EP0646245A1 - Appareil et procede permettant de realiser des operations individuelles impliquant la manipulation d'echantillons liquides - Google Patents

Appareil et procede permettant de realiser des operations individuelles impliquant la manipulation d'echantillons liquides

Info

Publication number
EP0646245A1
EP0646245A1 EP93912680A EP93912680A EP0646245A1 EP 0646245 A1 EP0646245 A1 EP 0646245A1 EP 93912680 A EP93912680 A EP 93912680A EP 93912680 A EP93912680 A EP 93912680A EP 0646245 A1 EP0646245 A1 EP 0646245A1
Authority
EP
European Patent Office
Prior art keywords
liquid
pipette
sample
container
containers
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.)
Withdrawn
Application number
EP93912680A
Other languages
German (de)
English (en)
Inventor
Henry Jorgensen
Axel Gronlund Nielsen
Soren Vindahl Andersen
Jorgen Dalsgaard
Gunner Sie
Flemming Hansen
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.)
N Foss Electric AS
Original Assignee
N Foss Electric AS
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 N Foss Electric AS filed Critical N Foss Electric AS
Publication of EP0646245A1 publication Critical patent/EP0646245A1/fr
Withdrawn legal-status Critical Current

Links

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
    • B01L3/5082—Test tubes per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1002—Reagent dispensers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices
    • G01N35/1072—Multiple transfer devices with provision for selective pipetting of individual channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00465—Separating and mixing arrangements
    • G01N2035/00524—Mixing by agitating sample carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0474—Details of actuating means for conveyors or pipettes
    • G01N2035/0482—Transmission
    • G01N2035/0484—Belt or chain
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1016—Control of the volume dispensed or introduced
    • G01N2035/102—Preventing or detecting loss of fluid by dripping
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices
    • G01N2035/1076—Multiple transfer devices plurality or independently movable heads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1081—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane
    • G01N35/1083—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane with one horizontal degree of freedom
    • G01N2035/1086—Cylindrical, e.g. variable angle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1081—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane
    • G01N35/1083—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane with one horizontal degree of freedom
    • G01N2035/1086—Cylindrical, e.g. variable angle
    • G01N2035/1088—Coaxial with a carousel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/025—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having a carousel or turntable for reaction cells or cuvettes

Definitions

  • the present invention relates to a novel and precise apparatus and method for performing unit operations involving handling of liquid samples, including very small liquid samples, such as samples of size of about 500 ⁇ l.
  • the invention is particularly useful in connection with qualitative or quantitative determinations, such as determinations of components in liquid samples, involving a reaction, such as a chemical, biological, biochemical or immunological reaction which can be detected by a chemical or physical measurement, for example: an optical measurement, such as measurement of absorbance, fluorescence or luminescence (e.g. bioluminescence as in the case of a luciferin/luciferase reaction) ; an electrochemical measurement, such as measurement of pH or redox potential; or another form of potentiometric or electrical measurement, such as measurement of conductivity or resistance.
  • a reaction such as a chemical, biological, biochemical or immunological reaction which can be detected by a chemical or physical measurement
  • an optical measurement such as measurement of absorbance, fluorescence or luminescence (e.g. bioluminescence as in the case of
  • the reaction in question may alternatively result directly or indirectly in a product whose amount is representative of that of the component to be determined and which can be detected by an optical determination; examples hereof are a fluorescein-carrying product, a coloured product, and a fluorescent product (fluorophore) .
  • a product whose amount is representative of that of the component to be determined and which can be detected by an optical determination; examples hereof are a fluorescein-carrying product, a coloured product, and a fluorescent product (fluorophore) .
  • the apparatus and method of the invention permit a high degree of automation with simultaneous attainment of very high accuracy.
  • the present invention relates to techniques where carry-over of volumes of sample, reactant or reagent is a critical factor, and where such carry-over should be reduced to a minimum, because any carry-over in any one operation results in an error which is the greater, the smaller the sample is compared with the carry-over volume.
  • One of the features of the present invention is that the number of objects which come into contact with the liquid samples in connection with a measurement is reduced. This is accomplished, e.g., by performing agitation of the samples in which reactions are performed in a manner which does not entail the introduction of any agitation bodies or other agitating or stirring means into the samples, and by performing dosing of liquids from pipettes which do not contact the samples.
  • the precision in the measurement is improved because the apparatus makes use of a novel principle for agitating a number of sample containers, a novel principle for avoiding cross-contamination when removing liquid from a number of samples using the same pipette, and a novel principle for increasing the precision when metering out or dosing small amounts of liquid.
  • Agitation or stirring of samples may in principle be carried out in two ways: using a stirring body or agitating the sample container.
  • a stirring body such as a stirring rod
  • carry-over may be a problem if the stirring body is not rinsed after each stirring.
  • stirring or agitation of the samples is carried out by agitating the sample containers.
  • This method is limited by the varia- tion in the size of the drop, remaining on the pipette after dosing the liquid.
  • This drop size may vary from a lower volume, where substantially no liquid is present on the outer surface of the pipette, to a volume such that a drop which was only slightly larger would fall from the pipette.
  • This variation in drop size may be reduced by reducing the outer diameter of the pipette, which would reduce the outer diameter of the drop.
  • the realistic outer diameter of a pipette has a lower limit.
  • a preferred type of measurement principle for determining the concentration of a component in a sample in the context of the present invention is optical measurement.
  • part of the sample should be transferred to a chamber in which no extraneous light may disturb the measurement.
  • a pipette or the like has to be brought into contact with the sample to remove an aliquot or part thereof. This operation may be a source of cross- contamination unless care is taken.
  • the present invention relates to an apparatus for performing unit operations involving the handling of liquid samples, the apparatus comprising:
  • a) holding means for holding the sample containers the holding means having container-receiving means adapted to receive each container in male-female relationship with the container-receiving means, and means for oscillatingly moving the holding means and thereby transferring movement from the holding means to the sample container to agitate liquid in the sample container received by the container-receiving means,
  • the means for adding liquid to the sample containers or removing liquid from the sample container comprise a plurality of pipettes arranged in pipette holding and moving means adapted to hold the pipettes and to move each individual pipette to a position relative to a sample container in which the pipette is capable of exerting its function of dosing liquid to the container or remove liquid from the container
  • the dosing pipette means are movable by pipette moving means which are adapted to confer to the pipette means an upward acceleration at the end of a pipetting procedure so as to release from the pipette tip any part of any liquid drop in excess of an amount determined by the upward acceleration,
  • the means for removing liquid from the sample containers comprise suction pipette means for sucking the liquid from the containers combined with flushing liquid supply means adapted to supply flushing liquid through an outer surface part of the suction pipette means.
  • a preferred embodiment of the apparatus of the invention is adapted for quantitative or qualitative determination of a component in liquid samples by performing, on individual liquid samples, a reaction which can be detected by a chemical or physical measurement or which directly or indirectly results in a product which is representative of the component and which can be detected by a chemical or physical measurement.
  • This preferred embodiment of the apparatus further comprises measuring means adapted to perform the chemical or physical detection of the reaction or the product resulting from the reaction.
  • the measurement in question is an optical measurement and the measuring means is an optical measuring means adapted to perform optical detection of the reaction or the product resulting from the reaction.
  • each of the above features a) -d) constitutes an important approach which can be utilized per se in connection with determinations of the above types, and as will become apparent from the disclosure herein, an apparatus according to the invention is ideally suited not only to performing unit operations in conjunction with associated chemical or physical measurements, but also simply for manipulating liquid samples with a high degree of precision for the purpose of, e.g., dilution, dividing into aliquots and/or mixing.
  • each of the above features, and additional features as explained below contribute to a unique exactitude in the manipulations and/or measurements involved in the operation of an apparatus according to the invention.
  • this reaction When a reaction is performed on the samples, this reaction may be a conventional chemical reaction generating a product which can be detected by an optical measurement, such as determination of UV-induced fluorescence, or it may be a biological or biochemical reaction, such as an enzymatic reaction on a substrate generating a fluorophore as a reaction product.
