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WO2007008483A2 - Capteur resonant mecanique hautement sensible a auto-confirmation - Google Patents

Capteur resonant mecanique hautement sensible a auto-confirmation Download PDF

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Publication number
WO2007008483A2
WO2007008483A2 PCT/US2006/025966 US2006025966W WO2007008483A2 WO 2007008483 A2 WO2007008483 A2 WO 2007008483A2 US 2006025966 W US2006025966 W US 2006025966W WO 2007008483 A2 WO2007008483 A2 WO 2007008483A2
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WIPO (PCT)
Prior art keywords
cantilever beam
cantilever
analyte
binding partner
immobilized
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/US2006/025966
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English (en)
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WO2007008483A3 (fr
Inventor
Richard A. Montagna
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Innovative Biotechnologies International Inc
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Innovative Biotechnologies International Inc
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Publication of WO2007008483A2 publication Critical patent/WO2007008483A2/fr
Anticipated expiration legal-status Critical
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • G01N21/1717—Systems in which incident light is modified in accordance with the properties of the material investigated with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance
    • 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
    • G01N21/55—Specular reflectivity
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0427—Flexural waves, plate waves, e.g. Lamb waves, tuning fork, cantilever

Definitions

  • the present invention relates generally to the field of chemical and biological sensors and, in particular, to a high sensitivity, mechanical resonant sensor for detecting and confirming the presence of chemical or biological material.
  • a difficulty common to many biosensors is the inability to distinguish a true positive reaction from a false positive reaction. It is well known in the art that, regardless of the sensitivity and specificity of a particular test, the Positive Predictive Value (PPV) of a test is a function of the prevalence of the analyte in the population of samples being analyzed.
  • PSV Positive Predictive Value
  • a test with a sensitivity of 99.9% (i.e., 99.9 percent of all true positive samples will be correctly scored as positive by the test) and a specificity of 99.9% (i.e., 99.9 percent of all true negative samples will be correctly scored as negative) will have a PPV ranging from as low as less than 10 percent in a low prevalence population to higher than 90% in a high prevalence population.
  • less than ten percent of the "positive” determinations will, in fact, be “true” positives while the remaining samples will, in fact, be “false” positives.
  • Regulatory agencies throughout the world, including the U.S. Food & Drug Administration recognize this difficulty and require that initially "positive” test results be confirmed by a more specific test method to assure that the initial positive test result is, indeed, a "true” positive and not a “false” positive.
  • Absorption is recognized as largely a result of Lennard- Jones potential, wherein at close distances, nearby molecules repel and at larger distances, the molecules are attracted to each other, hi many cases, absorption of molecules onto a surface can be readily reversed by merely heating the system or exposing the system to a vacuum.
  • the present invention relates to a system for detecting and confirming the presence of an analyte in a sample.
  • the system can include a light source, a resonating structure (such as a cantilever beam), and a photodiode.
  • the beam has a rigid end and a free end that resonates under ambient conditions.
  • the resonant frequency of the beam is a function of, inter alia, the mass on the beam.
  • the photodiode generates an output signal based on light reflected from the apex of the beam.
  • the photodiode output is a function of the frequency of vibration of the beam.
  • At least one surface of the beam includes an immobilized binding partner.
  • the binding partner is selected to bind with a particular analyte, or analytes, which in turn, increases the mass of the beam.
  • binding of the second analytical reagent will further increase the mass on the cantilever beam surface and thus cause an additional measurable decrease in resonant frequency.
  • binding partners on the second analytical reagent that differ from those used on the cantilever beam, it is unlikely that potential "false positive" binding events on the surface of the cantilever beam caused by the first binding partner will be duplicated by binding events facilitates by the second binding partner on the second analytical reagent.
  • the processor can be coupled to the photodiode and the processor can execute programming to determine the mass of analyte bound to the binding partner.
  • the processor can include a microprocessor or a spectrum analyzer coupled to the output signal.
  • the binding partner can be immobilized on a particular region of the beam, such as, for example near one end of the beam.
  • the beam can be fabricated of silicon nitride. A portion of the beam can be rigidly coupled to a support. The length of the beam may be in the range of 0.5 to 1000 ⁇ m.
  • the beam can be adjusted to vibrate in an out of plane mode.
  • the beam can operate in an atmosphere of thermal noise or it can operate in an atmosphere of air vibrations.
  • the beam can be tailored to detect an analyte including a pathogen, a microorganism, a bacteria, a virus or a subunit thereof.
  • the binding partner can include an antibody that binds to a particular analyte such as a cell, a cell fragment or subunit.
  • the binding partner can include a cellular receptor that binds to a ligand.
  • the analyte can be a ligand specific for a cellular receptor.
  • the analyte can be a metallic ion, or organic molecule, and the binding partner can be a chelator that binds to the metallic ion, or organic molecule.
  • the binding partner may include a deoxyribonucleic acid ("DNA”) sequence and the analyte include a complementary DNA sequence that hybridizes thereto under the operational conditions of the sensor.
  • DNA deoxyribonucleic acid
  • a method for detecting a pathogen is also described.
  • the method can include providing a cantilever beam, optically determining a first resonant frequency for the beam, exposing the cantilever beam to a mixture suspected of containing the pathogen, optically determining a second resonant frequency for the beam, and determining a mass difference for the beam.
  • the beam includes an immobilized binding partner for the pathogen on a surface thereof.
  • the first resonant frequency is determined at a time when the beam is excited by ambient conditions and the second resonant frequency is determined at a time after the beam is exposed to the mixture.
  • the mass difference is determined based on a difference between the first resonant frequency and the second resonant frequency.
  • the first resonant frequency can be determined by illuminating the beam using a laser light source and sensing light reflected by the beam using a photodiode.
  • the ambient conditions can include thermal mechanical noise and ambient air vibrations.
  • the cantilever beam can also be driven by a piezo electric oscillator.
  • the binding partner can be immobilized by immersing the beam in a first liquid mixture of the binding partner for a pathogen, removing unbound pathogen or other components of the mixture, e.g., by rinsing with water and drying the beam in an inert atmosphere.
