WO2008017829A1 - Procédé de détermination de la présence de substances d'intérêt dans des fluides - Google Patents

Procédé de détermination de la présence de substances d'intérêt dans des fluides Download PDF

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Publication number
WO2008017829A1
WO2008017829A1 PCT/GB2007/002994 GB2007002994W WO2008017829A1 WO 2008017829 A1 WO2008017829 A1 WO 2008017829A1 GB 2007002994 W GB2007002994 W GB 2007002994W WO 2008017829 A1 WO2008017829 A1 WO 2008017829A1
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WO
WIPO (PCT)
Prior art keywords
fluid
collection means
sensing area
assay apparatus
interest
Prior art date
Application number
PCT/GB2007/002994
Other languages
English (en)
Inventor
Patricia Connolly
John Gilmour
Andrew Mitchell
Nasr-Eddine Djennati
Paul Couchman
Original Assignee
Ivmd (Uk) Limited,
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 Ivmd (Uk) Limited, filed Critical Ivmd (Uk) Limited,
Publication of WO2008017829A1 publication Critical patent/WO2008017829A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7783—Transmission, loss

Definitions

  • the present invention relates to a method of determining the presence of substances of interest in fluids, and including although not exclusively the presence of substances of interest in biological fluids including measurement in a living body, such as a human body.
  • the invention relates to a method of determining the presence of substances of interest in a fluid without the requirement for complex electronics to analyse the fluid.
  • Probe which adheres to or binds to a molecule of interest.
  • the presence of the tagged probe is detected and the quantity of probe detected related to the concentration of the molecule under analysis.
  • Multiple probe systems with a capture probe or antibody and a second tagged probe or antibody to reveal the captured molecule are common.
  • Probes have been used with tags that are radioactive, enzymatic, fluorescent, chemiluminescent and spectrophotmetric or colourimetric. End points of tagged probe measurement can therefore be revealed in a variety of systems include spectrophotometric, electrochemical, radioactive, colourimetric, amperometric or potentiometric.
  • Magnetic beads have been employed in multiple probe systems as a solid phase for the capture probe, providing a highly mobile bead system with high surface area for capture probe attachment. Secondary probes or antibodies can then be added after molecular attachment to the capture probe and in the commonest application a magnetic field is then used to draw together the beads allowing a concentrate to form where the level of the tag can be measured.
  • sensing electronics to determine the concentration of the probe and hence determining the concentration of the molecule of interest.
  • This can be read directly by sensing increases in the magnetic field density at positions where the probe tags concentrate as described in WO2005/124345.
  • other properties of the tags may be utilised to enable the measurement of the probe concentration, for example light sensing electronics may be used if the tag is fluorescent.
  • light sensing electronics may be used if the tag is fluorescent.
  • suitable sensing electronics such as the production of light by chemiluminescent reactions with the tag).
  • a displacement assay is used in the analytical system and measurement of concentration is made by a complex oscillating coil system, and an antibody capture site for particles. This results in more complex manufacture for both the sensing system and the test element.
  • All of the above methods rely on at least some complex electronics either for controlling an applied magnetic field or for analysing the sample to determine the presence and concentration of the substance of interest.
  • the requirement for such complex electronics increases the cost of such systems and thus reduces the potential market for their application.
  • such electronics mean that users of the methods require a not inconsiderable amount of training before they may be utilised correctly.
  • a method of determining the presence or absence of one or more substances of interest in a fluid comprising the steps of: passing the fluid over a surface having a quantity of tag particles bound thereto, the tag particles free to adhere or bind to a substance of interest in the fluid or alternatively free to be displaced by a substance of interest in the fluid; introducing the fluid and any tag particles in the fluid into a sensing area; and providing a tag particle collection means in the sensing area so as to concentrate the tag particles by the collection means and thereby provide a visible indication of the presence or absence of a substance of interest.
  • the invention may be implemented in a classic 'displacement array' or 'flow displacement array' and the sensing area may be downstream of the surface with the tag particles bonded thereto.
  • the tag particles bound to the surface may be released into the fluid by becoming attached to the substance of interest or by being displaced by the substance of interest. This may occur though a specific bonding substance such as an antibody.
  • a sample of the fluid is introduced to this surface which contains the substance of interest in an unknown quantity. Competition for the binding site on the tag particle from the substance of interest in the sample will release the tag particles into solution in proportion to the concentration of the substance of interest in the sample.
  • the immobilised tag particles can be bound via any suitable bonding substance to substances of interest, multiple layers of different bonding substances can be used to create suitable sites for competition from substances of interest in the sample.
  • the sensing area may be a chamber such as a fluidics chamber.
  • the chamber may have a volume of less than 10 uL, preferably less than 5uL.
  • this method may be used in other assays with volumes greater than lOuL.
  • the sensing area may be an area of an assay strip.
  • the collection means may be in the shape of a word or symbol. The concentration of the tag particles on or around the collection means thus provides a visible pattern in the shape of the collection means, which may be easily and instantaneously read by the user.
  • the shape or word may alternatively be formed by a plurality of collection means mounted adjacent to one another.
  • the collection means may be in the shape of the word YES or similar. In embodiments wherein the absence of a substance of interest stimulates the release of tag particles, the collection means may be in the shape of the word NO or similar. This provides a clear and simple indication of the presence or absence of a substance of interest within the sample which can be rapidly read by even an unskilled user.
