Sa Ke city-Saco Maine) obtains from Biodesign International company with the polyclonal antibody (O and K antigen are had specificity) of vitamin H bonded, the colibacillary affinity purification of opposing.The concentration of the stock solution of biotin labeling antibody is 4 to 5mg ml ^-1Before use, described antibody was by Dilution ratio dilution in phosphate buffered saline buffer (PBS) (0.01M, pH 7.4) in 1: 10.

The rabbit antibody (after this be called for short " antihemagglutinin (anti-HA) antibody ") of opposing influenza A H5N1 hemagglutinin (HA) buy from Biodesign International company (maine state of u.s.a Sa Ke city-Saco, Maine).Cultivate the antibody of this affinity purification on one's body rabbit, with to the synthetic peptide (Genbank reception no.AAT76166) of 14 seed amino acids antagonism in the district in proteic corresponding to hemagglutinin.

The immunomagnetic isolation of bacterium and concentrated

Magnetic nano-particle antibody complex (MNAC) prepares in the aseptic polypropylene centrifuge tube of 1.7ml.The magnetic nano-particle (15 μ l) that biotin labeling polyclone goat-anti intestinal bacteria antibody (7.5 μ l) and streptavidin apply (coating) in the PBS BSA of 250 μ l at ambient temperature, (Maryland, USA Lao Leier city ATR company-Laurel Maryland) upward, mixed continuously 35 minutes with the velocity of rotation of 7rpm at the speed change rot.After antibody immobilization, magnetic nano-particle antibody complex (MNAC) mixed 15 minutes with the biotin solution (in PBS BSA) of 150 μ l, and is lip-deep not in conjunction with streptavidin so that blocking-up is present in magnetic nano-particle.Unnecessary vitamin H washes with PBS-BSA, and magnetic nano-particle antibody complex (MNAC) suspends in the PBS BSA of 450 μ l then.

The pure growth of Escherichia coli O 157: H7 is from 8.4 * 10 ²To 8.4 * 10 ⁸CFU ml ^-1Serial dilution in phosphate buffered saline buffer (PBS) (0.01M, pH 7.4), carry out.The aliquot pure growth of 50 μ l mixes with the magnetic nano-particle antibody complex (MNAC) of 450 μ l, when its immune response is 15 minutes.After described immune response, nanoparticle-bacterium mixture concentrates in the mannitol solution of 100 μ l then with the have a rest mannitol solution washing three times of 0.1M of magnetic resolution of interband.At last, will be attached to the Escherichia coli O 157 of the nanoparticle antibody complex (MNAC) that is suspended in the mannitol solution with the flow rate of 10 μ l/min with syringe pump: the H7 injection cell is to described stream unit.Virus detects the similar program of also following.

The biotinylation of H 5 N 1 avian influenza antibody

According to manufacturer's explanation, (obtain to carry out the biotinylation of H 5 N 1 avian influenza antibody from Illinois, USA Rockford city (Rockford, Illinois) PIERCE company) with " EZ-Link Sulfo-NHS Biotinylation Kit ".Concise and to the point speech, antihemagglutinin (anti-HA) antibody of 100 μ l and sulphonyl-NHS (N-hydroxy-succinamide) of 3 μ l-vitamin H (Sulfo-NHS-Biotin) solution (10mM) are mixed into phosphate buffered saline buffer (the PBS) (10mM of 200 μ l, pH 7.4) in, cultivated at ambient temperature then 60 minutes.Then, by using Slide-A-Lyzer dialysis card (Slide-A-Lyzer Dialysis Cassettes) that unnecessary vitamin H is removed.With 2-(4 '-hydroxyazobenzene) phenylformic acid (2-(4 '-Hydroxyazobenzene) Benzoic Acid, HABA) experimental measurement vitamin H bonded level is measured as every mole of antibody of 4 to 5 moles of vitamin Hs.

Magnetic nano-particle combines with antihemagglutinin (anti-HA) antibody

Biotin labeling H 5 N 1 avian influenza antibody (antihemagglutinin (anti-HA) antibody; 50 μ l) magnetic nano-particle (20 μ l) that applies with streptavidin in the phosphate buffered saline buffer (PBS) of 50 μ l at ambient temperature, (Maryland, USA Lao Leier city ATR company-Laurel Maryland) upward, mixed continuously 40 minutes with the velocity of rotation of 15rpm at the speed change rot.After antibody immobilization, the biotin solution of magnetic nano-particle and 100 μ l (1mg/ml is in phosphate buffered saline buffer (PBS)) was mixed 20 minutes, so that blocking-up is present in streptavidin unnecessary on the magnetic nano particle sub-surface.Unnecessary vitamin H is by the magnetic resolution eccysis.

The separation and the spissated magnetic nano-particle antibody conjugates that are used for avian influenza virus

Make and ten times of serial dilution degree (10 ^-2, 10 ^-3, 10 ^-4And 10 ^-5) the inactivation bird flu H 5 N 1 (original titre is 1 * 10 ⁷± 1log EID ₅₀/ ml) mix, from the cloaca swab specimen suspension of poultry the damping fluid of 500 μ l (the band Vitrum AB etc. ooze dextrose: every liter of distilled water of 60g dextrose), and use virus-free cloaca swab sample in contrast.Then, the nanoparticle that the swab sample of described 500 μ l and antibody apply (applying with described antihemagglutinin (anti-HA) antibody) mixes, when its immune response is 30 minutes.After described immune response, nanoparticle-viral mixture suspends in the mannitol solution of 150 μ l, 0.1 M, to carry out impedance measurement then with the have a rest mannitol solution washing of 300 μ l, 0.1M of magnetic resolution of interband.Unless otherwise indicated, the swab sample all is to contain 10^5 EID ₅₀The formulations prepared from solutions of/ml titre virus.

