EP2135626A1 - Streifen für multiparametrische Assays - Google Patents

Streifen für multiparametrische Assays Download PDF

Info

Publication number
EP2135626A1
EP2135626A1 EP08158549A EP08158549A EP2135626A1 EP 2135626 A1 EP2135626 A1 EP 2135626A1 EP 08158549 A EP08158549 A EP 08158549A EP 08158549 A EP08158549 A EP 08158549A EP 2135626 A1 EP2135626 A1 EP 2135626A1
Authority
EP
European Patent Office
Prior art keywords
strip
cap
well
wells
assay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP08158549A
Other languages
English (en)
French (fr)
Other versions
EP2135626B1 (de
Inventor
Françoise De Longueville
Sandrine Hamels
Eric Gancarek
Jens Liedecker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eppendorf Array Technologies SA
Original Assignee
Eppendorf Array Technologies SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eppendorf Array Technologies SA filed Critical Eppendorf Array Technologies SA
Priority to DE602008004752T priority Critical patent/DE602008004752D1/de
Priority to EP08158549A priority patent/EP2135626B1/de
Priority to AT08158549T priority patent/ATE496639T1/de
Publication of EP2135626A1 publication Critical patent/EP2135626A1/de
Application granted granted Critical
Publication of EP2135626B1 publication Critical patent/EP2135626B1/de
Not-in-force legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50855—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using modular assemblies of strips or of individual wells
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • 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
    • B01L2300/0636—Integrated biosensor, microarrays
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls

Definitions

  • the present invention is related to an assay strip having a 8-well format for performing biological reactions between target molecules and capture molecules, wherein these capture molecules present are preferably fixed upon the inner surface of a well of the strip in the form of a micro-array.
  • the present invention is also related to multiple strips for performing multiple assays in a 96-well format frame.
  • micro-array assays are performed on an individual basis along with the different steps of the method from the incubation with the sample to the scanning of the array and the data analysis. These methods are not adapted for handling multiple samples simultaneously. The ability to do so would be advantageous for routine assays requiring automated procedure, (preferably upon multi-wells plate) such as in clinical diagnostic laboratories or in high throughput screening.
  • reaction temperature being high for controlling the specificity when working with polynucleotides reaction.
  • evaporation will occur, if devices used for performing these reactions are not perfectly sealed.
  • a possible deformation of substrates bearing the reactive molecules at high temperatures will also hamper the specific detection of multiple targets at the end of the assay.
  • US patent 5,874,219 provides methods for processing multiple biological chip assays by providing a biological chip plate comprising a contiguous substrate with a plurality of probe arrays.
  • the support Surrounding the probe arrays, the support comprises a material that is resistant to the flow of liquid thereby forming a plurality of test wells, each test well defining a space for an introduction of a sample.
  • These probe arrays are made of small oligonucleotides which are hybridized at low temperature usually comprised between about 25°C and about 30°C.
  • the US patent 5,545,531 provides methods for making a device for processing multiple biological chip assays comprising the steps of providing a wafer comprising on its surface a plurality of probe arrays and applying a material resistant to a flow of a liquid sample so as to surround the probe arrays, thereby creating test wells.
  • the US patent application 20040191810 proposes a plate having conical wells for holding immersed micro-arrays.
  • the plate comprises a body with a plurality of conical wells having a frame for the attachment of the arrays.
  • the conical wells also contain inlets and outlets.
  • This frame has perforations along the sides of the micro-arrays that interact with the inlets and outlets to facilitate liquid pipetting and control bubbles.
  • An optically clear window is attached to the body for possible detection.
  • the US patent application 20040171167 proposes alternative methods for analysis of micro-arrays by packaging micro-array suspended in a well of a well plate. This configuration was proposed for easier handling as there is no micro-array at the bottom of the wells. Here also the array is first manufactured and then inserted inside the wells.
  • the US patent 7,220,573 describes an array assay device comprising a base dimensioned to hold a substrate having at least one array, a cover dimensioned to cover said substrate and a snap-fit for holding said cover and said base together and to enclose a space between said cover and said base.
  • this array assay device is substantially vapor and fluid tight, when the snap-fit hold said base and said cover together.
  • a first aim of the present invention is to provide an assay strip, a plate comprising multiple strips or an apparatus comprising strip(s), for performing multiple assays in parallel and possibly on multiple target molecules, possibly present (simultaneously) in a (biological) sample, in a miniaturized format that does not present the drawbacks of the state of the art.
  • a preferred aim of the present invention is to propose such assay including a sample injection, an incubation even at high temperature and a washing in a optimized device which avoids cross contamination between wells, avoids a bubble formation inside the wells and allows an easy, efficient and reproductive detection of target molecules bound on corresponding capture molecules thereafter.
  • the present invention is related to an assay strip C compatible with multi-well plate format and adaptable upon a frame D of a multi-well plate for performing a series of biological reaction between capture and target molecules, comprising:
  • the inlet of the cap strip B comprises a conical part and the inlet channel 10 of the cap strip B forms an angle lower than 85° and preferably lower than 75° with the flat inner bottom surface 5 of the well 2 upon which capture molecules are immobilized.
  • the inlet 10 and outlet 11 channels are linked by a ditch 13 at the bottom part 8 of the cap 6 and the adjacent wells are linked to each other by attache (s) located at or close to the top surface 3 of the wells 2.
  • the flat inner bottom surface 5 of all wells upon which capture probes are immobilized are preferably in the same focal plan.
  • the flat inner bottom surface 5 of each well is being separated from the external observation surface by an optically transparent solid support for detection of target molecules along an observation angle ( ⁇ ob) being a forbidden angle, and wherein said solid support has a refractive index higher than 1.33.
  • each well 3 has an external flat bottom surface which is not parallel to the flat inner bottom surface 5 and forms an angle between about 90° and about 62° with the normal to said flat inner bottom surface 5 preferably an angle between 85° and 70° with the normal to said flat inner bottom surface 5.
  • a side face of the solid support used for the detection of target molecules along the forbidden angle is between 0° and 36° preferably between 5° and 30° more preferably between 10° and 25° with the normal to the flat inner bottom surface 5.
  • the well 2 walls of the assay strip according to the invention have an opening angle of about 1.5° with the normal to the flat inner bottom surface 5.
  • an assay strip which further comprises an adapter F comprising at least two supercaps 23, preferably eight supercaps 23, each supercap having at least an inlet 24 fitting inside the cap strip inlet channel 10 or outlet channel 11 and having a conical part and possibly wherein the inlet 24 has preferably a length which is equal to the depth of the well 2 minus 0.5 and preferably minus 0.2 mm.
  • Another aspect of the invention is related to a multi-well plate (having frames preferably with squared shape E complementary to the well 2 with a multi-well chassis D, preferably having 96 frames (twelve x eight) for inserting, preferably at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve assay strip(s) C, (preferably composed of eight wells 2 sealed by eight caps 6).
  • the multi-well chassis D further comprises attachment feature(s) 17 complementary to attachment feature(s) 16 of the strip C and/or attachment feature(s) 19 complementary to mounting foot(s) 18 of the strip C.
  • the invention also relates to a multi-well plate for performing a series of biological reactions between capture molecules and (corresponding) target molecules, comprising:
  • Still another aspect of the invention is related to a method for a detection and/or quantification of a target molecule performed by the following steps:
  • the capture molecule binding the target molecule is present on an optically transparent solid support surface having a refractive index n1, said solid support being in contact with a medium having refractive index n2, whereby n1 > n2, said method comprising the steps of :
  • the target molecule is a polynucleotide sequence synthesized in the wells by copying and/or amplifying a polynucleotide sequence present in the sample solution inserted into the wells.
  • Another aspect of the invention is related to an apparatus, (preferably a high throughput screening apparatus of one or more target molecules) which comprise the assay strip or the multi-well plate according to the invention.
  • a last aspect of the invention is a kit of part comprising the strip A comprising capture molecules (probes) fixed (immobilized)upon the strip A (onto the inner bottom surface of the well) and cap strip B of the assay strip C or the multi well plate according to the invention and possibly sealing means for the cap strip, one or more washing solution(s) and one or more detecting solution(s).
