[go: up one dir, main page]

WO2010021639A1 - Détection de protéine attomolaire dans des matrices de type damle complexes avec des dosages de discrimination de force fluide semi-homogène - Google Patents

Détection de protéine attomolaire dans des matrices de type damle complexes avec des dosages de discrimination de force fluide semi-homogène Download PDF

Info

Publication number
WO2010021639A1
WO2010021639A1 PCT/US2008/087259 US2008087259W WO2010021639A1 WO 2010021639 A1 WO2010021639 A1 WO 2010021639A1 US 2008087259 W US2008087259 W US 2008087259W WO 2010021639 A1 WO2010021639 A1 WO 2010021639A1
Authority
WO
WIPO (PCT)
Prior art keywords
target
micrometer
assays
beads
stable
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.)
Ceased
Application number
PCT/US2008/087259
Other languages
English (en)
Inventor
Lioyd J. Whitman
Shawn P. Mulvaney
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.)
US Department of Navy
Original Assignee
US Department of Navy
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 US Department of Navy filed Critical US Department of Navy
Publication of WO2010021639A1 publication Critical patent/WO2010021639A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • Solid phase binding assays are far more common because of the advantages of anchoring the ligand- receptor interactions to a surface. Immobilizing the receptors simplifies many aspects of an assay, such as rinsing and exchange of reagents, and enables multiplexing by arraying different receptors across the surface.
  • the solid support can also be a limitation, both because of fouling and the inefficient diffusion of target molecules to the two- dimensional surface. See Sheehan, P.E., Whitman, LJ. , 2005. Nano Lett. 5, 803-807.
  • homogeneous assays are generally not limited by mass transport, they are more challenging to multiplex, both in terms of chemistry and detection.
  • a common solution to multiplexing in homogeneous assay systems has been the use of microbeads with unique fluorescent signatures, with each ligand-receptor pair anchored to a different color bead.
  • Solid phase assays proliferate because they have the flexibility to be either rapid, inexpensive, and simple to execute — e.g., lateral flow immunoassays as implemented in home pregnancy tests; or massively multiplexed and capable of deciphering complex relationships — e.g., large-scale nucleic acid microarrays as applied to gene expression profiling (Epstein et al., 2002 Anal. Chim. Acta 469, 3-36; Michalet et al., 2003 Annu. Rev. Biophys. Biomol. Struct. 32, 161-182).
  • the most sensitive multiplexed detection technology capable of both protein and nucleic acid detection is the bio-barcode assay (Hill and Mirkin, 2006, Nat. Protoc.
  • Bio-barcode assays that use microarray-based "scanometric" detection of these oligos have achieved the stated level of sensitivity in 2-5 hr.
  • barcode oligonucleotides have been fluorescently labeled and detected in solution, identifying a protein target (prostate specific antigen) in serum at 300 aM in only 90 min (Oh et al., 2006, Small 2, 103-108).
  • the fluid force discrimination (FFD) assay is an alternate approach to multiplexed detection that is simple, uses few reagents, is rapid ( ⁇ 25 min), and directly detects the biological target without amplification (Rife, et. al., U.S. Patent Application 11/424643; Mulvaney et al., 2007 Biosen. Bioelectron. 23, 191-200).
  • FFD assays have been used to demonstrate multiplexed, femtomolar nucleic acid hybridization and immunoassays in a solid phase microarray format. In FFD assays, biomolecular targets are captured onto a microarray and then labeled with microbeads.
  • microbead labels are preferentially removed by application of controlled, laminar fluidic forces.
  • the density of beads remaining on each spot after FFD indicates the target identity and concentration.
  • the use of microbead labels enhances the analytical sensitivity by enabling label detection at extremely low label densities ( «10 4 /cm ⁇ 2 ), down to single labels in a typical microarray spot ( ⁇ 100- ⁇ m diameter).