The preparation of swab sample

From the cloaca of poultry and swab sample and ten times of serial dilution degree (10 of tracheae ^-2, 10 ^-3, 10 ^-4And 10 ^-5) the inactivation bird flu H 5 N 1 (original titre is 1 * 10 ⁷± 1 logEID ₅₀/ ml) mix.Described swab is suspended in the damping fluid (wait and ooze dextrose) of 2ml, and the swab of not viruliferous cloaca of use or tracheae in contrast.

The immobilization program

For some experiments, be necessary to use following program, come the described microelectrode of precoating with the antibody of resisting bird flu H 5 N 1, so that on described microelectrode, make viropexisization, for analyzing subsequently.At first, (1mg/ml is at 10mM, in the phosphate buffered saline buffer of pH 7.4 (PBS)) is expelled to described biosensor with albumin A, cultivates at ambient temperature then about 1.5-2 hour.Then, further regulate described microelectrode with the antibody (cultivating at ambient temperature 2.5 hours) of opposing bird flu H 5 N 1.After with bovine serum albumin (BSA) (1%, at 10mM, in the phosphate buffered saline buffer of pH 7.4 (PBS), cultivated at ambient temperature 30 minutes) blocking-up, described chip can be for carrying out the swab sample testing.After each immobilization step, wash described electrode with the mannitol solution of 0.1M, carry out impedance measurement then.

Impedance measurement

With IM-6 impedance analysis device (Lafayette city, west, U.S. Indiana State Bioanalytical Systems Inc. company-West Lafayette, Indiana) the execution impedance measurement that has IM-6/THALES software.For all impedance measurements, described interdigitated array microelectrode (IDAM) has been applied the Sine Modulated alternating-current of 100mV voltage, and be size and phase angle that 10Hz to 1MHz range of frequency is measured impedance.Use the magnetic nano-particle antibody complex (MNAC) tested, from the detection total time that is sampled to measurement be 35 minutes (time that is used for immune response, washing and measurement was respectively 15,10,10 minutes).

In some embodiment (experiment of data presented in comprising corresponding to Figure 28 A and 29A), use portable handheld device, such as above-mentioned impedance biosensor 20, come image data.In a particular embodiment, described handheld device is in the frequency collection data of single-frequency rather than successive range.Described single-frequency is suitable between 1Hz to 1MHz, and is more suitable between 100Hz to 10kHz, and especially suitable between 1kHz to 10kHz.

An electrode of described interdigitated array microelectrode (IDAM) chip is connected to test probe and pickup probe, and another electrode then is connected to the reference electrode and the counter electrode of described impedance analysis device.When each end of test (EOT), described stream unit is with the sodium hydroxide solution washing 1 hour of 0.1M, with deionization water washing 30 minutes, with the salt acid elution 1 hour of 0.1M and at last with de-ionized water flushing 1 hour.The flow rate of using during the washing is 10 μ l/min.The mannitol solution that contains magnetic nano-particle antibody complex (MNAC) but do not contain Escherichia coli O 157: H7 uses in all tests in contrast.

Frequency when determining that band adheres to the bacterial cell sample of magnetic nano-particle antibody complex (MNAC) and the impedance measurement maximum difference between the control group has been drawn the graphic representation of normalized impedance variation (NIC) to frequency.Normalized impedance changes the value of (NIC) and obtains according to following formula (1):

Z _sample-Z _control

$\mathrm{NIC}=\frac{Z_{\mathrm{Sample}}-Z_{\mathrm{control}}}{Z_{\mathrm{control}}}\×100---\left(1\right)$

Z wherein _ControlBe the impedance magnitude of control group, and Z _SampleFor containing Escherichia coli O 157: the impedance magnitude of the sample of H7.Also, other carry out convertible calculating for comprising the sample of viral sample.

The preparation of hamburger sample

Hamburger is buied from local supermarket.Use laboratory Patting type homogenizer Stomacher 400 (prefecture, Britain Norfolk Seward company-Norfolk, UK), in the Whirl-pak plastics bag with 0.1% buffered peptone water of the 225ml heavy hamburger sample of 25g that homogenizes, for the time 2 minutes.After homogenate, with whizzer with 250 * g with described sample centrifuging 15 minutes, totally twice, so as to separate exist in the hamburger homogenate water than macroparticle.The supernatant layer of described food sample with ten times of dilutions between 7.9 * 10 ²To 7.9 * 10 ⁸CFUml ^-1Between Escherichia coli O 157: H7 culture inoculation.

