WO2006062312A1 - Lab-on-a-chip for an on-the-spot analysis and signal detection methods for the same - Google Patents
Lab-on-a-chip for an on-the-spot analysis and signal detection methods for the same Download PDFInfo
- Publication number
- WO2006062312A1 WO2006062312A1 PCT/KR2005/004084 KR2005004084W WO2006062312A1 WO 2006062312 A1 WO2006062312 A1 WO 2006062312A1 KR 2005004084 W KR2005004084 W KR 2005004084W WO 2006062312 A1 WO2006062312 A1 WO 2006062312A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lab
- pad
- chip
- signal
- biosensor system
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0645—Electrodes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0681—Filter
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0825—Test strips
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
Definitions
- the present invention relates to a lab-on-a-chip version of biosensor for an on- the-spot analysis whose analytical performances were remarkably improved, by incorporating commercial membranes, traditionally used for rapid diagnostics, into microfluidic channels engraved on the surface of a plastic chip, as follows: 1) reduction of sample size; 2) realization of variable functions for total analysis; and 3) transfer of medium by capillary action without the assistance of an external force.
- Rapid analytical devices based on chromatography using the lateral flow of medium through micro-pores present within the matrices of membrane pads have been conventionally applied for the diagnoses of various diseases and symptoms (References: S. H. Paek et al., 2000, Methods, Vol. 22, page 53-60; S. H. Paek et al., 1999, Biotechnol. Bioeng., Vol. 62, page 145-153; Y. Kasahara et al., 1997, Clin. Chimi. Acta, Vol. 267, page 87-102).
- one of the major drawbacks in routine, frequent application has been the induction of severe pain, in the case of using whole blood for specimens, because of a large amount of sampling.
- membrane pads can typically be cut smaller than 4 mm in width, which would make it difficult to hold them in a precise arrangement. This causes a low reproducibility of analysis and inaccuracy in detection.
- a device utilizing a flow- through mode References: A. E. Chu, 2001, U.S. Patent 6,284194 Bl
- the same problems must be addressed when membranes are of smaller sizes. These are probably the major reasons that products handling low capacity samples have not yet appeared in the market.
- the sample volume required by current, commercially available rapid analytical devices is typically in the range of 15 to 200 L (References: A. J. T ⁇ dos et al., 2001, Lab. Chip, Vol. 1, page 83-95).
- MEMS micro- electrical, mechanical systems
- membranes used for rapid analysis and micro-fluidic channels enabling the miniaturization of a device can be combined in order to achieve a practical lab-on-a-chip capable of handling quite a small sample.
- Many different commercially available membranes can perform various functions that may be needed for analyses, such as filtration, ion-exchange, reagent release, laminar flow, and absorption (References: S. H. Paek et al. 1999, Biotechnol. Bioeng., Vol. 62, page 145-153; Y. Kasahara et al., 1997, Clin. Chimi. Acta. Vol. 267, page 87-102).
- the membranes can be cut to widths of 1 mm or narrower, and then installed within the channels of a plastic chip. This approach facilitates precise arrangement and assembly of the small pieces of membranes together for the fabrication of a functional lab-on-a-chip.
- the present invention makes it the object to provide the said novel device that would offer three advantages hi addition to sample reduction: 1) realization of variable functions by selecting appropriate membranes mentioned; 2) implantation of membranes as parts of a complete channel for total analysis; and 3) transfer of medium by capillary action without the assistance of an external force.
- micro- to millimeter-sized micro-fluidic channels (21, 28) comprising parts for holding the said functional membrane pad(s) and parts for controlling the inlet(s) and outlet(s) of medium by capillary action;
- the functional membrane pad(s) (10) is placed within at least a part of the channels;
- the bottom plate is bonded to the top plate in order to compose micro-fluidic channels (21, 28) for delivering medium by capillary action.
- the top solid plate (20) can variably contain sample application pot (22), signal monitoring window (23), and enzyme substrate supply pot (25), and the bottom solid plate (30) can also include inlet/outlet pots of medium depending on the design of lab-on-a-chip.
- the said top solid plate (20) is made of organic polymers, such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polystyrene, and polycarbonate, and inorganic materials, such as glass, quartz, and ceramic.
- the bottom solid plate (30) is made of one of the same materials as for the top plate or, in addition, flexible solid matrices such as adhesive plastic film and rubber.
- the said micro-fluidic channel (21, 28) is formed on the inner surfaces of the top solid plate using various methods, for instances, photolithography, imprinting, laser and mechanical engraving.
- the channel may have a planar, smooth slant, or multilayer structure depending on the design of lab-on-a-chip.
- the said membrane pad(s) (10) is selectable from glass fiber membrane, cellulose membrane, nitrocellulose membrane, nylon membrane, and a synthetic polymer membrane.
- functional membrane is defined as a part ready to use for analysis in a lab-on-a-chip (40) after appropriate treatments of a raw membrane.