  • microbead assays are ideal for the analysis of samples in complex matrices because they do not suffer from the many forms of matrix interference that plague other labels (e.g., autofluoresence, electrochemical background).
  • FFD assays can be performed in a field-portable detection system incorporating magnetoresistive sensor array chips (Baselt et al., Biosens. Bioelectron. 13, 731-739, 1998; Edelstein et al., Biosen. Bioelectron. 14, 805-813, 2000; Miller et al., 2001; Mulvaney et al., 2007; Rife et al., Sens. Actuators, A 107, 209-21, 2003; Tamanaha et al., Biosensors and Bioelectronics 24 (2008) 1-13).
  • magnetoresistive sensor array chips Baselt et al., Biosens. Bioelectron. 13, 731-739, 1998; Edelstein et al., Biosen. Bioelectron. 14, 805-813, 2000; Miller et al., 2001; Mulvaney et al., 2007; Rife et al., Sens. Actuators, A 107, 209-21
  • SH semi-homogenous
  • FFD fluidic force discrimination
  • Previously microbead labels and FFD have been combined to achieve multiplexed, femtomolar nucleic acid hybridization and immunoassays in a microarray format (Mulvaney et al., 2007. Biosen. Bioelectron. 23, 191-200).
  • SH FFD assays the microbeads and any required intermediate receptors (e.g. secondary antibodies) are first mixed directly with a sample, allowing target analytes to be efficiently captured onto the beads.
  • SH target collection provides a 1000- fold improvement in the assay sensitivity, down to attomolar concentrations, as demonstrated by our detection of staphylococcal enterotoxin B (SEB) at 35 aM (1 fg/ml).
  • SEB staphylococcal enterotoxin B
  • SH assays are adaptable for extraction, preconcentration, and identification of analytes in complex sample matrices, including assays for SEB and ricin toxoid in serum and whole blood. Disclosed is a detailed model of the reaction kinetics that reveals how capturing the targets onto the beads in solution provides a significant kinetic advantage at low target concentrations where mass transport to a microarray surface is most limited.
  • the disclosed embodiment of FFD assays results in a 1000-fold improvement in analytical sensitivity — down to attomolar concentrations — using only two reagent mixtures and three assay steps that can be performed in as little as 10 min.
  • the microbeads and any required intermediate receptors e.g. secondary antibodies
  • the target- loaded beads are then specifically captured onto the microarray surface, with nonspecifically bound beads removed by FFD in buffer.
  • the assay results combined with detailed modeling of the reaction kinetics confirms that capturing the targets onto the beads in solution (vs.
  • SH assays are adaptable for extraction, preconcentration, and analysis of target in complex matrices, including assays for staphylococcal enterotoxin B (SEB) and ricin A chain (RCA) in serum and whole blood.
  • SEB staphylococcal enterotoxin B
  • RCA ricin A chain
  • a surface having primary amines the surface is reacted with succinimidyl A- formylbenzoate (SFB).
  • SFB succinimidyl A- formylbenzoate
  • SANH succinimidyl A- hydrazinonicotinate acetone hydrazone
  • the SFB-reacted surface is incubated with the NA-SANH solution under acidic conditions to configure said surface with stable SFB-SANH.
  • the stable surface is stored at 4° C and remains stable for at least 4 weeks.
  • the semi-homogenous binding assay method also includes an embodiment wherein a solution comprised of a target of interest is mixed with a micrometer-scale bead configured to bind specifically to the target or to an intervening receptor that binds specifically to the target and to the micrometer- scale bead.
  • a capture surface configured to specifically bind the target is exposed to the target solution.
  • a fluidic system is employed to remove non- specifically bound micrometer-scale particle labels from the capture surface, wherein said fluidic system applies a controlled and uniform laminar flow at the capture surface, said flow producing a Stokes force on said micron-scale bead of at least 1 pN. The Stokes force preferentially removes non- specifically bound micron sized labels from the capture surface.