Embodiment 2-Escherichia coli O 157: the impedance measurement of H7

Figure 17 is a Bode diagram, and its diagram is corresponding to being attached with 8.4 * 10 of magnetic nano-particle antibody complex (MNAC) ⁷CFU ml ^-1Escherichia coli O 157: H7 and contrast (contrast a carrying magnetic nanoparticle antibody complex (MNAC) but not with bacterium) record impedance.With sample injection to the micro-fluidic unit that comprises the non-marked biosensor that is used to measure impedance.In the N.F,USP MANNITOL of 0.1M, carry out impedance measurement.Resistance difference (normalized impedance with Figure 18 changes, NIC, expression) shows, shows ascendant trend (from 0.22% to 61%) in the frequency zones of 53Hz to 16kHz, then shows downtrending (61% to 6%) in the frequency zones of 16kHz to 1MHz.The maximum difference of impedance measurements occurs in the medium resistance district (1kHz to 50kHz) that seems big, and culminate at 16kHz (as shown in Figure 18).

Embodiment 3-detect impedance biosensor, relate to the Escherichia coli O 157 in pure growth and the hamburger sample: the limit of detection of the detection of H7

Figure 19 A, 19B, 19C and 19D show be present in pure growth and the hamburger sample, between 10 ¹To 10 ⁷CFUml ^-1Between the Escherichia coli O 157 of all concentration: H7 records impedance (using the non-marked biosensor to measure).Experiment finds, the impedance that is caused by bacterium is along with the cell number in the described sample increases linearly.Bacterium is 10 in the concentration of pure growth just to the polarization and the insulating effect on the surface of described biosensor ⁵CFUml ^-1Or when higher and in concentration be 10 with hamburger blended sample ⁶CFUml ^-1Or when higher, just begin to change impedance.The snapshot of the impedance when Figure 19 B and 19D are presented at frequency and are 16kHz, and real example in the concentration of pure growth 10 ⁵To 10 ⁷CFUml ^-1Between and the concentration of hamburger sample 10 ⁶To 10 ⁷CFUml ^-1Between the time, have resistance difference detectable, that have statistical significance between described control sample and pure growth and the hamburger sample really.

The impedance biosensor that embodiment 4-uses the antibody on it to be immobilized carries out the detection of avian influenza virus

The swab that may contain avian influenza virus (50-100nm diameter), non-avian influenza virus and other molecules by dipping to etc. ooze dextrose damping fluid, preparation poultry swab sample.Then, add 0.5% red blood cell suspension of 1ml to described swab sample solution, and mixed 1-5 minute, to form red corpuscle-viral mixture.Influenza virus and Avian pneumo-encephalitis virus can the aggegation red corpuscle, form virus-red corpuscle mixture.

The swab sample that will have described mixture is sent into microfluidic channel (dark 40 μ m, wide 100 μ m, long 10mm).Interdigitated array microelectrode (electrode finger widths and the electrode gap between referring to is all 15 μ m) is embedded described microfluidic channel, to be coated with the above interdigitated array microelectrode with albumin A bonded avian influenza virus polyclonal antibody then, produce " immobilization " described here biosensor.By described microfluidic channel the time, described avian influenza virus-red corpuscle mixture is captured by described immobilized avian influenza virus antibody (plain (anti-HA) antibody of anti-hemocyte cohesion), causes impedance to change.Apply the mannitol solution of 0.1M, so that the non-bird flu molecule that before measuring impedance loosely is adhered to is washed off.

Use the impedance analysis device, the frequency measurement impedance between 1Hz and 1MHz.Measuring result is described in Figure 20, and measuring result proves that described virus-red corpuscle mixture causes considerable impedance to increase.Figure 21 proves that avian influenza virus does not cause considerable impedance variations by oneself, causes considerable impedance variations but add red corpuscle to described biosensor after adding described virus.

Embodiment 5-uses the magnetic nano-particle antibody complex, detects avian influenza virus with impedance biosensor

Magnetic nano-particle (MNACs) is coupled to has specific polyclonal antibody (anti-hemocyte cohesion plain (anti-HA) antibody) to avian influenza virus.In described automatic magnetic sample, use described nanoparticle-antibody complex, so that from poultry swab sample, separate and enriched target virus.As described here, will be used as the red corpuscle that biomarker uses and the target of being captured virus is mixed, formation affinity part-coated nanoparticles-virus-red corpuscle mixture.As previously discussed, follow the impedance biosensor that described mixture is delivered to non-marked, passes through, to carry out impedance measurement.Measure the impedance variations of described affinity part-coated nanoparticles-virus-red corpuscle mixture.

As shown in Figure 22, use be attached to avian influenza virus H 5 N 1, coupling has hemocyte to condense the magnetic nano-particle (MNACs) of the polyclonal antibody of plain specificity (HA-specific), detection avian influenza virus H 5 N 1 it on.In described accompanying drawing, " N.F,USP MANNITOL " singly refers to damping fluid, and " virus " refers to have the sample of described nanoparticle-affinity part-viral mixture.

Embodiment 6-is to the specificity of bird flu

The composition of Avian pneumo-encephalitis virus and infectious bronchitis virus is mixed with the ratio of avian influenza virus H 5 N 1 with 1: 1.Then, cultivate described virus mixture with red corpuscle alternatively, in having the microfluidic channel that embeds interdigitated array microelectrode (described specific antibody is immobilization thereon), measure impedance then.Use has the IM-6 impedance analysis device (immobilized antibody impedance biosensor) of IL-6/THALES software, and (U.S. Indiana State west Lafayette city Bioanalytical Systems Inc. company-West Lafayette Indiana) carries out impedance measurement.