  • a type of reaction for which the apparatus and method of the invention are particularly suited is an immunological reaction, such as employed, for example, in an assay of the ELISA type (Enzyme Linked Immuno Sorbent Assay) for detection or determination of an antigen, antibody or hapten.
  • the ELISA method is based on the use of antibodies against the molecule or species to be determined or detected, and the antigen, hapten or antibody is detected by means of an enzyme which is covalently coupled (also denoted linked or conjugated) either (when an antigen or hapten is to be determined) to an antibody which is specific for the antigen or hapten in question, or (when an antibody is to be determined) to an antibody which is specific for the antibody in question.
  • An ELISA determination e.g. an ELISA determination suited for performance using an apparatus according to the present invention, may, e.g., be performed as follows:
  • the antigen, hapten (the latter generally in the form of a conjugate with, e.g., a protein) or antibody to be detected/- determined is bound or immobilized by allowing it to bind immunochemically to a so-called “catching" antibody (in the case of antigen or hapten determination) or an antigen (in the case of antibody determination) which is attached covalently or non-covalently to the surface of an appropriate material.
  • This reaction may be faster when the sample container is agitated or if the temperature is raised.
  • the latter attached antibody or antigen may be attached, for example, to the material of the inner surface of the sample container employed, or to insoluble, magnetic particles which are capable of being immobilized within the sample container by applying a magnetic field to the outside of the container.
  • Appropriate enzyme-linked antibodies (which may be specific antibodies, e.g. monoclonal antibodies) are then added to the sample and allowed to bind to the immobilized species (antigen, antibody or hapten) which is to be detec- ted/determined. This reaction may be faster'when the sample container is agitated or if the temperature is raised.
  • step E) A reagent that stops the enzymatic reaction in step E) is suitably added after an appropriate period of time.
  • step E) The measurement mentioned in step E) is performed.
  • step E) an alternative way of producing a product detectable by an optical measurement is to add an additional substrate to the reaction mixture formed in step E) .
  • the product of this new reaction may then be detected by an optical measurement.
  • "catching" antibodies or antigens may, instead of being attached to magnetic particles, be attached, bound or otherwise immobilized to the inner surface of the sample containers.
  • the accessible surface area of attached antibody or antigen will typically be of the order of 10 times that obtained when using sample containers having the antibody or antigen attached to the inner surface thereof.
  • completion of the reaction of an antigen which is to be determined with the attached "catching" antibodies, or of the enzyme-linked antibody with the bound antigen which is to be determined may take of the order of 20 hours, whereas completion of the corresponding reaction using magnetic particles may be achieved within about 15 minutes (Mi11an et al . , Clin. Chem.
  • operations A) through G) are performed in a sample container, most of these operations involving transfer of liquid to or from the container.
  • the individual samples must necessarily be quite small, such as about 500 ⁇ l, if a relatively large number of samples are to be handled efficiently and in view of the fact that the reagents employed in the various stages of the procedure are often quite expensive. This means that precise dosing, complete transfer and efficient rinsing must be ensured if the results obtained are to be accurate.
  • the present invention provides measures which permit the attainment of a very high accuracy in dosing, transfer, rinsing and flushing, and, at the same time, a high degree of precision with respect to the degree of completion of the reactions in question via efficient control of agitation and of sample temperature.
  • Control of sample temperature is made possible, in particular, by an efficiently thermostatable design obtainable due to the compact construction of the sample handling and measuring module of the apparatus according to the invention. This will become apparent from the following.
  • an aspect of the invention comprises obtaining increased precision in dosing liquids to sample container from pipettes by careful control of the size of any drop remaining at the pipette tip, thereby increasing the accuracy in the dosing by up to several orders of magnitude.
  • the agitation of the samples in sample containers is performed by oscillatingly moving the holding means in which the sample containers are received in male-female relationship in the container- receiving means.
  • the majority of the containers in an apparatus will typically be under agitation because a reaction is performed therein, one or a few of the containers are preferably not agitated (or not agitated to any substantial extent) , namely the container or containers which are close to or at the place where reagents are added thereto or liquid is withdrawn therefrom. Therefore, the agitation in these selected containers may be substantially reduced or prevented by ensuring that these selected containers are in a more loose engagement with the holding means.
  • the apparatus comprises means for moving at least one container axially in relation to the corresponding receiving means between a first position, in which the sample container is received in the holding means with a relatively close fit, and a second position in which the sample receiving part of the container may be moved transversely in relation to the receiving means.
  • the sample-containing part of the container When positioned in the second position, the sample-containing part of the container can be held substantially stationary while the opposite part of the container may move more freely.
  • the opposite part of the container can move considerably, it will be possible to dose liquid to the container or withdraw liquid from the container when the sample-containing part of the container is kept relatively static in the second position; this makes it possible to perform the dosing or withdrawal at a time where other containers are still being kept in the position where their contents are subjected to effective agitation.
  • a suitable design of the container and the container- receiving means, respectively, is one where the outer surface of each container defines a shoulder between its opposite ends, and each of the receiving means of the holding means defines a support surface for engaging with the shoulder of the container, such as illustrated in the drawing.
  • the holding means is suitably a plate-like member with a plurality of apertures for receiving the sample containers, where each aperture has a substantially circular outline, the diameter of the aperture at the ldwer surface of the holding means being smaller than at the upper surface thereof.
  • each aperture is at least partly defined by a frustro-conical surface part, the shoulder of each of the containers defining a substantially complementary frustro- conical surface part being in abutting engagement with the first mentioned frustro-conical surface part in the first position of the container.
  • a suitable top angle of the frustro-conical surface parts is within the range of 3°-18°, preferably 6°-13°, and more preferably within 9°-11°.
  • the top angle of the conical surface part of the container is normally smaller, preferably 0.1°-1° smaller, than the top angle of the conical surface part of the aperture.
  • the top angle of the conical surface part of the apertures is approximately 10.0° and the top angle of the conical surface part of the containers is approximately 9 . 1 ° .
  • the holding means is suitably adapted to oscillate or reciprocate the receiving means along an arcuate path, and the holding means thus preferably comprises a substantially horizontally arranged, substantially circular disc which is reciprocatingly movable around its central axis.
  • the preferred way of moving the holding means is to use a stepping motor drivingly connected to the disc, the disc preferably being mounted directly on the shaft of the stepping motor, and the stepping motor is preferably controlled so as to move with constant speed.
  • the forces acting on the liquid in the sample containers is the centrifugal force.
  • the magnitude of this force depends on the angular velocity of the liquid, again depending on the distance from the liquid to the centre of the movement.
  • the centrifugal force depends on the square of the angular velocity
  • the liquid parts positioned in the largest distance from the centre of the movement will respond strongest to the movement having the largest velocity, which means that the liquid in the sample container will perform a rotational movement when the movement if the holding means is larger in one direction than in the other.
  • the velocity in one direction is typically at least 2 times higher than in the other, preferably 3-4 times higher, but there is nothing to hinder the use of velocities more than e.g. 9 times higher in one direction than in the other.
  • a preferred embodiment of an apparatus comprises magnet holding means adapted to hold at least one set of magnets in a position where they induce a magnetic field in the sample- holding part of at least one sample container positioned in its second position.
  • the magnet holding means are suitably adapted to bring, in one position, the magnets in direct contact with the sample-holding part of the sample container positioned in its second position, and arrange the magnets in another position in which they do not contact the sample container.
  • sample receiving parts of a sample container positioned in its second position may be held substantially stationary while the rest of the sample container may follow the movements of the holding means.
  • Prevention of movement of the sample receiving parts of the sample container in its second position is preferably accomplished by contact of the magnets with the sample container and by elevation of the sample container by the elevating means.
  • the sample receiving parts of the sample container are not completely stationary, but are able to rotate about an axis through the sample held by the sample container. This enables the sample container to follow the movements of the holding means substantially without agitating the sample.
  • the magnets are able to contact the sample receiving parts of the sample container(s) to attract and immobilize any particles in the sample(s) in question having magnetic properties while other samples are agitated.
  • the magnet holding means comprise at least two sets of magnets, adapted to be brought in contact with at least two neighbouring sample containers.
  • one set of magnets can be utilized to pre- attract the magnetic particles in order to reduce the overall processing time in connection with a measurement.