  • a plurality of separate collection means may be provided, such that a rough quantisation of the amount of tag particles can be obtained by counting the number of collection means over which particles have gathered.
  • the sensing area may be adapted to aid the gradual collection of tag particles by the provision of a series of collection means along a direction of fluid flow to collect the tag particles in quantised amounts.
  • the individual collection means may be in the shape of integers. This allows a rough count to be made of the number of particles if say five collection means in the shape of integers 1 to 5 are provided in series but tag particles are visible only over collection means 1 to 3.
  • the collection means in the sensing area may be provided over a photovoltaic cell.
  • an indication means connected to the photovoltaic cell may be operable to indicate the presence or absence of a substance of interest in the fluid.
  • the indication means may comprise one or more LEDs. Additionally or alternatively, the indication means may comprise an LCD screen, which may be operable to display text such as
  • YES indicating the presence of a substance of interest and NO indicating the absence of a substance of interest.
  • an array of photovoltaic cells may be provided, such that a rough quantisation of the amount of tag particles over the photovoltaic cells may be determined.
  • the sensing area may be adapted to aid the gradual collection of tag particles by the provision of a series of collection means along a direction of fluid flow to collect the tag particles in quantised amounts.
  • a separate photovoltaic cell or photovoltaic cell array may be provided under each collection means.
  • the number of LEDs lit at any one time may be indicative of the amount of substance present.
  • the number of LEDs lit at any one time may be indicative of the amount of substance present.
  • the number of LEDs lit at any one time may be indicated by an alphanumeric figure indicated on the LCD screen.
  • one or more additional electronic sensing devices may be provided in addition to or in place of the photovoltaic cell. Such devices may be operable to sense other properties of the tag particles.
  • the collection means may comprise capture molecules bonded to a surface in the sensing area, the capture molecules adapted to bond with and thereby collect tag particles in the fluid.
  • suitable capture molecules may include antibodies, probes (ligands) or similar.
  • the collection means may additionally or alternatively comprise suitable traps or wells in a surface over which the fluid sample flows.
  • the tag particles may be magnetic tag particles.
  • the collection means may comprise one or more magnets mounted in or adjacent to the sensing area. If one magnet is provided, the magnet is preferably mounted on a surface adjacent to the sensing area and is most preferably comprised of permanent magnetic material printed on to said surface. In some embodiments, a plurality of magnets may be provided so as to collect tag particles on a surface which is not a surface adjacent to that on which the magnets are mounted. In embodiments wherein the collection means has a distinctive shape, the or each magnet may have a distinctive shape in order to provide a collection means having a distinctive shape.
  • the magnet may be aided in the trapping of the magnetic particles by the addition of a capture molecule at certain parts of the assay strip. Said capture molecules can be so placed to aid the kinetics of the assay.
  • Said capture molecules may in photovoltaic cell embodiments increase the electronic signal from the cell.
  • a secondary antibody capture site to "collect” the particles together for sensing. This makes the disposable element simpler and cheaper.
  • complex alignment systems between sensor and the sensing area to give accuracy and consistency. The magnet automatically concentrates all freed particles in the sensing area.
  • the photovoltaic cell may be supplemented by or replaced by one or more other, sensing devices including but not limited to a Hall effect sensor, a capacitive measurement circuit or a magnetoresistor.
  • Each device may be configured so as to operate an indicator means.
  • Such indicator means may be
  • the fluid may be a liquid or gas, and may be a biological fluid such as a body fluid.
  • Substances of interest may include naturally occurring substances, substances that are the result of a chemical or biological reaction, such as drug by-products, and substances introduced into a fluid sample.
  • the substance may be a compound, especially a molecule and could be, for example a protein, hormone or DNA section.
  • the or each tag particle may become attached to a substance of interest by means of a further substance, which shall be referred to as a bonding substance.
  • the or each tag particle may be coated with the bonding substance.
  • the bonding substance may be a protein, and in some embodiments it is an antibody or probe (ligand).
  • the or each tag particle may be coated with a material to facilitate adherence of a bonding substance to the tag particle.
  • a suitable coating material is polystyrene.
  • each tag particle may be arranged so that it can only become attached to or displaced by a single unit of a substance of interest, for example a single molecule.
  • each tag particle may be provided with a single antibody or capture probe.
  • the tag particles may be formed from gold, latex or other light absorbing substance. Particles of size in the range 5 nanometers to 100 micrometers may be used or in some embodiments particles of size in the range 5 nanometers to 50 micrometers may be used.
  • magnetic tag particles particles of non-zero magnetic susceptibility.
  • the or each magnetic particle may be ferromagnetic, diamagnetic, paramagnetic or superparamagnetic. A homogeneous or heterogeneous mixture of such particles may be employed.
  • the or each particle is formed from iron oxide. Particles of size in the range 5 nanometers to 100 micrometers may be used or in some embodiments particles of size in the range 5 nanometers to 50 micrometers may be used.
  • an assay apparatus for determining the presence or absence of one or more substances of interest in a fluid comprising: an assay strip having a first area with a plurality of tag particles bonded thereto, the tag particles free to adhere or bind to a substance of interest in the fluid or alternatively free to be displaced by a substance of interest in the fluid; and a sensing area, said sensing area having a collection means for concentrating the tag particles in the fluid by the collection means thereby providing a visual indication of the presence or absence of a substance of interest in the fluid.