  • the beam can be exposed by immersing the beam in a solution suspected of containing the pathogen, incubating the beam for a predetermined period of time, rinsing and drying the beam in an inert atmosphere.
  • the beam can be exposed to a buffered aqueous solution.
  • the pathogen can include a microorganism, microorganism fragment or subunit thereof.
  • a second beam can also be prepared by immersing in a second solution suspected of containing a second pathogen.
  • the beam can operate in a vacuum environment.
  • the mechanical properties of the beam can be tailored to detect a mass difference in the range of attograms (10 "18 g) to micrograms (10 "6 g).
  • the mechanical properties of the beam can be tailored to achieve a desired resonant frequency.
  • An array of analyte detectors is also disclosed.
  • An array can include a plurality of cantilever beams, a plurality of immobilized binding partners and a sensor responsive to light reflected by a particular beam. Each beam resonates at a particular frequency under ambient conditions. Each beam has an immobilized binding partner on a surface. Each binding partner binds to a predetermined analyte. The sensor generates an output signal based on a resonant frequency of a particular beam.
  • At least some beams can be made of silicon nitride.
  • a light source can be used to illuminate a beam.
  • At least some beams in the array can have different, or heterogeneous, binding partners.
  • At least some beams in the array can have the same, or homogeneous, binding partners.
  • a detector for an analyte is disclosed.
  • the detector includes binding means, cantilever means, sensor means and processor means.
  • the binding means are for binding with the analyte.
  • the cantilever means are for resonating under ambient conditions.
  • the binding means are immobilized on a portion of the cantilever means.
  • the cantilever means resonates in a first mode at a first resonant frequency.
  • the sensor means are for determining the first resonant frequency.
  • the processor means are for determining a mass of the analyte based on a difference between the first resonant frequency and a second resonant frequency after exposure of the binding means to the
  • the cantilever means can vibrate in an out of plane mode.
  • the cantilever means can include silicon nitride.
  • the sensor can include a photodiode.
  • the processor means can include a frequency analyzer.
  • the binding means can be immobilized at a location near an unsupported end of the cantilever means.
  • the cantilever means can be aligned substantially horizontally.
  • the cantilever means can be encapsulated in a vacuum.
  • the system can include a cantilever beam driver in communication with the cantilever beam.
  • the driver can vibrate the beam at a predetermined frequency and a sensor can monitor the amplitude of vibrations of the cantilever beam. The amplitude of vibrations will vary based on the mass of the analyte on the cantilever beam.
  • the driver can vibrate the cantilever beam over a range of frequencies and a peak vibration amplitude of the beam corresponds to the resonant frequency of the beam.
  • the system includes a second analytical reagent, to which are bound second binding partners that differ in structure and function from the first binding partners employed on the cantilever beam.
  • Figure 1 illustrates one embodiment of a system according to the present subject matter.
  • Figure 2 illustrates representative beam geometry
  • Figures 3A, 3B, 3C and 3D graphically illustrate a relationship between the number of cells and a frequency differential.
  • Figures 4A and 4B illustrate cantilever beams with bound cells on a surface.
  • Figures 5A and 5B illustrate a cantilever beam having a binding partner on a surface.
  • Figures 6A and 6B illustrate scanning electron micrographs showing a random distribution of bound cells immobilized on the surface of four different cantilever beams.
  • Figure 7 graphically illustrates resonant frequency as a function of detector output.
  • Figure 8 illustrates an array of cantilever beams.
  • Figure 9 illustrates a flow chart of a method pursuant to the present subject matter.
  • Figure 10 illustrates schematically a configuration for measuring the frequency response of a beam.
  • Figures 1 IA, 1 IB and 11C illustrate a surface micromachming fabrication process.
  • Figure 12 illustrates a test setup for use with a beam fabricated using surface micromachining.
  • Figure 13 illustrates frequency response of a beam operating in a vacuum.
  • Figure 14 illustrates a scanning electron micrograph of a single cell bound to an immobilized antibody layer on the surface of a beam.
  • Figure 15 illustrates the corresponding frequency response using the beam of Figure 14.
  • Figures 16A, 16B,16C and 16D illustrate alternative configurations for a resonant sensor pursuant to the present system.
  • Figures 17A-C illustrate the sequential binding of test analyte to the cantilever beam and the secondary analytical reagent to the immobilized analyte.
  • FIG. 1 illustrates one embodiment of a system according to the present subject matter.
  • system 100 is suitable for the detection of E. coli or other bacterial cells.
  • Cantilever beam 10 is affixed on one end to support 20. The other end of beam 10 is free to move in the directions indicated by arrow 15.
  • Beam 10 vibrates at a resonant frequency when driven by ambient environmental conditions or when driven externally, by, for example, a piezoelectric device.
  • laser 30 projects light 40, optionally through lens 35, onto the free end of beam 10. Lens 35 focuses the light onto the apex of beam 10.
  • Light 40 is reflected by beam 10.
  • a mirror 45 can redirect light 40 to illuminate sensor 50.
  • Sensor 50 generates an output signal 55 based on the vibrations of cantilever beam 10.
  • Spectrum analyzer 60 processes output signal 55 and yields useful information.
  • beam 10 is fabricated of silicon nitride.
  • beam 10 is fabricated of low stress silicon nitride.
  • Beam 10 may also be fabricated of other materials, including for example, silicon, silicon dioxide, silicon carbide, polysilicon, carbon, diamond like carbon (DLC) film, metal, gallium arsenide or other conductor or semiconductor material.
  • the material used for beam 10 is conducive to photolithography processes and etching to release beam 10 from the surrounding structure.
  • the material used for beam 10 is conducive to fabrication of structures having the scale and geometry as herein provided.
  • Beam 10 may be fabricated using either bulk or surface silicon micromachining technology.
  • beam 10 is substantially linear.
  • beam 10 may include a helical section or multiple anchor points with various modes of freedom to enable greater sensitivity.
  • Beam 10 can have different cross sectional shapes, including, for example, rectangular, square or round cross section.
  • beam 10 has a high aspect ratio, that is the length /, is longer than the width w, of beam 10.
