US20040040849A1 - Analysis - Google Patents

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Publication number
US20040040849A1
US20040040849A1 US10/463,647 US46364703A US2004040849A1 US 20040040849 A1 US20040040849 A1 US 20040040849A1 US 46364703 A US46364703 A US 46364703A US 2004040849 A1 US2004040849 A1 US 2004040849A1
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US
United States
Prior art keywords
substrate
analyte species
interface
velocity
potential difference
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Abandoned
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US10/463,647
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English (en)
Inventor
Silvia Valussi
Andreas Manz
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Forensic Science Service Ltd
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UK Secretary of State for the Home Department
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Application filed by UK Secretary of State for the Home Department filed Critical UK Secretary of State for the Home Department
Assigned to SECRETARY OF STATE FOR THE HOME DEPARTMENT, THE reassignment SECRETARY OF STATE FOR THE HOME DEPARTMENT, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VALUSSI, SILVIA, MANZ, ANDREAS
Publication of US20040040849A1 publication Critical patent/US20040040849A1/en
Assigned to FORENSIC SCIENCE SERVICE LTD. reassignment FORENSIC SCIENCE SERVICE LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SECRETARY OF STATE FOR THE HOME DEPARTMENT, THE
Priority to US11/811,101 priority Critical patent/US8097140B2/en
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus

Definitions

  • This invention concerns improvements in and relating to analysis, particularly to aspects of the collection of samples and/or preparation of samples.
  • Improvements in the efficiency of collection are desirable to extend the range of applications to which subsequent analysis techniques can be applied. Furthermore, improvements which provide a high concentration of the analyte species in the prepared sample can give rise to increased accuracy and/or speed and/or simplification in subsequent analysis techniques.
  • electro-phoretic velocity of the analyte species in the second substrate is balanced by or exceeded by the bulk flow velocity of the second substrate and the bulk flow velocity of the second substrate is in an opposing direction to the electrophoretic velocity of the analyte species in the second substrate.
  • the electrophoretic velocity of the analyte species in the second substrate being balanced by or exceeded by the bulk flow velocity of the second substrate, the bulk flow velocity of the second substrate being in an opposing direction to the electrophoretic velocity of the analyte species in the second substrate;
  • the method further including reversing the electrical potential difference across the at least a part of the substrate, the interface and the at least part of the second substrate, the collected analyte species moving away from the interface and through the second substrate as a result of the electrical potential difference being reversed.
  • the electrophoretic velocity of the analyte species in the second substrate being balanced by or exceeded by the bulk flow velocity of the second substrate, the bulk flow velocity of the second substrate being in an opposing direction to the electrophoretic velocity of the analyte species in the second substrate;
  • the method further including reversing the electrical potential difference across the at least a part of the substrate, the interface and the at least part of the second substrate, the collected analyte species moving away from the interface and through the second substrate as a result of the electrical potential difference being reversed;
  • the first and/or second and/or third aspects of the invention may include any of the following features, options or possibilities.
  • the analyte species may be one or more of a disease marker, a protein, a drug, a metabolite, a bio-chemical marker, a chemical residue, a chemical component of fingerprint or other body marks, a skin residue or an excretion or a plurality of such species or of different types of the same species.
  • the sample may be a blood sample, bodily fluid sample, a sample obtained by contact with the body or pre-prepared part of a body.
  • the substrate may be a gel, a polymer or porous membrane.
  • the at least part of the sample may be provided in the substrate by introducing the sample into the substrate or introducing the sample to a surface of the substrate.
  • a part of the substrate removed from the interface receives the at least part of the sample.
  • the sample may be provided in the substrate by contacting a part of the persons body with the substrate, for instance applying a fingertip, particularly the print thereof, to the substrate surface.
  • the second substrate may be a liquid volume, such as a buffer.
  • the second substrate may be a gel, polymer or porous membrane of a different conductivity to the substrate.
  • the interface between the substrate and the second substrate is provided on an opposing side of the substrate to the surface to which the sample is introduced.
  • the interface may have a smaller surface area than one or more other surfaces of the substrate, and particularly the surface of the substrate to which the sample is introduced.
  • the interface may be provided on a part of a side of the substrate, particularly a second side which opposes the first.
  • the non-interface part of the side of the substrate may be in contact with a support, for instance a glass support.
  • the substrate may have a pH of 9+/ ⁇ 1 pH unit.
  • the pH may be provided at a pH which maximises the electro-phoretic velocity of the analyte species in the substrate and/or minimises the electro-phoretic velocity of the analyte species in the second substrate and/or maximises the electro-osmotic velocity within the second substrate.
  • the substrate is preferably neutral, particularly in terms of its surface charge.
  • the substrate may be neutrally charged due to the use of neutral polymers and/or due to the neutralisation of the polymer charge by one or more additives. It is particularly preferred that no electro-osmotic flow occurs within the substrate.
  • the second substrate may have a pH of 9+/ ⁇ 1 pH unit.
  • the pH of the second substrate is equivalent to the pH of the substrate.
  • the buffer and/or the component the buffer is provided within are provided so as to provide a high electro-osmotic velocity.
  • the surface of the container may be conditioned, for instance through use of a pre-treatment with a strongly alkaline solution. Static and/or dynamic adjustment and/or coatings may be used to provide the desired level of electro-osmotic flow.
  • the electrical potential difference may be applied between a first electrode and a second electrode.
  • the first electrode is in contact with the substrate, for instance the surface of a substrate and particularly the surface of the substrate to which the sample is introduced.
  • the second electrode is preferably in contact with the second substrate, ideally a part of the second substrate removed from the interface.
  • the analyte species move towards the interface as a result of their electrophoretic velocity in the substrate.
  • the analyte species move from their location of introduction to the substrate to the interface.
  • the analyte species movement is not opposed by any electro-osmotic velocity within the substrate.
  • the bulk flow velocity may be caused and/or controlled by gravity, pressure or electro-osmotic properties.
  • the bulk flow velocity of the buffer equals or exceeds the electrophoretic velocity of the analyte species in the second substrate away from the concentrated band.
  • the electrophoretic velocity of the analyte species in the second substrate is balanced or exceeded by the electro-osmotic velocity of the second substrate is in an opposing direction to the electrophoretic velocity of the analyte species in the second substrate.
  • the electro-osmotic velocity of the second substrate equals or exceeds the electrophoretic velocity of the analyte species in the second substrate away from the concentrated band.
  • the method of collection is turned into a method of preparing a sample for analysis by reversing the polarity of electrical potential difference.
  • the reversed electrical potential difference may be applied at the same potential difference or at a different potential difference to the initial electrical potential difference.
  • the application of the reversed electrical potential difference may occur after a time period without an electrical potential difference applied.
  • the period without an electrical potential difference applied may be used to obtain diffusion of the analyte species away from the interface. Diffusion may occur away from the interface into the substrate, but more preferably occurs away from the interface into the second substrate.
  • the period without the application of electrical potential difference may be for any time from zero up.
  • the reversing of the polarity of the electrical potential difference causes the electro-osmotic flow to be away from the interface.
  • the electro-osmotic velocity conveys the analyte species away from the interface.
  • the analyte species may be conveyed away from the interface all at substantially the same speed or at the same speed, particularly if the electro-osmotic flow predominates on all the analyte species.
  • the concentration of the analyte species is maintained during movement away from the interface.
  • the bulk flow velocity of the buffer is the electro-osmotic velocity.
  • the electro-osmotic velocity exceeds the electro-phoretic velocity of the analyte species in the second substrate during this part of the process.
  • the method of preparation may be extended to a method of analysing an analyte species from the sample, by conveying the analyte species in the second substrate away from the interface to an analysis location.
  • the analysis location may be in the channel or container provided with the second substrate.
  • the analysis may involve a consideration of the mobility of one or more of the analyte species in the buffer. Analysis conditions may be applied during the analysis part of the process. Analysis as an integral part of the apparatus is preferred, but the analyte species could be conveyed to a separate analysis process or apparatus.
  • extraction, enrichment of analysis of the analyte species is provided in a single piece of equipment and/or in a single method. Collection, concentration and analysis may be similarly provided.
  • the analysis technique may involve electrophoresis, such as gel electrophoresis or capillary electrophoresis, and/or chromatography.
