CN101156059B

CN101156059B - Multiplex fluorescence detection device having fiber bundle for connecting multiple optical modules to public detector

Info

Publication number: CN101156059B
Application number: CN2006800114519A
Authority: CN
Inventors: 威廉姆·拜丁汉姆; 彼得·D·陆德外斯; 巴里·W·罗博莱
Original assignee: 3M Innovative Properties Co
Current assignee: Soring Italian Ag
Priority date: 2005-04-01
Filing date: 2006-03-24
Publication date: 2011-06-08
Anticipated expiration: 2026-03-24
Also published as: CN101189505A; CN101189505B; ZA200709381B; ZA200709382B; CN101156059A

Abstract

Techniques are described for the detection of multiple target species in real-time PCR (polymerase chain reaction). For example, a system is described that includes a data acquisition device and a detection device coupled to the data acquisition device. The detection device includes a rotating disk having a plurality of process chambers having a plurality of species that emit fluorescent light at different wavelengths. The device further includes a plurality of optical modules. Each of the optical modules is optically configured to excite the species and capture fluorescent light emitted by the species at different wavelengths. A fiber optic bundle coupled to the plurality of optical modules conveys the fluorescent light from the multiple optical modules to a single detector.

Description

Has the multiplex fluorescence detection device that a plurality of optical modules is connected to the fibrous bundle of common detector

Technical field

The present invention relates to assay system, and relate more specifically to be used to use fluorescent dye to detect the technology of a plurality of target substances.

Background technology

Often use optical disk system to carry out various biological, chemistry or biochemical assay.In canonical system, use rotatable dish as the medium that is used to store with the handling of fluids sample, this fluid sample is blood, blood plasma, serum, urine or other fluids for example.

One type analysis is polymerase chain reaction (PCR), and this polymerase chain reaction (PCR) is often used in nucleic acid sequence analysis.Particularly, PCR is often used in dna sequencing, clone, gene mapping and other forms of nucleic acid sequence analysis.

Usually, PCR relies on the DNA-replicase and at high temperature keeps stable ability.Have three Main Stage among the PCR: sex change, annealing and extension.During sex change, liquid sample is heated at about 94 ℃.During this process, double-stranded DNA " fusion " becomes single stranded DNA.During annealing, single stranded DNA is cooled to about 54 ℃.In this temperature, primer combination or " annealing " are to the end of the dna segment that will duplicate.Between extended peroid, sample is heated to 75 ℃.In this temperature, enzyme adds nucleotide to aim sequence, finally forms the complementary copy of dna profiling.New DNA chain becomes the new purpose that is used for next sequence of events or " cycle ".

Designed many existing P CR instruments determine in real time specific DNA and RNA sequence during PCR level.A lot of instruments are based on the use for fluorescent dye.Particularly, the fluorescence signal that during the amplification of PCR product, generates in proportion of a lot of traditional PCR in real time instrument detecting.

Traditional PCR in real time instrument is used to detect the distinct methods of different fluorescent dyes.For example, some traditional PCR instruments are used for differentiating each dyestuff with white light source and filter wheel combination on spectrum.White light source is the tungsten halogen lamp bubble, has several thousand hours life-span at most.Filter wheel is the electromechanical component of complicated easy wearing and tearing normally.

Summary of the invention

Usually, the present invention relates to be used for detecting the technology of a plurality of desired substances during PCR in real time (polymerase chain reaction), this PCR in real time refers to multiplex PCR here.Particularly, multiplex fluorescence detection device is described to comprise a plurality of optical modules.Can be optimized each optical module, be used to detect each fluorescent dye at separate wavelengths band place.In other words, optical module can be used to inquire a plurality of parallel reactors at the different wave length place.These reactions can for example take place in the single process chamber (for example well) of rotating disc.

Utilize the multiple-limb fibre bundle that a plurality of optical module optics are connected to single detector.By this way, by use a plurality of optical modules and single detector for example photomultiplier obtain to reuse.Optical element in each module can be selected as making that sensitivity is maximum and make the amount minimum that spectrum is crosstalked, and crosstalking for example is signal from a kind of dyestuff on another optical module.

In one embodiment, device comprises rotating disc, and rotating disc has a plurality of process chambers that keep each sample and a plurality of fluorescent dyes.Device further comprises a plurality of optical modules, and each optical module comprises the light source that is selected for different dyes.The zones of different of the light source excitation rotating disc of optical module, and catch the fluorescence of launching from dish.Fibre bundle is connected to a plurality of optical modules, thereby fluorescence is transferred to single detector from a plurality of optical modules.

In another embodiment, a kind of system comprises data acquisition facility.This system further comprises the pick-up unit that is connected to this data acquisition facility, wherein, pick-up unit comprises the rotating disc with a plurality of process chambers, each process chamber has a plurality of materials, a plurality of optical module, detecting device and the fibre bundle of the fluorescence of emission different wave length, wherein, each optical module is configured to encourage material optically and is caught the fluorescence of the different wave length of being launched by material, fibre bundle is connected to a plurality of optical modules, thereby fluorescence is transferred to detecting device from a plurality of optical modules.

In certain embodiments, data acquisition facility can calculate the time migration between optical module, and handles the data of self-test device based on time migration.

In a further embodiment, a kind of method comprises: rotation has the dish of a plurality of process chambers, and each process chamber has a plurality of materials, the fluorescence of these material emission different wave lengths; Utilize a plurality of light beams to encourage dish, thereby produce a plurality of emitted fluorescence bundles; Utilize a plurality of different optical modules to catch fluorescence beam, wherein, optical module is used for different wave length by optical arrangement; Utilize fibre bundle that fluorescence beam is transferred to single detector from a plurality of optical modules; And signal from detecting device output expression fluorescence beam.