As previously discussed, in the different steps that applies described fixing of biosensor antibody with reagent, carry out impedance measurement.In Figure 23 A and 23B, the impedance of the described empty biosensor of " naked gold (bare) " expression, " albumin A " is illustrated in and adds albumin A impedance afterwards, " anti-hemocyte cohesion plain (anti-HA) " impedance measurements when the described biosensor of expression is coated with the above antibody, " bovine serum albumin (BSA) " is illustrated in the impedance after the washing of PBS-BSA blocking-up damping fluid, " new city eqpidemic disease/infectious bronchitis+H5N1+ red corpuscle (NCD/IB+H5N1+RBC) " is illustrated in and adds described virus mixture and red corpuscle impedance (Figure 23 A) afterwards, " new city eqpidemic disease/infectious bronchitis+H5N1 (NCD/IB+H5N1) " is illustrated in and adds after the described virus mixture but the impedance (Figure 23 A) before adding red corpuscle, and " red corpuscle (RBC) " is illustrated in and adds the impedance of adding again after the described virus after the red corpuscle.

In Figure 23 A,, make described virus mixture then by described impedance biosensor with the described virus mixture of the pre-cultivation of red corpuscle.The result shows that even exist under the situation of Virus Pollution, described mixture can produce considerable impedance variations.

In Figure 23 B, before adding red corpuscle, make described virus mixture by described impedance biosensor, after capturing described virus, described biosensor adds red corpuscle then.Figure 23 B proves that described immobilization biological transmitter can detect the avian influenza virus in the hybrid virus sample, and erythrocytic interpolation has enlarged impedance variations.Significantly, even after adding red corpuscle, do not monitor the signal of Avian pneumo-encephalitis virus or infectious bronchitis virus.Embodiment 7-is through the detection of the virus in the tracheae swab after the centrifugation

Use standard program, obtain the tracheae swab from chick, and prepare tracheae swab sample as stated above.The grade that described swab is flushed to 2ml is oozed in the dextrose damping fluid, and with the speed of rotation centrifuging of 2000rpm 30 minutes.Then with 0.5% red blood cell suspension (grade of band Vitrum AB is oozed the dextrose damping fluid) of 1ml, under 4 ℃ of temperature condition, cultivate from the supernatant layer of described lotion 40 minutes, carry out then another take turns for the time 10 minutes velocity of rotation be the centrifuging of 1500rpm.The grade that settling is suspended in 0.5ml is oozed in the dextrose damping fluid, is used for impedance analysis.

By coating biosensor surface with albumin A, then coat biosensor surface with plain (anti-HA) antibody of anti-hemocyte cohesion, with the PBS-BSA blocking-up, described immobilization impedance biosensor is prepared then.The result shows in Figure 25 A and 25B.Figure 25 A is presented at not the impedance when adding virus to described tracheae swab.Impedance when Figure 25 B show to add virus to described tracheae swab.The existence of the virus in the described swab has increased impedance effectively.

Embodiment 8-is through the detection of the virus in the tracheae swab after the magnetic resolution

Use standard program, obtain the tracheae swab from chick, and prepare tracheae swab sample as stated above.The grade that described swab is flushed to 2ml band Vitrum AB is oozed in the dextrose damping fluid.Then, plain (anti-HA) antibody complex of anti-hemocyte cohesion that connects with the nanoparticle that produces with aforesaid method is cultivated " contrast " sample.Append to " tracheae swab+H5N1 " sample with H5N1 virus.Described tracheae swab is immersed 10 ⁵EID ₅₀The avian influenza virus solution of/ml, the grade that is flushed to 2ml is then oozed in the dextrose damping fluid.Final virus concentration is estimated as 10 ⁴-10 ⁵EID ₅₀/ ml.Then synthetic virus-nanoparticle-antibody complex is magnetically separated with the method for having narrated.The result of described non-marked impedance biosensor describes in Figure 26.Described result shows, compares with contrast swab sample, and when having avian influenza virus H 5 N 1 in described tracheae swab sample, impedance obviously increases.

Embodiment 9-is through the detection of the virus in the cloaca swab after the centrifugation

Use standard program, obtain the cloaca swab from chick, and prepare cloaca swab sample as stated above.The grade that will be flushed to 2ml with or without the described swab of virus is oozed in the dextrose damping fluid, and with the speed of rotation centrifuging of 2000rpm 30 minutes.For those samples that adds virus, the cloaca swab is dipped into 10 ⁵EID ₅₀The avian influenza virus solution of/ml, the grade that is flushed to 2ml is then oozed in the dextrose damping fluid.Then with 0.5% red corpuscle (grade at the band Vitrum AB is oozed in the dextrose damping fluid) of 1ml, under 4 ℃ of temperature condition, cultivate from the supernatant layer of described lotion 40 minutes, carry out then another take turns for the time 10 minutes velocity of rotation be the centrifuging of 1500rpm.The grade that settling is resuspended in 0.5ml is oozed in the dextrose damping fluid, is used for impedance analysis.