  • FIG. 1 is a depiction of the modified surface chemistry
  • FIG. 2 shows a sequential and a semi-homogenous FFD binding assay
  • FIG. 3 shows the relative performances of sequential and SH FFD assays for the detection of SEB in buffer
  • FIG. 4 the semi-homogeneous FFD detection signal for 35 pM and 35 fM SEB in buffer as a function of the homogeneous mixing time (1-20 min);
  • FIG. 5 shows a comparison of SH FFD assays for SEB in buffer and in serum
  • FIG. 6 shows the detection of 150 pM (10 ng/ml) RCA in whole blood with SH FFD assays.
  • DETAILED DESCRIPTION OF THE INVENTION Each assay was performed in an acrylic flow cell mounted on a microscope slide. The flow cell was 2.8 mm long x 2.2 mm wide x 100 ⁇ m high and had a tapered entrance and exit to insure uniform, laminar flow of reagents across the capture spots, as discussed in detail in (Mulvaney et al., 2007; Tamanaha et al., J. Micromechan. Microeng. 12, 347-347, 2002).
  • the flow cell design creates a very uniform fluid velocity across the middle of the channel where the assay occurs, ensuring consistent spot-to-spot application of fluidic shear forces.
  • the flow rates were controlled with a peristaltic pump (Instech Laboratories, Inc.).
  • NA succinimidyl 4-hydrazinonicotinate acetone hydrazone
  • SFB succinimidyl A- formylbenzoate
  • the assay buffer was comprised of one liter of 1 x phosphate buffered saline was mixed with 50 g of dehydrated skim milk (Carnation), 1 ml of Tween 20 (Aldrich), and 1 ml of 1 % thimersol (Aldrich).
  • Target solutions (1 ml) at the stated concentration were prepared in either buffer or canine serum (Innovated
  • the total image analysis time is currently ⁇ 2 min per spot, dominated by the time required to manually locate and focus on each capture spot. With automated image capture, under development, we expect the total image analysis time to be ⁇ 30 s per spot. Those skilled in the art would understand that other means may be used to count the beads.
  • the sequential assay shown is typical for labeled, solid phase assays (e.g., ELISAs) where an immobilized capture probe is exposed to a series of solutions to create a biomolecular "sandwich.”
  • ELISAs solid phase assays
  • an immobilized capture probe is exposed to a series of solutions to create a biomolecular "sandwich.”
  • the target is captured first, a secondary antibody (2° Ab) that recognizes a second epitope on the target follows, and then an antibody- conjugated microbead that recognizes the Fc portion of the 2° Ab completes the sandwich.
  • a 2° Ab facilitates multiplexing: if all 2° Ab are from the same source (i.e., rabbit anti-target), a single type of Ab-conjugated microbead (i.e., sheep anti-rabbit) can label all assays. Target identities and concentrations in the sample may then be determined from the density of bead labels at each capture spot.
  • a SH assay the biomolecular sandwich is partially formed by simultaneously mixing the target, the 2° Ab, and the microbeads in a single solution. The sandwich is completed in a second step by exposing the capture probe microarray to the target-loaded beads. Finally, in both assays the nonspecifically attached beads are preferentially removed with FFD and the remaining beads at each capture spot counted.
  • the relative performances of sequential and SH FFD assays for the detection of SEB in buffer are compared in FIG. 3.
  • the target and 2° Ab were separately introduced in stop flow with a 5 min incubation time for each. Detection was reproducible over 6 orders of magnitude, from 35 fM (1 pg/ml) to 35 nM (1 ⁇ g/ml). (At 3.5 fM, the signal is comparable to the background.)
  • the target, 2° Ab, and microbeads were combined in a 1 ml solution for 5 min. Note that this step results in a net time savings because the 2° Ab labeling was performed concurrently with target capture.
  • the SH FFD assays resulted in greater bead capture at all target concentrations.
  • the SH FFD assay is more sensitive by three orders of magnitude, with SEB easily detected at 35 aM (1 fg/ml).