  • a high aspect ratio beam is one having a ratio of length to width of approximately 3.75 or more.
  • typical dimensions for the length of beam 10 can be in the range of 0.5 to 1000 ⁇ m.
  • Typical dimensions for the width of beam 10 can be in the range of 0.1 to 50 ⁇ m.
  • a typical dimension for the thickness t, of beam 10 can be in the range of 0.05 to 4 ⁇ m. The aforementioned dimensions are not to be construed as limitations for the present system.
  • a coordinate system is also illustrated in Figure 2, with the z- axis aligned with t, the x-axis aligned with w, and the y-axis aligned with /.
  • beam 10 vibrates in the directions of arrow 15, or substantially along the z-axis. Arrow 15 extends normal to the plane of beam 10, and thus, the vibratory mode is said to be out of plane. Other modes of vibration may also be sensed. For example, vibrations in plane may be monitored with a suitable sensing apparatus. Vibrations in more than one plane can also be monitored.
  • Support 20 is coupled to one end of beam 10.
  • support 20 is illustrated as a rectangular housing.
  • Support 20 can be a heavier region of the substrate upon which cantilever beam 10 is fabricated, and is thus stable relative to the vibrations of cantilever beam 10.
  • Support 20 can be fabricated in conjunction with the fabrication of beam 10. Consequently, support 20 may also be fabricated of the same material used in the fabrication of beam 10.
  • support 20 may be fabricated in conjunction with other integrated electronic devices, components or circuitry. The other integrated electronic devices, components, or circuitry may be related or unrelated to the operation of detector system 100.
  • support 20 may be fabricated on the same substrate as digital logic gates, amplifiers, processors, memory cells, or other semiconductor devices.
  • Cantilever beam 10 vibrates at a first frequency determined by the geometry, the mass, the distribution of mass, and external forces acting on beam 10.
  • a change in the mass of beam 10 is detectable as a change in the resonant frequency of beam 10.
  • Figures 3A and 3B graphically illustrate this phenomena for a particular beam sensitized for detecting E. coli cells. The number of E. coli cells is shown on the abscissa and the differential frequency, measured in Hertz, is on the ordinate. The number of cells is proportional to the mass change of beam 10.
  • Figure 3 A corresponds to a cantilever beam 10 having dimensions of 100 micrometers (" ⁇ m”) in length, 20 ⁇ m in width and 320 nanometers (“nm”) in thickness and shows number of cells in the range of 0 to 900.
  • Figure 3B corresponds to a cantilever beam 10 having dimensions of 200 ⁇ m in length, 10 ⁇ m in width and 600 nm in thickness and shows number of cells in the range of 0 to 160.
  • the graphs show the substantially linear relationship between mass and frequency differential. Deviations from linearity are explained by such factors as nonuniform loading of beam 10 as well as nonuniform flexural rigidity of beam 10 resulting from variations in the distribution of the mass of beam 10.
  • FIGS 3 A and 3B The particular beam depicted in Figures 3 A and 3B was effective for detecting the presence of 16 E. coli cells.
  • Figures 3C and 3D illustrate measured frequency shift dependence relative to the number of bound E. coli cells for particular cantilever beams.
  • the figures show a linear regression fit to the data.
  • Beam 10, and the support structure may be fabricated using any of a number of semiconductor fabrication techniques.
  • An exemplary bulk micromachining fabrication process is as follows:
  • low stress silicon nitride is applied to a substrate by low pressure chemical vapor deposition ("LPCVD") to a thickness of 320 nm.
  • LPCVD low pressure chemical vapor deposition
  • silicon nitride can be applied by plasma enhanced low pressure chemical vapor deposition ("PECVD") to a depth of 600 nm.
  • PECVD plasma enhanced low pressure chemical vapor deposition
  • Other thicknesses, as well as other deposition technologies, are also contemplated.
  • the substrate can be a silicon wafer.
  • gallium such as gallium antimonide and gallium arsenide
  • indium such as indium antimonide, indium arsenide and indium phosphide
  • polycrystalline materials such as polycrystalline gallium arsenide and polycrystalline indium phosphide
  • the cantilever beam 10 and support 20 are defined by photolithography on a front side of the substrate wafer.
  • the exposed silicon nitride is etched in a reactive ion etch ("RIE") chamber using carbon tetrafluoride (“CF 4 ").
  • RIE reactive ion etch
  • CF 4 carbon tetrafluoride
  • a layer of oxide maybe deposited using PECVD to a thickness of 2 ⁇ m.
  • the wafer is etched using potassium hydroxide ("KOH").
  • KOH potassium hydroxide
  • the 2 ⁇ m layer of PECVD oxide is removed by buffered (6:1) oxide etch solution. The etch is continued until the cantilever is released from the surrounding structure.
  • the release of cantilever beam 10 is aided by using a high pressure carbon dioxide (CO 2 ) critical point dryer.
  • CO 2 carbon dioxide
  • the critical point dryer reduces, or prevents, morphological damage to the structure resulting from dehydration in the atmosphere due to surface tension at the liquid interface. If left unchecked, the surface tension of the solution may aggravate the situation and result in breaking of the cantilever structure.
  • FIGs 4A and 4B illustrate one embodiment of cantilever beam 1OA having a binding partner 115 on a surface.
  • support 2OA is represented as a base structure and is rigidly attached to further structure not depicted.
  • Beam 1OA has a first end 70 rigidly attached to support 2OA and a second end 80 that is cantilevered. In one embodiment, second end 80 is free to vibrate in an out of plane mode.
  • Binding partner 115 is immobilized on beam 1OA.
  • Binding partner 115 is conformally distributed, or coated, on all surfaces of the structure illustrated in Figure 4A. Binding partner 115 can be localized to a particular portion of beam 1OA, such as, for example, a region near second end 80.
  • Binding partner 115 can be distributed on an upper surface of beam 1OA. Binding partner 115 can be distributed on the exterior surfaces of beam 1OA. Binding partner 115 can be impregnated within the interior structure of beam 1OA. Binding partner 115 can be a surface coating on beam 1OA and thus, selectively bind to predetermined molecules.