  • the assay apparatus according to the second aspect of the present invention may contain any or all of the features described in respect of the method of the first aspect of the present invention, as desired or as appropriate.
  • the assay strip is preferably at least partially transparent.
  • Figure 1 is a schematic view of apparatus for implementing a magnetic embodiment of the present invention.
  • Figure 2 is a schematic view of apparatus for implementing a nonmagnetic embodiment of the present invention.
  • One embodiment of the present invention is illustrated by the assay depicted in Figure 1, the assay operable to test a sample for the presence of a substance of interest.
  • the following description will detail an example wherein the present invention is applied to the determination of the presence of hCG 16 (full name of hCG) in a urine sample.
  • a level of hCG above a particular threshold may provide an indication of pregnancy in the sample donor.
  • the assay apparatus comprises an assay strip 10 formed of substantially transparent material.
  • the liquid sample is introduced to the assay strip 10 and flows over an area 12 containing pre-deposited hCG 16 or hCG analogue 16a.
  • the pre- deposited hCG 16 or hCG analogue 16a is labelled with tag particles 14 which in this example are magnetic particles 14 that contain a specific probe for the hCG molecule.
  • tag particles 14 which in this example are magnetic particles 14 that contain a specific probe for the hCG molecule.
  • a sensing area 22 is provided at one end of the strip 10, the sensing area 22 being provided with collection means for collecting tag particles 14 that are released into the sample. Adjacent to the sensing area 22 is provided a magnet 20 placed beneath the assay strip 10, which in this example provides the collection means. The magnet 20 draws the complexes 18 through the solution towards the magnet 20. A photovoltaic cell 24 is been placed between the magnet 20 and the assay strip 10. It should of course be appreciated that the magnet 20 may be placed in any other suitable orientation relative to the strip 10 and the photovoltaic cell 24 or that the magnet 20 may be replaced by an array of magnets (not shown) also operable to concentrate magnetic tag particles 14 over the photovoltaic cell 24.
  • the photovoltaic cell 24 Before the sample is introduced to the assay strip 10, the photovoltaic cell 24 produces an output voltage proportional to the amount of incident ambient light. As free complexes 18 gather above the photovoltaic cell 24 drawn by the magnetic field of magnet 20, they provide a block to incident light reaching the photovoltaic cell 24 and thus the voltage output of the photovoltaic cell falls. At a threshold level of voltage drop, an indicator means (not shown) is activated, which provides a user with an indication of whether the sample tests positive for a threshold level of hCG 16.
  • the indicator means may comprise an LCD screen operable to display the messages 'Pregnant' or 'Not Pregnant' dependent upon the level of voltage drop compared to the threshold.
  • the indicator means may comprise on ore more LEDs which may be lit or not lit to indicate dependent upon the level of voltage drop compared to the threshold.
  • the threshold may be a preset value.
  • the threshold voltage drop may be monitored by comparison with the output of a further photovoltaic cell (not shown) in proximity to the photovoltaic cell 20.
  • the further cell is positioned such that the magnetic complexes 18 do not gather over it and cause its output signal to drop.
  • the additional photovoltaic cell may be used to generate power from ambient light to operate such circuitry and the indicator means.
  • a battery, electrochemical energy from metals in contact with the sample itself or any other local power source such as electromechanical energy, piezo electric energy may additionally be used to power the indictor means.
  • another photovoltaic cell may be provided as a safety feature, the additional photovoltaic cell operable to provide a check that ambient or supplied light is reaching the photovoltaic cells.
  • the magnetic particles 14 and hCG probe could have been separately introduced to the urine sample before the sample flowed over the bound hCG 16 or hCG analogue 16a.
  • the free particles 14, which have not bound to the hCG 16 in the sample will then bind to the surface deposited hCG 16 or surface deposited hCG analogue 16a and be removed from flow in the strip 10.
  • the amount of complexes 18 drawn to the magnet 20 are in proportion to the amount of hCG 16 in the donor's sample.
  • the tag particles 14 are not necessarily magnetic tag particles. Accordingly, no magnet is provided and the collection means are comprised of tag capture molecules 16b. These tag capture molecules 16b may (in the present example) be hCG 16 or hCG analogues 16a, if desired. The tag capture molecules 16b cause the tags (at least) of complexes 18 to be collected over the photovoltaic cell 24. The captured tags 14 thus cause a dip in the output voltage of the photovoltaic cell 24 which can be utilised as described above to provide an indication of the presence or absence of hCG in the fluid sample.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Theoretical Computer Science (AREA)
  • Mathematical Physics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un appareil de dosage comportant une bandelette (10) de dosage présentant une première zone sur laquelle est collée une pluralité de particules (14) marqueuses, les particules marqueuses étant libres d'adhérer ou de se coller à une substance d'intérêt située dans le fluide ou, en variante, libre d'être déplacées par une substance d'intérêt dans le fluide. Une zone (22) de détection est également aménagée sur la bandelette, comprenant un moyen (20) de collecte destiné à concentrer les particules marqueuses dans le fluide à l'aide du moyen de collecte, donnant ainsi une indication visuelle de la présence ou de l'absence d'une substance d'intérêt dans le fluide. Le moyen de collecte peut prendre la forme d'un mot ou d'un symbole.
PCT/GB2007/002994 2006-08-08 2007-08-07 Procédé de détermination de la présence de substances d'intérêt dans des fluides WO2008017829A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0165738.2 2006-08-08
GB0615738A GB0615738D0 (en) 2006-08-08 2006-08-08 Method of determining the presence of substance if interest in fluids