  • the improvement in sensitivity in a SH assay is attributed to the greater efficiency of target capture onto a bead by homogeneous mixing as compared to laminar flow delivery to a microarray surface.
  • diffusion of the target and 2° Ab to the capture probes on the microarray surface is known to be a limiting step (Sheehan and Whitman, 2005).
  • SH assays are more efficient because of the homogeneous sampling of target molecules and 2° Ab by the microbead labels, with each target molecule in the 1 ml SH mixture having numerous opportunities to bind to a 2° Ab and be captured onto a bead.
  • the flow cell volume is ⁇ 2 ⁇ l, such that — on a simple per volume basis — SH assays have a 500-fold sampling advantage.
  • sequential assays could compensate for this sampling advantage by flowing target solution over the capture array, reasonable flow rates would add significant time to the assay; for example, flowing 1 ml of solution at 5 ⁇ l/min would require 200 min.
  • the advantage of SH assays becomes most pronounced. Whereas the resulting difference in final bead density between the sequential and SH SEB assay at 35 nM is only 6%, at 35 fM it grows to 36%.
  • FIG. 4 shows the semi-homogeneous FFD detection signal for 35 pM ( ⁇ ) and 35 fM (O) SEB in buffer as a function of the homogeneous mixing time (1-20 min).
  • the 35 fM data has been fit to the model described in the text, yielding the rate constants shown.
  • the signal is well above background with ⁇ 1 min of mixing, reaches about 50% of the maximum within 5 min, and is approaching saturation for 35 fM at 20 min.
  • 1 min of homogeneous mixing ⁇ 10 min total assay time
  • 1 min of homogeneous mixing may be sufficient, or longer mixing times may be used to achieve additional sensitivity.
  • FFD assays are physical labels and therefore compatible with complex sample matrices.
  • FFD assays have been successfully performed in bacterial growth medium, milk, saliva, feces, urine, plasma, serum, and blood diluted 10-fold (Mulvaney et al., 2007).
  • the only matrix requirement is that the chemical composition does not prevent biomolecular recognition.
  • SH assays maintain this exceptional compatibility with complex matrices as demonstrated by the preliminary dose response curve for the detection of SEB in serum.
  • FIG. 5 shows a comparison of SH FFD assays for SEB in buffer (same data as FIG. 3) and in serum.
  • the background represents the average bead count over a negative capture spot (goat anti-ricin).
  • SH assays are not only compatible with complex matrices, but when combined with magnetic microbeads also provide a capability for sample extraction and preconcentration.
  • Paramagnetic microbeads were used so that the assays can be incorporated into a compact detection system based on magnetoelectronic sensor chips.
  • magnetic microbeads are used to homogeneously capture and magnetically extract targets of interest from complex samples for subsequent analysis. Magnetic extraction has been shown to be effective even from whole blood, one of the most complicated matrices to assay. The abundance of proteins, lipids, and cellular material found in whole blood both foul surfaces and create steric barriers that hinder target-probe binding (Tian et al., Anal. Biochem. 283, 175-191, 2000).
  • FIG. 6 shows the detection of 150 pM (10 ng/ml) RCA in whole blood with SH FFD assays.
  • the optical micrographs (250 ⁇ m x 250 ⁇ m) show bead capture within goat anti-ricin and negative capture probe (sheep anti-SEB) spots.
  • PCR Polymerase chain reaction
  • other asymmetric amplification schemes enable the detection of ⁇ 10 copies of DNA in typical sample volumes of ⁇ 100 ⁇ l (Decaro et al., 2006; Hoffmann et al., 2006; Mackay, 2004).
  • PCR polymerase chain reaction
  • these amplification schemes add complexity and sources of error to the analysis, especially when analyzing samples in complex matrices or outside a controlled laboratory setting.
  • the bio-barcode assay addresses many of these shortcomings by using magnetic extraction combined with internal amplification to achieve PCR-like sensitivity in complex matrices.