  • Each set of magnets is suitably adapted to contact the sample container from substantially opposite sides and with opposite magnetic poles so as to generate a suitable magnetic field in the sample container.
  • the apparatus according to the invention preferably comprises, as means for adding liquid samples to the sample container, pipettes fed by continuous liquid strings extending from containers containing the liquids to the tip of the pipettes, the transport of liquid preferably being performed by means of peristaltic pump means.
  • the present invention also relates to a novel way of performing precise pipetting of liquid into a container by means of a pipette, comprising subjecting the pipette to a movement with an upward acceleration at the end of a pipetting procedure so as to release from the pipette tip any part of any liquid drop in excess of an amount determined by the upward acceleration and allowing any such released amount to be added to the liquid in the container.
  • the precision of the pipetting is normally limited by the volume interval of drops able to remain on the tip of the pipette. By introducing this movement of the tip of the pipette, this volume interval is reduced whereby the precision of the dosing is increased.
  • the pipette is moved upward to a first position, whereupon the pipette is subjected to downward movement, which movement is reversed by means of the upward acceleration.
  • This jerk-movement, and results illustrating the advantages obtained are described in Example 2.
  • the pipette is preferably kept free of contact with the liquid in the container.
  • the means for removing liquid from the sample containers comprises flushing pipette means combined with flushing liquid supply means adapted to supply flushing liquid to an outer surface part of the flushing pipette means.
  • the flushing pipettes are preferably designed so that the flushing liquid supply means is adapted to supply flushing liquid to the outer surface part of the flushing pipette through at least one orifice in the flushing pipette means positioned at a distance above the tip of the flushing pipette.
  • the flushing liquid supply means suitably comprises an annular cavity surrounding the flushing pipette means along a part of the length of the flushing pipette means.
  • the sample container When performing e.g. an ELISA-type determination, the sample container, from which liquid has been removed, is preferably flushed to additionally rinse the contents of the sample container.
  • This flushing method preferably comprises immersing the pipette into the liquid, - sucking the liquid sample into the pipette, and flushing the outer surface part of the pipette having been in contact with the container by means of a flushing liquid.
  • the flushing pipette used in an apparatus according to the invention is able to perform this flushing without being removed from the sample container. This means that the flushing pipette comprises both means for removing liquid and means for introducing flushing liquid into the sample container.
  • the flushing liquid is supplied through at least one orifice in the pipette positioned above the outer surface part of the pipette which has been immersed in the liquid. In this way the flushing pipette is efficiently flushed, and as the flushing liquid is introduced in the sample container, the parts of the sample container, previously in contact with the sample, are also flushed.
  • the flushing preferably comprises at least one flushing sequence where flushing liquid is supplied in an amount which is equal to or preferably larger than the amount of liquid sample which has been withdrawn so as to secure that total outer surface part of the pipette which has been immersed in liquid is contacted by the flushing liquid.
  • the pipettes of the apparatus according to the invention are very suitably held and moved by means of pipette holding and moving means adapted to hold a plurality of pipettes and to move any one of the pipettes to a position in which the pipette tip is capable of entering a sample container.
  • a preferred embodiment of an apparatus showing this feature comprises a number of pipette-holding segments each of which is capable of holding at least one pipette, a segment support means comprising a guide surface on which the segments, arranged adjacent to each other, are supported and guided, means for sidewardly moving the segments, a' movable member comprising a support surface adapted to support a segment, the movable member being able to occupy a first position in which its support surface replaces and constitutes part of the guide surface, and means for moving the movable member in downward and upward directions, each segment being sidewardly movable by the segment moving means to a position where the guide surface is replaced with the support surface of the movable member, whereby the segment becomes movable with ' the movable member, the movable member being movable between the first position and a second position in which the tip of a pipette held by the segment is capable of entering a sample container.
  • the guide surface is preferably in a substantially horizontal plane, and the downward movement is preferably a substantially vertical movement.
  • the movable member preferably comprises an upper guide surface part adapted to engage an upper surface part of a segment supported by the support surface part of the movable member.
  • the means for supporting the segment comprises a groove having substantially vertical- parallel edges, the movable member being movable from its first position to its second position along the groove, the segment held by the movable member being fixed in sideward direction by the edges of the groove when the movable member is not in its first position.
  • the guide surface of the segment support means preferably constitutes part of a substantially circular surface, the remaining part of the substantially circular surface being constituted by the support surface of the movable member when the movable member is in its first position.
  • the sideward movement of the segments is suitably obtained by means of a rotatable top part provided with means for engaging the segments supported by the support surface so that the segments are moved sidewards in a substantially circular path when the rotatable top part is rotated, the engaging means being in slidable engagement with the segments allowing a segment which is moved downward with the movable member to become disengaged from the top part.
  • the invention also relates to a sample container, in particular for use in the apparatus described above, the sample container having the features described above.
  • the diameter of the upper substantially cylindrical surface part of the container is preferably larger than the diameter of the lower cylindrical surface part.
  • the diameter of the lower substantially cylindrical surface part is normally in the range of 10-15 mm, preferably in the range of 11.5-
  • the lower cylindrical part preferably comprises a downwardly extending substantially hemispherical bottom part.
  • the sample container is suitably made of polyolefin, such as polypropylene.
  • Fig. 13 illustrates a preferred embodiment of a sample container, also giving the dimensions of the container.
  • the container, or at least the sample-containing part thereof, is preferably not transparent to light.
  • the method permits the use of larger samples, and at the same time permits a much better agitation and thereby diffusion in the reactions performed in the containers. Because of the use of larger samples than in ELISA machines, combined with high dosing accuracies and high diffusion reproducibilities, genuinely quantitative and, indeed, highly accurate ELISA determinations become possible.
  • the method of the invention is unique in that all reactions are performed without transfer of samples between different containers, and needing shorter time because of the very efficient agitation of the individual containers, at the same time, as a very special feature, permitting dosing and withdrawal from containers arranged in the same holding means as the containers which are subjected to agitation, and even permitting the simultaneous agitation of some containers and dosing to or withdrawal from, other containers.
  • Fig. 1 shows an apparatus according to the invention in its entirety
  • Fig. 2 is a detailed partly broken-away view of a pipette tower
  • Fig. 3 shows the flow of liquids in the apparatus
  • Fig. 4 is a detailed partly broken-away view of the bottom part of the apparatus comprising elevating means, heating means, and a motor moving the holding means,
  • Fig. 5 shows a sample container placed in the lower position in which it is in a relatively close fit with the holding means
  • Fig. 6 shows an elevated sample container which is positioned in its second position and is contacted by a set of magnets
  • Fig. 7 shows an elevated sample container contacted by a set of magnets, the sample container being in one of its extreme positions during agitation of other sample containers which are not elevated,
  • Fig. 8 shows the forces acting on the contents of a sample container when agitated
  • Fig. 9 shows a graph illustrating the position of a sample holding means versus time during agitation
  • Fig. 10 shows a partly broken-away view of a dosing pipette and a flushing pipette
  • Fig. 11 shows forces acting on a drop of liquid hanging from the tip of a dosing pipette
  • Fig. 12 shows the movement of a dosing pipette while the pipette is subjected to the jerk movement
  • Fig. 13 is a cross sectional view of a sample container suitable for use in the apparatus according to the invention.
  • Fig. 1 shows a preferred embodiment of an analysis apparatus according to the invention, generally designated 10, comprising a controlling/calculating module 12, a sample handling and measuring module 14 and a keyboard or other interface 16.
  • the controlling/calculating module 12 comprises power supply (not shown) and controlling/calculating electronics 11.
  • the sample handling and measuring module 14 comprises a reagent supply module 18, two pump modules 20, each com ⁇ prising up to four substantially identical pumps 22, e.g. peristaltic pumps, a valve module 24 comprising three substantially identical valves 26, a turntable 27, a pipette tower 34 and an optical measuring module 42.
  • a cover 15 permits efficient thermostating of the turntable 27, the pipette tower 34, and the optical measuring module 42.
  • the turntable 27 includes a disc-shaped holding means or turntable disc 28 comprising a plurality of holes 30 con ⁇ stituting container-receiving means for holding a number of substantially equally spaced sample containers 32.