When device can be carried out PCR in real time, this device can also be analyzed this biologically when the biologically of any kind takes place.This device can be independently or as the temperature of selection group ground each reaction of modulation, and this device can come each stage of supporting reactions by comprise valve between two chambeies.By the laser instrument of use, thereby during reaction open this valve to the pulse of valve transmission of power.

In certain embodiments, this device can be of portable form, to allow in remote areas or to operate in the laboratory temporarily.This device can comprise that the data that are used for real-time analysis reaction obtain computing machine, maybe this device can be by the wired or wireless communication interface with data communication to another device.

In certain embodiments, the light source of the optical module that adopts in the pick-up unit of the present disclosure can comprise light emitting diode, laser diode or its combination.In addition, in certain embodiments, the detecting device of pick-up unit of the present disclosure can comprise photomultiplier cell, amplify photodiode, avalanche photodide, phototransistor or its combination.

In accompanying drawing and following description, set forth one or more embodiments of the detail of the present invention.Other features of the present invention, target and advantage are according to description and accompanying drawing and what is claimed is conspicuous.

Description of drawings

Fig. 1 is the block diagram that the exemplary embodiment of multiplex fluorescence detection device is shown;

Fig. 2 is the synoptic diagram that exemplary optics module is shown, and this optical module is corresponding to any one of a plurality of optical modules of the fluorescence detection device of Fig. 1;

Fig. 3 is the block diagram that illustrates in greater detail the example embodiment of multiplex fluorescence detection device;

Fig. 4 is the block diagram of single detector that is connected to four optical fiber of fibre bundle;

Fig. 5 is the process flow diagram that the exemplary operation of multiplex fluorescence detection device is shown;

Fig. 6 and 7 illustrates the absorption and the emission spectrum of the common fluorescent dye that can be used to multiplex PCR;

Fig. 8 A and 8B are illustrated in the raw data of utilizing single detector to obtain from two exemplary optics modules during the pcr analysis;

Fig. 9 is the figure that the data after regulating at time migration are shown;

Figure 10 A and 10B illustrate the detection limit that is used for the data that receive from two exemplary optics modules.

Embodiment

Fig. 1 is the block diagram that the exemplary embodiment of multiplex fluorescence detection device 10 is shown.In the example that illustrates, device 10 has four optical modules 16, and these optical modules 16 provide four " passages " for the optical detection of four kinds of different dyes.Especially, device 10 has four optical modules 16, and these optical modules 16 encourage the zones of different of rotating disc 13 at any given time, and collects from the fluorescent energy of the different wave length of dyestuff emission.As a result, can use optical module 16 to inquire multiple, the parallel reactor that in sample 22, takes place.

Multiple reaction can for example side by side take place in the single chamber of rotating disc 13.Each optical module 16 is inquired sample 22 and is collected the fluorescent energy of different wave length when dish 13 rotates.For example, the driving source in module 16 can sequentially be activated enough periods, thereby collects the data of corresponding wavelength.That is to say that optical module 16A can be activated the regular period, to be collected in the data of first wavelength coverage, this first scope is at selecting corresponding to first dyestuff of first reaction.Then can the inactivation driving source, and can activate driving source in module 16B, with at second wavelength coverage place inquiry sample 22, this second scope is at selecting corresponding to second dyestuff of second reaction.This process continues up to the data that capture from all optical modules 16.In one embodiment, each driving source in optical module 16 is activated about 2 seconds initial period to reach steady state (SS), then is the inquiry cycle, and the 10-50 that this inquiry cycle continues dish 13 changes.In other embodiments, can be to driving source ordering (for example 1 or 2 millisecond) shorter or longer cycle to obtain.In certain embodiments, can side by side activate optical module more than one, thus inquiry when in dish 13 rotations, carrying out for sample 22.

Though described single sample 22, dish 13 can comprise a plurality of chambeies that keep sample.Optical module 16 can be inquired some or all of different cavity at the different wave length place.In one embodiment, dish 13 comprises 96 chambeies of opening around the circle spacing of dish 13.Utilize 96 chambeies and four optical modules 16, device 10 can obtain data from 384 different materials.

In one embodiment, optical module 16 comprises driving source, and this driving source is high energy light emitting diode (LED), and they can buy in a plurality of wavelength, and has the long life-span (for example 100,000 hours or more).In another embodiment, traditional based on halogen bulb or mercury bulb can be used as driving source.

As shown in FIG. 1, each optical module 16 can be connected in the branch of fibre bundle 14.Fibre bundle 14 is provided for not losing from optical module 16 reception fluorescence signals the compliant mechanism of sensitivity.Usually, fibre bundle comprises placed side by side and combines and be encapsulated in a plurality of optical fiber in the flexible protective casing at end place.Perhaps, fibre bundle 14 can comprise the major diameter multi-mode fiber of the separation of lesser amt, these fibers be glass or plastics, and have total end.For example, for the device of four optical modules, fibre bundle 16 can comprise four independently multi-mode fibers, and each fiber has the core diameter of 1mm.The public end of bundle comprises four fibers that combine.In this example, the aperture of detecting device 18 can be 8mm, and this aperture is greater than the aperture that enough is used for being connected to four fibers.

In this example, fibre bundle 14 is connected to single detector 18 with optical module 16.The fluorescence that bearing optical fiber is collected by optical module 16 and effectively with the light transmission of catching to detecting device 18.In one embodiment, detecting device 18 is photomultipliers.In other embodiments, detecting device can comprise a plurality of photomultiplier transit elements, and a photomultiplier transit element is used for each optical fiber of single detector.In other embodiments, can use one or more solid-state detectors.