  • the strip assay according to the invention is configured in order to obtain a chamber that is vapor and fluid tight. Therefore, the side wall(s) of the cap 6 comprise a pad (or feature 14) to seal the cap 6 inside the well 2, so that the (flat) bottom surface 5 of the well 2 and the bottom surface 8 of the cap 6 are essentially parallel to each other.
  • the cap strip is sealable and removable from the strip.
  • the cap strip is made of a flexible material such as thermoplastic elastomer (TPE) to better seal the wells.
  • TPE thermoplastic elastomer
  • the (linear) thermal expansion coefficient of the cap strip material is higher by at least 5 and better 10 and even better more than 20 10 -6 /K at 20 °C in m/m.K x 10 -6 than the thermal expansion coefficient of the well material.
  • the linear thermal expansion is the one-dimensional length change with temperature.
  • the cap strip B has been designed to avoid bubble formation either during the introduction of the sample and/or during the incubation.
  • the diameter of the outlet channel 11 is at least 1 mm preferably about 1.2 mm to allow bubble to get out of the chamber.
  • the inlet channel 10 and outlet channel 11 are preferably linked by a ditch 13 or guiding area located on the bottom part 8 of the cap 6 in order to guide bubble to the exit (outlet channel 11).
  • This ditch is preferably not flat with the upper part being oriented to the outlet and the lower part to the inlet channel, so as to guide the air outside the chamber when injecting solution (through the inlet channel).
  • the strip is best designed in order to fit the pipette in the inlet and inject liquids without touching the array, preferably the inlet of the cap strip B comprises a conical (injection) part so that it can accommodate a pipette being inserted into such conical part.
  • the tip of the conical part is sticking out in the well chamber and more preferably is close to, but not touching the surface.
  • Washing is also best obtained in an automatic process by having a pipette in the inlet and/or in the outlet opening comprising preferentially a conical part so that it can aspirate the liquid of the wells.
  • the inlet channel 10 of the cap strip B forms an angle lower than 85° and preferably lower than 75° with the flat inner bottom surface 5 of the well 2 upon which capture molecules are immobilized. In this way injection flow will be directed on a side of the surface, where the capture molecules and the targets are not fixed, so as to avoid any possible detachment by the injecting flow.
  • the invention also includes the possibility for having a cover or adapter F having an appropriate injection part which fit into the cap inlet and being placed on the cap strip during the injection and/or the aspiration of the liquids from the wells.
  • the cover is designed as such as an injection part does not touch the surface of the wells containing the capture probes, but is made such as to be very close to this surface.
  • the liquid influx is best dispersed by solid part(s) being present in the injection section of the inlet channel and which splits the liquid flux.
  • the orientation of the liquid influx on the side of the well in order to avoid direct flux of injection liquid on immobilized target molecules.
  • the outflow of the liquid from the injection inlet is oriented with angle lower than 85° and better lower than 75° compared to the inner flat surface of the well comprising the immobilized probes.
  • the assay strip C comprises an adapter F comprising at least two supercaps 23, preferably eight supercaps 23, each supercap having at least an inlet 24 fitting inside the cap strip inlet channel 10 or outlet channel 11 and having a conical shape ( figure 5 ).
  • the inlet 24 has a length which is equal to the depth of the well 2 minus 0.5 and preferably minus 0.2 mm.
  • the capture molecules (probes) are immobilized (fixed) on the (flat) inner bottom surface 5 of the well 2 in discrete regions in the form of a micro-array having a density of at least 4, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 600, 1000 or more discrete region per cm 2 .
  • the sealing of the cap strip B into the strip A prevent or avoid drying out of this array.
  • this (flat) inner bottom surface of all wells upon which capture molecules are immobilized are in the same focal plan.
  • each well has an external (flat) surface which is parallel to the (flat) inner surface and all surfaces are in the same focal plan.
  • the distance (tolerance) between this micro-array comprising capture molecules and the well 2 side walls is higher than 1 mm, for allowing an efficient delivery of capture molecules upon this (flat) bottom surface 5 of the well 2 by a spotting arrayer.
  • the distance of at least 1 mm also avoids border effects during incubation with the sample (capillary movements along the walls.
  • the pitch between adjacent wells 2 is about 9 mm.
  • the assay strip C of the invention allows many tests upon many arrays to be set up and processed together; because they allow much higher throughput of test samples and greatly improves the efficiency for performing assays on biological micro-arrays.
  • the assay strip C allows proper formation of a tight chamber resulting from the sealing of two complementary parts strip A, cap strip B by avoiding the use of glue that can interfere with biological reaction which is difficult to remove from the support and which can induce a fluorescent background.
  • the volume of liquid above capture molecules present on a (flat)bottom surface 5 of the well 2 is small and remains homogeneous on the overall bottom surface 5 comprising these immobilized capture molecules during the different processing steps, this requirement improves the reproducibility of the assay, particularly when these capture molecules are present in a micro-array format.
  • a sealing of a cap strip B on a strip A, to form the assay strip C of the invention is also configured to avoid contamination of a sample solution contained in a first well by another sample solution possibly contained in an adjacent well.
  • the bottom surface 5 of the wells 2 has preferably a square shape with round shape corner to avoid capillary effects bringing solution out of the well.
  • the corner radius is preferably of about 1.5 mm.
  • the top surface 7 of the cap strip B comprises a trapping well 15 around the inlet and outlet channels 10, 11 where the exceeding solution is trapped.
  • the assay strip C according to the invention allows easy access to the bottom surface 5 of fixed by capture molecules as required for the scanning of the array.
  • the chamber can be dissociated and re-associated according to the constraints of the fabrication or of the multiple steps of a detection method. These sealing and opening step have to be easily performed either manually or by using a robotic automate that can be also used for the formation of the array on this bottom surface 5 and for a scanning of this bottom flat surface 5 comprising the capture molecules.
  • the assay strip C can be subjected to a plurality of processing steps, including genetic amplification such as PCR, hybridization, washing, staining and scanning steps.
  • the cap strip B can be removed after a sample incubation, thus making the strip A easily accessible for successive working and labeling either manually or by using an automatic device.
  • adjacent wells 2 of the strip A are preferably linked to each other by attache(s) located at or close to the top surface 3 of the wells 2 in order to allow a tight insertion into a multi-well plate.
  • the assay strip C according to the invention is compatible with a multi-well format. Therefore, another aspect of the present invention is related to a multi-well plate comprising a multi-well chassis D and at least one, preferably twelve assay strip(s) C according to the invention.
  • this chassis D comprises 96 square frames E (12 x 8) for inserting up to 12 assay strip (s) C made of square shape wells 2 according to the invention.
  • Dimension of the chassis D are preferably about 85.5 mm (W) x 127.8 mm (L) x 14.8 mm (H).
  • the chassis D is made of a polymer being preferably resistant at 100°C. The polymer is preferably transparent polycarbonate.