  • the pipette tower 34 comprises a rotatable top part 35 holding a plurality of pipette holding segments 36 each adapted to hold one or two pipettes .
  • Two types of pipettes are shown namely a dosing pipette 38 and a withdrawal and flushing pipette 40.
  • the top part 35 of the pipette tower 34 is rotatably mounted on a stationary part 33 of the pipette tower 34 by means of a bolt 451 received in a thread 462 (Fig. 2) positioned in a rotatable shaft 413.
  • Rotation of the top part 35 is accomplished by activating a motor 404 having a shaft 409 which, via a toothed belt drive 410, rotates the shaft 413 and thereby the top part 35.
  • the rotation of the top part 35 is monitored by optical detectors 408 and 414 detecting apertures 405 and an aperture 407 positioned close to the outer edges of discs 406 and 412, respectively, fixed to the shafts 409 and 413.
  • Each of the optical detectors 408 and 414 comprises a light emitting part (not shown) and a light detecting part (not shown) which are oppositely positioned in two oppositely positioned arms of the detector so that light emitted from the light emitting part will be detected by the light detecting part if the light is not obstructed.
  • the disc 412 comprises only one aperture 407 enabling the detector 408 to generate a signal when the top part 35 is in a certain position - the starting position.
  • the motor 404 may be activated to rotate the top part 35 controlled by the controlling/calculating electronics 11, on the basis of the mechanical advantage of the belt drive 410 and the signals obtained from the optical detectors 408 and 414.
  • the top part 35 may be rotated to move any of the pipette holding segments 36 to a position where it engages a slide member 422.
  • the lower parts of the pipette holding segments 36 are supported by a frustro-conical surface part 450, formed in the stationary part 33, with which frustro conical surface part 450 complementary surface parts 452 constituting a sector of a frustro conical surface, formed on each pipette holding segment 36, engage.
  • the upper part of each pipette holding segment 36 has a surface part 448 constituting a sector of a frustro conical surface and engaging with a corresponding frustro conical surface part (not shown) of an element (not shown) to be fastened to the stationary part 33.
  • Surface parts 453 and 449 each constituting sectors of a frustro conical surface and corresponding to the conical surface parts 450 and the upper frustro conical surface part (not shown) of the stationary part 33, are formed in the slide member 422, and, in the upper position of the slide member 422, cooperate with these frustro conical surface parts of the stationary part 33 to support the pipette holding segments 36 in such a way that they are able to move along the circumference of the stationary part 33.
  • a condition for the circumferential movement of the pipette holding segments 36 is that the slide member 422 is in an upper position. When the slide member 422 is not in the upper position, a catch (not shown) mounted on the stationary part 33 of the pipette tower 34 engages the top part 35 and prevents rotation thereof.
  • the slide member 422, carrying the pipette holding segment 36 may be moved along a groove 460 extending along a longitudinal axis of the stationary part 33.
  • the slide member 422 is moved downward to an intermediary position, and the slide member 422 will not occupy its upper position until a new pipette holding segment 36 is to be selected.
  • edges 461 of the groove 460 prevent the pipette holding segment 36 from moving along the circumference of the stationary part 33, whereby the pipette holding segment 36 is substantially fixed in relation to the slide member 422.
  • pins 454, fixed to the top part 35 engage holes 456 in the pipette holding segments 36, thereby forcing the pipette holding segments 36 to rotate with the top part 35.
  • the movement of the slide member 422 is performed by means of a motor 424 rotating a shaft 426 which, by means of a toothed belt drive including a toothed belt 420, transports the slide member 422, which is fixed to the toothed belt 420.
  • the positioning of the slide member 422 in its upper and intermediary positions is monitored and controlled by the controlling/calculating electronics 11 on the basis of signals generated by an optical detector 430 detecting apertures 432 and 434 in a disc 428 fixed to the shaft 426.
  • the aperture 432 is detected when the slide member 422 is in the upper position, and the aperture 434 is detected when the slide member 422 is in the intermediary position which is preferably slightly lower than the upper position. Movement of the slide member 422 and thereby the pipette holding segment 36 below the intermediary position of the slide member 422 is accomplished by means of a sine-shaped voltage generated by the controlling/calculating electronics 11.
  • the motor 424 is preferably a stepping motor, in which case the number of periods and the frequency of the voltage transmitted to the motor 424, define the position and velocity of the slide member 422.
  • rotation of turntable disc 28 is accomplished by activating a motor 502 having a shaft 504 on which the holding means 28 is fixed (Fig. 4) .
  • a disc 506, which is fixed to the shaft 504, comprises a number of apertures 512 positioned at a small distance from the edge of the disc 506, and one aperture 514 positioned in the edge of the disc.
  • the apertures 512 are detectable by an optical detector 508 and the aperture 514 is detectable by an optical detector 510.
  • the spacing of the apertures 512 corresponds to the spacing of the holes 30 in the holding means 28.
  • the holes 30 are preferably frustro-conical having a smaller diameter at the lower surface of the holding means 28 than at the upper surface.
  • the sample containers 32 preferably comprise a shoulder substantially matching the frustro- conical apertures 30. To ensure easy removal of the sample containers 32 the angle of the shoulder of the sample container 32 is preferably 0.3° smaller than the angle of the frustro-conical apertures.
  • the holding means 28 may either be moved in a reciprocating manner, so as to agitate the contents of the sample con- tainers 32 received in the holes 30, or the holding means 28 may simply be rotated an angle corresponding to the angle between two adjacent holes 30, so as to advance a sample container 32 to the position previously occupied by an adjacent sample container 32.
  • All movements of the holding means 28 are controlled by the controlling/calculating electronics 11 to which the output from the optical detectors 508 and 510 are input, and in which the voltage controlling the motor 502 is generated.
  • the holding means 28 When the holding means 28 is rotated an angle corresponding to the angle between two adjacent holes 30, the holding means 28 is rotated from a position where the optical detector 508 detects an aperture 512 to a position where the optical detector 508 detects a next adjacent aperture 512.
  • the control of the motor when moving the holding means 28 in a reciprocating manner to agitate the contents of the sample containers 32, is accomplished by generating a sine-shaped
  • magnet holding arms 552 and 554 each of which holds two magnets 560, are pivotable around shafts 553 and are provided with arms 555 and 557, in which frustro-conical surface parts 556 and 558, respectively, are formed.
  • the surface parts 550 engage the frustro-conical surface parts 556 and 558, whereby the magnet holding arms 552 and 554 are forced away from each other, thus counteracting the magnetic attraction therebetween.
  • the conical surface parts 550, 556, and 558 disengage so that the magnet holding arms 552 and 554 are able to approach and contact, due to magnetic attraction, any sample containers 32 elevated by the elevating means 524 and 526.
  • the magnets 560 are positioned so that two magnets, one fixed to each of the magnet holding arms 552 and 554, contact the sample containers 32, elevated by the elevating means 524 and 526, from opposite sides (Fig. 6 and 7) .
  • the magnet holding arms 552 and 554 approach due to the magnetic forces, as the magnets 560 are fixed to the magnet holding arms 552 and 554 in such an orientation so that the magnets 560 attract each other.
  • An additional set of opposite magnets 570 is arranged in a fixed position in a frame of the apparatus clockwise of the magnets 560 (Fig. 4) .
  • This set of magnets 570 is not able to approach and contact the sample containers 32. However, it is arranged at a sufficiently small distance from each side of a sample container 32, positioned adjacent to the magnets 570 to be able to attract any elements having magnetic properties in the sample container 32.
  • the elevating means 524 and 526, the additional set of magnets 570, the holding means 28, and the pipette tower 34 are positioned in relation to each other in ' such a way that the sample container 32, to be elevated by elevating means 526, and the neighbouring sample container 32 exposed to the additional set of magnets 570 are able to receive pipettes held by the pipette holding segment 36, engaged in the slide member 422.
  • the first set of magnets 560 contacting the sample container 32, elevated by the elevating means 524, are included to reduce the processing time, and increase the efficiency of the attraction of the elements, having magnetic properties, attracted by the second set of magnets 560, as the attraction and binding of elements having magnetic properties starts one position before the position in which the magnetic fixation of the elements is actually utilized.