As previously discussed,, then coat biosensor surface, with the PBS-BSA blocking-up, described immobilization impedance biosensor is prepared at last with plain (anti-HA) antibody of anti-hemocyte cohesion by coating biosensor surface with albumin A.The result shows in Figure 27, and to use the tracheae swab to obtain the result similar.Figure 27 A is presented at not the impedance when adding virus to described cloaca swab.Impedance when Figure 27 B show to add virus to described cloaca swab.The existence of the virus in the described swab has increased impedance significantly.

The susceptibility of embodiment 10-immobilization impedance biosensor

Use standard program, obtain the cloaca swab, and prepare cloaca swab sample by the avian influenza virus H 5 N 1 that makes described swab be exposed to different concns from chick.Described cloaca swab is immersed a series of ten times of dilution avian influenza virus solution, and the grade that then described cloaca swab is flushed to 2ml is oozed the dextrose damping fluid.The grade that described swab is flushed to the band Vitrum AB of 2ml is oozed in the dextrose damping fluid, uses whizzer with the speed of rotation centrifuging of 2000rpm 30 minutes then.Then with 0.5% red corpuscle (grade at the band Vitrum AB is oozed in the dextrose damping fluid) of 1ml, under 4 ℃ of temperature condition, cultivate from the supernatant layer of described lotion 40 minutes, carry out then another take turns for the time 10 minutes velocity of rotation be the centrifuging of 1500rpm.The grade that settling is suspended in the band Vitrum AB of 0.2ml is oozed in the dextrose damping fluid, is used for impedance analysis.

As previously discussed,, then coat biosensor surface, with the PBS-BSA blocking-up, described immobilization impedance biosensor is prepared then with plain (anti-HA) antibody of anti-hemocyte cohesion by coating biosensor surface with albumin A.The result shows in Figure 28.Figure 28 A display frequency is 10, the bar graph of the impedance data that records during 400Hz.Described data presentation, along with the viral dilution degree drops at 1/10,000 o'clock from 1/100,000, or about 10 ³EID ₅₀/ ml, impedance obviously increases.This susceptibility is enough to be used in the on-the-spot test of infected poultry.The impedance variations of the different dilution viruses when Figure 28 B then is presented at certain range of frequency test.

The susceptibility of embodiment 11-non-marked impedance biosensor

Use standard program, obtain the cloaca swab, and prepare cloaca swab sample by the avian influenza virus H 5 N 1 that makes described swab be exposed to different concns from chick.Described cloaca swab is immersed a series of ten times of extent of dilution, the avian influenza virus solution of ultimate density 1/100,1/1000,1/10000 and 1/100000, the grade that then described cloaca swab is flushed to 2ml is oozed the dextrose damping fluid.Then, plain (anti-HA) antibody complex of anti-hemocyte cohesion that connects with the nanoparticle that produces with aforesaid method is cultivated described swab.Then cultivate described synthetic virus-nanoparticle-antibody complex, with aforesaid method described synthetic virus-nanoparticle-antibody complex is magnetically separated then with 0.5% red blood cell suspension (grade at the band Vitrum AB is oozed in the dextrose damping fluid) of 1ml.

The detected result of described non-marked impedance biosensor is described in Figure 29 A and 29B.Figure 29 A display frequency is 4, the bar graph of the impedance data that records during 150Hz.Described data presentation, along with viral dilution to 1/100,000 o'clock, or about 10 ²EID ₅₀During/ml, impedance obviously increases.This susceptibility is enough to be used in the on-the-spot test of infected poultry.The impedance variations of the different dilution viruses when Figure 29 B then is presented at certain range of frequency test.

Embodiment 12-uses the non-marked impedance biosensor to detect avian influenza virus

Also use magnetic nano-particle-antibody complex described here and non-marked impedance biosensor to detect H 5 N 1 avian influenza.Concise and to the point speech, the nanoparticle that streptavidin is applied is linked to plain (anti-HA) antibody of biotinylated anti-hemocyte cohesion, and the mixture with avian influenza virus H 5 N 1 or Avian pneumo-encephalitis virus and infectious bronchitis virus adds described nanoparticle-antibody complex to then.Make described mixture by described impedance biosensor.The result describes in Figure 24, and the result shows that it can detect H5N1 virus clearly.Described result shows that also impedance is not subjected to the influence of the existence of non-target virus.

As shown in Figure 30, the concentration of the avian influenza virus H 5 N 1 in cloaca swab sample equals or exceeds 100EID ₅₀During/mL, obtain the positive signal of the existence of the described virus of indication.Figure 24 shows, when frequency is 10kHz, with (virus-free) control group or contain Avian pneumo-encephalitis virus and the sample of infectious bronchitis virus relatively, the impedance that contains the sample of avian influenza virus H 5 N 1 obviously increases.Described result shows that susceptibility is captured responsive 100 times of enzyme linked immunosorbent assay (ELISA) than existing antigen.The maximum resistance variation of 100 to 800k Ω betides the range of frequency of 1kHz to 100kHz.