- the lab-on-a-chip (40) therefore, can be constructed to accomplish desired functions by selecting membranes among available ones carrying out filtration, ion-exchange, reagent release, laminar flow, absorption, enzyme reaction, antigen- antibody binding, and nucleic acid hybridization.
- the lab-on-a-chip (40) from this invention is utilized for analyses of a variety of analytes including metabolic substances, proteins, hormones, nucleic acids, cells, drugs, food contaminants, environmental pollutants, and biological weapons. They are detected with high specificity and sensitivity by employing bio-receptors, such as enzyme, antibody, and oligonucleotide, placed within the micro-pores of the functional membranes. Such a biological interaction among analyte and bio-receptor is converted to a physical signal (e.g., color, luminescence, fluorescence, electric current, voltage, conduction, or magnetism), resulting from the interaction itself or via a signal generator usually labeled to one of the reaction partners, readily measurable using a relatively simple detector.
- a physical signal e.g., color, luminescence, fluorescence, electric current, voltage, conduction, or magnetism
- the micro-fluidic channels may comprise a vertical micro-fluidic channel (21) and a horizontal micro- fluidic channel (28) crossing with one another, wherein the horizontal micro-fluidic channel (28) may comprise a substrate supply channel (24) and a horizontal flow absorption channel (26).
- the vertical micro-fluidic channel (21) may be integrated with sample application pad (12), signal generator conjugate release pad (13), cell filtration pad (14), signal generation pad with immobilized capture binding component (15), and vertical flow absorption pad (16); and the horizontal flow absorption channel (26) may be prepared to be wholly integrated with a horizontal flow absorption pad (17).
- the horizontal flow absorption pad (17) is remained in a spatially separated state at first and then physically connected to the signal generation pad (15), belong to the vertical arrangement pads, after the completion of the vertical flow reaction.
- the horizontal flow absorption channel (26) may also be prepared in a combined structure of connection fine-capillary channels (42), having a defined width and length, with parts integrated with a horizontal flow absorption pad (17), wherein the fine-capillary channels (42) is located between the signal generation pad with immobilized capture binding component (15) and the horizontal flow absorption pad (17) and may have a dimension of 1 to 900 ⁇ m width and 0.1 to 10 mm length.
- the movement of the horizontal absorption pad (17) for signal generation is not required as shown in Figure 2B (right).
- the signal generator conjugate release pad (13) may comprise the conjugate of a signal generator with a binding component for detection, or a binding component for detection and the conjugate of a signal generator with a secondary binding component specific to the binding component for detection.
- the signal generator is horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, or arthromyces ramosus peroxidase
- the substrate solution may comprise a chromo genie substrate component specific to the signal generator, and, at the time of signal generation, a color change detectable with naked eyes is shown as signal resulting from enzyme-substrate reaction.
- the substrate solution may comprise a silver compound, and, at the time of signal generation, a color change detectable with naked eyes or conductivity change is measured as signal resulting from chemical catalytic reaction.
- the substrate solution may comprises luniinol or other luminescent substrate components specific to the signal generator, an enzyme, and at the time of signal generation, a light signal is measured as signal resulting from enzyme-substrate reaction.
- the substrate solution may comprise luminol or other luminescent substrate components specific to the signal generator, and at the time of signal generation, a light signal is measured as signal resulting from chemical catalytic reaction.
- the substrate solution may comprise an electrochemical signal- generating component specific to the signal generator, an enzyme, and, at the time of signal generation, electric conductivity change, current change, or voltage change is measured as signal resulting from enzyme-substrate reaction.
- the electrochemical signal may be detected using an electrode either directly screen-printed onto the signal generation pad or physically combined with the pad by means of an external force.
- a detector measuring a signal produced from the chip is also an essential component of the biosensor system.
- the signal can be measured based on, for examples, colorimetry, luminometry, fluorometry, electrochemistry, or magnetometry, depending on the signal to be measured.
- a colorimetric detector (50) can be constructed to measure a color change of bio-receptor- dispensed lines on the signal generation pad (15) of lab-on-a-chip using a charge- coupled device (CCD) camera (51).
- CCD charge- coupled device
- lab-on-a-chip in this invention can be applied for the analyses of a number of analytes
- biological affinity-based analyses such as immunoassay based on antigen-antibody binding are selected for illustrating the utility of the lab-on-a-chip.
- Enzyme-linked immunosorbent assay is an analytical method that utilizes solid-phase immune reactions to detect an analyte in sample via an enzyme labeled to an immuno-reagent as a signal generator (References: G. G. Guilbault, 1968, Anal. Chem., Vol. 40, page 459-471).
- a binding reaction partner antigen or antibody
- microtiter plates consisting of multiple, small-volume capacity wells made of plastic (e.g., polystyrene).
- the enzymes used as signal generators in ELISA are huge, proteinacious molecules, which catalyze each specific substrate (References: L. J. Kricka, 2002, Ann Clin. Biochem., Vol. 39, page 114-129).