  • FIG. 1 illustrates the trapping stage of the present invention
  • FIG. 2 illustrates the electro-osmotic injection stage of the present invention
  • FIG. 3 illustrates a microchip based system for fingerprint collection
  • FIG. 4 illustrates the mobilisation of the concentration band arising from the concentration achieved by the present invention
  • FIG. 5 a illustrates in plan view a chip using the present invention
  • FIG. 5 b illustrates in side view the chip of FIG. 5 a
  • FIG. 6 illustrates an alternative chip design.
  • a variety of biological based investigations require the collection of as much as possible of an analyte species from a sample in which that analyte species is potentially widely dispersed. As high a recovery as possible of the analyte species from that raw sample is desirable to maximise the amount of analyte available for analysis and maximise the number of raw samples on which meaningful analysis can be performed.
  • the concentration of analyte species in the prepared sample is also desirable for the concentration of analyte species in the prepared sample to be as high as possible. This will lead to a higher concentration in the media used for subsequent analysis.
  • analyte species for instance biochemical markers
  • identification of chemical substances present in a range of biological materials can yield valuable forensic intelligence on the lifestyle, sex and physiology of a perpetrator of a crime or person of interest.
  • Such forensic intelligence is complementary to that obtained from DNA analysis, and so enables a broader description of the perpetrator and/or person of interest to be established.
  • WO 01/89667 and others provide improved methods for loading substrates, such as gels.
  • substrates such as gels.
  • These prior art teachings us various chemical combinations and voltage profiles to increase the concentration of the prepared sample as it is loaded from a liquid buffer into the substrate to give the loaded sample.
  • the present invention is concerned with improvements in efficiency and/or concentration which can be achieved prior to this in the generation of the prepared sample from the raw sample.
  • the techniques for improving the position when loading prepared samples into substrates can also be applied to the present invention, but it is preferred to perform the analysis in the buffer phase.
  • the fingerprint is applied to a first surface 1 of a gel 3 , as illustrated in 3 .
  • a potential difference is applied across the gel 3 between the first, contact surface 1 to the other surface 5 using electrodes, not shown. The potential difference causes the chemical residues to migrate from the first surface 1 through the gel 3 towards the second surface 5 .
  • the gels best suited to the present invention there is no endoelectro-osmotic flow due to the careful selection of the gels used.
  • the gels feature polymers which are neutral or alternatively for which any charge present has been neutralised, for instance through the use of additives.
  • Agar rose gels having such properties are widely available.
  • the analyte species migrates through the gel 3 at a velocity, V1, that corresponds to its electrophoretic velocity in the gel.
  • the second surface 5 of the gel 3 defines an interface between the gel 3 and a buffer 13 of equivalent pH.
  • the electro-osmotic flow within the buffer 13 is high, and contributes most to the velocity of the analyte species in the buffer 13 .
  • Electro-osmotic flow is normally towards the cathode the provision of high electro-osmotic flow within the buffer is ensured by suitable design or treatment of the capillary and supporting system.
  • a highly alkaline solution may be used to pre-treat the channel and so condition it to have a generally positive charge associated with the internal surface.
  • dynamic adjustments or static adjustments could be used to achieve the same characteristics.
  • the electrophoretic velocity of the analyte species in the buffer 13 is considerably smaller that its electrophoretic velocity in the gel 3 , principally due to the high difference between the conductivity of the gel 3 and the buffer 13 , the electrophoretic velocity in the buffer 13 is at least equalled by, but opposed in direction, by the electro-osmotic velocity.
  • V2 equals zero or is negative. The result of this is that the analyte species cannot move significantly beyond the interface and a concentrated mass of the analyte species builds up with time at the interface between the gel 3 and the buffer 13 .
  • the potential difference is removed and diffusion of the concentrated analyte species is allowed. Diffusion can occur in both directions, but it is significantly faster in the direction of the buffer 13 . As a result this stage allows the concentrated analyte species to move from the interface into the initial section 15 of the buffer 13 . Controlling the diffusion time allows effective diffusion out of the gel 3 , whilst limiting dispersion of the analyte species within the buffer 13 and hence dilution of the prepared sample.
  • the mobilisation of the concentrated, prepared sample and its transfer to the next stage of any technique is achieved by applying a potential difference of reversed polarity compared to that use in the collection/concentration stage.
  • This is a significant different when compared with “stacking” techniques, therefore, in which the same polarity is used to concentrate and then move on the analyte species.
  • the electro-osmotic injection for use in the subsequent analysis technique is thus generated.
  • the electro-osmotic flow is far and away the dominant contributor to the velocity of the analyte species in the buffer 13 , but on this occasion the polarity promotes the flow of the concentrated analyte species away from the gel/buffer interface and through the buffer 13 .
  • Laminar flow results in the integrity of the concentrated, prepared sample of the analyte species being maintained within the buffer 13 .
  • the prepared sample can then be subject to separation and/or processing within the structure provided by the chip or other component embodying the process.
  • the concentration and collection occurs at the interface 100 between the narrow channel 102 containing the buffer 13 and the gel 3 .
  • the analyte species are moved along the channel 102 and the analysis is performed within the channel 102 for instance, by measuring differences in mobility for different analyte species in the electro-osmotic injection plug.
  • the present method achieves a focussing and concentration of the analyte species rather than stacking.
  • the voltage switched is used to mobilise the concentration band after the concentration and diffusion parts of the process.
  • extraction, enrichment and analysis can all be performed in one device using the present invention.
  • the relative dimensions of the substrate/gel and interface and buffer are also not critical in the present technique as the interface morphology is not relied upon to create an electrical potential drop or the like.
  • the technique is also applicable to even small electro-phoretic velocity, and as a consequence can be used for any charged component, not just those bearing a very significant charge.
  • a measure of the efficiency of the electro-osmotic injection achieved can be seen in FIG. 4 by virtue of the concentration peak for the analyte species in that part of the buffer compared with its surroundings.
  • FIG. 5 an illustration of the use of such a technique to analyse fingerprints is provided.
  • the fingerprint is applied to the top surface 200 of the gel 202 , is collected from across the whole surface 202 and concentrated at the gel 200 to buffer 204 interface. This is at the top of a channel 206 .
  • the analyte species arising from the fingerprint are allowed to diffuse and then are drawn away from the interface by a reversal of the polarity of the potential difference.
  • the concentrated analyte species are moved along channel 208 and analysed there. Alternatively they can then be directed along channel 210 to form the feed to an analytical technique, not shown.
  • FIG. 6 A further chip design is provided in FIG. 6 which offers still greater versatility and performance in applying the method of the present invention. It is preferred in this case that a further channel is provided.
  • This channel provides an optional buffer inlet and ideally leads to close to the interface between the gel and the channel into which the analyte species are moved after collection. By introducing buffer to this channel at the time of the reversal of the polarity of the electrical potential the electric field strength/electrical potential can be maintained. This gives a faster movement of the analyte species away from the interface.
  • the channel is shown in relief in FIG. 6 for the purposes of clarity.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Electrostatic Separation (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
US10/463,647 2002-06-18 2003-06-17 Analysis Abandoned US20040040849A1 (en)