It can be favourable using single detector 18, because allow to use detecting device (for example photomultiplier cell) super-sensitive and that possibility is expensive like this, keeps minimum cost simultaneously, because only need single detector.Here single detector is discussed; Yet, can comprise one or more detecting devices, be used to detect the dyestuff of bigger quantity.For example, can add four the other optical modules 16 and second detecting device, thereby allow to detect eight different wavelength from a dish emission to system.

Optical module 16 can remove from device, and can easily exchange with other optical modules, and these other optical modules are optimized at the inquiry of different wave length.For example, optical module 16 can be installed in the position of housing by physics.Each optical module 16 can be inserted in the relevant position of housing by the guiding piece (for example recessed channel) that easily mates along the one or more marks (for example directing pin) with optical module.Each optical module comprises optics output port (shown in Figure 2), is used to be connected to a branch of fibre bundle 14.This optics output port can have the thread end of the screw thread coupling that is connected to branch.Perhaps, can use the form (for example, have O shape ring and catch slidably being connected of pin) of " connecting fast ", should " connect " form fast and allow fibrous bundle 14 is slidably engaged onto in the optics output port and from this optics output port.In addition, each optical module 16 can have one or more electrical contacts, is used for being electrically connected to when inserting fully control module 23.The u.s. patent application serial number No.11/174 that the exemplary removable optical modules that is used for using together with rotating disc 13 was submitted on July 5th, 2005 is described among 754 " the MULTIPLEX FLUORESCENCEDETECTION DEVICE HAVING REMOVABLE OPTICAL MODULES " by name.

The modular organization permission device of device 10 easily is applicable to all fluorescent dyes in given analysis environments, and this analysis environments is multiplex PCR for example.Other chemical treatments that can be used in the device 10 comprise Invader (Third Wave, Madison, Wisconsin), transcriptive intermediate amplification (GenProbe, San Diego, California), fluorescence labeling enzyme linked immunological absorption chemical examination (ELISA) or fluorescence in situ hybridization (FISH).The modular organization of device 10 can provide additional advantage, because can be by to the corresponding driving source (not shown) and the excitation of the wavelength that is used for little specific purpose scope and detect light filter and select, thereby optionally encourage and detect corresponding dyestuff in multiple reaction, optimize the sensitivity of each optical module 16.

For the purpose of example, device 10 is shown as 4 looks multiple layout, but passage more or less can use with suitable fibre bundle 14.This modular designs allows user by adding other optical modules 16 and a branch of fibrous bundle 14 is inserted in the new optical module to pedestal 20 simply, comes field upgrade device 10 easily.Optical module 16 can have integrated-optic device, this integrated-optic device identification optical module, and calibration data is downloaded in the internal control optical module or other internal electronic devices (for example control module 23) of device 10.

In the example of Fig. 1, sample 22 is accommodated in dish 13 the chamber, and this dish 13 is installed on the rotation platform under the control that is in control module 23.Output signal and data that sensor slot trigger 27 provides control module 23 to use are obtained, and are used for during disc spins data are obtained with the position, chamber carrying out synchronously.Sensor slot trigger 27 can be machinery or optical sensor.For example, this sensor can be to send the laser instrument of light beam to dish 13, and control module 23 use sensors, and this sensor is by the light of the groove in the dish 13, to be positioned at the chamber on the dish.Optical module 16 can be installed in rotation platform 25 tops by physics.As a result, in any one time, optical module 16 and different chamber overlappings.

Pick-up unit 10 also comprises the heating element (not shown) of the temperature of the sample 22 that is used on the chopper wheel 13.This heating element can comprise the based on halogen bulb that is included in the reflective coating.Reflection cavity is shaped as the radiation from bulb is focused in the radial component (radialsection) of dish 13.Usually, dish 13 heating zone when dish 13 rotations just as a ring.In this embodiment, the shape of reflective coating can be the combination that allows fine-focused ellipse and spherical geometry.In other embodiments, reflective coating can have different shapes, perhaps the bulb bigger zone of radiation widely.In other embodiments, reflective coating can be shaped as the radiation from bulb is focused on the single zone of dish 13, for example comprises the single process chamber of sample 22.

In certain embodiments, heating element can add hot-air and the hot-air of promotion above one or more samples, thereby regulates temperature.In addition, can directly sample be heated by the utilization dish.In this case, heating element can be arranged in platform 25, and is thermally coupled on the dish 13.Can be as control module 23 controls at the resistance in the heating element, the selection zone of heating plate.For example, the zone comprises one or more chambeies, also can be whole dish.The u.s. patent application serial number No.11/174 that is used for " HEATING ELEMENT FOR A ROTATING MULTIPLEXFLUORESCENCE DETECTION DEVICE " by name that the exemplary heating element that uses together with rotating disc 13 submits on July 5th, 2005 is described in 691.

Selectively or additionally, device 10 can also comprise the cooling element (not shown).Comprise that in device 10 fan is to provide cold air air at room temperature just to dish 13.Need cool off the temperature of suitably regulating sample and after finishing experiment, sample is stored.In other embodiments, cooling element can be included in platform 25 and coil thermal coupling between 13, and platform 25 can be waited when needed and reduced its temperature.For example, some biological samples can be stored at 4 degrees centigrade, to reduce enzymatic activity or protein denaturation.

Pick-up unit 10 can also be controlled at the reactive material that comprises in the process chamber.For example, it can be favourable some materials being loaded in the process chamber to generate first reaction and to add another material to sample after a while after the first reaction termination.Can add laser aiming valve (laser homingvalve) system, the valve that inner holding chamber and process chamber is separated with control, thereby the interpolation that the control material arrives the chamber during the rotation of dish 13.This laser aiming valve system can be positioned in the optical module 16 or with optical module and separate.Below dish 13, directly can the laser sensor that be used for respect to dish 13 locating laser devices below laser instrument.