- the catalytic action amplifies the signal, which, depending on its chemical properties, can be measured using a simple detector based on colorimetry, luminometry, and electrochemistry, for example (References: A. Morrin et al., 2003, Biosens. Bioelectron., Vol. 18, page 715-720; R. J. Jackson et al., 1996, J. Immunol. Methods., Vol.
- POCT version of ELISA is developed by employing the method of cross-flow chromatography (References: J. H. Cho et al., 2005, Anal. Chem., Vol. 77, page 4091 - 4097). This would demonstrate a widespread application of immunosensors to various analytes with minimal costs and, potentially, dimensions.
- the concept was originally developed to use enzymes as signal generators in immuno-chromatographic assay by sequentially accomplishing antigen-antibody bindings and catalytic reactions to generate signals.
- a lab-on-a-chip is constructed in this invention to achieve a semiautomatic switching of the sequential processes for a complete analysis and a miniaturization of the immunosensor.
- This chip is fabricated as stated above by incorporating a conventional immuno-strip into a plastic chip with elaborately devised channels on the surfaces.
- Lab-on-a-chip immunosensor system To fabricate a lab-on-a-chip installed with membrane pads for ELISA, fluidic channels are devised by mechanically etching the surfaces of the top solid plate (20).
- the chip consists of two distinct flow channels in the vertical (21) and horizontal directions (28; Figure IA).
- the vertical compartment (21) is carved to tightly fit a 2 mm- wide immuno-strip (11), essentially the same as that of a conventional rapid test kit (References: J. G. Schwartz et al., 1997, Am. J. Emerg. Med., Vol. 15, page 303-307; R. H. Christenson et al., 1997, Clin. Biochem., Vol.
- a sample application pot (22) and a signal monitoring window (23) are provided by drilling.
- an enzyme substrate supply channel (24) and a horizontal flow absorption channel (26) are horizontally arranged on each lateral side of the signal generation pad (15) of the strip, respectively.
- a supply pot (25) and two air ventilation holes (27) are located at the inlet and near the outlet, respectively.
- the immuno-strip (11) is comprised of four different, commercially available membranes, furnishing various functions of sample application (12), enzyme conjugate release (13), cell filtration (14), signal generation (15), and vertical flow absorption (16). They are lengthily disposed in order, partially superimposed on one another, and mounted on a plastic film. This strip is fixed in the vertical channel (21) of the chip. The position of the horizontal flow absorption pad (17), on the other hand, is variable.
- the cross-flow chromatographic analysis for an analyte is performed.
- the analyte is spiked in a human serum to prepare a standard solution, which is then transferred into the sample application pot (22) of the chip (40; Figure 2A). It is migrated in the vertical direction by capillary action ( Figure 2A, left), and dissolves the detection antibody labeled with an enzyme (e.g., HRP), which triggers bindings between this enzyme conjugate and the analyte molecules in the liquid phase.
- an enzyme e.g., HRP
- a solution containing a chromogenic substrate for HRP e.g., insoluble TMB
- a chromogenic substrate for HRP e.g., insoluble TMB
- the horizontal flow absorption pad (17) is connected to the lateral side of the signal generation pad (15; Figure 2 A, right).
- a color signal at the site of the immobilized antibody is produced in proportion to the analyte concentration.
- a control is also run to monitor the consistency of the assay using a secondary antibody, recognizing the detection antibody, immobilized at a site on the signal generation (see the color signal and control in Figure 2A, right).
- connection capillary channels can be further expanded in horizontally connecting a multiple vertical channels in parallel toward the substrate flow.
- a detector Figure 3A is built based on image capture using a digital camera.
- the chip with colored signals is placed under the camera, and the color densities which appeared on the signal generation pad are digitized in the vertical direction using a software program.
- the data are collected and stored in the Microsoft Excel program installed on a personal computer.
- a PDA-based portable prototype detector is further demonstrated as shown in Figure 3B.
- Fig. 1 shows construction of an analytical lab-on-a-chip for ELISA adopting the concept of cross-flow chromatography.
- Fig. 3 shows a schematic diagram of detector for the color signal produced from the lab-on-a-chip sensor (A) and a PDA-based portable prototype detector built as an example (B).
- Fig. 4 shows calibration curve of the lab-on-a-chip and signal detector system for cTnl.
- the signal and control are quantified by integration of the color densities under the respective peaks. Each standard deviation of replicate measurements is indicated.
- Connection capillary channels (2-mm long ) 50 Colorimetric detector 51 : Charge-coupled device (CCD) camera
- Polymethylmetacrylate was obtained from LG Chem (PMMA IF870, Seoul, Korea).
- a stock of cardiac troponin (cTn) I-T-C complex, cTnl single molecule for immunization, and a monoclonal antibody (Clone 19C7) specific to cTnl were supplied by Hytest (Turku, Finland).
- Human anti-mouse antibody (HAMA) blocker (mouse IgG fraction) and a cardiac marker control were obtained from Chemicon International (Temecula, California) and Cliniqa (Fallbrook, California), respectively.