  • binding partner 115 includes molecules 90 that bind to complementary molecules on target cells in a "lock and key" fashion.
  • binding partner 115 includes a plurality of antibody molecules, herein represented as a plurality of "Y" shaped characters 90.
  • Figure 4B illustrates beam 1OA having binding partner 115 at a time when complementary molecules 110 have bound with the antibody molecules 90 of binding partner 115.
  • Binding partner 115 can bind to one or more target substances in a reversible or essentially irreversible fashion.
  • essentially irreversible bonds may include those arising by van der Waal forces, ionic bonds, or by formation of covalent bonds.
  • binding does not occur by simple physical absorption of the target by beam 10 or binding partner 115 thereon.
  • Binding partner 115 is selected to bind to a desired target substance, or substances, wherein said bound target substance, or substances, is then detected by system 100.
  • one protein such as an antibody
  • a binding partner 115 on beam 10 for purposes of detecting a second protein (such as an antigen).
  • other pairs include using a receptor for detecting a ligand such as using a cellular receptor to detect a ligand that binds to such receptor, using a protein for detecting a peptide, using a protein for detecting a DNA 5 using a first DNA sequence to detect a second DNA sequence, using a metallic ion to detect a chelator, and using an antibody, or an antibody fragment, for detecting an antigen or analyte.
  • a receptor for detecting a ligand such as using a cellular receptor to detect a ligand that binds to such receptor
  • a protein for detecting a peptide using a protein for detecting a DNA 5 using a first DNA sequence to detect a second DNA sequence, using a metallic ion to detect a chelator, and using an antibody, or an antibody fragment, for detecting an antigen or analyte.
  • the aforementioned examples bind to each other in a "lock and key" fashion by ionic bonding, co
  • a peptide may be the binding partner on beam 10 for use in detecting a protein.
  • the binding partner 115 immobilized on cantilever beam 10 can be DNA and thus, the present system is responsive to the substantial DNA complement.
  • the bound, or "hybridized” DNA sequences can then be treated or "washed” under various conditions of stringency so that only DNA sequences that are highly complementary (e.g., that has high sequence identity) will be retained on beam 10.
  • the binding partner 115 can also bind to a plurality of substances, in which case, system 100 will indicate detection of any substance binding to cantilever beam 10. In addition, more than one binding partner 115 may be immobilized on a particular cantilever beam 10 to enable detection of multiple molecules. Multiple binding partners 115 may be immobilized in the same or different regions of cantilever beam 10. [0053]
  • the binding partner 115 can include an antibody for detection of an antigen, or binding partner 115 includes an antigen for detection of an antibody. Examples of antigens include proteins, oligopeptides, polypeptides, viruses, and bacteria.
  • antigens include OMP 3 , OMP b and OMP 0 , commonly referred to as outer membrane protein “a” “b” and “c", respectively.
  • the interaction includes one or more amino acid interactions wherein the amino acids are spatially arranged to form two complementary surfaces in three dimensions. Each surface includes one or more amino acid side chains or backbones.
  • the binding partner 115 can include an antibody for detection of a hapten, or binding partner 115 includes a hapten for detection of an antibody.
  • Haptens tend to be much smaller than antigens and include such compounds as transition metal chelators, multi-ring phenols, lipids, and phospholipids.
  • the interaction includes an intermolecular reaction of a surface of the hapten with one or more amino acids of the antibody, wherein the amino acids of the antibody are spatially arranged to form a complementary surface to that of the hapten.
  • binding partner 115 interacts with the targeted substance in a manner beyond that of simple absorption of analyte into a matrix of some type.
  • the interaction of binding partner 115 with the target substance is characterized by rapid bonding, preferably bonding that is not reversible under ambient conditions, thus reducing the time required for reliable detection using system 100.
  • Hybrid antibodies are also contemplated for either the target substance or binding partner 115.
  • a portion of a first antibody may be cleaved and a second antibody may be bonded to the remaining portion of the first antibody, thus forming a hybridized antibody.
  • Such an antibody may subsequently bind with two forms of antigens or haptens.
  • a third antibody may be bonded to the remaining portion of the first antibody, thus enabling subsequent bonding to additional antigens or haptens.
  • the use of hybridized antibodies in system 100 yields a detector sensitive to multiple substances and may be desirable for certain applications where detection of two or more analytes is desired.
  • Binding partner 115 is affixed, or immobilized, to the surface of beam 10 using any of a number of techniques, including absorption, covalent bonding with or without linker or spacer molecules or complexation.
  • a cantilever beam 10 is prepared for detection of E. coli cells using the following method:
  • E. coli cells were affinity purified (by means of affinity chromatography) and isolated from a serum pool from goats immunized with whole cells of E. coli serotype 0157. ⁇ 7 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD).
  • a phosphate buffer saline (0.1M NaH 2 PO 4 , 0.2M Na 2 HPO 4 ), (hereinafter "PBS"), was prepared from 1.38 g NaH 2 PO 4 H 2 O and 2.84 g Na 2 HPO 4 , diluted to 100ml with deionized water.
  • PBS phosphate buffer saline
  • coli antibody concentration of lmg/ml was prepared by adding 1 ml of 0.3M Sodium Phosphate Buffer (pH 7.4) to 1 mg vial of antibodies. The vial was rotated until total dissolution was achieved and the solution was then incubated at 37 0 C for 30 minutes.
  • the anti-E. coli antibodies maybe immobilized on the surface of beam 10 to an average thickness of 40 nm. Thicknesses greater than or less than 40 nm are also contemplated.
  • E. coli O157:H7 cells were cultured in Luria broth (a nutrient broth used to support the growth of E. coli) and enumerated (colony forming units ("CFU") per mL) on Luria agar.
  • CFU colony forming units
  • Cells were heat-inactivated by immersing 1 mL aliquots of cell culture (in 1.5 mL ⁇ ppendorf tubes) in boiling water for approximately 90 seconds. Inactivation of the cells was confirmed by spread plating 100 ⁇ L of the heat-treated cell culture onto Luria agar and plates were read after an incubation period of 24 hours at 37 0 C.