  • the bio-barcode approach combines homogeneous collection of target molecules onto microbeads with subsequent microarray detection of the identifying barcode labels. Because of the simplicity and efficiency of the labels in FFD assays, however, SH FFD assays are able to achieve comparable attomolar sensitivities in a notably simpler and faster format, using only two reagent mixtures (target + 2° Ab + bead solution and buffer) and three assay steps.
  • Homogeneous capture of target and secondary label onto the microbeads enhances the assay sensitivity in two ways: primarily through improved ligand-receptor capture; and, to a lesser extent, through assisted delivery of on-bead target to the microarray surface.
  • performance is limited by diffusion of target molecules to the microarray surface, which can be enhanced somewhat by flow.
  • the accumulation rate of target species on the surface depends on flow cell dimensions, sensor size, the concentration of the target, and the physical properties of the sample solution (viscosity, salinity, etc.). Even if a microarray system is designed with these principles in mind, overcoming the diffusion limit requires assisted target delivery (Morozov et al., J. Am. Chem. Soc.
  • the homogeneous collection step also improves assay efficiency via bead-assisted target delivery.
  • gravity is used to enhance delivery of the target to the microarray surface.
  • the relatively dense magnetic microbeads will quickly settle from solution onto the microarray.
  • a bead at the top of the flow cell will reach the microarray surface in ⁇ 1 min.
  • the three-dimensional search for a binding site is then reduced to a two-dimensional search as the bead continues to sample the surface through Brownian motion.
  • the reduction of dimensionality due to bead settling is partially responsible for the greater sensitivity of SH assays. It is also possible for beads to be more rapidly pulled to the surface using an applied magnetic field, a technique that works well for nanometer-diameter beads (de Boer et al., Biosen. Bioelectron. 22, 2366-2370, 2007;
  • Microarrays are excellent strategies for multiplexing, but are susceptible to surface fouling and are limited by mass transport and the reaction rate of target species with the microarray capture probes.
  • homogeneous assays enable very efficient target labeling, but multiplexing greatly increases the complexity of the chemistry and detection.
  • semi-homogeneous fluidic force discrimination assays we combine the capture efficiency of homogeneous mixing, the ease of multiplexing with microarrays, and the sensitivity and specificity enhancements provided by FFD.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention porte sur un procédé de test de discrimination de force fluide semi-homogène dans lequel des billes de la taille du micron et tous récepteurs intermédiaires requis (par exemple, des anticorps secondaires) sont tout d'abord mélangés directement avec un échantillon, permettant à des analytes cibles d'être efficacement capturés sur les billes. Les billes chargées cibles sont ensuite capturées spécifiquement sur une surface de microréseau avec des billes liées de manière non spécifique retirées par des forces fluides laminaires, commandées. L'invention porte également sur un procédé de chimie de surface pour fixer de la neutravidine à une surface de nitrure. Une surface ayant des amines primaires est amenée à réagir avec du succinimidyl-4-formylbenzoate (SFB). La neutravidine est amenée à réagir avec de l'hydrazone d'acétone de succinimidyl-4-hydrazinonicotinate (SANH). La surface ayant réagi avec SFB est incubée avec la solution NA-SANH dans des conditions acides pour configurer ladite surface avec SFB-SANH stable. La surface stable est stockée à 4°C et reste stable pendant au moins 4 semaines.
PCT/US2008/087259 2008-08-19 2008-12-17 Détection de protéine attomolaire dans des matrices de type damle complexes avec des dosages de discrimination de force fluide semi-homogène Ceased WO2010021639A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9006708P 2008-08-19 2008-08-19
US61/090,067 2008-08-19