In one embodiment, this laser instrument is near infrared (NIR) laser instrument with at least two power settings.Be provided with down in low-power, the laser positioning sensor can come the position of indication laser above the valve of chamber by the NIR light that coils the groove in 13 by identification.In case laser instrument is in suitable position, thereby the short pulse of control module 23 indication laser output high-power energies heats valve and opens valve.The valve of opening can allow the internal flow sample to flow from inner chamber towards outer process chamber then and implement second reaction.In certain embodiments, dish 13 can comprise a plurality of valves sequentially to generate a plurality of reactions.When using a plurality of chambeies valve, can also use group more than one laser instrument and laser sensor.The u.s. patent application serial number No.11/174 that is used for " VALVECONTROL SYSTEM FOR A ROTATING MULTIPLEX FLUORESENCEDETECTION DEVICE " by name that the exemplary laser aiming valve control system that uses together with rotating disc 13 submits on July 5th, 2005 is described in 957.

Data-acquisition system 21 can sequentially or concurrently be collected data for each dyestuff from installing 10.In one embodiment, data-acquisition system 21 is sequentially collected data from optical module 16, and utilizes from the trigger delay of each optical module of sensor slot trigger 27 measurements, comes the space overlapping is proofreaied and correct.

An application that is used for device 10 is a PCR in real time, but the technology of here describing can be extended to the platform of other uses in the fluoroscopic examination at a plurality of wavelength place.Device 10 can be used for separation, amplification and the detection of nucleic acid in conjunction with the microfluid of rapid thermal cycles, use heating element and centrifugal driving.By using multi-fluorescence to detect, can detect and analyze a plurality of desired substances concurrently.

For real-time PCR, use fluorescence is measured the amplification amount in of three common technology.First kind of technology be to use dyestuff for example SybrGreen (Oregon), the fluorescence of this dyestuff obtains increasing after being attached on the double-stranded DNA for Molecular Probes, Eugene.Second kind of technology used fluorescence labeling probe (hybridization probe, hair clip probe etc.), and the fluorescence of this fluorescence labeling probe changes behind the aim sequence that is attached to amplification.This technology type is similar to and uses the double-stranded DNA combination dye, but more special, because this probe will only be attached to certain segment of aim sequence.The third technology is to use hydrolysis probes (Taqman ^TM, Applied BioSystems, Foster City California), wherein the exonuclease of polymerase divides (cleave) quencher molecule in the extension stage of PCR from probe, makes that it is a fluorescence-activation.

In every kind of method, fluorescence is linear proportional with amplification purpose concentration.The output signal (or selectively utilizing control module 23 to take a sample and communicate by letter) that data-acquisition system 21 is come self-detector 18 at PCR reaction period measurements, thereby near observing amplification in real time.In multiplex PCR, utilize the different dyes of measuring independently to come a plurality of purposes are carried out mark.In general, each dyestuff will have different absorptions and emission spectrum.For this reason, optical module 16 can have driving source, lens and relevant light filter, and these driving sources, lens and relevant light filter are at random selected to be used at the different wave length place sample 22 being inquired.

Some examples that are applicable to the suitable structure technology used together in conjunction with the present invention or material can be in for example U.S. Patent No. 6 of common " ENHANCED SAMPLE PROCESSINGDEVICES SYSTEMS AND METHODS " (Bedingham etc.) by name that transfer the possession of, 734,401 and the U.S. Patent Application Publication No.US 2002/0064885 of by name " SAMPLE PROCESSING DEVICES " in be described.The U.S. Provisional Patent Application sequence number No.60/214 that is called " THERMAL PROCESSINGDEVICES AND METHODS " that other available apparatus structures can be submitted in for example on June 28th, 2000,508, the U.S. Provisional Patent Application sequence number No.60/214 of " the SAMPLE PROCESSING DEVICES; SYSTEMS AND METHODS " by name that submitted on June 28th, 2000,642, the U.S. Provisional Patent Application sequence number No.60/237 of " the SAMPLE PROCESSING DEVICES; SYSTEMS AND METHODS " by name that submitted on October 2nd, 2000,072, the U.S. Provisional Patent Application sequence number No.60/260 of " the SAMPLE PROCESSING DEVICES; SYSTEMS AND METHODS " by name that submits to January 6 calendar year 2001,063, the U.S. Provisional Patent Application sequence number No.60/284 of " the ENHANCED SAMPLE PROCESSINGDEVICES; SYSTEMS AND METHODS " by name that submits to April 18 calendar year 2001,637 and the U.S. Patent Application Publication No.US 2002/0048533 of " SAMPLE PROCESSING DEVICES ANDCARRIERS " by name in find.Other potential apparatus structure can find in the U.S. Patent No. 6,627,159 of for example " CENTRIFUGAL FILLING OFSAMPLE PROCESSING DEVICES " (Bedingham etc.) by name.

Fig. 2 is the synoptic diagram that exemplary optics module 16A is shown, and it is corresponding to any optical module 16 of Fig. 1.In this example, optical module 16A comprises high power excitation source LED 30, collimation lens 32, excitation light filter 34, dichroic filter 36, condenser lens 38, detects light filter 40 and lens 42, thereby fluorescence is focused on the optics output port 19, and this optics output port 19 is connected in the branch of fibre bundle 14.

Then, being utilized collimation lens 32 from the exciting light of LED 30 carries out alignment, utilizes excitation light filter 34 to filter, transmit by dichroic filter 36 and utilizes condenser lens 38 to be focused in the sample 22.The fluorescence that obtains by sample emission was utilized identical condenser lens 38 and carries out optically focused, filters by dichroic filter 36 reflections and by detecting light filter 40 before a branch that is focused the fibre bundle 14 that is connected to the optics output port.Then, fibre bundle 14 transports light to detecting device 18.