  • Heat-treated cells were then pelleted by centrifugation (2040 x g for approximately 10 minutes), and re-suspended in PBS. Serial 10-fold dilutions of the re-suspended cells were performed in PBS from 10 9 to 10 6 CFU/mL.
  • FIG. 5A schematically illustrates the resulting beam 10.
  • element 91 represents the upper surface of beam 10 and element 92 represents antibodies bound to beam 10.
  • the cantilevers were immersed into a solution of PBS with suspended E. coli cells ranging in concentration from 10 6 -10 9 E. coli cells/ml for approximately 15 minutes. The cantilevers were subsequently rinsed in. a 0.05% solution of Tween7 (ICI Americas, Inc.), rinsed in water, and then blown dry with nitrogen.
  • Figure 5B represents the resulting beam 10.
  • Figure 5B represents a scanning electron micrograph showing a random distribution of bound E. coli cells immobilized on the surfaces of several cantilevers.
  • elements 93 and 94 represent bound E. coli cells.
  • Figures 6A and 6B illustrate scanning electron micrographs showing a random distribution of bound cells immobilized on the surface of four different cantilever beams.
  • S ⁇ M scanning electron microscope
  • the samples were prepared by evaporating a thin (under 10 run) layer of Au/Pd.
  • Figures 6A and 6B were prepared in a similar manner, using 10 E. coli cells per ml, and show a random distribution of the cells.
  • binding partner 115 can be covalently bonded to a surface of beam 10. Binding partner 115 can also be non-covalently bonded to a surface of beam 10. Binding partner 115 can be bonded by absorption to a surface of beam 10. In particular, amino chemistry, carboxyl chemistry, and carbohydrate chemistry techniques may be used to bond binding partner 115 to a surface of beam 10.
  • Exposure of cantilever beam 10 to the test solution may be achieved by any suitable means.
  • one representative method includes immersing cantilever beam 10 in a buffered aqueous medium containing different concentrations of E. coli cells ranging from 10 6 to 10 9 E. coli cells/ml.
  • the beam may be incubated in the medium at room temperature for a period of time and then rinsed, to remove unbound components of the medium, and dried in a nitrogen atmosphere. The incubation period can be approximately fifteen minutes.
  • Rinsing may be in a solution of Tween7 (ICI Americas, Inc.) to remove any loosely bound cells. Rinsing may also be in deionized water.
  • the E. coli cells are not dissolved in the water.
  • cantilever beam 10 resonates under ambient conditions.
  • the geometry and dimensions of beam 10 enable measurable differences in resonant frequency without the use of external oscillatory driving forces.
  • Ambient conditions include any thermal mechanical noise.
  • Ambient conditions include airborne vibrations.
  • System 100 can operate in a vacuum, thus reducing the influence of airborne vibrations.
  • cantilever beam 10 can resonate under external forces. External driving of cantilever beam 10 yields a greater amplitude of oscillation. In one embodiment, the driving frequency is swept through a range of frequencies and oscillation amplitude of cantilever beam 10 is measured.
  • An example of external force includes the forces exerted by a piezoelectric driver. Magnetic drivers are also contemplated wherein a changing magnetic field drives beam 10. Other field forces may also be used to drive beam 10.
  • the support structure for cantilever beam 10 can be externally driven at a particular frequency.
  • analyzer 60 derives information from sensor 50.
  • Sensor 50 can include a split photodiode or two separate photodetectors and analyzer 60 can comprise a spectrum analyzer.
  • a common spectrum analyzer presents signal amplitude information as a function of frequency.
  • the light reflected from cantilever beam 10 falls first, on one portion of photodiode 50, and second, on another portion of photodiode 50.
  • This alternating illumination of photodiode 50 generates an alternating output signal.
  • Analyzer 60 receives the alternating output signal and displays information corresponding to the resonant frequency of cantilever beam 10.
  • Figure 7 is a compilation of five representative displays appearing on the screen of analyzer 60.
  • the abscissa marks the resonant frequency, in kilohertz, ("kHz") and the ordinate marks the output of sensor 50, herein labeled as Optical Detector Output and calibrated in arbitrary units.
  • the output of sensor 50 is a varying voltage, and thus, the ordinate corresponds to output voltage.
  • the output of sensor 50 can be a varying resistance, in which case, the ordinate may correspond to resistance.
  • the lower pair of curves, marked 200 and 205 in Figure 7, corresponds to a resonating beam 10 having detected 44 cells of E. coli bacteria in a test solution.
  • Curve 200 illustrates detector output as a function of frequency for beam 10 at a time after affixation of immobilized binding partner 115 as herein described and prior to exposure to the test solution.
  • Curve 205 illustrates the same beam 10 after exposure to the test solution. Resonance occurs at the frequency corresponding to the peak output. The difference between the resonant frequency of curve 200 and that of curve 205 is a function of the mass difference. La the case of curves 200 and 205, that difference in mass represents the binding of 44 cells of E. coli bacteria to beam 10.
  • Curves 210 and 215 illustrate a typical resonant frequency differential using a similar cantilever beam 10 according to the present subject matter when detecting the presence of 82 cells of E. coli bacteria in a test solution.
  • Curves 220 and 225, curves 230 and 235, and curves 240 and 245 illustrate typical results with test media having 325, 453 and 800 cells of E. coli bacteria, respectively.
  • Figure 8 illustrates one embodiment of the present subject matter having an array of four cantilever beams, marked herein as 1OB, 1OC, 10D, and 10 ⁇ .
  • each of the four beams 1OB, 1OC, 10D, and 10 ⁇ are arranged in a linear manner and coupled rigidly to common support 20B.
  • Each beam is shown having an immobilized binding partner 115B, 115C, 115D, and 115E on a surface of a beam. More or less cantilever beams assembled on a common support are also contemplated.
  • each of the plurality of cantilever beams is arranged along one edge of a linear support. Other configurations are also contemplated.
  • each of the plurality of beams can be arranged on two or more edges of a geometrically shaped support or each of the plurality of beams can be arranged in a circular or oval configuration.
  • One dimensional and two dimensional configurations for the arrangement of cantilever beams are contemplated.
  • each of the binding partners 115B, 115C, 115D, and 115E may be distinct from each other.