  • this frame E has squared shape (format) which is obtained at low cost, but allow an easy insertion of the assay strip(s) C, especially the wells 2 of strip(s) A into the frame E of the plate.
  • the square is about 8 x 8 mm.
  • the chassis D has preferably an asymmetric shape for receiving the assay strip C in a single orientation.
  • it may present insertion features 17 and 19 fitting with complementary features 16 and 18 of the strip A in a predetermined position into the chassis D as provided in figures 2 and 4 .
  • the extremities or holding pads 22, 22' of the strip A and/or the extremities or holding pads 12, 12' of the cap strip B have different shapes (i.e. one pad is square and the other one is circle) to fit with this requirement.
  • the square pad may be used to apply a code bar.
  • This insertion system provides an adequate alignment of the top surfaces of different assay strips C positioned in a same plane above the frames E.
  • the extremities of the strip A may comprise a mounting foot 18 to maintain strip A in the multi-well chassis D.
  • the mounting foot 18 is inserted in the attachment feature 19 of the multi-well chassis D.
  • the extremities of the strip A are asymmetric and one of them comprises the attachment feature 16 that is complementary to the attachment feature 17 of the multi-well chassis D, as provided in figure 2 .
  • the assay strip according to the invention is also adapted for easy automation of reaction inside these strips.
  • the assay strips C are aligned in the same orientation in the multi-well chassis D by means of the mounting foot 18 inserted in the attachment feature 19 of the multi-well chassis D and also by means of complementary attachment features 16 and 17.
  • each well 2 is easily inserted and maintained in the frames E of the multi-well chassis D.
  • Examples of automatic handling elements are: pipette tips which are inserted into the inlet 10 and outlet 11 channels of the strip caps B. Automation of the different steps of the process makes the assays much more reproducible and thus leads to a better quantification of the tests which is a requirement of assays on micro-arrays where the quantification of the results are obtained by the detection of signals on very small surfaces.
  • the multi-well chassis contains 12 inserted assay strips, each one being inserted into the chassis by means of asymmetrical extremity. This feature was found useful for stable and unambiguous insertion of the assay strip and to have all flat surfaces aligned on the same height so that they are in the same focus for the detection step.
  • the number of assay strips C inserted in the multi-well chassis D is adapted according to the customer need and the number of samples to be processed in parallel.
  • the distance between the external bottom surface of the assay strip C or the wells 2 of the strip A and a support surface (table surface) is of at least 1 mm.
  • the surface of the chassis D in contact with a support (table surface) is preferably aligned with the external bottom surface 1 of the strip A or of the well 2. This surface 1 is opposite to the inner bottom surface 5 comprising the immobilized capture molecules.
  • the strip A is inserted in the multi-well plate at a position which avoid any contact between this external bottom surface 1 and the solid support preferably, in this position the external bottom surface is located at the distance of at least 1 mm from this solid support surface.
  • the contact is made only between the solid support surface and the chassis D of the multi-well plate.
  • the bottom surface 1 of strip A or of the well 2 may present also an ring format 20 of polymer in a height of at least 1 mm. This feature avoids scratches of the external bottom 1 of the well 2 due to contact with various tables surfaces.
  • An ring 20 of polymer of at least 1 mm high surrounding the external bottom surface 1 of the well 2 is depicted in figure 2 .
  • the wells 2 of the strip A and the caps 6 of the cap strip B have preferably a square shape for an easy introduction into the frames E of the multi-well chassis D.
  • other shapes or others sections of the wells could be selected by the person skilled in the art.
  • the assay strip C according to the invention may further comprises means for sealing inlet and outlet channels 10, 11 at the top surface 7 of the cap strip B so as to obtain a sealing of inlet port and an outlet port of this inlet channel and outlet channel.
  • Sealing typically comprise the aluminium sealing foil, plastic foils or plugs.
  • the capture molecules are selected from the group consisting of nucleotide sequences (polynucleotide sequences or oligonucleotide sequences), proteins, peptides (preferably antibodies or hypervariable portions thereof) polysaccharides or a mixture thereof. These capture molecules are capable of binding complementary target molecules which are preferably selected from the group consisting of nucleotide sequences, proteins, peptides, haptens or a mixture thereof.
  • the capacity volume of the chamber formed in the assay strip C according to the invention is comprised between about 10 ⁇ l and about 200 ⁇ l, preferably about 100 ⁇ l.
  • the capacity volume of the well 2, when not sealed by the cap is comprised between about 100 ⁇ l and about 1000 ⁇ l.
  • the wells 2 of the strip A have a square shape to maximize the spotting area of the capture molecules, particularly when they are spotted in the form of a micro-array.
  • the well walls have an opening angle of about 1.5° with the normal to the (flat) inner bottom surface 5.
  • the (flat) bottom surface 5 of the well 2 has a square shape with the corners being curved and having a curve shape corresponding to a circle of 1.5 mm of radius.
  • the assay strip C according to the invention is made of two parts: the strip A and the cap strip B which are advantageously made of different polymeric materials.
  • the strip A is made of a rigid polymer while the cap strip B is made of flexible polymer.
  • the strip A is preferably made of a rigid low fluorescence material, preferably selected from the list consisting of cycloolefin, polycarbonate, polyacrylate and polyethylene, more preferably a cycloolefin polymer.
  • the cap strip B is preferably made of a flexible polymer, preferably thermoplastic elastomer (TPE).
  • TPE thermoplastic elastomer
  • the bottom surface 5 onto which the capture molecules are immobilized is preferably transparent to light and distortion free for purposes of imaging the surface.
  • the transmission of the light of wavelength between 400 and 600 nm is higher than 50% and even higher than 90% and even higher than 99%.
  • This material should preferably also be non-fluorescent in order to minimize the background signal level and allow detection of low level signals from low intensity features on the surface.
  • the intrinsic fluorescence of the material should be lower than 10 % and even lower than 1% of he fluorescent emitted by the location where the target is bound.
  • the capture molecules are preferably covalently immobilized on Zeonex TM (330R) using the chemistry described in the EP Patent application 06112775.9 . incorporated herein by reference.
  • the well B bottom thickness is preferably of less than 1 mm with 50 ⁇ m tolerance between the first well and the last well of one strip.
  • the bottom surface 5 of the well 2 is about 40 mm 2 .
  • the bottom surface 5 of the well 2 has a square shape with a length of about 6 mm and height of about 4 mm with a tolerance of 50 ⁇ m.
  • such surface comprises round shape corners (having preferably a radius of about 1.5 mm) to avoid capillary effect.
  • the strip A has width of about 8 mm, a length of about 82 mm and height of about 5 mm (without the o-ring 20) or 6 mm (with the o-ring 20) with a 100 ⁇ m tolerance.
  • the cap strip B has width of about 8 mm, a length of about 82 mm and height of about 1 mm.
  • the top surface 7 of the cap strip comprises at least one inlet channel 10 and at least one outlet channel 11 which are separated from each other by a pitch of about 4.5 mm.
  • the diameter of inlet channel 10 or outlet channel 11 is about 1.2 mm.