Embodiment 13-uses magnetic nano-particle and detects avian influenza virus with use non-commercialization antibody immobilization impedance biosensor

Use resists-H5 antibody at the non-commercialization polyclone that rabbit produces on one's body, carries out another papova test experience.Use the standard test scheme, reorganization H5 polypeptide (is obtained from Connecticut, USA Protein Science Inc. company-Meriden, Connecticut; A/Vietnam/1203/2004) cultivate described antibody.Anti--H5 antibody from described rabbit passes through the ammonium sulfate precipitation purifying, to acetate buffer and phosphate buffered saline buffer dialysis, uses for biosensor then.

In some experiment (referring to the result shown in Figure 31 A-31D), described antibody chain is received and bird flu H 5 N 1 particle bonded magnetic nano-particle.Described antibody labeling nanoparticle is combined with the H5N1 virus of different concns (or only to be combined with damping fluid, in contrast), further handled before being applied to the non-marked biosensor then.In other experiment, (the results are shown in Figure 31E), coat described interdigitated array microelectrode (IDAM) biosensor that is arranged in micro-fluidic chamber, produce " immobilization " biosensor with described antibody.

The described H5 type of biotinylation antibody is so that be attached to the nanoparticle that streptavidin applies.According to manufacturer's explanation, carry out the biotinylation of anti--H5 antibody with " EZ-Link Sulfo-NHS Biotinylation Kit ".Concise and to the point speech, antihemagglutinin (anti-HA) antibody (4.9mg/ml) of 100 μ l and the sulphonyl of 3 μ l-N N-Hydroxysuccinimide-vitamin H (Sulfo-NHS-Biotin) solution (10mM) are mixed into phosphate buffered saline buffer (the PBS) (10mM of 200 μ l, pH 7.4) in, cultivated at ambient temperature then 60 minutes.Then, by using Slide-A-Lyzer dialysis card (Slide-A-Lyzer Dialysis Cassettes) that unnecessary vitamin H is removed.With HABA (2-(4 '-hydroxyazobenzene) phenylformic acid) experimental measurement vitamin H bonded level, be measured as every mole of antibody of 4 to 5 moles of vitamin Hs.

Biotin labeling is anti--magnetic nano-particle (20 μ l) that H5 antibody (50 μ l) and streptavidin apply in the phosphate buffered saline buffer (PBS) of 50 μ l at ambient temperature, (Maryland, USA Lao Leier city ATR company-Laurel Maryland) upward, mixed continuously 40 minutes with the velocity of rotation of 15rpm at the speed change rot.After antibody immobilization, the biotin solution of magnetic nano-particle and 100 μ l (1mg/ml is in phosphate buffered saline buffer (PBS)) was mixed 20 minutes, so that blocking-up is present in streptavidin unnecessary on the magnetic nano particle sub-surface.Unnecessary vitamin H is by the magnetic resolution eccysis.

Make ten times of serial dilutions (10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5And 10 ^-6) the inactivation bird flu H 5 N 1 (its original titre is 1 * 10 ⁷± 1log EID ₅₀The dilution of the solution of/ml) be suspended in the damping fluid (wait and ooze dextrose) of 500 μ l, and use does not contain viral damping fluid in contrast.Then, the nanoparticle that the sample of described 500 μ l and antibody apply mixes, when its immune response is 30 minutes.After described immune response, nanoparticle-viral mixture suspends in the mannitol solution of 150 μ l, 0.1M, to carry out impedance measurement then with the have a rest mannitol solution washing of 300 μ l, 0.1M of magnetic resolution of interband.

Use albumin A, make described antibody be attached to described biosensor.At first, (1mg/ml is at 10mM, in the phosphate buffered saline buffer of pH 7.4 (PBS)) is expelled to described micro-fluidic chamber with albumin A, cultivates at ambient temperature then 1.5-2 hour.Then, further regulate described microelectrode with anti--H5 antibody (245 μ g/ml) (cultivating at ambient temperature 2.5 hours).After with bovine serum albumin (BSA) (1%, at 10mM, in the phosphate buffered saline buffer of pH 7.4 (PBS), cultivated at ambient temperature 30 minutes) blocking-up, described chip can be for carrying out sample testing.After each immobilization step, wash described electrode with the mannitol solution of 0.1M, carry out impedance measurement then.

With IM-6 impedance analysis device (Lafayette city, west, U.S. Indiana State Bioanalytical Systems Inc. company-West Lafayette, Indiana) the execution impedance measurement that has IM-6/THALES software.For all impedance measurements, described interdigitated array microelectrode (IDAM) has been applied the Sine Modulated alternating-current of 100mV voltage, and be size and phase angle that 10Hz to 1MHz range of frequency is measured impedance.

The result of impedance measurement shows in Figure 31 A-31E.Figure 31 A-31C shows three independently measurements to sample, and these are measured and compare 10 ⁵EID ₅₀/ ml virus (" H5N1 "), washings (" N.F,USP MANNITOL ") or only contain etc. oozes the impedance of the control sample (" contrast ") of dextrose damping fluid.Figure 31 D show to use with the sample (mark " 10^1EID50/ml " is to " 10^5EID50/ml ") of above-mentioned viral dilution series preparation and the described comparison of oozing the impedance that dextrose damping fluid (" contrast ") records such as virus-free.Figure 31 E is presented at after each step of described antibody labeling program, in the impedance measurement of on the immobilization biological transmitter, carrying out under the situation of not using nanoparticle.