  • the dashed lines on the upper surface of the cantilever beams are aligned on different axis and may be interpreted as denoting different binding partners, hi this manner, the cantilever beam system of Figure 8 may be used to determine if a test sample includes any element that bind with binding partners 115B, 115C, 115D, or 115E.
  • Any suitable technique may be used to immobilize a particular binding partner 115 to a particular cantilever beam 10 within an array of cantilever beams.
  • a photoactivation technique is used to immobilize a particular binding partner 115 to a particular cantilever beam 10.
  • light activation of a particular cantilever beam 10 can activate a photosensitive chemical coating and enable subsequent bonding of the particular binding partner 115 to the activated cantilever beam 10.
  • a laser light source can be used to activate a particular cantilever beam 10.
  • Light of a particular wavelength can also be used to activate, and affix, a binding partner 115 to a particular cantilever beam 10.
  • Cantilever beam 10 may be a single beam or it may be a single beam in an array of other cantilever beams 10.
  • a desired binding partner 115 is applied to a particular cantilever beam 10 in an array using a manifold.
  • the manifold can include a series of capillaries having a first end aligned to transfer a fluid to each of a plurality of cantilever beams and a second end that enables introduction of the fluid on a macro level, hi similar fashion, a tubule may be used to immobilize a particular binding partner on a single cantilever beam 10 or to a single cantilever beam 10 in an array of other cantilever beams 10.
  • at least two cantilever beams can be prepared with the same immobilized binding partner 115.
  • the system offers broad area coverage for detecting a particular cell.
  • using a common binding partner 115 on multiple cantilever beams 10 provides redundancy.
  • each beam 10 in the plurality of beams in an array can have a different geometry.
  • a first beam 10 may have a high aspect ratio and a second beam 10 may have a low aspect ratio.
  • each beam 10 has a different resonant frequency.
  • the binding partner 115 coating on each beam 10 may be the same or it may be different.
  • Detecting the frequency response of cantilever beam 10 may be achieved by any suitable means.
  • An optical sensor can be used to detect the frequency response.
  • One embodiment of an optical sensor includes laser light reflected by a portion of the cantilever beam 10 wherein the reflected light is detected by a photodiode.
  • the laser light may emanate from a laser diode.
  • Preferably the light is substantially monochromatic and collimated.
  • a single laser light source illuminates multiple cantilever beams.
  • a single photodiode, or other sensor monitors the frequency response of multiple cantilever beams.
  • Each cantilever beam 10 in an array may be monitored individually by a single laser light and a single sensor.
  • Each cantilever beam 10 in an array can be aligned to reflect light to a particular one of a plurality of photodiodes.
  • the photodiode can include a plurality of photodiodes arranged in a manner to provide output signals that corresponds to the frequency response of a cantilever beam 10.
  • the frequency response of a particular cantilever beam 10 within a plurality of cantilever beams may be discerned using electronic means.
  • cantilever beam 10 Other means of deriving, or analyzing, the frequency response of a cantilever beam 10 are also contemplated.
  • movement of the cantilever beam 10 is detected based on a change in capacitance.
  • cantilever beam 10 serves as one electrode of a capacitor and a second electrode is held in a fixed position near the cantilever beam. Capacitance between the first and second electrode will vary as a function of the movement of cantilever beam 10.
  • movement of cantilever beam 10 may be used to change the thickness, or amount, of dielectric material between beam 10 and a stationary electrode. Changes in dielectric thickness, or amount, are measurable as a frequency response.
  • piezoelectric or piezoresistive methods are used to detect the movement of cantilever beam 10.
  • Piezoelectric detection involves generating an electric signal when the material is subjected to stress and piezoresistive detection involves sensing changes in resistance based on a stress in cantilever beam 10.
  • Magnetic detection involves conductor movement relative to a magnetic field. Current in the conductor may be sensed.
  • Cantilever beam 10 can serve as the moving conductor in a stationary magnetic field.
  • the output of the sensor can be digitized and communicated to a processor.
  • the processor executes programming to discern the differential frequency, and thus the mass difference.
  • Each of the aforementioned methods of detecting the frequency response may be used in an embodiment of the present system.
  • multiple optical sensors may be used for an array of a plurality of cantilever beams.
  • a single optical sensor may be used to monitor an array of a plurality of cantilever beams.
  • an array of cantilever beams is fabricated wherein some beams are tailored to detect a first type of cell and a second set of beams are tailored to detect a second type of cell.
  • the aspect ratio of a cantilever beam may be selected to respond with greater sensitivity to a cell having a particular mass.
  • Geometric dimensions, the method of fabrication, and the material selected for the cantilever beam are some of the parameters that may be tailored to achieve a desired sensitivity.
  • the environment in which beam 10 operates has an effect on the sensitivity of the present subject matter. In the viscous regime, for example, the atmospheric pressure operating on beam 10 will produce a dampening effect due to the viscosity of the air.
  • the quality factor Q of cantilever beam 10 is proportional to the inverse square root of the atmospheric pressure.
  • a beam operating in an environment of atmospheric pressure of 1 atm (approximately 760 mm Hg) and at room temperature (approximately 25 EC) may have a quality factor Q of between 5 and 8. With a Q in this range, a particular beam 10 can detect approximately 44 bound cells of bacteria, such as E. coli bacteria. Sensitivity increases with increased quality factor Q. Increased sensitivity of the present subject matter can enable detection of both single E. coli bacteria and single monoatomic layers.
  • the quality factor is inversely proportional to the pressure. Therefore, when operated in a vacuum of 1 mTorr at room temperature, the quality factor Q is on the order of 10 4 for one embodiment.
  • the present subject matter can detect a mass in the range of 14.8xlO "15 grams, and when operated in a standard atmosphere, can detect a mass 100 times larger.
  • the mass distribution on the length of beam 10 will affect sensitivity.
  • the resolution of the frequency spectra is related to the width of the peak, and thus, the quality factor Q. Resolution can be 0.1Hz when operating in a vacuum and 10 Hz in standard atmosphere.