The specific absorption and the emission band of the multiple dyestuff that utilizes based on optical module 16A are selected LED 30, collimation lens 32, excitation light filter 34, dichroic filter 36, condenser lens 38, are detected light filter 40 and lens 42.By this way, at different dyestuffs, a plurality of optical modules 16 can be configured and be carried in the device 10.

Table 1 has been listed the exemplary elements that can be used in the 4 channel and multiple fluorescence detection devices 10 of multiple fluorescent dye.FAM, HEX, JOE, VIC, TET, ROX are the trade marks of Applera, Norwalk, California.Tamra is AnaSpec, San Jose, the trade mark of California.Texas Red is the trade mark of Molecular Probe.Cy 5 is Amersham, Buckinghamshire, the trade mark of United Kingdom.

Table 1

Optical module	LED?	The excitation light filter	Detect light filter	Dyestuff
					1?	Blue	475nm?	520nm?	FAM，Sybr?Green?
2?	Green	530nm?	555nm?	HEX，JOE，VIC，TET?
					3?	Orange	580nm?	610nm?	TAMRA，ROX，Texas REd?
4?	Red	630nm?	670nm?	Cy5?

An advantage of described modular multiple detection architecture is the dirigibility of optimizing for the detection of extensive multiple dyestuff.The user can have the collection (bank) of a plurality of different optical modules as can be known, these a plurality of different optical modules can be inserted in the device 10 as required, can use any time N in these modules, wherein, N is the maximum quantity of the passage of device support.Therefore, device 10 and optical module 16 can use with any fluorescent dye and PCR detection method.Can use bigger fibre bundle to support the sense channel of larger amt.In addition, a plurality of fibre bundles can use with a plurality of detecting devices.For example, two 4 branch optical fiber bundles can use with eight optical modules 16 and two detecting devices 18.

Fig. 3 is the functional block diagram of multiplex fluorescence detection device 10.Particularly, Fig. 3 shows the electrical connection between the device element and the general paths of the light by these elements.In the example of Fig. 3, device 10 comprises at least one processor 44 or other steering logics, storer 46, coil motor 48, light source 30, excitation light filter 34, lens 38, detects light filter 40, collector lens 42, detecting device 18, sensor slot trigger 27, communication interface 50, heating element 54, laser instrument 55 and power supply 52.As shown in Figure 3, lens 38 and collector lens 42 do not need to be electrically connected to another element.In addition, light source 30, light filter 34 and 40, lens 38 and optical module 16 of collector lens 42 expressions.Though do not have shown in Figure 3ly, as described above, device 10 can comprise other optical module 16.In this case, each other optical module can comprise basically and the element of arranging similarly shown in Figure 3.

The certain path of passing through a plurality of elements of light in Fig. 3.In case launched light by light source 30, this light enters excitation light filter 34 and leaves as the light of separate wavelengths.This light is scioptics 38 then, and at lens 38 places, light leaves pick-up unit 10 and is activated at sample 22 in the process chamber (not shown).Sample 22 responds by sending fluorescence at the different wave length place, and at this moment this light enters lens 38 and filters by detecting light filter 40.The bias light that needs the wavelength outside the fluorescence that light filter 40 is removed from sample 22.Remaining light is sent out by collector lens 42 and entered the branch of fibre bundle 14 before being detected by detecting device 18.Detecting device 18 then amplifies the light signal that receives.

Processor 44, storer 46 and communication interface 50 can be the parts of control module 23.Processor 44 console panel motors 48 are with rotation or rolling disc 13 as required, thereby collect fluorescence information or move fluid by coiling 13.Processor 44 can use the dish positional information that receives from sensor slot trigger 27, with the position in the chamber on the identification dish 13 during rotation, and makes obtaining synchronously for the fluorescence data that receives from dish.

Processor 44 can also be controlled at light source 30 in the optical module 16 and when switch on and cut off the power supply.In certain embodiments, processor 44 control excitation light filters 34 and detection light filters 40.At the sample of radiation, processor 44 can change light filter according to just, thereby allows the exciting light of different wave length to arrive sample, perhaps allows the fluorescence of different wave length to arrive collector lens 42.In certain embodiments, can one or two light filter be optimized at the light source 30 of particular optical module 16, and this one or two light filter can not be changed by processor 44.

Collector lens 42 is connected to a branch of fibre bundle 14, and fibre bundle 14 provides from the collector lens to the detecting device 18 optical path for light.Processor 44 can control detection device 18 operation.Though detecting device 18 (constantly) always detects all light, some embodiment can use other obtaining mode.Processor 44 can determine when detecting device 18 collects data, and other configuration parameters of detecting device 18 can be set by programming.In one embodiment, detecting device 18 is photomultipliers, and this photomultiplier is caught fluorescence from the light that is provided by collector lens 42.In response, detecting device 18 generates the output signal (for example analog output signal) of the light that expression receives.Though do not illustrate among Fig. 3, detecting device 18 can receive the light from other optical modules 16 of device 10 simultaneously.In this case, output signal 19 is electrically represented the combination of the optics input that received from a plurality of optical modules 16 by detecting device 18.

Processor 44 can also be controlled the data stream from device 10.Data storage can be used to analyze in storer 46, data for example come the sampling fluorescence of self-detector 18, from the sample temperature and the disc spins information of heating element 54 and related sensor.Processor 44 can comprise following any one or a plurality of: microprocessor, digital signal processor (DSP), special IC (ASIC), field programmable gate array (FPGA) or other DLC (digital logic circuit).In addition, processor 44 is provided for the operating environment of firmware, software or their combination, and these firmwares, software or their combination are stored in computer-readable medium for example on the storer 46.