- iV-succimmidyl-3-(2-pyridyldithio) propionate SPDP
- succinimidyl 4- (iV-maleimidomethyl) cyclohexane-l-carboxylate SCC
- DTT dithiothreitol
- Goat anti-mouse antibody, casein (sodium salt type, extracted from milk), human serum (frozen liquid), Triton X-IOO, Sephadex G-15, and G-100 were supplied by Sigma (St. Louis, MO).
- Nitrocellulose (NC) membrane (12- m pore size) and glass fiber membrane (Ahlstrom 8980) were obtained from Millipore (Bedford, MA).
- cTnl A monoclonal antibody specific to cTnl was raised through the adoption of a standard protocol.
- cTnl (30 g) was emulsified with Complete Freund's adjuvant and injected into the peritoneal cavity of a 6-week old Balb/c mouse. After 3 weeks, the mouse was immunized with the same amount of cTnl emulsified with Incomplete Freund's adjuvant. An identical procedure was repeated 2 weeks later, and the final immunization was conducted after the same period with cTnl dissolved in 10 mM phosphate buffer, pH 7.4, (PB) containing 140 mM NaCl (PBS).
- PB mM phosphate buffer
- mice splenocytes were collected and fused with murine plasmacytoma (sp2/0 Ag 14) as a fusion partner.
- Fused hybridoma cells were screened based on HAT selection, and a cell clone producing antibody specific to cTnl (BD Clone 12) was finally screened by immunoassay using antigen-coated microtiter plates.
- This antibody was produced as ascitic fluid from a Balb/c mouse and was then purified on a protein G column (5 mL, HiTrap protein G HP; Amersham Biosciences, Piscataway, NJ). The eluted IgG fractions were pooled, concentrated, dialyzed against PBS, and frozen as aliquots until later use.
- the coupled SPDP linker was activated using DTT, and both modified proteins were fractionated by means of Sephadex G- 15 gel chromatography.
- the antibody was then immediately combined with the HRP in a 5 molar excess and reacted overnight at 4 0 C. This mixture was purified on a Sephadex G-IOO gel column (1O x 200 mm).
- the purified conjugates were quantified by the Bradford method (References: R. C. Duhamel, 1983, Coll. Relat. Res. 1983, Vol. 3, page 195-204), and stored as aliquots after snap freezing.
- Each sample application pad was a glass fiber membrane (2 x 15 mm; Ahlstrom 8980) pre-treated with polyvinyl alcohol by the manufacturer.
- a conjugate release pad was fabricated by transferring 8 L of a conjugate solution onto a glass membrane (2 x 5 mm; Rapid 24Q).
- the conjugate solution was prepared by diluting the HRP labeled- antibody (2.5 g/mL) with 100 mM PB containing 0.5% casein (Casein-PB), HAMA blocker (150 g/mL), ascorbic acid (5 mM), Triton X-100 (0.5%, v/v), and trehalose (20%, w/v).
- a signal generation pad was made by dispensing (1.5 L/cm) the monoclonal antibody (Clone 19C7; 2 mg/mL) in PBS onto a site at 10 mm from the bottom of NC membrane (2 x 25 mm) using a microdispenser (Bio Jet 3000, Biodot, Irvine, CA).
- Fluidic channels were made by mechanically engraving the surfaces of a polyacrylamide chip (32 x 76 x 2 mm), essentially enabling us to comprise the immuno- strip in the vertical position as a part of fluidic channels and to deliver an aqueous solution crosswise (see Figure IA for the overall structure).
- An immuno-strip mounting channel was arranged in the center of the chip by carving the surface to a width of 2 mm, a length of 51 mm, and variable depths, adapting the different thicknesses of each membrane pad of the strip.
- the bottom of the channel was drilled in an oval shape (5 x 10 mm) to provide a sample application pot with a maximum sample holding capacity of 100 L.
- a signal monitoring window was furnished by slitting the chip surface (1 x 18 mm) corresponding to the ceiling of the signal generation pad of the strip.
- an enzyme substrate supply channel and a horizontal flow absorption channel were installed on each opposing side of the vertical channel.
- a substrate supply channel with a depth of 0.8 mm was formed in a shape of a circular triangle expanded to the vertical channel as shown in Figure 1.
- a substrate supply pot (7-mm diameter) was installed by drilling the surface at an inlet of the channel.
- Two air ventilation holes (1-mm diameter) were also made at both of the end projection areas near the outlet of the channel.
- a horizontal flow absorption channel for the flow was built to specific dimensions: a width of 14 mm, length of 12 mm, and a depth of 1 mm.
- the etched plastic chip was integrated with the immuno-strip and a horizontal flow absorption pad by installing them into the vertical channel and the horizontal flow absorption channel, respectively.
- the absorption pad was prepared by attaching the cellulose membrane (14 x 12 mm) to a plastic film using a double-sided tape.
- the integrated chip was closed by covering with a laminating film and then bonding an intact plastic chip of the same size using double-sided tape.
- the chip was finally kept in a desiccator maintained at room temperature until use.
- a stock of cTnl (1 mg/mL; I-T-C complex form) was serially diluted with human serum to prepare samples at pre-determined concentrations.