Claims (52)

1, a kind of impedance biosensor that is used for detecting the pollutent of starting material, described biosensor comprises:

Seat board;

Input unit, described input unit is by described seat board support;

Output equipment, described output equipment is by described seat board support;

Micro-fluidic unit, described micro-fluidic unit are by described seat board support, and described starting material can engage with described micro-fluidic unit; And

Impedance analysis device, described impedance analysis device be by described seat board support, and can operate the impedance of measuring described starting material, so that the existence of detection of contamination.

2, the impedance biosensor described in claim 1 further comprises interdigitated array microelectrode, and described interdigitated array microelectrode is connected to described impedance analysis device.

3, the impedance biosensor described in claim 2, wherein said interdigitated array microelectrode is disposed in the described micro-fluidic unit.

4, the impedance biosensor described in claim 1, wherein said impedance biosensor is a handheld device.

5, the impedance biosensor described in claim 1, wherein said impedance biosensor is a portable equipment.

6, the impedance biosensor described in claim 1 further comprises injection port, and described injection port fluidly engages with described micro-fluidic unit, and described starting material can be introduced described biosensor by described micro-fluidic unit.

7, the impedance biosensor described in claim 6, comprise that further the waste material storing chamber used starting material with storage, described waste material storing chamber fluidly engages with described micro-fluidic unit, and described starting material flows to described waste material storing chamber from described micro-fluidic unit.

8, a kind ofly be used for detecting and the impedance biosensor of the pollutent of quantitative starting material, described biosensor comprises:

Injection port, described starting material can be introduced described biosensor by described injection port;

Micro-fluidic unit, described micro-fluidic unit comprises:

Inlet,

Outlet,

Microchannel, described microchannel are extended between described inlet and described outlet and are comprised sensing chamber, and

A plurality of microelectrodes, described a plurality of microelectrodes are positioned at described sensing chamber,

Wherein said starting material is introduced described micro-fluidic unit by described inlet, and from described inlet flow through described microchannel and described sensing chamber, flows out described micro-fluidic unit by described outlet then;

The waste material storing chamber is used for storing and has used starting material, and described starting material flows to the waste material storing chamber from described micro-fluidic unitary outlet;

Discharge outlet, described discharge outlet fluidly join described waste material storing chamber to, and the described starting material that used discharges by described discharge outlet from described waste material storing chamber; And

The impedance analysis device can be operated described impedance analysis device and measure the impedance of the described starting material in the described sensing chamber, so that detect the existence of the pollutent in the described starting material and the amount of the pollutent in the quantitative described starting material.

9, a kind ofly be used for detecting and the portable impedance biosensor of the pollutent of quantitative starting material, described biosensor comprises:

Seat board, described seat board comprises first part and second section, described first part and described second section are movably connected together, so that determine the seat board cavity when linking together;

Keypad, described keypad is supported by the described first part of described seat board;

Indicating meter, described indicating meter is supported by the described first part of described seat board;

Battery socket, described battery socket are limited to the described second section of described seat board;

Lid, described lid can be connected to the described second section of described seat board movably on the position, so that cover described battery socket;

Injection port, described injection port is limited to described seat board, can described starting material be introduced described biosensor by described injection port;

Micro-fluidic unit, described micro-fluidic unit support by seat board in cavity, comprising:

Inlet,

Outlet,

Microchannel, described microchannel are extended between described inlet and described outlet and are comprised sensing chamber, and

A plurality of microelectrodes, described a plurality of microelectrodes are positioned at described sensing chamber,

Wherein said starting material is introduced described micro-fluidic unit by described inlet, and from described inlet flow through described microchannel and described sensing chamber, flows out described micro-fluidic unit by described outlet then;

The waste material storing chamber, described waste material storing chamber is limited by seat board, is used for storing having used starting material, and described starting material flows to the waste material storing chamber from described micro-fluidic unitary outlet;

Discharge outlet, described discharge outlet is limited by described seat board, and fluidly joins described waste material storing chamber to, and the described starting material that used discharges by described discharge outlet from described waste material storing chamber; And

The impedance analysis device, described impedance analysis device is supported in the cavity by described seat board, and can operate the impedance of measuring the described starting material in the described sensing chamber, so that detect the existence of the pollutent in the described starting material and the amount of the pollutent in the quantitative described starting material.

10, a kind of method that is used for detecting the pollutent of starting material, described method comprises:

A) partly contact described starting material to form target with the affinity that can be attached to described pollutent, wherein said affinity partly is coupled to magnetic nano-particle; And

B) detect described target with the biosensor described in the claim 1, the detection of wherein said target can be indicated the existence of the pollutent in the described starting material.

11, the method described in claim 10 further is included in before the step b) described target from described starting material magnetic resolution.

12, the method described in claim 10 further is included in before the step b) described target from described starting material centrifugation.

13, the method described in claim 10 further is included in step b) and by filtering described target is separated from described starting material before.

14, the method described in claim 10, wherein said affinity part by be coupled to described magnetic nano-particle and also can be attached to described magnetic nano-particle in conjunction with the joint of described affinity part.

15, the method described in claim 14, wherein said joint are selected from the group that comprises albumin A, Protein G, Fc acceptor and anti--Fc antibody.

16, the method described in claim 10, wherein said affinity part by select from comprise vitamin H-streptavidin, vitamin H-avidin 9 bletilla IgG-anti--the bridge joint mixture of the group of IgG, be attached to described magnetic nano-particle, the second section that the part of wherein said bridge joint mixture is coupled to described magnetic nano-particle and described bridge joint mixture is coupled to described affinity part.

17, the method described in claim 10, wherein said starting material is selected from the group that comprises food, animal, environmental sample and clinical sample.

18, the method described in claim 17, wherein said food select from comprise fruit, vegetables, without the group of processed food, cooked product, beef product, pork product, poultry prod, seafood prods and milk preparation.

19, the method described in claim 18, wherein said poultry prod is selected from the group that comprises carcass, carcass wash-down water sample, boneless chicken, broken poultry meat sample and poultry pie.

20, the method described in claim 17, wherein said environmental sample is selected from the group that comprises water sample, air sample and soil pattern.

21, the method described in claim 17, wherein said clinical sample select from comprise urine sample, blood sample, movement sample, from the swab of skin surface, the group of swab, cloaca swab, tracheae swab and tissue sample from organ surface.

22, the method described in claim 10, wherein said pollutent is selected from the group that comprises prokaryotic organism, eukaryote, virus and polypeptide.

23, the method described in claim 22, wherein said prokaryotic organism are selected from the group that comprises intestinal bacteria, Escherichia coli O 157: H7, salmonella and monocytosis Li Site bacterium.

24, the method described in claim 22, wherein said virus is selected from comprising all subclass of influenza virus, H 5 N 1 avian influenza, Avian pneumo-encephalitis virus and infectious bronchitis or the group of bacterial strain.

25, the method described in claim 22, wherein said polypeptide is selected from the group that comprises toxin and Protein virus.

26, the method described in claim 10 further comprises the quantity of determining the described pollutent in the described starting material.

27, a kind of method that is used for detecting the virus of starting material, described method comprises:

A) contact described starting material with red corpuscle, wherein said virus can be attached to described red corpuscle to form mixture; And

B) detect described mixture with biosensor, the detection of wherein said mixture can be indicated the existence of the described virus in the described starting material.

28, the method described in claim 27 comprises that further the affinity can be attached to described virus partly contacts described starting material, and wherein said affinity partly is coupled to magnetic nano-particle, to form magnetic target.

29, the method described in claim 27, wherein said virus is selected from the group that comprises influenza, H 5 N 1 avian influenza and Avian pneumo-encephalitis virus.

30, the method described in claim 27, wherein said starting material is selected from the group that comprises food, animal, environmental sample and clinical sample.

31, the method described in claim 27, the described biosensor that wherein is used to detect described target is an impedance biosensor.

32, the method described in claim 31, wherein said impedance biosensor are the biosensors described in the claim 1.

33, the method described in claim 27 further comprises the quantity of determining the described virus in the described starting material.

34, a kind of method that is used for detecting the pollutent of starting material, described method comprises:

A) partly contact described starting material to form target with the affinity that can be attached to described pollutent, wherein said affinity partly is coupled to magnetic nano-particle; And

B) detect described target with impedance biosensor, the detection of wherein said target can be indicated the existence of the pollutent in the described starting material.

35, the method described in claim 34, wherein said affinity partly are red corpuscle.

36, the method described in claim 34, wherein said affinity partly are antibody.

37, the method described in claim 34, wherein said impedance biosensor comprise interdigitated array microelectrode.

38, the method described in claim 37, wherein said interdigitated array microelectrode is configured in the micro-fluidic unit, and the step that detects described target further comprises described sample is delivered to described micro-fluidic unit.

39, the method described in claim 34 further is included in before the step b) described target from described starting material magnetic resolution.

40, the method described in claim 34 further is included in before the step b) described target from described starting material centrifugation.

41, the method described in claim 34 further is included in step b) and by filtering described target is separated from described starting material before.

42, the method described in claim 34, wherein said pollutent are bacterium or virus.

43, the method described in claim 34, wherein said pollutent is an avian influenza virus H 5 N 1.

44, the method described in claim 34, wherein said biosensor have the affinity part that is attached on it.

45, the method described in claim 34, wherein said affinity partly are not attached to described interdigitated array microelectrode biosensing device.

46, a kind of method that is used for detecting the pollutent of starting material, described method comprises:

A) partly contact described starting material to form target with the affinity that can be attached to described pollutent, wherein said affinity partly is coupled to magnetic nano-particle;

B) described target is separated from described starting material;

C) described target is delivered to micro-fluidic unit, disposes interdigitated array microelectrode biosensing device in the described micro-fluidic unit; And

D) detect described target with described interdigitated array microelectrode biosensing device, the existence of the pollutent in the described starting material is indicated in the detection of wherein said target.

47, the method described in claim 46 wherein comprises described target with described target from described starting material magnetic resolution from described starting material separation steps.

48, the method described in claim 46, wherein step a) further comprises with red corpuscle and contacts described starting material.

49, the method described in claim 46 further is included in step c) and contacts described target with red corpuscle before.

50, the method described in claim 46 further is included in step d) and before red corpuscle is delivered to described micro-fluidic unit.

51, the method described in claim 46, wherein said interdigitated array microelectrode biosensing device has the affinity part that is attached on it.

52, the method described in claim 51, wherein said affinity partly are antibody.

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