  • Fig. 3 shows a detailed schematic view of the flow of the liquid substances in an apparatus as illustrated in Fig. 1.
  • Peristaltic pumps 102, 104, 106 and 108 pump liquid from reagent liquid containers 120, 122, 124 and 126 through liquid sensors 116 and lines 170, 172, 174 and 176 to dosing pipettes 38, held in pipette holding segments 350, 352, 354 and 356, respectively.
  • a pump 110 pumps liquid from a flushing liquid container 128 through a line 180 and, depending on a valve 140, through a line 182 to a liquid supply line 210 (Fig. 10) in withdrawal and flushing pipette 40 held by the pipette holding segment 356, or through a line 186 to a valve 142 which controls the flow of the liquid through lines 184 and 188 to liquid supply lines 210 in withdrawal and flushing pipettes 40 held by the pipette holding segments 354 or 352 respectively.
  • a pump 112 pumps liquid from a valve 144, through a line 190 to a liquid container 130.
  • This liquid is, depending on the valve 144, pumped through a line 192 and a liquid removing part 212 (Fig. 10) of a withdrawal and flushing pipette 40 held by the pipette holding segment 354, or through a line 194 and a liquid removing part 212 of a withdrawal and flushing pipette 40 held by the pipette holding segment 352.
  • a pump 114 pumps liquid from a liquid removing part 212 of a withdrawal and flushing pipette 40, held by the pipette holding segment 356, to the liquid container 130 through a line 200, a measuring cuvette 300, and a line 202 to the liquid container 130.
  • the liquid sensors 116 sense the presence of liquid in the lines 170, 172, 174, 176, 180, 190, 192, 200 and 202. Absence of liquid in one of these lines may lead to incorrect results as liquid may not be dosed when intended. A "no-liquid signal" from a liquid sensor 116 may give rise to the apparatus 10 informing the operator which sample containers 32 have not received all relevant liquid or from which sample containers 32 not all relevant liquid has been removed, and therefore which measurements should be repeated.
  • the optical measuring module 42 (Fig. 3) comprises a light emitter 302, preferably a halogen lamp, a collimating lens 304 transforming part of the light emitted from the light emitter 302 into a parallel beam of light, and a focusing lens 310 focusing part of the colli ated light onto the measuring cuvette 300.
  • the lenses 304 and 310 are preferably chosen so that the light, focused inside the measuring cuvette 300, forms an image having the same size as a light emitting element 303 in the light emitter 302. While the light is collimated, it is launched through a heat filter 306 removing IR light emitted from the light emitter 302.
  • the IR light might otherwise destroy a band pass filter 308 which transmits light in a predetermined waveband able to excite a fluorophore in a sample contained in the measuring cuvette 300.
  • fluorescent substances in the sample will emit fluorescence having a wavelength longer than the exciting light trans ⁇ mitted through the filter 308.
  • Part of this fluorescence is collimated by a collimating lens 312 and focused on to a light detector 318 by a focusing lens 317. While the fluorescence is collimated, it is launched through filters 314 and 316 together forming a band pass filter removing light having a wavelength not being in a wavelength region of the fluorescence emitted from the exited sample.
  • the collimating and focusing lenses 312 and 317 are pre ⁇ ferably selected so that the fluorescence launched on to the light detector 318 has the same size as the light emitting element 303 in the light emitter 302.
  • the light detector 318 is preferably selected so that the light sensitive surface (not shown) of the detector 318 has the same size as the light emitting element 303. The above-mentioned selections are performed to increase the signal to noise ratio of the optical measurement.
  • a reference light detector 320 is positioned close to the measuring cuvette 300.
  • the reference light detector 320 is positioned in a way so that it receives a small amount of the light launched on to the measuring cuvette 300. In this way, variations in the amount of exciting light launched onto the measuring cuvette 300 may be compensated for when determining the amount of fluorescence emitted from the sample contained in the measuring cuvette 300.
  • the determination of the concentration of a substance to be determine ⁇ is performed in the controlling/calculating electronics 11 based on a calibration of the optical measurement and the amount of liquids added to the sample containers 32.
  • the solid black arrows in Fig. 3 show the direction of controlling/monitoring signals in the apparatus. It is seen that all pumps, valves, and motors are controlled by the controlling/calculating module 12, that the optical detec- tors, the liquid sensors and the light detectors generate signals transmitted to the controlling/calculating module 12, and that the interface 16 exchanges information with the controlling/calculating module 12.
  • the apparatus 10 may be used for e.g. measuring the contents of a fluorescent agent produced by an immunological or another process taking place in the sample containers.
  • An ELISA analysis based on the so-called “sandwich-technique" using two different monoclonal antibodies against - in this specific case - Salmonella antigens may be performed as described in the following (see Example 1, below, for an explanation of the terms Wash, Calibrator, Conjugate, Substrate, Immunospheres and Stop) ; the antibodies used react slightly with the native flagella, but strongly with a heat- treated extract of Salmonella . Therefore, the samples are preferably heat-treated at 100°C for 15 minutes before performing the ELISA. This heat-treatment kills/inactivates the Salmonella and other pathogenic bacteria.
  • the ELISA is able to detect 10 5 cells per ml, and the samples should therefore comprise at least this number of cells per ml.
  • the automated ELISA analysis on an apparatus according to the invention typically takes approximately 75 minutes, but can be performed manually.
  • step 1 one test tube with Wash (negative control) and one test tube with 500 ⁇ l Calibrator (a heat-treated suspension of Salmonella t phimurium diluted to a concentration of approximately 10 6 cells per ml) are placed in a sample disc together with test tubes containing 500 ⁇ l heat-treated samples. The samples are warmed up to 35°C and the analysis starts.
  • Calibrator a heat-treated suspension of Salmonella t phimurium diluted to a concentration of approximately 10 6 cells per ml
  • step 2 90 ⁇ l I ⁇ rnninospheres are added to each sample and incubated for 12 ⁇ minutes. Salmonella antigens present in the sample will react with antibody and form immunecomplexes on the surface of the Immunospheres. The total surface area of the microspheres is very large, and this allows a fast and efficient binding between antibodies and antigens to take place.
  • step 3 Non-bound material is washed away with Wash while the Immunospheres are retained in the side of the test tube by magnetic force. 130 ⁇ l Conjugate is then added and incubated 12-M minutes. The conjugated antibody binds to the immunosphere-antigen complex. This creates a "sandwich” with Salmonella antigens as the "filling".
  • step 4 Non-bound conjugated antibody (Conjugate) is washed away with Wash.
  • step 5 200 ⁇ l Substrate is added and incubated 12 ⁇ minutes.
  • the enzyme bound to the particles will split the substrate to, inter alia.
  • 4-methylumbelliferyl a fluorescent compound which is measured by a fluorescence detector (excitation 365 nm, emission 450 nm) .
  • step 6 The enzyme reaction is stopped by adding 200 ⁇ l Stop.
  • step 7 The concentration of 4-methylumbelliferyl is now measured by the detector and the concentration is proportional to the amount of Salmonella antigen present in the sample.
  • the fluorescence signal for the individual sample is compared to a cut-off value, defined as 25% of the calibrator signal. Samples with a signal higher than or equal to the cut-off value are considered positive. Samples with a signal below the cut-off value are considered negative.
  • Magnetic particles to which antibodies are bound may be made in a manner known per se by coating paramagnetic particles, such as DYNABEADS ® with the antibody in question. While, in the above example of an immunological process, the element bound to the magnetic elements is an antibody, it will, of course, as already mentioned, also be possible to have an antigen bound to the magnetic elements, etc. in cases where the component to be determined is an antibody.
  • the apparatus After turning on the apparatus 10, the apparatus performs a self-test of the controlling/calculating electronics 11 comprised in the controlling/calculating module 12.
  • the starting position, position 1 is the position of the container receiving means 30 in the holding means 28 which is positioned directly above the elevating means 526 when the optical detector 510 detects the aperture 514. From this position the positions for the sample containers 32 are numbered in counter clockwise direction.
  • Step 1 adding the samples
  • the samples are added to the sample containers in positions 4 to 30, which is the total number of container receiving means 30 in the holding means 28 in a preferred embodiment of the apparatus according to the invention.
  • sample containers 32 are placed in the container receiving means 30, and an amount of Calibrator is manually transferred to a sample container 32 in position three, seen counter clockwise from a starting position (position 1) of the holding means 28.
  • the Calibrator is preferably a liquid containing a known concentration of the substance to be determined. This liquid will then be treated as a real sample, and as the Calibrator will be transferred to the measuring cuvette 300, the measured fluorescence, corresponding to the known concentration of the substance to be determined, may then be used for calibrating the optical measurement. This calibration will be described below.
  • sample containers contain:
  • the contents of the container 120 are agitated to ensure a homogeneous composition of the contents.
  • the agitation of the contents of the container 120 is performed by activating a motor 132, connected to a suction pipe 134 in the liquid container 120 by an eccentric 136 which moves the suction pipe 136 so as to agitate the liquid in the liquid container 120.
  • the apparatus performs a "priming", where the lines leading liquids to the dosing pipettes 38 are filled with liquid.
  • a "priming" where the lines leading liquids to the dosing pipettes 38 are filled with liquid.
  • small volumes of the corresponding liquids are dosed into the sample container 32 in position 1 in the holding means 28.
  • the corresponding liquid sensor 116 of a type known per se, is used for ensuring that when the pump 102 is activated, liquid is dosed into the sample container 32.
  • the pump 102 is activated to pump a predefined volume of liquid, hereby ensuring that liquid is dosed into the sample con ⁇ tainer 32 in position 1 in the holding means 28.
  • a similar procedure is performed for priming the liquids in the liquid containers 122, 124, and 126.
  • a volume of the liquid from liquid container 128 is dosed into the empty sample container 32 in position 2 in the holding means 28, this liquid constituting a blind sample.
  • This blind sample which does not contain any of the sub ⁇ stance to be determined, is treated as a normal sample. Due to this, the blind sample may be used for supervising the reacticn and compensating for a "blind level" ' .
  • the processes preferably take place at a controlled temperature. Therefore, after the self test, the control- ling/calculating electronics 11 activates two fans 382 and three sets comprising two heating elements 380 and a tempe- rature sensor 384, positioned in the sample handling and measuring module 14.
  • the temperature sensors 384 transmit signals to the controlling/calculating module 12 so that the desired temperature in the sample handling and measuring module 14 may be obtained and maintained.
  • the apparatus 10 is ready to start the immunological process in the sample containers 32 holding the samples, to perform the UV-measurement on the fluorescent agent of the process, and to determine the amount of this fluorescent agent in the sample.
  • the Immunospheres having magnetic properties, are added to the samples to attach to the substance to be determined.
  • the first sample container holding a "sample” (position number 2 in the holding means 28 as the Calibrator and the blind sample are treated as samples) is, by activating the motor 502, positioned in the position where it is exposed to the additional set of magnets 570.
  • the sample containers 32 positioned above the elevating means 524 and 526 are elevated by activating the motor 534, rotating the shaft 532 an angle of 180°, whereby the elevating means 526 are elevated and the magnet holding arms 552 and 554 approach and contact the sample containers 32 in question.
  • the pipette 38 held by the pipette holding segment 350, is moved up to a higher position A (Fig. 12 and example 2) , whereafter the slide member 422 is given a velocity down toward the sample container 32.
  • a position B higher than the surface of the sample in the sample container 32, this velocity is reversed, giving the tip of the dosing pipette 38 an acceleration in an upward direction.
  • the force F a applied to the drop may have a size so large that the drop breaks up. Due to the direction of the applied force F a , the released drop will fall from the pipette 38 to be added to the sample positioned below the pipette 38.
  • the critical volume of a drop being the maximum volume that a drop may have without breaking due to the applied force, depends on the size of the applied force F a .
  • the movement of the slide member 422 is achieved by ac ⁇ tivating the motor 424 and controlling the' motor on the basis on a signal generated by the controlling/calculating electronics 11. This jerk movement is repeated; this time the velocity of the pipette 38, held by the pipette holding segment 350, is reversed at a position C higher than position B. Then the slide member 422 is again positioned in the intermediate position.
  • FIG 11 A translation of the signals transferred to the motor 424 is shown in figure 11.
  • the theoretical movement pattern of the dosing pipette 38 is shown where the y-axis is the position (height) of the tip of the pipette 38, and the x- axis is the time.
  • the y-axis is the position (height) of the tip of the pipette 38
  • the x- axis is the time.
  • elevating means 524 and 526 are elevated and the contents of the sample containers 32, not elevated (Fig. 5), are agitated by activating the motor 502, such as will be described below.
  • Figure 5 shows a sample container 32 which is not elevated. This sample container 32 is placed in a "close fit" in which the sample container 32 substantially follows the movement of the holding means 28.
  • Figure 6 shows an elevated sample container 32 contacted by the magnets 560. It is seen that the sample container 32 is no longer in the "close fit" seen in fig. 5, the sample container 32 is able to move relative to the holding means 28.
  • Figure 7 shows an elevated sample container 32 during agitation of the samples held by the sample containers 32 which are not elevated. It is seen that the lower, sample- containing part of the sample container 32 is contacted and substantially fixed by the magnets 560 and the elevating means 524 or 526.
  • the elevated sample container 32 When moving the holding means 28 in an oscillating manner, the elevated sample container 32 is able to rotate about an axis positioned in the liquid sample held by the sample container 32 in such a way that the top part of the sample container 32 may substantially follow the movement of the holding means, while the lower part of the sample container 32 may remain substantially fixed in relation to the magnets 560 and the elevating means 524 or 526.
  • this feature enables the magnets to attract and hold any elements having magnetic properties, while other samples in the holding means are agitated. Due to this substantial prevention of the agitation of a sample in an elevated container 32, it will also be possible, if desired, to add liquid to or withdraw liquid from a thus elevated container 32 while the samples in the other containers 32 are agitated.
  • the agitation of the samples in the containers 32 is pre ⁇ ferably performed in such a way that the holding means 28 is given a larger velocity in one direction than in the other direction.
  • the force acting on the liquid in the sample containers 32 is the centrifugal force. This force depends on the square of the angular velocity of the holding means 28, which means that if the velocity of the holding means 28 in one direction is, e.g., twice the velocity of the other direction, the force acting on the liquid is four times higher in one direction than in the other.
  • the motor 302 is preferably a stepping motor which may be controlled by a sine-shaped voltage. To ensure that the holding means 28, after one agitation period, returns to the starting position, care is taken that the motor 302 receives the same number of periods of the sine- shaped voltage in the two directions. When giving the holding means 28 e.g. twice the velocity in one direction than in the other, the frequency of the sine-shaped voltage is twice as high in the one direction than in the other.
  • Fig. 9 The position of the holding means 28 during agitation, as a function of time, may be seen from Fig. 9, where the vertical dashed lines show the periods of the agitation cycle of an apparatus according to the invention.
  • the y-axis represents the position of the holding means 28 and the x- axis represents the time.
  • the time passing between lines A and B corresponds to the time used for the faster movement in one direction.
  • the time passing between lines B and C corresponds to the time used for the slower movement in the other direction of the holding means 28. From Fig. 9, it can be seen that the time used for the slower movement is approximately 3.4 times larger than the time used for the faster movement. It can also be seen that the acceleration in the faster movement is higher than the acceleration in the slower movement.
  • Step 2 is repeated once for each sample holding sample container 32 in the holding means 28.
  • the slide member 422 is positioned in the upper position whereby the catch (not shown) disengages the top part 35 which is now able to rotate. Then the top part 35 is rotated to its starting position by activating the motor 404 and monitoring the rotation of the top part 35 by means of the detectors 408 and 414.
  • the magnets 560 fixed to the magnetic holding arms 552 and 554 brought into contact with the sample containers 32 when elevated, are utilized to attract and hold the magnetic particles, to which the substance to be determined is bound, while the liquid in the sample con ⁇ tainers 32 is withdrawn, whereupon flushing is performed.
  • the second substance is added to the sample containers 32.
  • step 2 the first sample, to receive liquid in step 2 (position 2) , has been thoroughly agitated, and the reaction, in which the substance to be determined binds to the magnetic particles, has been allowed to proceed for a predetermined period of time.
  • This sample container is now, by activation of the motor 502, positioned in the position directly above the elevating means 524.
  • the elevating means 524 and 526 are again elevated, con ⁇ tacting the sample containers 32 positioned directly above the elevating means 524 and 526, and the magnets 560 con ⁇ tacting the sample containers 32 attract and hold the magnetic particles present in the sample containers 32.
  • the top part 35 of the pipette tower 34 is rotated so that the pipette holding segment 352 is brought into engagement with the slide member 422.
  • This pipette holding segment 352 holds two pipettes: a withdrawal and flushing pipette 40 and a dosing pipette 38 positioned so that the withdrawal and flushing pipette 40 is lowered into the sample container 32 elevated by the elevating means 526, so that, in the way of rotation of the sample holding means 28, the liquid in the sample container is withdrawn and the sample container is flushed, and then, after a rotation of one step by the sample holding means 28, an amount of the liquid from the liquid container 122 may be dosed from pipette 38, held by the pipette holding means 352.
  • the withdrawal of liquid from and the flushing of a sample container 32 is performed by lowering the withdrawal and flushing pipette 40 to a position in which it is able to remove substantially all liquid from the sample container 32.
  • the pump 112 is activated whereby substantially all liquid is removed from the sample container
  • the substrate to be determined is not removed from the sample container 32.
  • the pump 112 When the pump 112 has been activated for a given period of time, it is turned off and the pump 110 is activated to introduce liquid from the liquid container 128 into the sample container 32.
  • the valves are, of course, controlled so that the liquid pumped from the liquid container 128 is in fact transferred to the sample container 32 in question.
  • the pump 110 When a given volume, preferably larger than the amount of liquid removed, of the liquid from liquid container 128 has been is introduced in the sample container 32, the pump 110 is turned of.
  • liquid is again withdrawn from the sample container 32, whereupon liquid from the liquid container 128 is again introduced into the container.
  • the magnetic particles may tend to stick to the sides of the sample container 32 for approximately two positions even though the sample container is agitated. After this, the agitation of the sample container 32 will resuspend the magnetic particles, and the substance bound to which may then react with the added Conjugate.
  • This step is repeated until all sample-containing sample containers 32 have been subjected to withdrawal of liquid and flushing and a volume of the Conjugate contained in liquid container 122 has been added thereto.
  • Conjugate added in step 3 is typically added in an amount larger than the amount actually binding to the substance to be determined, some of the dosed Conjugate is unbound.
  • This unbound Conjugate has to be removed from the sample containers 32 as the next reaction is not able to distinguish between bound and unbound elements.
  • the Conjugate chemically splits the Substrate, thus forming a fluorescent product.
  • the amount of fluorescent product generated in this reaction depends on the total amount of the Conjugate, bound as well as (undesired, and to be removed) unbound.
  • a third amount of flushing liquid is dosed after the second dose of flushing liquid has been withdrawn, and the holding means 28 is rotated to flush the next sample container 32.
  • this flushing step is repeated after the holding means 28 has performed a full circle where the contents of the sample containers 32 have been resuspended and mixed with the third amount of flushing liquid. Now substantially no unbound, previously trapped, part of the Conjugate will be trapped when the magnetic particles are again attracted by the magnets and the flushing is repeated, whereby substantially all of the unbound Conjugate may be flushed away.
  • the Substrate to be chemically activated or split by the Conjugate is added.
  • Substrate contained in container 124 is added while the next sample container 32 is subjected to withdrawal of liquid and flushing.
  • a pipette holding segment 356 is used in which two pipettes 38 and 40 are positioned in such a manner that the sample containers 32 first receive an amount of liquid from the liquid container 126 (Stop) , so as to stop the reaction started in step 3, from the dosing pipette 38, whereafter, after rotating the holding means 28 one position, step 7 is performed wherein the optical measurement is performed.
  • the last step is the determination of the fluorescence induced in the sample.
  • the amount of fluorescence depends on the concentration of the substance to be determined in the original samples.
  • the elevating means 524 or 526 are lowered, the holding means is rotated one position, and the elevating means 524 and 526 are again elevated. Then the samples which are not elevated, including the sample which has just received the liquid in step 6, are agitated. Due to the magnets 570 and the fact that the magnetic particles are not resuspended until they have been agitated two times, the magnetic particles which were previously attracted by the magnets 560 are not resuspended while the sample is agitated.
  • the un-attracted contents of the sample container 32 are transferred to the measuring cuvette 300 through a withdrawal and flushing pipette 40 held in the pipette holding segment
  • Removing the liquid from the sample container 32 and trans ⁇ ferring this liquid to the measuring cuvette 300 is performed by activating the pump 114 which removes liquid from the sample containers 32 through line 200 to the measuring cuvette 300 and further through line 202 to the liquid container 130.
  • the samples contained in the sample containers 32 in positions 2 and 3 in the holding means 28 are, in fact, a blind sample and a calibrator liquid, respectively, both samples containing known concentrations of the substance to be determined.
  • these liquids are, in turn, transferred to the measuring cuvette 300 in the optical measuring module 42 where the light emitter 302 is activated.
  • the pump 112 pumps liquid through line 200 into the measuring cuvette 300.
  • the pump 112 is stopped so that the measurement is not performed on the first liquid to enter the measuring cuvette 300. This is to ensure that the liquid present in the measuring cuvette is not contaminated or diluted by any liquid present in the flushing pipette 40, line 200, or the measuring cuvette 300.
  • the intensity of the light emitted from the light emitter 302 is adjusted by adjusting the voltage fed to the light emitter 302.
  • the light launched on to the measuring cuvette 300 excites the calibrator liquid in the cuvette 300 so that fluorescence is detected by the light detector 318.
  • the intensity of the light emitted from the light emitter 302 is adjusted so that the light detector 318 detects a predetermined intensity of fluorescence.
  • the voltage fed to the light emitter 302 will now be held at a constant value as the intensity of the light launched on to the measuring cuvette 300 varies only slightly, and may be compensated for as described below.
  • a fraction of the light launched on to the measuring cuvette 300 is detected by the reference light detector 320.
  • the detector 318 detects the predetermined intensity of fluorescence
  • the output (reference output) from the detector 320 is stored in a memory (not shown) in the controlling/calculating module 12.
  • This reference output value df the detector 320 is later on used in the determination of the fluorescence emitted from the sample contained in the measuring cuvette 300, for compensating for slightly deviating intensities of exciting light launched on to the measuring cuvette 300.
  • the UV-measurement is calibrated, and the sample con ⁇ tainer 32 and the withdrawal and flushing pipette 40, held in the pipette holding segment 356, are flushed by activating the pump 112 and ensuring that the valve 144 transfers liquid from the liquid container 130 through line 182 to the liquid supply line 212 of the withdrawal and flushing pipette 40 held by the pipette holding segment 356.
  • the pump 112 After dosing an amount of liquid, the pump 112 is stopped, and the pump 114 is activated to remove the liquid from the sample container 32 through lines 200, 202 and measuring cuvette 300, so as to also flush these elements.
  • the flushing steps are repeated to obtain a sufficient degree of flushing of the pipette 40, the sample container 32, line 200, and the measuring cuvette 300. .
  • the motor 424 is activated, as described above, to elevate the slide member 422 and the pipette holding segment 356 holding pipettes 38 and 40, to the intermediate position.
  • the UV-measurement performed on the samples is performed in the same manner as the calibration.
  • Part of the fluorescence emitted from the excited sample and part of the light launched on to the measuring cuvette 300 are measured by the light detector 318 and the reference detector 320 respectively.
  • the controlling/calculating module 12 on the background of the calibration, is able to determine the amount of the fluorescent agent, and thereby the amount of the substance to be determined, in the sample.
  • the rest of the sample is removed from the sample container 32 by again activating the pump 114.
  • the sample container 32, the withdrawal and flushing pipette 42, line 200, and the measuring cuvette 300 are flushed as described above.
  • a value corresponding to the amount of the substance to be determined in the original sample is calculated. This value is compared to a selectable cut-off value, and samples having values higher than this cut-off value may contain high levels of the substance to be determined.
  • the rinsing is performed by removing the liquid containers 120, 122, 124, and 126, and inserting a container (not shown) containing a rinse liquid.