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Application Number Priority Date Filing Date Title
US11/811,101 US8097140B2 (en) 2002-06-18 2007-06-08 Liquid sample analysis methods

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GBGB0213979.8A GB0213979D0 (en) 2002-06-18 2002-06-18 Improvements in and relating to analysis
GB0213979.8 2002-06-18

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US (2) US20040040849A1 (ja)
EP (1) EP1514100B1 (ja)
JP (1) JP2004132952A (ja)
AU (1) AU2003275927A1 (ja)
GB (1) GB0213979D0 (ja)
WO (1) WO2003106986A2 (ja)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050034990A1 (en) * 2003-08-12 2005-02-17 Crooks Richard M. System and method for electrokinetic trapping and concentration enrichment of analytes in a microfluidic channel
EP2393596B1 (en) 2009-02-09 2016-09-28 Whitespace Enterprise Corporation Microfluidic devices and methods of providing a storable sample
US9047605B2 (en) * 2010-07-27 2015-06-02 Theodosios Kountotsis System and method for instantaneous fingerprint recognition and analysis resulting in targeted output
US20130323737A1 (en) 2010-08-10 2013-12-05 Bioaccel Method and system for analyzing a sample
US20160187293A1 (en) 2011-06-10 2016-06-30 The Arizona Board of Regents Acting for and on behalf of University of Arizona Electrophoresis system
DE102012219156A1 (de) 2012-10-19 2014-04-24 Albert-Ludwigs-Universität Freiburg Integriertes mikrofluidisches bauteil zur anreicherung und extraktion biologischer zellbestandteile