  • the strip A When the strip A is sealed with the cap strip B (assay strip C) it prevents less than 10% evaporation of liquid present in the chamber after 24 hours incubation at 65°C, preferably less than 5% evaporation of liquid volume. Furthermore, the sealed assay strip C resists to agitation and may be incubated in adequate temperature in a mixing apparatus like the thermomixer from Eppendorf AG (Hamburg, Germany).
  • the target molecules may be labeled to allow their detection.
  • the labelled associated detections are numerous. A review of the different labelling molecules is given in W0 97/27317 .
  • the most frequently used and preferred labels are fluorochromes like Cy3, Cy5 and Cy7.
  • Radioactive labelling, cold labelling or indirect labelling with small molecules recognised thereafter by specific ligands are common methods.
  • the resulting signal of target fixation on the array is either fluorescent, colorimetric, diffusion, electroluminescent, bio- or chemiluminescent, magnetic, electric like impedometric or voltametric ( US-A-5,312,527 ).
  • a preferred label is the use of the gold labelling of the bound target in order to obtain resonance light scattering (RLS) detection or silver staining which is then easily detected and quantified by a scanner.
  • RLS resonance light scattering
  • Gold particles of 10-30 nm are required for silver amplification while particles of 40-80 nm are required for direct detection of gold particles by RLS or by Photothermal Heterodyne Imaging.
  • gold particles of 10-30 nm are amplified by silver enhancement preferably using the silverquant analysis platform including the Silverquant kit for detection, the Silverquant Scanner for slide scanning and Silverquant Analysis software for image quantification and data analysis (Eppendorf, Germany). Due to the non linear detection of the presence of silver, the data analysis requires a linearization of data before data processing. The data are then processed according to the invention. An algorithm of curve fitting is applied to a positive detection curve spotted on the array. Then each spot signal is linearized in 'concentration units' using the fitting curve.
  • Assays on biological reactions are performed according by the use of the strip and cap strip according to the present invention and having the feature as provided here above in the following way.
  • the solution containing the target to be detected and/or quantified are inserted into the strip containing immobilized capture molecules and comprising a cap strip (B) of the assay strip (C) according to any of the preceding embodiments.
  • the cap strips are then sealed and the strip incubated under the selected reaction conditions for the binding reaction between the target and its capture molecule to take place.
  • the sample solution is removed preferably trough the cap strip and washing solution inserted in the well.
  • reagents are incorporated into the wells to provide a detecting signal and the signal at the target location is detected in order to detect and/or quantify the presence of the target in the sample solution.
  • the detection of the array based signal can be performed by direct illumination of the bound target and detection of an emitted or transmitted or refracted or absorbed light.
  • the light emitted from the bound target molecule can be aslo detected at a forbidden angle for the light emitted from the soluble target molecule.
  • This method allows detecting and/or quantifying a biological target molecule present on an (optically transparent) solid support surface having a refractive index n1, this solid support being in contact with a medium having refractive index n2, whereby n1 > n2, this method comprising the steps of a) illuminating the target molecule, thereby causing the target molecule to emit light and b) detecting light emitted from the target molecule through said support at an observation angle ⁇ obin relative to the normal to the said solid support surface in the support, such that 90° > ⁇ obin > sin -1 (n2/n1).
  • the observation angle ( ⁇ ob) is within the forbidden angle range, but close to the critical angle ( ⁇ c ) to be able to reconstitute the image of the support. This is especially useful when different capture molecules are present on the surface of the support and have to be differentiated from each other as in the case of microarray.
  • the observation angle is lower than 85°, more preferably lower than 80° and still more preferably lower than 70°.
  • the observation angle is within the forbidden angle and in the range of the critical angle plus 10°, preferably plus 5° and more preferably plus 3°.
  • the observation angle is between about 62.4° and about 65° when the solid support has a refractive index of around 1.5 as for glass.
  • the required observation angle ⁇ obin is given by the formula 90° > ⁇ obin > ⁇ c . In other words, the observation angle should be at least 62.4° .
  • the (flat) inner bottom surface 5 of each well is being separated from an external observation surface by an optically transparent solid support for detection of target molecules along an observation angle ( ⁇ ob) being a forbidden angle, and wherein this solid support has a refractive index higher than 1.33.
  • each well 2 of a strip A has an external flat bottom surface which is not parallel to the (flat) inner bottom surface 5 and forms an angle of between 90° and 62° with the normal to this (flat) inner bottom surface 5.
  • the observation angle is within the forbidden angle and is such that the signal coming from a solution is not significantly detected compared to an emitted light of the bound target molecules.
  • a side face of the solid support used for the detection of target molecules along the forbidden angle is between about 0 and about 36° with the normal to the (flat) inner bottom surface 5.
  • the well presents a ridge channel on the opposite side of the support compared to the observation area having immobilized capture molecules.
  • the illuminating and/or the detecting light are in the Total Internal Reflection (TIR) method using the support as the wave guide.
  • TIR Total Internal Reflection
  • the invention provides an apparatus for carrying out the process of the invention requiring: an optically transparent solid support having refractive index n1 designed to comprise, when in use, at least one target molecule bound on capture molecules present on said solid support surface (bound target molecule) and wherein the refractive index of the solid support is higher than the refractive index n2 of the medium, where the binding of the target molecule on the capture molecule occurs; a light source to produce a light beam directed on the target molecule; a detector for measuring light emitted from the target molecule, said emitted light being detected through said optically transparent solid support at an observation angle ⁇ obin relative to the normal to the said solid support surface in the support, such that 90° > ⁇ obin > sin -1 (n2/n1).
  • the detection of the signals at the target molecule location is performed online during the target molecule binding to its capture molecule.
  • the target molecule is a polynucleotide sequence synthesized in the wells by copying and/or amplifying a polynucleotide sequence present in the sample solution inserted into the wells.
  • the first and preferably the second removals of the solutions from the well after the incubation are performed with the cap strip still present on the strip and the following liquid handlings are then performed without the cap strips being present on the strips.
  • the assay method of this invention automates the handling steps so as to allow multiple assays to be performed concurrently.
  • this invention employs automated fluid handling systems for concurrently performing the assay steps in each device. Fluid handling allows uniform treatment of samples in the devices. Microtiter robotic and fluid-handling devices are available commercially, for example, from Tecan AG, Biomek or EPmotion (Eppendorf AG).
  • handling of the assay strip C is performed by using a (multi)pipette or syringe and/or 96-wells plate format automate.
  • Samples solutions are preferably introduced into the assay strip C through inlet port of an inlet channel 10.
  • the air present in the chamber 1 of the device C is moved out of the chamber by the outlet channel 11.
  • the inlet port of the inlet channel 10 and the air duct port of the outlet channel 11 are located in a depression on the cap strip where the exceeding solution is trapped if needed when injecting a too high volume of solution thus avoiding contamination from one strip to the other.
  • the steps after the incubation step with the sample are preferably performed without removing the cap strip B by just removing sealing means applied on the inlet elements of the cap strip top surface 7.
  • Drying of the surface area comprising the capture molecules is also possible using the assay strip of the invention. Air aspiration is performed by one of the port.
  • the cap strip B is preferably removed to allow a scanning of the well 2 bottom surface 5 from the top.
  • Scanning of multiple assay strips C inserted into the multi-well chassis D may be performed by several means.
  • Scanners adapted for multi-well plate reading may be used. These scanners have usually a limited focusing tolerance. Alternatively, the scanning may be performed by using a scanner having independent focusing system for each assay strip.
  • Asymmetric holding pads 22, 22' of the strip A as depicted in figure 1 are advantageously used to insert the strip into the multi-well plate chassis D having complementary structures. These support structures may be used to mount or position the strip assays C in the same orientation into the multi-well chassis D.
  • Each frame E of the multi-well chassis D matches the shape of a well of the strip A that has preferably a square shape to avoid rotation of the assay strip C once positioned into the frame E.
  • Asymmetric and unidirectional positioning of the assay strips C into multi-well chassis allows unique horizontal orientation.
  • the assay strips C are also aligned vertically into the frames E due to the presence of discontinuous bottom surface 1 of the strip A allowing each well to be inserted into an individual frame and making the overall structure rigid and stable.
  • the assay strips which are inserted in the multi-well chassis D may be easily aligned by an automatic handling device or on detection or imaging system. This feature ensures proper orientation and alignment for liquid handling and scanning of the target molecules bound to complementary capture molecules.
  • the plate of the invention can be introduced into a holder in the fluid-handling device.
  • This robotic device is programmed to set appropriate reaction conditions, such as temperature, add samples to the well 2 of assay strip C through inlet channel 10, incubate the test samples for an appropriate time, remove un-reacted samples, wash the wells 2, add substrates as appropriate and perform detection assays.
  • the particularity of the reaction conditions depends upon the purpose of the assay. For example, the assay may involve testing whether a sample contains target molecules that react to a probe under a specified set of reaction conditions. In this case, the reaction conditions are chosen accordingly.
  • removals of the solutions from the wells 2 after the incubation step is performed with the cap strip (B) present on the strip (A) and the next liquid handlings are then performed in the strips (A) in absence of the cap strips (B).
  • the present invention is related to an apparatus (high throughput screening apparatus) which comprise the assay strip C or the multi-well plate comprising the assay strip C or the well(s) 2 of the assay strip C according to the invention, and possibly multi pipettes and other element of a 96 well plate format or an automate.
  • Another aspect of the present invention is related to an apparatus (high throughput screening apparatus) which comprise the assay strip C and the multi-well plate according to the invention, and a 96-well format PCR thermocycler.
  • An example of compatible PCR cycler is the Mastercyler® (Eppendorf AG).
  • Such apparatus allow automated PCR and/or hybridization on capture molecules into the assay strip C of the invention.
  • a multi-well plate cover (preferably in plastic) may be used between the assay strip C and the heating cover of the thermocycler to exert a homogeneous pressure over all assay strips and maintain a tight sealing during the PCR.
  • Example 1 micro-array spotting at the bottom (flat) surface of the strip A of the assay strip C
  • Aldehyde functions were introduced into a cycloolefin (Zeonex TM 330R) strip A using the chemistry described in the EP Patent application 07106545.2 incorporated herein by reference using 0.1% of dextran polyaldehyde.
  • the dimensions of the strip A were the following: 8.07 mm (W) x 82 mm (L) x 4.9 mm (H) without bottom o-ring 20 (5.9 mm with o-ring).
  • the strip A comprises 8 wells 2.
  • the capture probes of the array were those of the DualChip GMO kit (Eppendorf AG, Hamburg, Germany) and were described in the EP Patent application 05447115.6 incorporated herein by reference.
  • the capture molecules (probes) were single stranded polynucleotide which were aminated on the 5' end. These probes were able to identify genetically modified events by screening simultaneously multiple genetic elements:
  • the PCR were performed in a final volume of 25 ⁇ l containing: 1X Buffer Biotools including 2 mM MgCl 2 , 0.2 ⁇ M of each primer with one of the primer being biotinylated, 200 ⁇ M of each dATP, dCP, dGTP and 400 ⁇ M of dUTP, 1.25 U of Taq DNA polymerase Biotools, 0.5 U of UNG (ref 71960, USB, Cleveland, Ohio, USA) and containing 100 ng of Genomic DNA that was extracted from RRS 0.1% sample (ERM-BF410f, IRMM, Geel, Belgium) using a CTAB-based method (Rogers and Bendich, 1985) and quantified using the "Quant-it TM PicoGreen dsDNA assay kit" (Invitrogen, USA) as described in the manual.
  • DNA sample were from RRS GMO which contains the following 5 genetic elements: P35S, T-nos, EPSPS, Lectin (Soybean
  • Samples were first incubated at 22°C for 10 min and then denatured at 94 °C for 5 min. Then 35 cycles of amplification were performed consisting of 30 sec at 94 °C, 40 sec at 56 °C and 1 min at 72 °C and a final extension step of 10 min at 72 °C.
  • each well 9 ⁇ l of each PCR product was mixed in a 1.5 ml microtube with 5 ⁇ l of SensiHyb solution and 4 ⁇ l of hybridization control. 5 ⁇ l of 0.5 N NaOH was added and incubated for 5 min at room temperature. Then 50 ⁇ l of Genomic HybriBuffer was added and 100 ⁇ l of the hybridization mix solution was injected inside a well 2 of a strip A of the invention. The strip A was sealed with the cap strip B and hybridization was performed for 1h at 60°C. The hybridization was immediately followed by the detection step. The colorimetric detection was performed using the Silverquant detection kit (Eppendorf, Hamburg, Germany).
  • the cap strip B was removed from the strip A and wells were washed 2 times with Post Hybridization Buffer for 1 min and 3 times with Washing Buffer for 1 min.
  • the wells were incubated for 10 min at temperature in Pre-Blocking Buffer and for 45 min at room temperature in the Diluted Gold-Conjugate (100 ⁇ l/well). After incubation, the wells were washed 4 times with Washing Buffer for 1 min, then once with Rinsing Buffer for 1 min. Then, an equal volume of Silverquant A and B solutions were incubated in the wells for 5 min at room temperature. After incubation, the wells were washed 2 times with distilled water for 30 sec at room temperature and air dried.
  • the strip A was then inserted into the holder adapter of the Silverquant scanner (Eppendorf, Hamburg, Germany) and scanning of the array was performed according to the instruction manual.
  • the Silverquant analysis software (Eppendorf, Hamburg, Germany) was used for processing the data of the array. The result is presented in figure 6 .
  • the five genetic elements P35S, T-nos, EPSPS, Lectin, and Rbcl) present in RRS were effectively detected on the array present in the 6 wells of the assay strip of the invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measurement Of Radiation (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
EP08158549A 2008-06-19 2008-06-19 Streifen für multiparametrische Assays Not-in-force EP2135626B1 (de)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DE602008004752T DE602008004752D1 (de) 2008-06-19 2008-06-19 Streifen für multiparametrische Assays
EP08158549A EP2135626B1 (de) 2008-06-19 2008-06-19 Streifen für multiparametrische Assays
AT08158549T ATE496639T1 (de) 2008-06-19 2008-06-19 Streifen für multiparametrische assays

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP08158549A EP2135626B1 (de) 2008-06-19 2008-06-19 Streifen für multiparametrische Assays

Publications (2)

Publication Number Publication Date
EP2135626A1 true EP2135626A1 (de) 2009-12-23
EP2135626B1 EP2135626B1 (de) 2011-01-26

Family

ID=39720412

Family Applications (1)

Application Number Title Priority Date Filing Date
EP08158549A Not-in-force EP2135626B1 (de) 2008-06-19 2008-06-19 Streifen für multiparametrische Assays

Country Status (3)

Country Link
EP (1) EP2135626B1 (de)
AT (1) ATE496639T1 (de)
DE (1) DE602008004752D1 (de)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012112815A3 (en) * 2011-02-18 2013-01-03 Ivera Medical Corporation Medical implement cleaning system
US8834650B2 (en) 2006-07-21 2014-09-16 Ivera Medical Corporation Medical implement cleaning device
US8832894B2 (en) 2011-07-19 2014-09-16 Ivera Medical Corporation Cleaning device for male end of intraveneous set
US9259284B2 (en) 2007-02-12 2016-02-16 3M Innovative Properties Company Female Luer connector disinfecting cap
WO2017072289A1 (en) * 2015-10-30 2017-05-04 Lonza Limited Modular well plates
US9895526B2 (en) 2006-03-08 2018-02-20 Ivaxis, Llc Anti-contamination cover for fluid connections
US9907617B2 (en) 2013-03-15 2018-03-06 3M Innovative Properties Company Medical implement cleaning device
US9999471B2 (en) 2012-06-04 2018-06-19 3M Innovative Properties Company Male medical implement cleaning device
US10406525B1 (en) 2018-04-13 2019-09-10 Inscripta, Inc. Automated cell processing instruments comprising reagent cartridges
US10508288B1 (en) 2017-09-30 2019-12-17 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems comprising flow-through electroporation devices
US10519437B1 (en) 2017-06-30 2019-12-31 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10532324B1 (en) 2018-08-14 2020-01-14 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US10590375B2 (en) 2018-03-29 2020-03-17 Inscripta, Inc. Methods for controlling the growth of prokaryotic and eukaryotic cells
US10689669B1 (en) 2020-01-11 2020-06-23 Inscripta, Inc. Automated multi-module cell processing methods, instruments, and systems
US10752874B2 (en) 2018-08-14 2020-08-25 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US10787683B1 (en) 2017-08-28 2020-09-29 Inscripta, Inc. Electroporation cuvettes for automation
US10858761B2 (en) 2018-04-24 2020-12-08 Inscripta, Inc. Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells
US10907125B2 (en) 2019-06-20 2021-02-02 Inscripta, Inc. Flow through electroporation modules and instrumentation
US10920189B2 (en) 2019-06-21 2021-02-16 Inscripta, Inc. Genome-wide rationally-designed mutations leading to enhanced lysine production in E. coli
US10927385B2 (en) 2019-06-25 2021-02-23 Inscripta, Inc. Increased nucleic-acid guided cell editing in yeast
US10954485B1 (en) 2018-08-14 2021-03-23 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11142740B2 (en) 2018-08-14 2021-10-12 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US11225674B2 (en) 2020-01-27 2022-01-18 Inscripta, Inc. Electroporation modules and instrumentation
US11268088B2 (en) 2020-04-24 2022-03-08 Inscripta, Inc. Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells via viral delivery
US11787841B2 (en) 2020-05-19 2023-10-17 Inscripta, Inc. Rationally-designed mutations to the thrA gene for enhanced lysine production in E. coli
US11965154B2 (en) 2018-08-30 2024-04-23 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0688602A2 (de) * 1994-06-22 1995-12-27 Nunc A/S Mikrotitriersystem
US5874219A (en) 1995-06-07 1999-02-23 Affymetrix, Inc. Methods for concurrently processing multiple biological chip assays
WO2002072423A1 (en) * 2001-03-09 2002-09-19 Biomicro Systems, Inc. Microplate lid
EP1391242A2 (de) * 2002-05-24 2004-02-25 Corning Incorporated Mikrosäulenbasierte Anordnung zur Hochdurchsatzanalyse
WO2004094060A1 (en) * 2003-04-22 2004-11-04 Chip-Man Technologies Oy Analysis and culture apparatus
US20050048642A1 (en) * 1997-10-27 2005-03-03 Nalge Nunc International Corporation Multi-slide assembly including slide, frame and strip cap, and methods thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0688602A2 (de) * 1994-06-22 1995-12-27 Nunc A/S Mikrotitriersystem
US5874219A (en) 1995-06-07 1999-02-23 Affymetrix, Inc. Methods for concurrently processing multiple biological chip assays
US20050048642A1 (en) * 1997-10-27 2005-03-03 Nalge Nunc International Corporation Multi-slide assembly including slide, frame and strip cap, and methods thereof
WO2002072423A1 (en) * 2001-03-09 2002-09-19 Biomicro Systems, Inc. Microplate lid
US7390463B2 (en) 2001-09-07 2008-06-24 Corning Incorporated Microcolumn-based, high-throughput microfluidic device
EP1391242A2 (de) * 2002-05-24 2004-02-25 Corning Incorporated Mikrosäulenbasierte Anordnung zur Hochdurchsatzanalyse
WO2004094060A1 (en) * 2003-04-22 2004-11-04 Chip-Man Technologies Oy Analysis and culture apparatus