  • the sensitivity of an embodiment of the present system is a function of the slope of the relationship as illustrated in Figures 3A, 3B 5 3C, and 3D.
  • the sensitivity is approximately 6.81 and 5.115 Hz/pg, respectively.
  • E. coli bacteria cell as well as a single monoatomic layer can be detected, as explained herein.
  • HMDS hexamethyldisiloxane
  • Figure 9 illustrates a flow chart of a method pursuant to the present system. Beginning at 305, it is assumed that cantilever beam 10 has been fabricated and suitable differential frequency response detection resources are aligned. At 310, a binding partner 115 is immobilized onto cantilever beam 10. The immobilized binding partner 115 couples to the complementary molecule and securely holds the complementary molecule with respect to cantilever beam 10. At 315, a first resonant frequency is determined for cantilever beam 10 along with binding partner 115. At 320, binding partner 115 on cantilever beam 10 is exposed to the test sample suspected of containing the complementary molecule.
  • this entails immersing the cantilever beam 10 in the test sample which can be a solution, dispersion, or suspension in an organic or inorganic liquid such as water.
  • a second resonant frequency is determined for cantilever beam 10, binding partner 115, and any complementary molecules that have been immobilized by binding partner 115.
  • the method continues by determining a mass difference based on the first and second resonant frequencies. Determining a mass difference may be accomplished using a look up table or by executing programming on a suitable processor. The method ends at 335.
  • beam 10 may be fabricated by methods other than bulk micromachining.
  • beam 10 may also be fabricated using surface micromachining techniques.
  • bulk micromachining entails removal of a substrate by etching whereas surface micromachining entails a sequence of depositions followed by selective removal of material.
  • the material removed is defined by lithographic techniques.
  • Bulk micromachining techniques are complicated by backside alignment concerns as well as thickness variations across the surface of the wafer.
  • Surface micromachining may ameliorate such issues and enable fabrication of more complex structures having smaller dimensions. Surface micromachining may also yield a more sensitive resonator.
  • the frequency response of a surface micromachined device may be measured using interferometric means.
  • interferometric measurement entails laser light directed at surface of beam 10. A first portion of the incident light is reflected by the surface of beam 10 and a second portion of the light passes through the beam and is reflected by the underlying substrate. Vibrations of beam 10 produce an interference pattern of varying light intensity in the total reflected beam. A single photodetector cell can transduce the variations in the intensity of the reflected light. The output signal from the photodetector can be displayed on a spectrum analyzer.
  • Figure 10 One configuration for test apparatus is illustrated in Figure 10.
  • Figure 10 also includes a magnified view of a portion of the test apparatus.
  • laser 30 projects incident light 40, through beam splitter 37, to beam 10.
  • Light reflecting from the top surface of beam 10 is transmitted to beam splitter 37 and further reflected onto photodetector 50a.
  • a portion of the incident light falling on the resonator passes through beam 10, passes through air gap 17 beneath beam 10, and is reflected by substrate 12 located under beam 10.
  • Light reflected by substrate 12 again passes through beam 10 and is reflected by beam splitter 37 and is incident on photodetector 50a.
  • the intensity of the reflected beam is modulated by the interference of the light ray, or beam, reflecting off the surface of vibrating beam 10 and the light beam reflected off the underlying substrate.
  • the interference patterns thus generated can be detected by photodetector 50a and an output signal from the photodetector is then applied to spectrum analyzer 60.
  • a profile view of the resonator is also illustrated in Figure 12.
  • substrate 12 is fabricated of silicon
  • beam 10 is fabricated of polysilicon and between substrate 12 and beam 10 is a layer of silicon dioxide.
  • beam 10 is semi-transparent to the incident light. In other words, a portion of incident light is reflected by beam 10 and a portion of incident light is transmitted through beam 10.
  • the thickness t of beam 10 is sufficiently small to render beam 10 at least partially transparent.
  • a vacuum chamber can enhance the mechanical quality factor, and thus enhance sensitivity.
  • chamber 14 provides an evacuated environment for beam 10.
  • the vacuum environment increases the mechanical quality factor Q, thus increasing beam 10 sensitivity and ability to detect smaller masses.
  • the configuration shown in Figure 10 may be used to generate frequency response data using a single photocell.
  • the bulk micromachined configurations normally employs either two photodetectors or a conventional split cell photodiode.
  • the voltage differential between the two sensing elements provides the signal for driving the spectrum analyzer.
  • the sensitivity of such a resonator is limited by the high speed capabilities of the instrumentation amplifiers coupled to the photodetectors, or split cell photodiode, and driving the spectrum analyzer. For example, the bandwidth of typical operational amplifiers, that is the 3 dB drop point, is in the range of several megahertz. This limitation of frequency response thus limits the sensitivity of the resonator.
  • the frequency response is not sensitive to a voltage differential, and thus, higher speed single-ended operational amplifiers can be utilized, m this case, the frequency responses may be in the gigahertz range. Thus, smaller, more sensitive, resonators may be possible. It has been demonstrated that using surface micromachined devices and single photodetectors, sensitivity can be in the range of attogram detection.
  • 1 IA, 1 IB, and 11C begins with silicon substrate 12.
  • an 800 nm layer of silicon dioxide, layer 22 is thermally grown on silicon substrate 12 in an atmosphere of pyrogenic steam.
  • Undoped poly-silicon, forming beam 10 is then deposited on top of the sacrificial oxide layer 22.
  • silicon nitride may also be used for forming beam 10.
  • the poly- silicon is then thermally annealed at 1050° C to alleviate stresses from the residual film.
  • a conductive 30 nm layer of chromium, layer 24 is thermally evaporated. This conductive layer reduces charging effects in the subsequent e-beam lithographic definition of the resonating beam.
  • PMMA polymethylmethacrylate
  • PMMA layer 26 is exposed using an electron beam at a dose current of
  • FIG. 1 IB illustrates a representative profile view.
  • FIG 11C illustrates the result of subsequent steps.
  • Polysilicon is selectively removed using reactive ion etching.
  • PMMA layer 26 is stripped using oxygen plasma and chrome (Cr) 24 is removed using a wet etch.