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US5302264A (en) * 1992-09-02 1994-04-12 Scientronix, Inc. Capillary eletrophoresis method and apparatus
US5660703A (en) * 1995-05-31 1997-08-26 The Dow Chemical Company Apparatus for capillary electrophoresis having an auxiliary electroosmotic pump
US5858195A (en) * 1994-08-01 1999-01-12 Lockheed Martin Energy Research Corporation Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis
US5958202A (en) * 1992-09-14 1999-09-28 Perseptive Biosystems, Inc. Capillary electrophoresis enzyme immunoassay
US6319379B1 (en) * 1999-08-23 2001-11-20 The Regents Of The University Of California Modified electrokinetic sample injection method in chromatography and electrophoresis analysis
US20020003089A1 (en) * 2000-05-23 2002-01-10 Devault James D. Electrophoretic stacking of DNA and other samples prior to electrophoresis
US6475441B1 (en) * 1997-06-09 2002-11-05 Caliper Technologies Corp. Method for in situ concentration and/or dilution of materials in microfluidic systems
US6613580B1 (en) * 1999-07-06 2003-09-02 Caliper Technologies Corp. Microfluidic systems and methods for determining modulator kinetics
US20030186255A1 (en) * 2001-06-06 2003-10-02 Li-Cor, Inc. Single molecule detection systems and methods
US6695009B2 (en) * 2000-10-31 2004-02-24 Caliper Technologies Corp. Microfluidic methods, devices and systems for in situ material concentration
US6749735B1 (en) * 2000-03-16 2004-06-15 David Le Febre Electromobility focusing controlled channel electrophoresis system
US6764817B1 (en) * 1999-04-20 2004-07-20 Target Discovery, Inc. Methods for conducting metabolic analyses
US20050161326A1 (en) * 2003-11-21 2005-07-28 Tomoyuki Morita Microfluidic treatment method and device
US7005050B2 (en) * 2001-10-24 2006-02-28 The Regents Of The University Of Michigan Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode-defined sample injection
US7060171B1 (en) * 2001-07-31 2006-06-13 Caliper Life Sciences, Inc. Methods and systems for reducing background signal in assays

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US5089099A (en) * 1990-11-30 1992-02-18 Varian Associates, Inc. Field amplified polarity switching sample injection in capillary zone electrophoresis
DE69331996T2 (de) 1992-09-14 2002-12-05 Purdue Research Foundation, West Lafayette Chemische Analyse durch Elektrophorese
US20020070166A1 (en) 2000-12-07 2002-06-13 Board Of Governors Of The University Of Alberta Sample purification on a microfluidic device

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Publication number Priority date Publication date Assignee Title
US5275710A (en) * 1990-05-14 1994-01-04 Labintelligence, Inc. Gel electrophoresis system including optical stage, sample applicator and sample retriever
US5302264A (en) * 1992-09-02 1994-04-12 Scientronix, Inc. Capillary eletrophoresis method and apparatus
US5958202A (en) * 1992-09-14 1999-09-28 Perseptive Biosystems, Inc. Capillary electrophoresis enzyme immunoassay
US5858195A (en) * 1994-08-01 1999-01-12 Lockheed Martin Energy Research Corporation Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis
US5660703A (en) * 1995-05-31 1997-08-26 The Dow Chemical Company Apparatus for capillary electrophoresis having an auxiliary electroosmotic pump
US6475441B1 (en) * 1997-06-09 2002-11-05 Caliper Technologies Corp. Method for in situ concentration and/or dilution of materials in microfluidic systems
US6764817B1 (en) * 1999-04-20 2004-07-20 Target Discovery, Inc. Methods for conducting metabolic analyses
US6613580B1 (en) * 1999-07-06 2003-09-02 Caliper Technologies Corp. Microfluidic systems and methods for determining modulator kinetics
US6319379B1 (en) * 1999-08-23 2001-11-20 The Regents Of The University Of California Modified electrokinetic sample injection method in chromatography and electrophoresis analysis
US6749735B1 (en) * 2000-03-16 2004-06-15 David Le Febre Electromobility focusing controlled channel electrophoresis system
US20020003089A1 (en) * 2000-05-23 2002-01-10 Devault James D. Electrophoretic stacking of DNA and other samples prior to electrophoresis
US6695009B2 (en) * 2000-10-31 2004-02-24 Caliper Technologies Corp. Microfluidic methods, devices and systems for in situ material concentration
US20030186255A1 (en) * 2001-06-06 2003-10-02 Li-Cor, Inc. Single molecule detection systems and methods
US7060171B1 (en) * 2001-07-31 2006-06-13 Caliper Life Sciences, Inc. Methods and systems for reducing background signal in assays
US7005050B2 (en) * 2001-10-24 2006-02-28 The Regents Of The University Of Michigan Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode-defined sample injection
US20050161326A1 (en) * 2003-11-21 2005-07-28 Tomoyuki Morita Microfluidic treatment method and device

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US8097140B2 (en) 2012-01-17
GB0213979D0 (en) 2002-07-31
EP1514100A2 (en) 2005-03-16
AU2003275927A8 (en) 2003-12-31
US20070240990A1 (en) 2007-10-18
EP1514100B1 (en) 2012-11-14
AU2003275927A1 (en) 2003-12-31
WO2003106986A2 (en) 2003-12-24
WO2003106986A3 (en) 2004-02-19
JP2004132952A (ja) 2004-04-30

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Effective date: 20051206

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION