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WO2005033687A1 - Procede de detection de proteines par microcircuits integres et capillaires d'un ordre inferieur au ug/ml - Google Patents

Procede de detection de proteines par microcircuits integres et capillaires d'un ordre inferieur au ug/ml Download PDF

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Publication number
WO2005033687A1
WO2005033687A1 PCT/US2004/001276 US2004001276W WO2005033687A1 WO 2005033687 A1 WO2005033687 A1 WO 2005033687A1 US 2004001276 W US2004001276 W US 2004001276W WO 2005033687 A1 WO2005033687 A1 WO 2005033687A1
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Prior art keywords
sample
dye
sds
fluorescent dye
protein
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Ceased
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PCT/US2004/001276
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English (en)
Inventor
James P. Landers
Braden P. Giordano
Lianji Jin
Dean Burgi
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UVA Licensing and Ventures Group
University of Virginia UVA
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University of Virginia UVA
University of Virginia Patent Foundation
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Publication of WO2005033687A1 publication Critical patent/WO2005033687A1/fr
Anticipated expiration legal-status Critical
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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
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • 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

  • the present invention relates to protein detection and quantification using microfabricated devices and capillaries.
  • the present invention provides methods for separation and detection, in a microfabricated device or capillary, of proteins in a sample without requiring pre- or post-column protein labeling or modification for eventual laser-induced fluorescence (LLF) detection.
  • LEF laser-induced fluorescence
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • CE capillary electrophoresis
  • No. 5,126,021 to Grossman discloses a capillary electrophoresis element which includes a capillary electrophoresis tube containing a low viscosity uncharged polymer solution, for separating nucleic acids.
  • U.S. Pat. No. 5,264,101 to Demorest et al. discloses the use of a hydrophilic polymer solution, which is characterized by a molecular weight of 20 to 5,000 kD, and a charge between 0.01 and 1% as measured by the molar percent of total monomer subunits to total polymer subunits, where the charge is opposite to the charge of the surface of the capillary in which the polymer is used.
  • Jin et al. uses a fluorescent dye, NanoOrange ® , which also binds to SDS-micelles and protein-SDS complexes, however, a dilution step was not required prior to detection, and separation and detection is possible on both the capillary and microchip platforms.
  • the work by Bousse et al. is limited to the microchip due to the microchannel structure necessary for the dilution step.
  • Harvey et al. J. Chromatogr. B 2001, 754, 345-356 have used NanoOrange ® as an on-column dynamic labeler of proteins in CE under non-denatured conditions, achieving a limit of detection of 212 ng/mL for human serum albumin.
  • Liu et al. (Anal. Chem. 2000, 72, 4606-4613) utilized the same fluor for post- column labeling of non-denatured proteins on a microchip with a unique cross-t mixing design positioned prior to the detection point with sensitivity as low as 1.21 ⁇ g/mL.
  • the present invention describes the development of conditions more amenable to dynamic labeling with a fluorescent dye, particularly a merocyanine dye, especially those disclosed in U.S. Patent No. 5,616,502 to Haughland et al., which is incorporated herein by reference, for capillary and microchip electrophoresis-SDS analysis. Buffer components including sieving polymer, ionic strength, and SDS concentration were considered with a mind towards increasing detection sensitivity. These conditions were translated to the microchip platform for rapid protein separation, detection, and quantification.
  • the present invention further provides a method for laser-induced fluorescence (LIF) detection of proteins in capillary electrophoresis or microfabricated devices without pre- or post-column modification of the proteins.
  • the method consists of incorporating a fluorescent dye, particularly a merocyanine dye, into the matrix, buffer, or sample.
  • a fluorescent dye particularly a merocyanine dye
  • the proteins denatured by SDS, binds to the fluorescent dye through hydrophobic interaction.
  • SDS is not present to promote dye-protein interaction, "trasient denaturing" can be used to optimize uptake of the fluorescent dye by the protein.
  • Heat shock labeling involves labeling with the fluorescent dye, on-capillary or on-microchip, by heat denaturing the protein in the absence of SDS or other surfactants.
  • the protein can be detected when the dye is excited by a radiation source, such as an argon-ion laser.
  • a radiation source such as an argon-ion laser.
  • the fluorescent dye binds to proteins on a per-mass basis when capillary and microchip conditions are equivalent to SDS-PAGE, which allows for protein quantification.
  • Figure 1 shows the separation of an 8-component protein-sizing ladder at a total concentration of 32 ⁇ g/mL.
  • Separation buffer was 250 mM Tris, 250 mM CHES, pH 8.7 with 0.1% w/v SDS and 5% w/v 100,000 MW PEO.
  • Capillary was 50 micron diameter, 27 cm long (20 cm to detection window).
  • Sample was injected for 10 seconds at a field strength of 370 V/cm. Separation field strength was 370 V/cm.
  • Figure 2 shows A) the separation observed when dye is added to the sample, while none is included in the separation buffer.
  • Figure 3 shows a comparison in peak intensity between 0.04% w/v SDS containing separation buffer and 0.1% w/v SDS containing separation buffer on a capillary. Injection and separation fields as described in Figure 1.
  • Sample is the 8- component sizing ladder at a total protein concentration of 16 ⁇ g/mL.
  • Sample is injected using a cross-t configuration at -lOOV/cm.
  • Sample is separated at -300 V/cm.
  • Figure 4 shows separation of the 8-component ladder on-chip with 0.04% w/v containing separation buffer using the 0.04% SDS containing separation buffer, 0.8% merocyanine dye included.
  • Figure 5 shows: Lower trace - microchip electropherogram of bovine serum albumin (66 kDa) using 0.04% w/v SDS separation buffer with 0.8% v/v merocyanine dye; and Upper trace - separation of BSA with 1.6% merocyanine dye added to sample prior to separation.
  • Figure 6 shows a sample matrix components comparison between capillary and microchip: A) 25 mM Tris-CHES, ImM DTT on capillary; B) 25 mM Tris-CHES, ImM DTT on microchip; C) SDS containing sample matrix on capillary; D) SDS containing sample matrix on microchip; E) SDS containing sample matrix with dye included on capillary; F) SDS containing sample matrix with dye included on microchip; G) 25 mM Tris-CHES, ImM DTT with dye included on capillary; and H) 25 mM Tris-CHES, 1 mM DTT with dye included on microchip.
  • Figure 7 shows a separation of solid-phase extraction purification of proteins from human semen.
  • Figure 8 shows comparison of CZE analysis of human sera to partially-denatured sera using the 0.04% merocyanine dye containing run buffer.
  • Sera is diluted 1-500 in 0.5% SDS, with 1% merocyanine dye included. Separation conditions are as shown in Figure 4.
  • Figures 8 A and 8B show the normal serum profile, 8C and 8D the profile of a sample with elevated ⁇ -region, and 8E and 8F the profile with an elevated ⁇ -region.
  • Figure 9 shows a pictorial representation of capillary and microchip electrokinetic injection.
  • the present invention provides methods for electrophoretically separating, detecting, and quantifying proteins in a capillary or microfabricated device.
  • Microfabricated or micro fluidic devices are used to perform the separation of the present invention.
  • "Microfabricated” or “microfluidic,” as used herein, refers to a system or device having fluidic conduits or microchannels that are generally fabricated at the micron to submicron scale, e.g., typically having at least one cross-sectional dimension in the range of from about 0.1 ⁇ m to about 500 ⁇ m.
  • the microfluidic system of the invention is fabricated from materials that are compatible with the conditions present in the particular experiment of interest. Such conditions include, but are not limited to, pH, temperature, ionic concentration, pressure, and application of electrical fields.
  • the materials of the device are also chosen for their inertness to components of the experiment to be carried out in the device.
  • the devices can include an optical or visual detection element.
  • the devices are generally fabricated, at least in part, from transparent materials to allow, or at least, facilitate detection and/or detection.
  • transparent windows of, e.g., glass or quartz may be incorporated into the device for these types detection elements.
  • the device generally comprises a solid substrate, typically on the order of a few millimeters thick and approximately 0.2 to 12.0 centimeters square, microfabricated to define at least one inlet reservoir, at least one outlet reservoir, and a microchannel flow system, preferably a network of flow channels, extending from the at least one inlet reservoir to the at least one outlet reservoir.
  • the device contains at least a main electrophoresis channel and at least one intersection channels forming a cross-t intersection with the main electrophoresis channel. More than one cross-t intersections may be appropriate for injecting various samples, buffers, and reactants.
  • a variety of manufacturing techniques are well known in the art for producing microfabricated channel systems.
  • the system generally includes a voltage controller that is capable of applying selectable voltage levels, sequentially or, more typically, simultaneously, to each of the reservoirs, including ground.
  • Such a voltage controller is implemented using multiple voltage dividers and multiple relays to obtain the selectable voltage levels. Alternatively, multiple independent voltage sources are used.
  • the voltage controller is electrically connected to each of the reservoirs via an electrode positioned or fabricated within each of the plurality of reservoirs.
  • Use of electrokinetic transport to control material movement in interconnected channel structures was described, e.g., in WO 96/04547 to Ramsey, which is incorporated by reference.
  • Modulating voltages are concomitantly applied to the various reservoirs to affect a desired fluid flow characteristic, e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate direction of travel) flow of labeled components toward a waste and/or collection reservoir.
  • modulation of the voltages applied at the Various reservoirs can move and direct fluid flow through the interconnected channel structure of the device.
  • Another way to control flow rates is through creation of a pressure differential.
  • a cell suspension is deposited in a reservoir or well at one end of the channel, and at sufficient volume or depth, that the cell suspension creates a hydrostatic pressure differential along the length of the channel, e.g., by virtue of its having greater depth than a well at an opposite terminus of the channel.
  • the reservoir volume is quite large in comparison to the volume or flow through rate of the channel, i.e., 1 ⁇ L reservoirs or larger as compared to a 100 ⁇ m channel cross section.
  • Another pressure based system is one that displaces fluid in the micro fluidic channel using, e.g., a probe, piston, pressure diaphragm, or any other source capable of generating a positive or negative pressure.
  • a pressure differential is applied across the length of the channel.
  • a pressure source is optionally applied to one end of the channel, and the applied pressure forces the material through the channel.
  • pressure applied at the inlet reservoir would force the cell mixture contained therein through the microchannel, and into the outlet reservoir.
  • the pressure is optionally pneumatic, e.g., a pressurized gas or liquid, or alternatively a positive displacement mechanism, i.e., a plunger fitted into a material reservoir, for forcing the material along through the channel.
  • Pressure can, of course, also be due to electrokinetic force, thermal expansion, or a variety of other methods and devices.
  • a vacuum source i.e., a negative pressure source
  • a vacuum source can be placed in the outlet reservoir to draw a cell suspension from the inlet reservoir.
  • Pressure or vacuum sources are optionally supplied external to the device or system, e.g., external vacuum or pressure pumps sealably fitted to the inlet or outlet of the channel, or they are internal to the device, e.g., microfabricated pumps integrated into the device and operably linked to the channel, such as those disclosed in WO 97/02357 to Anderson et al., which is incorporated herein by reference.
  • the main electrophoresis channel may or may not be filled with a sieving matrix.
  • a sieving matrix be used.
  • the matrix can be, but is not limited to, dextran, polyacrylamide (PAA), polyethylene oxide (PEO), hydroxypropyl cellulose, and/or combinations thereof.
  • the proteins can be subjected to electrophoresis in their native, partially denatured, or in completely denatured form.
  • the proteins can be denatured by a surfactant, such as routinely done with SDS; reducing agent, such as 2-mercaptoethanol and/or dithiothreitol (DTT); pH; heat; and/or other methods known in the art.
  • the protein is bound to SDS at a ratio of 0-1.4 g SDS/g protein.
  • the protein-SDS complex is then heated for about 10 minutes at about 95-100°C. If the proteins are to be analyzed in their reduced form, a reducing agent can be added prior to heating.
  • the SDS concentration is preferably below the micelle concentration (C mc ). Depending on the ionic character of the system, the SDS concentration is preferably about 0.5 to about 8mM.
  • the proteins are detected using a fluorescent dye, particularly a merocyanine dye.
  • the dyes disclosed by U.S. Patent No. 5,616,502 to Haughland et al., which is incorporated herein by reference, is most appropriate for the present invention.
  • Several fluorescent dye properties are highly desirable for use with the present invention.
  • the dye binds preferentially to SDS-protein complex, when compare to its binding with free SDS in solution.
  • the binding of the fluorescent dye to free SDS is preferably at least 10% lower than its binding to SDS-protein complex, resulting in low background fluorescence.
  • the fluorescent dye binds to the protein through hydrophobic and/or electrostatic interaction. Although the interaction is hydrophobic, the protein needs not be hydrophobic. Only the region of the protein that binds to the fluorescent dye has to be hydrophobic, while the overall protein is not required to be hydrophobic.
  • the fluorescent dye can be in the sample, the matrix, or the buffer. Importantly, however, the fluorescent dye must come into contact with the proteins on-column, i.e., no pre- or post-column modification.
  • the fluorescent dye is used in the buffer and/or the matrix, it should be in concentrations of about 0.01-25%, more preferably about 0.1%- 1.0%, and most preferably about 0.2-0.8 % (v/v).
  • the fluorescent dye preferably has an excitation wavelength of about 450-500 nm, most preferably about 470-480 am, and an emission wavelength of about 520-660 nm, most preferably about 570-600 nm.
  • the matrix is selected so that it has little of no binding with the fluorescent dye.
  • the binding of the fluorescent dye to the matrix should be at least 10% lower than its binding to the protein or the SDS-protein complex, resulting in low background fluorescence.
  • the protein is SDS-denatured when electrophoresis is performed with the dye in the buffer or the matrix.
  • the addition of the fluorescent dye to the separation buffer and sample matrix allows for a simple method for non-covalent labeling of protein-SDS complexes for sizing.
  • the fluorescent dye binds to the SDS-protein complex on a per-mass basis regardless of the type of protein under denaturing conditions. This is very important because it allows for protein quantification.
  • SDS binds to proteins in a per-mass fashion: for every gram of protein, about 1.4 grams of SDS is bound to the peptide backbone. This amounts to a single SDS molecule binding to the backbone of two amino acids.
  • SDS is used at the sub-micellular concentrations ( ⁇ C mc ).
  • the protein can also be injected into the electrophoresis column/microfabricated device in its native form. In this approach, the proteins are labeled on-column and then ran under completely non-denaturing conditions.
  • Transient denaturing This involves labeling with the fluorescent dye on-capillary or on-microchip by denaturing the protein in the absence of SDS or other surfactants. This is preferably carried out on-column in a zone that can be heated, either by Joule heating provided the appropriate buffer conditions and electric fields are utilized or by inputting energy (laser, IR, microwave, and the like). Heating the native protein causes it to unfold. Adding the fluorescent dye in the heated zone it will incorporate the dye into the protein during the refolding process upon cooling (the heated zone disperses over time as equilibrium is achieved).
  • the dye and protein can exist in separate zones, with the dye outside of the heated zone and, during electrophoresis, the unfolded protein is allowed to migrate out of the heated zone, into the dye zone which may or may not be at a lower temperature - refolding of the protein, thus leads to incorporation of the dye.
  • the binding of the fluorescent dye to the protein would be a combination of interaction with hydrophobic residues, the peptide backbone, and just being in the right place at the right time and being caged within the refolded protein. While the refolded protein may not necessarily take on the original tertiary structure, it will have bound sufficient dye for detection and be amenable to separation.
  • the proteins can be detected and/or quantify through the optical or visual detection element on the microfabricated device.
  • a light source such as a laser, which emits light in the wavelengths known to induce fluorescence of the fluorescent dye, is focused onto the optical or visual detection element.
  • the excitation/emission wavelengths for merocyanine dye are 450-500/520-660 nm, respectively.
  • the 488 nm line of an Argon-ion laser can be used for excitation of the fluorophore and emission can be filtered through a 590DF35 band-pass filter for dection of fluorescence.
  • Various lenses and optics can be used to focus and gather excited and emitted lights.
  • the emitted light can be detected and quantify by a photoreceptor, such as a photomultiplier tube, a photodiode, a CCD array, and the like, and can be converted into electrical signals to be sent to a microprocessor for data recording and analysis.
  • a photoreceptor such as a photomultiplier tube, a photodiode, a CCD array, and the like.
  • Example 1 Experimental Method Apparatus Beckman P/ACE 5510 (Beckman Instruments, Inc., California) with a LIF detector was used to obtain capillary electrophoresis data.
  • the microchip electrophoresis system was assembled in-house.
  • a laser beam of 488 nm was emitted from an Argon Ion laser source (Laser Physics, Utah) and reflected to the beam splitter (505DRLP02;
  • Tris[hydroxymethyl]aminomethane (TRIS), 2-[N-cyclohexylamino]ethane sulfonic acid (CHES), Dextran (2,000,000 MW), and Hydroxyethylcellulose (250,000 MW) were purchased from Sigma- Aldrich (St. Louis, MO). Sodium dodecylsulfate and an 8-component protein ladder obtained from Bio-Rad (Hercules, CA). Polyethylene oxide (100,000, 200,000, and 600,000 MW) was purchased from Acros (Pittsburgh, PA). Fluorescent dye (merocyanine dye) was purchased from Molecular Probe (Oregon) as
  • NanoOrange ® was used according to the product instruction manual.
  • merocyanine dye stock solution 500X was added to the separation buffer at IX concentration unless otherwise specified.
  • microchip Fabrication The microchip was fabricated by using standard photolithography and wet chemical etching technique.
  • a film mask containing the microchip design was prepared on a negative film using an image setter.
  • the microchip design a traditional cross-T type, was transferred onto the glass wafer (Nanofilm, California) with positive photoresist by UV exposure.
  • the channels were etched with HF solution.
  • Etched plate was thermally bonded to a drilled cover plate in a programmable furnace (Ney Dental Inc., California).
  • the configuration of a single channel microchip is 7.5 cm from injection cross to the outlet, 0.5 cm from injection cross to inlet and sample and sample waste.
  • capillary and Microchip Separations Unless otherwise specified, capillaries and microchips were flushed for 10 minutes with 1M HNO , followed by a 12 minute rinse with IX merocyanine dye containing run buffer. Proteins (dissolved in 25 mM Tris-CHES, 0.1% SDS, ImM DTT and heated at 94°C for 10 minutes then cooled to room temperature) were electrokinetically injected into the capillary or microchip. Separations were performed under reverse polarity (inlet was cathode) with field strength of 370 V/cm.
  • Example 2 Results and Discussion Evaluation of Sieving Polymers
  • Our initial goal in developing a separation buffer more amenable to labeling with merocyanine dye was the difficulty in handling the commercially available system, specifically with respect to viscosity. Loading of separation buffer in both capillaries and microchips was 'difficult. In addition, this buffer provided a high background fluorescence and sensitivity was not enhanced by going from UV to LIF detection. A number of polymers were evaluated in this work, including dextran, PEO, HPC, and HEC.
  • Figure 2a shows the electropherogram for a 100 ⁇ g/mL ladder with 0.2%
  • microchip injection to result in a concentrating of SDS-dye and MC-dye (as seen in Figures 6E, F, and H) on both sides of the sample matrix (the side towards the inlet and the side towards the outlet) - in capillaries, the side of the sample matrix facing the outlet is disrupted during injection and the concentrating effect is seen only on the side towards the inlet.
  • a capillary pressure injection with the microchip electrokinetic injection; unfortunately due to the viscosity of the separation buffer, pressure injection results are not reproducible. What this means, is that for LIF detection of proteins using dye, the conditions for achieving maximum sensitivity are system dependent.
  • FIG. 3 Microchip Electrophoresis - Detection Sensitivity and Range
  • the buffer system described in Figure 3 was utilized in a traditional cross-T injection configuration microchip for on-chip protein analysis. On-chip sensitivity was improved by going to a 0.8% merocyanine dye concentration. interesting, use of this dye concentration in capillary separations resulted in increases in the noise levels with no appreciable increase in signal.
  • Figure 4 illustrates the protein ladder (64 ⁇ g/mL) as separated on a microchip. A log (MW) versus 1/MT plot was associated with an R 2 value of 0.9957, indicating that the ability to size proteins on the microchip is retained.
  • BSA at various concentrations was included in sample matrix and electrophoresed.
  • the peak area of BSA was determined and correlated to concentration.
  • detector response was not linear with respect to concentration and there was variation in peak area for the same BSA concentration greater than 5%.
  • FIG. 7 shows the protein sizing profile observed from a sample of proteins extracted from human semen.
  • the sample is dissolved in an 8M Guanadine HC1 buffer and is flowed through a silica bed, in the form of a sol-gel.
  • Biological components i.e. proteins, DNA, lipids
  • the proteins and other cellular components i.e.
  • lipids and sugars are washed off of the silica surface in a 40% solution of isopropanol.
  • DNA is eluted from the sol-gel using aqueous buffer such as Tris-EDTA.
  • the sample from Figure 7 was diluted 1:10 in sample matrix and heat denatured. Since the sample is a crude extract, it has in the range of 10,000 individual proteins and peptides at various concentrations.
  • dye was not included in the sample (addition of dye to the sample matrix would result in a more sensitive separation).
  • the electropherogram shows a series of primary peaks on top of what appears to be a drifting baseline; in fact they are low abundance proteins not well resolved in this separation.

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Abstract

La présente invention concerne la détection et la quantification de protéines par des dispositifs micro-usinés et des capillaires. L'invention concerne en particulier des procédés de séparation et de détection, dans un dispositif micro-usiné ou un capillaire, de protéines présentes dans un échantillon, sans nécessité de recourir à un marquage ou une modification protéique pré ou post-colonne pour une éventuelle détection par fluorescence induite par laser (LIF). Le procédé consiste à accomplir une électrophorèse avec un colorant fluorescent présent dans le tampon et/ou la matrice, en vue de marquer de manière décelable les protéines.
PCT/US2004/001276 2003-01-17 2004-01-20 Procede de detection de proteines par microcircuits integres et capillaires d'un ordre inferieur au ug/ml Ceased WO2005033687A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1947453A4 (fr) * 2005-10-19 2012-02-29 Nat Inst Of Advanced Ind Scien Procédé d analyse de protéine
US11207677B2 (en) 2018-03-07 2021-12-28 University Of Virginia Patent Foundation Devices, systems, and methods for detecting substances
WO2022047269A1 (fr) * 2020-08-28 2022-03-03 Regeneron Pharmaceuticals, Inc. Caractérisation de virus adéno-associé à l'aide d'une électrophorèse capillaire à micropuce
WO2022219538A1 (fr) * 2021-04-14 2022-10-20 Dh Technologies Development Pte. Ltd. Détection de fluorescence native pour l'analyse de protéines dans une électrophorèse capillaire

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5626502A (en) * 1996-06-07 1997-05-06 Novey; Richard T. Power steering adapter for outboard powerheads of various size
US20010001061A1 (en) * 1997-02-21 2001-05-10 Prusiner Stanley B. Assay for disease related conformation of a protein
US6475364B1 (en) * 1999-02-02 2002-11-05 Caliper Technologies Corp. Methods, devices and systems for characterizing proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5626502A (en) * 1996-06-07 1997-05-06 Novey; Richard T. Power steering adapter for outboard powerheads of various size
US20010001061A1 (en) * 1997-02-21 2001-05-10 Prusiner Stanley B. Assay for disease related conformation of a protein
US6475364B1 (en) * 1999-02-02 2002-11-05 Caliper Technologies Corp. Methods, devices and systems for characterizing proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BUSWELL A.M. ET AL.: "Critical analysis of lysozyme refolding kineticts", BIOTECHNOL. PROG., vol. 18, June 2002 (2002-06-01), pages 470 - 475, XP002983470 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1947453A4 (fr) * 2005-10-19 2012-02-29 Nat Inst Of Advanced Ind Scien Procédé d analyse de protéine
US11207677B2 (en) 2018-03-07 2021-12-28 University Of Virginia Patent Foundation Devices, systems, and methods for detecting substances
WO2022047269A1 (fr) * 2020-08-28 2022-03-03 Regeneron Pharmaceuticals, Inc. Caractérisation de virus adéno-associé à l'aide d'une électrophorèse capillaire à micropuce
US12320814B2 (en) 2020-08-28 2025-06-03 Regeneron Pharmaceuticals, Inc. Characterization of adeno-associated virus using microchip capillary electrophoresis
WO2022219538A1 (fr) * 2021-04-14 2022-10-20 Dh Technologies Development Pte. Ltd. Détection de fluorescence native pour l'analyse de protéines dans une électrophorèse capillaire

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