  • This container has a shape so that the pumps 102, 104, 106, and 108 may pump liquid from the container (not shown) through lines 170, 172, 174, and 176 to the dosing pipettes 38 held by the pipette holding segments 350, 352, 354, and 356.
  • this liquid is then pumped into empty sample con ⁇ tainers 32 in position 1 and 2 in the holding means 28. This liquid is then sucked into lines 192, 194, 190, 200, and 202 through the liquid removing parts 212 of the withdrawal and flushing pipettes 40 held in the pipette holding segments 352, 354, and 356. This is accomplished by positioning the withdrawal and flushing pipettes 40 in the sample containers 32 holding the rinse liquid after turn, and filling the lines with the rinse liquid.
  • the liquid is allowed to react for a period of time where ⁇ after all lines are emptied into the container 130. Now the apparatus is rinsed and ready for a new measurement.
  • this apparatus Due to the increased dosing accuracy, the agitation, the temperature control, and low cross-contamination, this apparatus is excellently suited also for other types of chemical or immunological measurements.
  • the precision of the measurement depends both on the precision when dosing the reactants, and the conditions under which the reaction takes place.
  • determination of enzymes may also be performed on an apparatus according to the present invention.
  • a simple determination of an enzyme may comprise the following steps:
  • Step 1 will be conceptually of the same type as previously described above in connection with the determination of Salmonella antigens, and will involve adding the samples containing the enzyme to be determined to the sample containers and preparing the apparatus for the measurement.
  • step 2 a substance or species reacting with the enzyme to be determined is added.
  • the product of this reaction may be measurable by, e.g., an optical measurement.
  • Step 3 will then be the determination of the product(s), enabling a calculation of the concentration of the enzyme in the original sample to be determined.
  • Step 2 may be replaced by more complicated reactions, e.g. reactions involving more reactants, but the pattern of the determination will be as described.
  • EXAMPLE 1 When a preferred embodiment of an apparatus according to the invention is used for an ELISA type determination of flagellin e.g. from Salmonella bacteria, the liquids con ⁇ tained in the liquid containers are:
  • Liquid container Liquid 120 Immunospheres
  • the Calibrator is a dissolved heat extract of S. typhimurium. Fully grown culture in M-Bouillon (DIFCO) , dissolved approx. 1:3,000 in M-Boullion.
  • the optical elements are:
  • Light emitter (302) 10 W halogen bulb Heat filter (306) : Melles griot KG1 Filter (308) BP 365, 365 nm ⁇ 10 nm Filter (314) LWP 420, 420 nm ⁇ 3 nm Filter (316) SWPBL720, 720 nm ⁇ 10 nm Reference detector (320) Hamamatsu S1133-05 Light detector (318) : Hamamatsu S1227-16BR Measuring cuvette (300) : Glass tube, 1.1 mm light path, 10 ⁇ l volume.
  • This apparatus is able to measure fluorescence varying 4 decades.
  • a standard, related to the fluorescent substance used in the measurement of flagellin e.g. from Salmonella bacteria, is representative of the concentration of the fluorescent element (4-Methylumbelliferyl- ⁇ -D-galactoside, abbreviated: 4-MU) in the sample.
  • 4-MU 4-Methylumbelliferyl- ⁇ -D-galactoside
  • a preferred embodiment of an apparatus according to the invention is able to measure 2.5 nmol - 25 ⁇ mol 4-MU.
  • fluorescence corresponding to a standard value typically approx. 2500 nmol 4-MU or more
  • fluorescence corresponding to a standard value typically approx. 2500 nmol 4-MU or more
  • this apparatus is in fact able to perform quite pre ⁇ cise measurements as it has a measuring range of 4 decades, (2.5 nmol - 25 ⁇ mol 4-MU) and a signal to noise ratio of 6.8:1 at 2.5 nmol 4-MU.
  • the relative standard deviation of the light detection of the detector unit is less than 1%, typically in the order of 0.5%.
  • the calibration of the measurement is typically performed so that the measurement is linear for typical values and slightly nonlinear for the highest values of fluorescence where there is no doubt that the original sample has a too high content of flagellin.
  • the cross con ⁇ tamination is as low as 0.01%.
  • a determination of flagellin in a sample is performed as described above:
  • the relative standard deviation obtained when performing a measurement as described above wherein all samples have been replaced with calibrator liquid is less than 3%, typically 1- 2%.
  • a test has been made wherein the dosing accuracy of a pipette having an inner diameter of 0.56 mm and a wall thickness of 0.25 mm without using the jerk-movement is compared to the accuracy of a pipette having an inner diameter of 0.56 mm and a wall thickness of 0.15 mm using the jerk-movement.
  • an amount of the liquid containing the particles having magnetic properties, used in the immuno ⁇ logical measurements is dosed into a number of sample containers.
  • the weight of the sample containers is measured before and after the addition of the liquid whereby the dosed amount may be determined.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
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  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

Un appareil permettant de réaliser des opérations individuelles impliquant la manipulation d'échantillons liquides comprend un certain nombre de récipients à échantillon (32), pourvus chacun d'une partie dans laquelle un échantillon liquide peut être contenu, ainsi qu'un dispositif (38) servant à ajouter du liquide dans lesdits récipients à échantillon et un dispositif (40) servant à enlever du liquide desdits récipients. Lorsque le support (28) retenant lesdits récipients à échantillon subit un mouvement oscillant, certains des échantillons peuvent être remués puisque leur récipient est agité, tandis que les échantillons se trouvant dans les autres récipients ne sont pas remués de façon sensible étant donné que la partie contenant l'échantillon de ces récipients n'est elle-même pas agitée de façon sensible. Le mouvement oscillant s'effectue, de préférence, à différentes vitesses, dans les deux sens. On obtient une haute précision, lors du dosage d'une quantité de liquide à partir d'une pipette (38), par le fait que la pipette est soumise à une accélération vers le haut à la fin de l'opération de pipettage, de façon que toute partie d'une goutte de liquide en excès, par rapport à une quantité déterminée, soit libérée de la pointe de la pipette par ladite accélération vers le haut. L'on obtient un faible transfert d'éléments résiduels dû au fait que l'on rince un certain nombre de récipients à échantillon et que l'on en enlève du liquide au moyen de la même pipette d'aspiration (40), grâce à l'utilisation d'un dispositif à pipette d'aspiration pour aspirer le liquide des récipients conjointement avec un dispositif d'alimentation en liquide de rinçage (40) conçu pour amener du liquide de rinçage à travers une partie superficielle extérieure (214) du dispositif à pipette d'aspiration. L'invention concerne en outre un dispositif porte-pipettes (36) et de déplacement de pipettes (422) adapté servant à maintenir une pluralité de pipettes et à mettre n'importe quelle pipette dans une position dans laquelle elle peut introduire une dose de liquide dans un récipient ou enlever du liquide d'un récipient.
EP93912680A 1992-06-03 1993-06-03 Appareil et procede permettant de realiser des operations individuelles impliquant la manipulation d'echantillons liquides Withdrawn EP0646245A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK746/92 1992-06-03
DK74692A DK74692D0 (da) 1992-06-03 1992-06-03 Apparat
PCT/DK1993/000195 WO1993025913A1 (fr) 1992-06-03 1993-06-03 Appareil et procede permettant de realiser des operations individuelles impliquant la manipulation d'echantillons liquides

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EP0646245A1 true EP0646245A1 (fr) 1995-04-05

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EP (1) EP0646245A1 (fr)
AU (1) AU4311093A (fr)
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WO (1) WO1993025913A1 (fr)

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EP2347254A2 (fr) 2008-09-16 2011-07-27 Ibis Biosciences, Inc. Unités de traitement d'échantillons, systèmes et procédés associés
ES2610468T3 (es) 2010-07-23 2017-04-27 Beckman Coulter, Inc. Sistema o método para incluir unidades analíticas
EP2776844B1 (fr) 2011-11-07 2020-09-30 Beckman Coulter, Inc. Détection de récipient d'échantillon
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AU4311093A (en) 1994-01-04
DK74692D0 (da) 1992-06-03
WO1993025913A1 (fr) 1993-12-23

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