Cited By (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9895526B2 (en) 2006-03-08 2018-02-20 Ivaxis, Llc Anti-contamination cover for fluid connections
US8834650B2 (en) 2006-07-21 2014-09-16 Ivera Medical Corporation Medical implement cleaning device
US8999073B2 (en) 2006-07-21 2015-04-07 Ivera Medical Corporation Medical implement cleaning device
US9259284B2 (en) 2007-02-12 2016-02-16 3M Innovative Properties Company Female Luer connector disinfecting cap
US10195000B2 (en) 2007-02-12 2019-02-05 3M Innovative Properties Company Female luer connector disinfecting cap
WO2012112815A3 (en) * 2011-02-18 2013-01-03 Ivera Medical Corporation Medical implement cleaning system
US8832894B2 (en) 2011-07-19 2014-09-16 Ivera Medical Corporation Cleaning device for male end of intraveneous set
US9999471B2 (en) 2012-06-04 2018-06-19 3M Innovative Properties Company Male medical implement cleaning device
US9907617B2 (en) 2013-03-15 2018-03-06 3M Innovative Properties Company Medical implement cleaning device
WO2017072289A1 (en) * 2015-10-30 2017-05-04 Lonza Limited Modular well plates
US11203751B2 (en) 2017-06-30 2021-12-21 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US11034953B1 (en) 2017-06-30 2021-06-15 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10954512B1 (en) 2017-06-30 2021-03-23 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10689645B1 (en) 2017-06-30 2020-06-23 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10519437B1 (en) 2017-06-30 2019-12-31 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10894958B1 (en) 2017-06-30 2021-01-19 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10787663B1 (en) 2017-06-30 2020-09-29 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10584334B1 (en) 2017-06-30 2020-03-10 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10584333B1 (en) 2017-06-30 2020-03-10 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10738301B1 (en) 2017-06-30 2020-08-11 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10647982B1 (en) 2017-06-30 2020-05-12 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
US10787683B1 (en) 2017-08-28 2020-09-29 Inscripta, Inc. Electroporation cuvettes for automation
US10508288B1 (en) 2017-09-30 2019-12-17 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems comprising flow-through electroporation devices
US10557150B1 (en) 2017-09-30 2020-02-11 Inscripta, Inc. Automated nucleic acid assembly and introduction of nucleic acids into cells
US10851389B2 (en) 2017-09-30 2020-12-01 Inscripta, Inc. Modification of cells by introduction of exogenous material
US10822621B2 (en) 2017-09-30 2020-11-03 Inscripta, Inc. Automated nucleic acid assembly and introduction of nucleic acids into cells
US10590375B2 (en) 2018-03-29 2020-03-17 Inscripta, Inc. Methods for controlling the growth of prokaryotic and eukaryotic cells
US10883077B2 (en) 2018-03-29 2021-01-05 Inscripta, Inc. Methods for controlling the growth of prokaryotic and eukaryotic cells
US10717959B2 (en) 2018-03-29 2020-07-21 Inscripta, Inc. Methods for controlling the growth of prokaryotic and eukaryotic cells
US10406525B1 (en) 2018-04-13 2019-09-10 Inscripta, Inc. Automated cell processing instruments comprising reagent cartridges
WO2019200004A1 (en) * 2018-04-13 2019-10-17 Inscripta, Inc. Automated cell processing instruments comprising reagent cartridges
US10799868B1 (en) 2018-04-13 2020-10-13 Inscripta, Inc. Automated cell processing instruments comprising reagent cartridges
US10478822B1 (en) 2018-04-13 2019-11-19 Inscripta, Inc. Automated cell processing instruments comprising reagent cartridges
US10858761B2 (en) 2018-04-24 2020-12-08 Inscripta, Inc. Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells
US10625212B2 (en) 2018-08-14 2020-04-21 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US10835869B1 (en) 2018-08-14 2020-11-17 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US10532324B1 (en) 2018-08-14 2020-01-14 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11685889B2 (en) 2018-08-14 2023-06-27 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US11365383B1 (en) 2018-08-14 2022-06-21 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US10752874B2 (en) 2018-08-14 2020-08-25 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US10954485B1 (en) 2018-08-14 2021-03-23 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11268061B2 (en) 2018-08-14 2022-03-08 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US10744463B2 (en) 2018-08-14 2020-08-18 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11142740B2 (en) 2018-08-14 2021-10-12 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US11072774B2 (en) 2018-08-14 2021-07-27 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11965154B2 (en) 2018-08-30 2024-04-23 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US11118153B2 (en) 2019-06-20 2021-09-14 Inscripta, Inc. Flow through electroporation modules and instrumentation
US11015162B1 (en) 2019-06-20 2021-05-25 Inscripta, Inc. Flow through electroporation modules and instrumentation
US10907125B2 (en) 2019-06-20 2021-02-02 Inscripta, Inc. Flow through electroporation modules and instrumentation
US11078458B2 (en) 2019-06-21 2021-08-03 Inscripta, Inc. Genome-wide rationally-designed mutations leading to enhanced lysine production in E. coli
US10920189B2 (en) 2019-06-21 2021-02-16 Inscripta, Inc. Genome-wide rationally-designed mutations leading to enhanced lysine production in E. coli
US11066675B2 (en) 2019-06-25 2021-07-20 Inscripta, Inc. Increased nucleic-acid guided cell editing in yeast
US10927385B2 (en) 2019-06-25 2021-02-23 Inscripta, Inc. Increased nucleic-acid guided cell editing in yeast
US10689669B1 (en) 2020-01-11 2020-06-23 Inscripta, Inc. Automated multi-module cell processing methods, instruments, and systems
US11225674B2 (en) 2020-01-27 2022-01-18 Inscripta, Inc. Electroporation modules and instrumentation
US11268088B2 (en) 2020-04-24 2022-03-08 Inscripta, Inc. Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells via viral delivery
US11591592B2 (en) 2020-04-24 2023-02-28 Inscripta, Inc. Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells using microcarriers
US11787841B2 (en) 2020-05-19 2023-10-17 Inscripta, Inc. Rationally-designed mutations to the thrA gene for enhanced lysine production in E. coli

Also Published As

Publication number Publication date
ATE496639T1 (de) 2011-02-15
DE602008004752D1 (de) 2011-03-10
EP2135626B1 (de) 2011-01-26

Similar Documents

Publication Publication Date Title
EP2135626B1 (de) Streifen für multiparametrische Assays
US9428800B2 (en) Thermal cycling apparatus and method
EP1322413B1 (de) Verfahren und vorrichtung zur behandlung von biomolekülmatrizen
EP1487580B1 (de) Hybridisierungsvorrichtung
US20030138969A1 (en) Closed substrate platforms suitable for analysis of biomolecules
US20110003699A1 (en) Thermal Cycler for Microfluidic Array Assays
AU2003217261A1 (en) Hybridization device and method
EP1718411B1 (de) Vorrichtung zur analyse einer wechselwirkung zwischen target- und sondenmolekülen
US20050106607A1 (en) Biochip containing reaction wells and method for producing same and use thereof
EP2135674A1 (de) Vorrichtung für multiparametrische Assays
WO2003036298A2 (en) Closed substrate platforms suitable for analysis of biomolecules
JP2007304094A (ja) 分析チップ
JP5145752B2 (ja) 分析チップ

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20090923

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA MK RS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

AKX Designation fees paid

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REF Corresponds to:

Ref document number: 602008004752

Country of ref document: DE

Date of ref document: 20110310

Kind code of ref document: P

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602008004752

Country of ref document: DE

Effective date: 20110310

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20110126

LTIE Lt: invalidation of european patent or patent extension

Effective date: 20110126

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110507

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110526

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110526

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110427

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110426

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110426

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20110531

Year of fee payment: 4

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20110727

Year of fee payment: 4

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20110606

Year of fee payment: 4

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

26N No opposition filed

Effective date: 20111027

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602008004752

Country of ref document: DE

Effective date: 20111027

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110619

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

BERE Be: lapsed

Owner name: EPPENDORF ARRAY TECHNOLOGIES SA

Effective date: 20120630

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20120619

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20130228

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602008004752

Country of ref document: DE

Effective date: 20130101

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120630

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120619

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120630

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110630

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120702

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120630

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130101

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110619

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110126