Storer 46 can comprise the one or more storeies that are used to store multiple information.For example, a storer can comprise specific configuration parameters, executable instruction, and storer can comprise the data of collection.Therefore, processor 44 can use the data that are stored in the storer 46, is used for control device operation and calibration.Storer 46 can comprise one or more in random access storage device (RAM), ROM (read-only memory) (ROM), electricallyerasable ROM (EEROM) (EEPROM), flash memory or the like.

Processor 44 can additionally be controlled heating element 54.Based on the instruction that comprises in the storer 46, heating element 54 can be selectively driven according to the heating curves of expectation, to control the temperature in one or more chambeies.Usually, heating element heats coiling 13 radial component in disc spins.Heating element 54 can comprise based on halogen bulb, and is used for heat energy is focused on reverberator on dish 13 the specific region.In other embodiments, heating element 54 can sequentially heat one or more chambeies.It is static that this embodiment requires to work as heating chamber hour indicator 13.In any embodiment, heating element 54 can must be opened and turn-off as required very soon.

Laser instrument 55 is used to operation valve and opens, and this valve is opened and allowed to allow the content of inner chamber flow to another chamber of coiling on 13, for example process chamber.Processor 44 and support hardware drive laser 55 are optionally opened the specific valve that comprises in the dish 13.Processor 44 can interact with the laser sensor below the dish 13, is used for determining the position of laser instrument with respect to the expectation valve.When being in suitable position, processor 44 output signals come guided laser device 55 to generate the energy pulse of aiming valve.In some cases, this pulse can continue about 0.5 second, and other embodiment can comprise shorter or longer opening the time.Can be by utilizing processor 44 to control laser energy and duration of pulse with communicating by letter of laser instrument 55.

Processor 44 uses communication interface 50 to communicate with data-acquisition system 21.Communication interface 50 can comprise that the combination of single method or method transmits data.Certain methods can comprise and be used for carrying out USB (universal serial bus) (USB) port or the IEEE 1394 that hardware connects with higher data transmission rate.In certain embodiments, memory storage directly can be attached to one of these ports, thereby be used for the data storage of aftertreatment.These data can be carried out pre-service and are ready for and check by processor 44, and perhaps raw data may intactly be handled before analyzing beginning.

Can also connect by radio frequency (RF) communication or Local Area Network and finish and the communicating by letter of pick-up unit 10.In addition, can obtain connectivity by direct connection or by Network Access Point, this Network Access Point for example can be hub or a router of supporting wired or wireless communication.For example, pick-up unit 10 can be at certain RF frequency transmitting data, thereby is received by destination data deriving means 21.Data acquisition facility 21 can be multi-purpose computer, notebook computer, hand-held computing device or isolated plant.In addition, a plurality of data acquisition facilities can side by side receive data.In other embodiments, data acquisition facility 21 can be included as an integrated detection together and obtain system with pick-up unit 10.

In addition, pick-up unit 10 can for example come software, firmware and calibration data from the remote-control device down loading updating in the Internet via network.Communication interface 50 can also make processor 44 can monitor any fault of inventory report.If generation operational issue, processor 44 can output error messages, thereby get rid of by providing service data to come assisting users to carry out fault.For example, processor 44 can provide information to help user's tracing trouble heating element or stationary problem.

Power supply 52 is to the element transmission operand power of device 10.Power supply 52 can use from the electric power of standard 115 voltaism sockets or comprise battery or electricity-generating circuit generates operand power.In certain embodiments, can recharge the operation that prolongs to allow to battery.For example, device 10 can be of portable form to detect in emergency circumstances the biological sample in the disaster area for example.

Fig. 4 is the functional block diagram of single detector 18 that is connected to four optical modules of fibre bundle.In this embodiment, detecting device 18 is photomultipliers.Each branch of fibre bundle 14, optical fiber 14, optical fiber 14B, optical fiber 14C and optical fiber 14D are connected to the optics input interface 55 of detecting device 18.By this way, the light that is carried by any optical fiber 14 is provided to the single optics input interface 55 of detecting device 18.In certain embodiments, each branch of fibre bundle 14 can have different diameters, length or neither same.For example, optical fiber 14A may be bigger than the diameter of other optical fiber of light transmitting fiber 14, thereby to the more light of detecting device 18 transmission.Optics input interface 55 provides the light of gathering to electron-multiplier 56.Anode 58 is collected electronics and is generated the corresponding simulating signal as output signal.

In other words, as shown, optical fiber 14 is engaged in the input optical aperture that is used for detecting device 18.Thus, detecting device 18 can be used to side by side detect the light from each branch of fibre bundle 14.Optics input interface 55 provides light to electron-multiplier 56.For photomultiplier, at first clash into luminous negative electrode from the photon of optical fiber, this luminous negative electrode discharges photoelectron then.Photoelectron is connected (cascade) by clashing into a series of dynodes then, with launch more photoelectron after each dynode contacts.In fact the electronics group that obtains has doubled by the primary optical signal of optical fiber 14 original transmission.The electronics of accelerating is finally collected by anode 58.To be transferred to current/voltage amplifier 59 as analog output signal from the electric current of anode 58, this analog output signal is represented the optical fluorescence signal of the sample that next free a plurality of optical modules 16 provide.

Control optical module 23 comprises modulus (A/D) converter 60, and this analog to digital converter 60 is sampled digital data stream, just digital signal with analog signal conversion.Processor 44 receives this digital signal and sampled data is stored in the storer 46, the data acquisition facility 21 that is used to communicate by letter, as previously mentioned.In certain embodiments, A/D converter 60 can be contained in the detecting device 18 rather than controls in the optical module 23.

By this way, can use single detector 18 to collect from all light of fibre bundle 14 and the signal of these all light of generation expression.In case this signal is amplified by amplifier 59 and is converted to digital signal, it can be become and the corresponding data of light of being collected by each independent optical module 16 by digital separation.Can utilize frequency range with whole (just whole) Signal Separation each detection signal for each fluorescence of expression.These frequencies can be utilized by data acquisition facility 21 digital light filter that use or in device 10 and separate.

In other embodiments, amplifying signal can use the simulation light filter to utilize frequency to separate and send to split tunnel before the A/D converter 60.Each passage can be separated the ground digitizing then and send to data acquisition facility.Under any situation, this single detector can be caught all fluorescence information from each optical module 16.Data acquisition facility 21 can be drawn in real time and analyze then from coiling the signal that each chamber of 13 obtains and not need a plurality of detecting devices.

In certain embodiments, detecting device 18 can not be a photomultiplier.Usually, detecting device 18 can be the analog or digital pick-up unit of any kind, and this analog or digital pick-up unit can catch from optical delivery mechanism for example a plurality of branches of fibre bundle 14 light and can generate the expression transmitted of the light of this seizure.Other embodiment can comprise as the detecting device that amplifies photodiode or phototransistor.

Fig. 5 is the process flow diagram that the operation of multiplex fluorescence detection device 10 is shown.At first, the user comes designated program parameter (62) on data acquisition facility 21 or via the interface with control module 23.For example, these parameters can comprise speed and the period that is used for rotating disc 13, are defined for the temperature curve and the sample position on dish 13 of reaction.

Then, the user will be coiled 13 and will be loaded into (64) in the pick-up unit 10.After having fixed device 10, user's start-up procedure (66) causes control module 23 to begin rotating disc (68) with the speed of appointment.After dish began to rotate, two concurrent flow processs may take place.

At first, pick-up unit 10 begins to detect the fluorescence (70) of the exciting light of the one or more reactions generations in next free one or more samples.18 pairs of fluorescence signals from each sample of detecting device amplify, and these signals are synchronized to the time (72) of each respective sample and emitting fluorescence.During this flow process, processor 44 is kept at storer 46 and the data acquisition facility 10 of can in real time these data communication being communicated by letter with the data of catching, thereby monitors the program of operation and the program of handling in addition (73).Perhaps, processor 44 can save the data in the device 10 and finish up to program.Processor 44 continues to detect the fluorescence of sample and preserve data and finish (74) up to program.In case the rotation (76) of control module 23 stop discs is finished in operation.

During this flow process, the temperature of control module 23 monitor temperature (78) and adjustment disk or each sample obtains to be used for the purpose temperature (80) of this time.Control module 23 persistent surveillances and control this temperature are finished (82) up to program.In case operation is finished, control module 23 remains on the purpose storing temperature with the temperature of sample, normally 4 degrees centigrade (84).

The operation of device 10 can be different with the example of Fig. 5.For example, during program, can regulate the dish revolution of per minute, and can use laser instrument 55 to open valve between the chamber on the dish, thereby allow multiple reaction.These steps can be carried out with any order in operation according to the program of user's qualification.

Example

Fig. 6 and 7 illustrates for multiplex PCR the absorption and the emission spectrum of operable general fluorescent dye in device 10.In these examples, the absorption maximal value of dyestuff changes in the scope of 480-620nm, and the emission maximum that obtains changes in the scope of 520-670nm.The signal that is used for each dyestuff in Fig. 6 is numbered as FAM 88, Sybr 90, JOE 92, TET 94, HEX 96, ROX 98, Tx Red 100 and Cy5 102.Signal in Fig. 7 is FAM 104, Sybr 106, TET 108, JOE 110, HEX 112, ROX 114, TxRed 116 and Cy5 118.FAM, HEX, JOE, VIC, TET, ROX are the trade marks of Applera, Norwalk, California.Tamra is AnaSpec, San Jose, the trade mark of California.Texas Red is the trade mark of Molecular Probes.Cy 5 is Amersham, Buckinghamshire, the trade mark of United Kingdom.

In an example, 96 chambeies dish is filled FAM and the ROX dyestuff that is diluted in the variable concentrations in the Standard PC R reaction buffer.Four of each dyestuff duplicate with the 2x dilution series that begins from 200nM FAM and 2000 nM ROX and add.Each sample volume is 10 μ L.Chamber 82 has the potpourri of the 2000 nM Rox of the 200 nM FAM of 5 μ L and 5 μ L.Device 10 is built as two channel and multiple PCR pick-up units with two optical modules 16 that are used to detect dyestuff.

First optical module (FAM optical module) comprises that blue-ray LED, 475nm excitation light filter and 520nm detect light filter.Second optical module (ROX optical module) comprises that green light LED detects light filter together with 560nm excitation light filter and 610nm.The another kind of selection is to comprise orange LED and encourage light filter at 580nm, thereby detection is optimized to ROX.

Carry out pcr analysis, and it is intrafascicular to be multiplexed to bifurcation fiber from the fluorescence signal of sample.This fibrous bundle and single detector especially photomultiplier (PMT) carry out interface.The national instrument data of the Visual Basic Data Acquisition Program interface that utilizes and carry out on multi-purpose computer obtains (DAQ) plate and collects data.When being rotated with 1000 rev/mins (specified), dish obtains data.Sequentially use FAM optical module and ROX optical module to inquire sample.Each scanning is made up of average 50 rotations.Raw data from two optical modules is presented among Fig. 8 A and the 8B.

Figure in Fig. 8 A is by energising obtains to the LED in the FAM optical module, and the figure in Fig. 8 B is by energising obtains to the LED in the ROX optical module.

During analyzing, the data of collection are clearly shown that: at any one time, the time migration that is associated with optical module physically is positioned at above the different cavity.By determining to calculate off-set value being used for the particular cavity time migration between in this case the

optical module

1 and 2 in chamber 82 just.In other words, the time delay between this time migration indication data of catching for the data of catching by the FAM optical module in identical chamber and by the ROX optical module.

Fig. 9 illustrates the figure that the skew that is used for each chamber deducts integrated data.FAM is by Hash mark bar (hash marked bar) indication, and ROX is by opening bar (open bar) mark, and the ROX data are placed on the FAM data.Data illustrate, and do not have the signal from the ROX dyestuff on optical module 1, and do not have the signal from the FAM dyestuff on optical module 2.On optical module 1, have higher background, this background is adjusted by the optimization group of light filter.Thereby these data are analyzed the restriction (LOD) of determining detection, be described as equaling the signal of baseline noise level.What the baseline noise level was defined as ten times of cavity scannings on average adds 3 times of standard deviation.

Determine LOD by the integrated signal of drawing with respect to the linear least square match standard of the concentration of FAX and ROX.The LOD of FAM and ROX optical module is calculated as 1 and 4nM respectively, shown in Figure 10 A and 10B.

Claims

1. pick-up unit comprises:

Motor is used for rotating disc, and this dish has a plurality of process chambers, and described each process chamber keeps respective sample and a plurality of fluorescent dye;

A plurality of optical modules, wherein, each described optical module comprises the light source that is selected for the different dyes in the described dyestuff and is used for catching lens from described dish emitted fluorescence;

Detecting device; And

Fibre bundle is connected to described a plurality of optical module, thereby described fluorescence is transferred to described detecting device from described a plurality of optical modules.

2. pick-up unit according to claim 1, wherein, each described optical module further comprises the excitation light filter and detects light filter.

3. pick-up unit according to claim 1, wherein, the light source of described optical module comprises light emitting diode or laser diode.

4. pick-up unit according to claim 1, wherein, described device comprises at least four optical modules.

5. pick-up unit according to claim 1, wherein, described pick-up unit further comprises the sensor slot trigger, described sensor slot trigger provides output signal, is used for process chamber position and the data that provided by described detecting device are carried out synchronously.

6. pick-up unit according to claim 1, wherein, described fibre bundle comprises a plurality of fiber optic taps, described a plurality of fiber optic taps all stop at the place, aperture of described detecting device.

7. pick-up unit according to claim 1, wherein, described detecting device is photomultiplier cell, amplification photodiode, avalanche photodide or phototransistor.

8. detection system comprises:

Data acquisition facility; And

Be connected to the pick-up unit of described data acquisition facility, wherein, described pick-up unit comprises:

Motor is used to rotate the dish with a plurality of process chambers, and each described process chamber has a plurality of materials of the fluorescence of emission different wave length;

A plurality of optical modules, wherein, described each optical module is also caught the fluorescence of the different wave length of being launched by described material for the described material of excitation by optical arrangement;

Detecting device; And

9. system according to claim 8, wherein, described data acquisition facility calculates the time migration between described optical module, and handles data from described pick-up unit based on described time migration.

10. system according to claim 8, wherein, described each optical module further comprises the excitation light filter that is selected for different wave length and detects light filter.

11. system according to claim 8, wherein, described pick-up unit further comprises the sensor slot trigger, and described sensor slot trigger provides output signal, is used for process chamber position and the data that provided by described detecting device are carried out synchronously.

12. system according to claim 11,

Wherein, each optical module can be installed in each position of described pick-up unit by physics, and

Wherein, each optical module can be inserted in the relevant position along the guiding piece that cooperates with one or more marks of described optical module.

13. system according to claim 12, wherein, each optical module comprises the optics output port of the branch that is used to be connected to fibre bundle.

14. system according to claim 12, wherein, each optical module has one or more electrical contacts, is used for being electrically connected when described optical module is fully inserted into described position control module.

15. system according to claim 8, wherein, described fibre bundle comprises a plurality of fiber optic taps, and described a plurality of fiber optic taps all stop at the place, aperture of described detecting device.

16. system according to claim 8, wherein, described detecting device is photomultiplier cell, amplification photodiode, avalanche photodide or phototransistor.

17. a fluorescence detection method comprises:

Rotation has the dish of a plurality of process chambers, and described each process chamber has a plurality of materials of the fluorescence of emission different wave length;

Utilize a plurality of light beams to encourage described dish, thereby generate a plurality of emitted fluorescence bundles;

Utilize a plurality of different optical modules to catch described fluorescence beam, wherein, described optical module by optical arrangement for being used for different wave length;

Utilize fibre bundle that described fluorescence beam is transferred to single detector from described a plurality of optical modules; And

Signal from the detected light beam of described detecting device output expression.

18. method according to claim 17 wherein utilizes a plurality of light beams to encourage described dish to finish by described light beam is sent by the excitation light filter, can finish by described fluorescence beam being sent by detecting light filter and catch described fluorescence beam.

19. the output signal that provides from the sensor slot trigger further is provided method according to claim 17, is used for process chamber position and the data that provided by described detecting device are carried out synchronously.

20. method according to claim 17, wherein, described single detector comprises photomultiplier cell, amplifies photodiode or phototransistor.

21. method according to claim 17 wherein, produces and catches light beam from least four optical modules.

22. method according to claim 17 further comprises:, encourage the different material of polymerase chain reaction with the fluoroscopic examination that utilizes a plurality of wavelength at each selection wavelength of described a plurality of light beams.