Publications (1)

Publication Number Publication Date
WO2010021639A1 true WO2010021639A1 (fr) 2010-02-25

Family

ID=41707375

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/087259 Ceased WO2010021639A1 (fr) 2008-08-19 2008-12-17 Détection de protéine attomolaire dans des matrices de type damle complexes avec des dosages de discrimination de force fluide semi-homogène

Country Status (1)

Country Link
WO (1) WO2010021639A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120147A1 (fr) 2014-02-06 2015-08-13 Scanogen Inc. Unités de détection et procédés de détection d'un analyte cible
US12474330B2 (en) 2018-05-25 2025-11-18 Scanogen Inc. Detection units and methods for detecting a target analyte

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040253744A1 (en) * 2003-06-10 2004-12-16 Rife Jack C. Fluidic force discrimination

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040253744A1 (en) * 2003-06-10 2004-12-16 Rife Jack C. Fluidic force discrimination

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MULVANEY ET AL.: "Attomolarprotein detection in complex sample matrices with semi- homogeneous fluidic force discrimination assays.", BIOSENS BIOELECTRON, vol. 24, no. 5, 1 January 2009 (2009-01-01), pages 1109 - 1115 *
PIERCE.: "Reacti-Bind NeutrAvidin Coated 96-Well Plates.", March 2006 (2006-03-01), Retrieved from the Internet <URL:http://www.piercenet.com/files/0611as4.pdf> [retrieved on 20090731] *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120147A1 (fr) 2014-02-06 2015-08-13 Scanogen Inc. Unités de détection et procédés de détection d'un analyte cible
EP3058372A4 (fr) * 2014-02-06 2017-06-07 Scanogen Inc. Unités de détection et procédés de détection d'un analyte cible
US10179930B2 (en) 2014-02-06 2019-01-15 Scanogen Inc. Detection units and methods for detecting a target analyte
US11505818B2 (en) 2014-02-06 2022-11-22 Scanogen Inc. Detection units and methods for detecting a target analyte
US12291741B2 (en) 2014-02-06 2025-05-06 Scanogen Inc. Detection units and methods for detecting a target analyte
US12474330B2 (en) 2018-05-25 2025-11-18 Scanogen Inc. Detection units and methods for detecting a target analyte

Similar Documents

Publication Publication Date Title
Mulvaney et al. Attomolar protein detection in complex sample matrices with semi-homogeneous fluidic force discrimination assays
Huang et al. Lateral flow biosensors based on the use of micro-and nanomaterials: a review on recent developments
Mulvaney et al. Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics
Zhao et al. Chemiluminescence immunoassay
Akama et al. Droplet-free digital enzyme-linked immunosorbent assay based on a tyramide signal amplification system
Tekin et al. Ultrasensitive protein detection: a case for microfluidic magnetic bead-based assays
CA2565166C (fr) Detection d&#39;analytes cibles fondee sur des codes a barres biologiques
US20020119470A1 (en) Magnetic bead-based array for genetic detection
US20060040286A1 (en) Bio-barcode based detection of target analytes
US20080085508A1 (en) Non-nucleic acid based biobarcode assay for detection of biological materials
JP2005524849A (ja) ラマン分光分析のフィンガープリントを備えた分析物質検出用のナノ粒子プローブ
WO2007075891A2 (fr) Analyses multiplex effectuees au moyen de particules magnetiques et de particules non magnetiques
CN101523156A (zh) 用于分析物检测的比色生物条形码扩增测定
WO2011087916A2 (fr) Dosages médiés par la force
KR20230169418A (ko) 개선된 분석 방법
KR20090060143A (ko) 자성 나노 입자와 주파수 혼합 자기 판독기를 이용한 생체물질의 정량적 검출방법
US8492084B2 (en) Method and apparatus for assaying test substance in sample
US20090005255A1 (en) Method for detecting target biological material using dna barcodes
CN113791207A (zh) 高灵敏度的免疫检测方法及其应用
EP4165389B1 (fr) Procédés et appareil de détection de molécules
US20120058548A1 (en) Detection of biotargets using bioreceptor functionalized nanoparticles
RU2006140262A (ru) Способы и устройство для выявления вирусных инфекций
WO2010021639A1 (fr) Détection de protéine attomolaire dans des matrices de type damle complexes avec des dosages de discrimination de force fluide semi-homogène
KR100952891B1 (ko) 광분해성 올리고머를 이용한, 복수의 생체분자에 대한신호의 동시 검출 방법
WO2008140620A2 (fr) Détection de biomolécules ultrasensibles à l&#39;aide de nanoparticules d&#39;or cochargées par de l&#39;adn double brin et de molécules de capture co-immobilisées

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08876803

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08876803

Country of ref document: EP

Kind code of ref document: A1