- the serum itself was regarded as the negative sample.
- the responses of the lab-on-a-chip to the analyte concentrations were obtained using the standard samples of cTnl.
- the samples were added into different lab-on-a-chip, the immune reactions were processed for 15 min and, sequentially, the signal generation was processed for 5 min after the enzyme substrate was supplied.
- the chip with colored signals as shown in Figure 2 was placed under a digital camera (FA185A#ABA, Hewlett-Packard, Palo Alto, CA) built within a detector and illuminated from the bottom using a light source (SR0307A-5230, Seho Robot, South Korea) as shown in Figure 3.
- the image of the signal generation pad was captured and the color densities which appeared on the pad were digitized in the vertical direction using software programmed in C ++ language, installed on a personal computer.
- the data were collected and stored in the Microsoft Excel program.
- the measured optical densities were first subtracted from the mean value of the background colors present between the signal and control peaks.
- the normalized optical densities under the signal peak were then integrated so that a numerical signal value could be assigned.
- the same procedure was repeated three times, and the mean values at each concentration were used to plot a graph of the dose-response curve.
- the dose-response curve of the sensor using standard samples of cTnl was plotted in a semi-log graph as shown in Figure 4.
- the signal varied in a sigmoidal shape, while the control was kept approximately constant regardless of the dose of analyte.
- the sigmoidal curve can be converted to a straight line by means of the log-logit transformation (References: A. DeLean et al., 1978, Am. J. Phys., Vol. 235, page 97-102), which is then used for quantifying the analyte in unknown samples.
- the detection limit of the lab-on-a-chip sensor was found to be approximately 0.1 ng/mL, and the quantification limit was found to be 0.25 ng/mL when the selected cTnl was used as a calibrator.
- the present invention provides a membrane-implanted lab-on-a-chip offering a minimal sample requirement and analytical functions necessary for simultaneously measuring multiple prognostic or diagnostic indicators.
- the chip led the sample flow through the channel merely by capillary action without using an external driving force, which would allow the use of the device for on-the-spot-analysis. Since the device is a miniaturized version for sample reduction that would alleviate, in case of clinical diagnosis, a refusal against finger prick, it would be suitable for a frequent testing of symptoms and diseases with a high sensitivity and at an economical price.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Clinical Laboratory Science (AREA)
- Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007545362A JP2008523386A (en) | 2004-12-09 | 2005-12-01 | Lab-on-chip and signal detection methods for field analysis |
US11/720,177 US20080019866A1 (en) | 2004-12-09 | 2005-12-01 | Lab-On-A-Chip For An On-The-Spot Analysis And Signal Detection Methods For The Same |
EP05819074A EP1834179A1 (en) | 2004-12-09 | 2005-12-01 | Lab-on-a-chip for an on-the-spot analysis and signal detection methods for the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2004-0103462 | 2004-12-09 | ||
KR1020040103462A KR100635110B1 (en) | 2004-12-09 | 2004-12-09 | Lab-on-a-chip for an on-the-spot analysis and signal detector for the same |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2006062312A1 true WO2006062312A1 (en) | 2006-06-15 |
Family
ID=36578105
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2005/004084 WO2006062312A1 (en) | 2004-12-09 | 2005-12-01 | Lab-on-a-chip for an on-the-spot analysis and signal detection methods for the same |
Country Status (6)
Country | Link |
---|---|
US (1) | US20080019866A1 (en) |
EP (1) | EP1834179A1 (en) |
JP (1) | JP2008523386A (en) |
KR (1) | KR100635110B1 (en) |
CN (1) | CN101073008A (en) |
WO (1) | WO2006062312A1 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1990638A1 (en) * | 2007-05-11 | 2008-11-12 | Koninklijke Philips Electronics N.V. | Flow-through biosensor |
EP1992948A1 (en) * | 2007-05-14 | 2008-11-19 | Koninklijke Philips Electronics N.V. | Biosensor device, method of manufacturing a biosensor device and method of detecting target molecules in an analyte |
EP2017006A1 (en) * | 2007-07-20 | 2009-01-21 | Koninklijke Philips Electronics N.V. | Microfluidic methods and systems for use in detecting analytes |
US7964370B2 (en) * | 2008-10-17 | 2011-06-21 | Actherm Inc | Analytical strip and detecting method using the same |
RU2493258C1 (en) * | 2012-03-22 | 2013-09-20 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Орловский государственный аграрный университет" (ФГБОУ ВПО "Орел ГАУ") | Method to detect number of microorganisms in air |
EP3064943A4 (en) * | 2013-10-31 | 2017-05-31 | Konica Minolta, Inc. | Antigen detection method using sandwich immunoassay method |
US9952211B2 (en) * | 2008-06-29 | 2018-04-24 | Realbio Technologies Ltd. | Liquid-transfer device particularly useful as a capturing device in a biological assay process |
CN108527744A (en) * | 2017-03-03 | 2018-09-14 | 南开大学 | Controllable nano materials synthesis reactor based on micro flow chip |
US10598572B2 (en) | 2011-12-22 | 2020-03-24 | Realbio Technologies, Ltd. | Sequential lateral capillary flow device for analyte determination |
Families Citing this family (52)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100723424B1 (en) | 2006-04-07 | 2007-05-30 | 삼성전자주식회사 | Microfluidic device and method for concentrate and lyse cells or viruses, and method for manufacturing the microfluidic device |
KR100811532B1 (en) * | 2006-07-18 | 2008-03-07 | 한양대학교 산학협력단 | Micro bio chip for immunoreaction and manufacture method thereof and immunoreaction detecting method using micro bio chip |
KR100742229B1 (en) | 2006-08-10 | 2007-07-24 | 이상훈 | Microchip for protein fixation using nano fiber sheet |
KR100808415B1 (en) * | 2006-09-07 | 2008-02-29 | 엘지전자 주식회사 | Chip for analyzing matter and matter analysis apparatus having the same |
KR100868769B1 (en) * | 2007-06-07 | 2008-11-17 | 삼성전자주식회사 | Microfluidic chip and fabricating method of the same |
KR101066598B1 (en) * | 2007-12-17 | 2011-09-22 | 한국전자통신연구원 | Method of Multiplex Detecting on Microfluidic Chip |
US20110039033A1 (en) * | 2008-01-31 | 2011-02-17 | Erika Merschrod | Method of depositing a polymer micropattern on a substrate |
KR101541458B1 (en) | 2008-07-03 | 2015-08-04 | 삼성전자주식회사 | Method for Mixing Micro-fluids and Micro-fluidic Mixing Device |
US20100070188A1 (en) * | 2008-08-08 | 2010-03-18 | Neal Solomon | Intelligent medical device system for on-demand diagnostics |
JP5162031B2 (en) * | 2008-10-09 | 2013-03-13 | 紅電醫學科技股▲分▼有限公司 | Liquid inspection method |
BRPI0823169A2 (en) * | 2008-10-17 | 2015-06-23 | Actherm Inc | Fluid Test Strip and Method |
KR101059838B1 (en) * | 2008-11-28 | 2011-08-29 | 한국세라믹기술원 | Microfluidic device using photosensitive ceramic sheet and manufacturing method thereof |
KR100941069B1 (en) * | 2008-12-22 | 2010-02-09 | 한국전자통신연구원 | Microfluidic dilution device |
CA2755347A1 (en) * | 2009-03-23 | 2010-09-30 | Actherm Inc. | Analytical strip and the manufacturing method thereof |
WO2010129302A1 (en) * | 2009-04-28 | 2010-11-11 | Innovative Laboratory Technologies, Inc. | Lateral-flow immuno-chromatographic assay devices |
KR101148769B1 (en) * | 2009-06-02 | 2012-05-24 | 주식회사 인포피아 | appratus, method and test strip for multi-testing |
KR101058743B1 (en) * | 2009-06-04 | 2011-08-24 | 주식회사 인포피아 | Cholesterol Test Strip and Cholesterol Detection Method Using the Same |
KR101203385B1 (en) | 2009-06-04 | 2012-11-21 | 주식회사 인포피아 | Test strip improved spreadability of plasma or serum |
US9248249B2 (en) * | 2009-06-08 | 2016-02-02 | Covidien Lp | Point-of-care pathogen monitoring devices and systems |
US8012770B2 (en) * | 2009-07-31 | 2011-09-06 | Invisible Sentinel, Inc. | Device for detection of antigens and uses thereof |
CN102612555A (en) | 2009-10-09 | 2012-07-25 | 因威瑟堡善迪诺有限公司 | Device for detection of antigens and uses thereof |
KR101117242B1 (en) * | 2009-12-15 | 2012-03-16 | 한국전기연구원 | Appaeatus for detecting biochemistry material using flourescence |
WO2011088247A1 (en) * | 2010-01-13 | 2011-07-21 | Nomadics, Inc. | In situ-dilution method and system for measuring molecular and chemical interactions |
KR101198447B1 (en) | 2010-10-13 | 2012-11-06 | 한국과학기술원 | Plastic based biosensor and manufacturing method for the same |
CN103189524B (en) | 2010-11-01 | 2015-01-28 | 3M创新有限公司 | Biological sterilization indicator and method of using same |
JP5934231B2 (en) | 2010-11-01 | 2016-06-15 | スリーエム イノベイティブ プロパティズ カンパニー | Biological sterilization indicator |
EP3608021A3 (en) | 2011-01-27 | 2020-04-22 | Invisible Sentinel, Inc. | Analyte detection devices, multiplex and tabletop devices for detection of analytes, and uses thereof |
KR101198111B1 (en) | 2011-05-31 | 2012-11-09 | 에스디 바이오센서 주식회사 | Sensor strip |
CA2890892C (en) | 2011-11-28 | 2023-09-12 | Immunoprofile, Llc | Point of care immunization testing system |
JP6190395B2 (en) | 2012-03-09 | 2017-08-30 | インビジブル・センチネル,インコーポレーテッド | Method and composition for detecting multiple analytes with a single signal |
EP2839296A4 (en) * | 2012-04-18 | 2015-11-25 | Univ Texas | Method for the detection and quantification of analytes using three-dimensional paper-based devices |
KR101380368B1 (en) * | 2012-09-18 | 2014-04-10 | 포항공과대학교 산학협력단 | Microfluidic chips having flow cells for absorbance measurements and absorbance measurement apparatus having thereof |
US9990464B1 (en) | 2012-10-09 | 2018-06-05 | Pall Corporation | Label-free biomolecular interaction analysis using a rapid analyte dispersion injection method |
KR20150106493A (en) * | 2014-03-11 | 2015-09-22 | 포항공과대학교 산학협력단 | Microfluidic chips having flow cells using standard addition method and absorbance measurement apparatus having thereof |
KR20150107945A (en) * | 2014-03-13 | 2015-09-24 | 포항공과대학교 산학협력단 | Microfluidic chip based absorbance measurement apparatus using standard addition method |
EP3161483A4 (en) * | 2014-06-25 | 2017-12-20 | ImmunoProfile, LLC | Point of care immunization testing system - detection methods |
WO2016164738A1 (en) | 2015-04-08 | 2016-10-13 | Board Of Regents, The University Of Texas System | Methods and systems for the detection of analytes |
US9733188B2 (en) * | 2015-09-21 | 2017-08-15 | International Business Machines Corporation | Enhancing on-chip fluorescence detection |
WO2017058813A1 (en) * | 2015-09-28 | 2017-04-06 | Essenlix Corp. | Methods and systems for point-of-care sample analysis |
US11125746B2 (en) * | 2016-12-14 | 2021-09-21 | Reliant Immune Diagnostics, Inc. | Two-sided flow-through immunoassay |
KR102088277B1 (en) * | 2018-04-27 | 2020-03-13 | 주식회사 유니언스진 | 3D paper based Microfluidic device for Thioredoxin detection by Enzyme linked immunosorbent assay |
CN109161478B (en) * | 2018-06-26 | 2022-01-25 | 东南大学 | Gold/luminol nanocomposite-based biochip and preparation method and application thereof |
EP3814773A4 (en) * | 2018-06-29 | 2021-07-28 | Siemens Healthcare Diagnostics, Inc. | Contoured sample path for fluid analyzer |
CN109655611A (en) * | 2018-12-20 | 2019-04-19 | 天津瑞普生物技术股份有限公司 | Micro-fluidic chip immunodiagnosis kit and preparation method thereof |
KR102235753B1 (en) * | 2019-05-09 | 2021-04-05 | 경북대학교 산학협력단 | Method and manufacturing system for a lan-on-a-chip using a laser |
CN110849868B (en) * | 2019-10-21 | 2022-09-13 | 江苏大学 | Intelligent detection device and method for potassium bromate in flour based on micro-fluidic chip |
CN111077303A (en) * | 2019-12-25 | 2020-04-28 | 兰州大学 | Vertical channel array microchip and imaging device and method for immunodetection |
CN116867889A (en) * | 2020-12-02 | 2023-10-10 | 苏州新格元生物科技有限公司 | Reagent exchange method, device and system |
CN113083384A (en) * | 2021-03-26 | 2021-07-09 | 河南理工大学 | Droplet type micro-fluidic chip suitable for nucleic acid hybridization detection and preparation method thereof |
WO2022210312A1 (en) * | 2021-04-01 | 2022-10-06 | 富士フイルム株式会社 | Testing device |
KR102585166B1 (en) * | 2021-05-24 | 2023-10-06 | 한국전자통신연구원 | Multi-layered biosensor chip and biomarker measuring apparatus using the same |
CN114295606B (en) * | 2021-11-10 | 2023-06-09 | 扬州大学 | Microfluidic biological logic gate for detecting ocean copper ions |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040214253A1 (en) * | 2003-04-25 | 2004-10-28 | Paek Se Hwan | Membrane strip biosensor system for point-of-care testing |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5135716A (en) | 1989-07-12 | 1992-08-04 | Kingston Diagnostics, L.P. | Direct measurement of HDL cholesterol via dry chemistry strips |
US6806543B2 (en) * | 2002-09-12 | 2004-10-19 | Intel Corporation | Microfluidic apparatus with integrated porous-substrate/sensor for real-time (bio)chemical molecule detection |
-
2004
- 2004-12-09 KR KR1020040103462A patent/KR100635110B1/en not_active IP Right Cessation
-
2005
- 2005-12-01 WO PCT/KR2005/004084 patent/WO2006062312A1/en active Application Filing
- 2005-12-01 CN CNA2005800422290A patent/CN101073008A/en active Pending
- 2005-12-01 EP EP05819074A patent/EP1834179A1/en not_active Withdrawn
- 2005-12-01 US US11/720,177 patent/US20080019866A1/en not_active Abandoned
- 2005-12-01 JP JP2007545362A patent/JP2008523386A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040214253A1 (en) * | 2003-04-25 | 2004-10-28 | Paek Se Hwan | Membrane strip biosensor system for point-of-care testing |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1990638A1 (en) * | 2007-05-11 | 2008-11-12 | Koninklijke Philips Electronics N.V. | Flow-through biosensor |