Publications (1)

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WO2008017829A1 true WO2008017829A1 (fr) 2008-02-14

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WO (1) WO2008017829A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011128696A1 (fr) * 2010-04-14 2011-10-20 Bio Amd Holdings Limited Appareil d'immunoessai comprenant un canal microfluidique

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4668868A (en) * 1986-02-20 1987-05-26 Noller Hans T Apparatus for performing fluoroimmunoassays of biological specimens
EP0421294A2 (fr) * 1989-10-05 1991-04-10 Abbott Laboratories Dispositif immunochromatographique amélioré d'action automatique
US6136549A (en) * 1999-10-15 2000-10-24 Feistel; Christopher C. systems and methods for performing magnetic chromatography assays
WO2005124345A2 (fr) * 2004-06-19 2005-12-29 Hall Effect Technologies Limited Procede pour determiner la presence et/ou la concentration de substances d'interet dans des liquides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4668868A (en) * 1986-02-20 1987-05-26 Noller Hans T Apparatus for performing fluoroimmunoassays of biological specimens
EP0421294A2 (fr) * 1989-10-05 1991-04-10 Abbott Laboratories Dispositif immunochromatographique amélioré d'action automatique
US6136549A (en) * 1999-10-15 2000-10-24 Feistel; Christopher C. systems and methods for performing magnetic chromatography assays
WO2005124345A2 (fr) * 2004-06-19 2005-12-29 Hall Effect Technologies Limited Procede pour determiner la presence et/ou la concentration de substances d'interet dans des liquides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011128696A1 (fr) * 2010-04-14 2011-10-20 Bio Amd Holdings Limited Appareil d'immunoessai comprenant un canal microfluidique
CN102905789A (zh) * 2010-04-14 2013-01-30 超微生物控股有限公司 包括微流通道的免疫测定设备
US9678064B2 (en) 2010-04-14 2017-06-13 Bio Amd Holdings Limited Immunoassay apparatus incorporating microfluidic channel

Also Published As

Publication number Publication date
GB0615738D0 (en) 2006-09-20

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