EP1992948A1 (en) * | 2007-05-14 | 2008-11-19 | Koninklijke Philips Electronics N.V. | Biosensor device, method of manufacturing a biosensor device and method of detecting target molecules in an analyte |
WO2008139367A1 (en) * | 2007-05-14 | 2008-11-20 | Koninklijke Philips Electronics N.V. | Biosensor device, method of manufacturing a biosensor device and method of detecting target molecules in an analyte |
EP2017006A1 (en) * | 2007-07-20 | 2009-01-21 | Koninklijke Philips Electronics N.V. | Microfluidic methods and systems for use in detecting analytes |
WO2009013658A3 (en) * | 2007-07-20 | 2009-03-12 | Koninkl Philips Electronics Nv | Microfluidic methods and systems for use in detecting analytes |
US11280785B2 (en) | 2008-06-29 | 2022-03-22 | Realbio Technologies Ltd. | Liquid-transfer device particularly useful as a capturing device in a biological assay process |
US9952211B2 (en) * | 2008-06-29 | 2018-04-24 | Realbio Technologies Ltd. | Liquid-transfer device particularly useful as a capturing device in a biological assay process |
US7964370B2 (en) * | 2008-10-17 | 2011-06-21 | Actherm Inc | Analytical strip and detecting method using the same |
US10598572B2 (en) | 2011-12-22 | 2020-03-24 | Realbio Technologies, Ltd. | Sequential lateral capillary flow device for analyte determination |
RU2493258C1 (en) * | 2012-03-22 | 2013-09-20 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Орловский государственный аграрный университет" (ФГБОУ ВПО "Орел ГАУ") | Method to detect number of microorganisms in air |
US10591473B2 (en) | 2013-10-31 | 2020-03-17 | Konica Minolta, Inc. | Antigen detection method using sandwich immunoassay method |
EP3064943A4 (en) * | 2013-10-31 | 2017-05-31 | Konica Minolta, Inc. | Antigen detection method using sandwich immunoassay method |
CN108527744A (en) * | 2017-03-03 | 2018-09-14 | 南开大学 | Controllable nano materials synthesis reactor based on micro flow chip |
CN108527744B (en) * | 2017-03-03 | 2021-03-30 | 南开大学 | Controllable nano material synthesis reactor based on microfluidic chip |
Also Published As
Publication number | Publication date |
---|---|
US20080019866A1 (en) | 2008-01-24 |
KR100635110B1 (en) | 2006-10-17 |
EP1834179A1 (en) | 2007-09-19 |
CN101073008A (en) | 2007-11-14 |
KR20060064807A (en) | 2006-06-14 |
JP2008523386A (en) | 2008-07-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20080019866A1 (en) | Lab-On-A-Chip For An On-The-Spot Analysis And Signal Detection Methods For The Same | |
US7300802B2 (en) | Membrane strip biosensor system for point-of-care testing | |
US6316205B1 (en) | Assay devices and methods of analyte detection | |
AU2007280929B2 (en) | Analysis device for biological sample | |
AU2001253354B9 (en) | Diagnostic device with multiple independent flow paths | |
EP0852005B1 (en) | Competitive immunoassay device | |
JP5043186B2 (en) | Fine channel type sensor composite structure | |
US8137905B2 (en) | Apparatus and method for determining an analyte in a fluid | |
US20070087450A1 (en) | Immuno-gold lateral flow assay | |
JP2009501908A (en) | Microfluidic device and method for making and using the same | |
EP2426193A1 (en) | Automated immunoassay cassette, apparatus and method | |
CA2324096A1 (en) | Integrated assay device and methods of production and use | |
Shen et al. | An enhanced centrifugation-assisted lateral flow immunoassay for the point-of-care detection of protein biomarkers | |
JPH04290961A (en) | Device for effecting rapid and easy manual assay | |
KR101916608B1 (en) | Biochemical-immunological hybrid biosensor and sensor system including the same | |
WO2011014673A1 (en) | Automated lateral flow immunoassay cassette with improved flow properties | |
KR20020066792A (en) | Rapid Diagnosis Kit for Diabetes Using Immunochoromatography and Detection Method | |
WO2021053206A1 (en) | Immunoassay analyzer, immunoassay kit and method for detecting analyte in liquid sample | |
Kawde et al. | Moving enzyme-linked immunosorbent assay to the point-of-care dry-reagent strip biosensors | |
KR20040018893A (en) | Rapid Diagnosis Kit and Detection Method for Hemoglobin A1c Using Immunochoromatography | |
WO2014168580A1 (en) | Double-chamber bi-directional reverse flow device |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 11720177 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2007545362 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 200580042229.0 Country of ref document: CN |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005819074 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2005819074 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 11720177 Country of ref document: US |