  • the structure is then immersed in a solution of hydrofluoric acid which etches the exposed silicon dioxide layer 22, thereby undercutting and freeing beam 10.
  • Figure 12 illustrates a test setup for use with beam 10 fabricated according to the above procedure.
  • Piezo driver 32 is used to determine the resonant frequency of the beam when operating in a vacuum bound by vacuum chamber 14.
  • Piezo drive 32 is helpful with small resonators since the amplitude of vibration due to ambient noise is very small (less than 0.01 nm).
  • the cantilever is then coated with a self-assembled monolayer 28 and the resonant frequency is measured. The frequency shift is then correlated to the added mass.
  • HMDS vapor deposited hexamethyldisilazane
  • This sample was prepared in a following manner, first after the undercutting of the oxide with hydrofluoric acid ("HF"), the surface was devoid of any adsorbed water vapors. Sample was then placed inside a vacuum chamber and the first resonance peak was obtained. Sample was dehydrated at 15O 0 C for 1 hour and immediately placed into the HMDS deposition oven. Sample was then placed into the vacuum chamber and the frequency shift reported in Figure 13 was observed.
  • HF hydrofluoric acid
  • Figure 14 illustrates a scanning electron micrograph of a single cell bound to the immobilized antibody / layer on the surface of beam 10.
  • a thin layer, under 10 nm, of Au/Pd was evaporated onto beam 10 to reduce charging effects during SEM imaging.
  • Figures 16 A, 16B, 16C, and 16D illustrate alternative suitable structures, hi Figure 16 A, the structure includes four linear members 500A, 500B, 500C, and 500D extending radially from center element 510.
  • center element 510 is approximately 1 ⁇ m in length and linear members 500A, 500B, 500C, and 500D are 200 nm thick and 150 nm in width.
  • Linear members 500A, 500B, 500C, and 500D are immobilized at one end by attachment to external structure. The inner ends of the four linear members support center element 510 which is free to resonate in an out of plane mode.
  • the resonant frequency of the structure can be determined by optical means operating on light reflected, or refracted, from center element 510.
  • Figure 16B illustrates another embodiment also having a resonating center element and four supporting linear members. Center element 530 in Figure 16B is approximately 4 ⁇ m in length.
  • linear elements 520 A, 520B, 520C, and 520D include orthogonal portions.
  • Figure 16C two arrays of linear members, or strings, are illustrated.
  • String 550 is a representative string and is immobilized by attachment to structure 540 and 545. In this figure, the width of the strings shown in the left array is 200 nm and those of the right array are 120 nm, and, for each, the thickness is 50 nm.
  • the length of the strings range between 7 ⁇ m and 16 ⁇ m in Figure 16C.
  • a drum-shaped, or disk-shaped, structure is illustrated.
  • the center of drum 565 is unsupported and the perimeter of drum 565 is supported by external structure 560.
  • Optical detection means can be used to detect resonance of the drum structure.
  • the center of the drum may be immobilized and the perimeter is free to resonate.
  • Other structures and other dimensions may be employed to yield a structure having a desired sensitivity to mass differentials at a particular resonant frequency. Structures other than cantilever beams can be used for detecting mass differentials as herein described.
  • FIG. 17 One method to self confirm the analytical results obtained by a resonating cantilever beam is shown in Figure 17.
  • the cantilever beam, with the first binding partner immobilized upon it will oscillate at a defined resonant frequency (Initial Native Frequency, Figure 17A).
  • the mass of the cantilever beam Upon binding of the target analyte via the first binding partner on the cantilever beam, the mass of the cantilever beam will be increased resulting in a decrease in resonant frequency of the cantilever beam ("First" Alarmed Frequency, Figure 17B).
  • the second analytical reagent is introduced.
  • Binding of the second analytical reagent with the target analyte via the second binding partner will result in a further increase in mass on the cantilever beam thereby further reducing the resonant frequency ("Confirmatory" Alarm Frequency, Figure 17C).
  • binding partners on the second analytical reagent that differ from those used on the cantilever beam, it is unlikely that potential "false positive" binding events on the surface of the cantilever beam caused by the first binding partner will be duplicated by binding events facilitates by the second binding partner on the second analytical reagent.

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Abstract

L'invention concerne un système et un procédé destinés à détecter une masse sur la base d'un différentiel de fréquence d'une structure micro-usinée résonante, telle qu'une console. Une console à rapport de forme élevé est revêtue d'un partenaire de liaison immobilisé se couplant à une cellule ou une molécule prédéterminée. Une première fréquence de résonance est déterminée pour la console comprenant le partenaire de liaison immobilisé. Lors d'une exposition de la console à une solution se liant avec le partenaire de liaison, la masse de la console augmente. Une deuxième fréquence de résonance est déterminée et la fréquence de résonance différentielle fournit une base pour la détection de la cellule ou molécule cible. Lors de la détection d'un événement de liaison provoquant une augmentation de masse et une réduction subséquente de la fréquence de résonance de la console, un second réactif analytique est introduit, ce réactif contenant un autre partenaire de liaison capable de reconnaître ledit analyte et de se lier à ce dernier. Lors d'une liaison du second réactif analytique, une troisième fréquence de résonance est déterminée et la fréquence de résonance différentielle fournit une confirmation de la présence de la cellule ou molécule cible. La console peut être soumise à une action extérieure ou à un bruit ambiant. La réponse de fréquence de la console peut être déterminée par voie optique au moyen d'une lumière réfléchie et de deux photodétecteurs ou par interférence au moyen d'un photodétecteur unique.
PCT/US2006/025966 2005-07-08 2006-06-30 Capteur resonant mecanique hautement sensible a auto-confirmation Ceased WO2007008483A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008106469A1 (fr) * 2007-02-26 2008-09-04 University Of Florida Research Foundation, Inc. Capteurs de force de cellule vivante et procédés d'utilisation de ceux-ci
US7691583B2 (en) 2000-07-12 2010-04-06 Cornell Research Foundation, Inc. High sensitivity mechanical resonant sensor
US8826724B2 (en) 2010-12-24 2014-09-09 Honeywell International Inc. Carbon dioxide sensor

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DE3342525A1 (de) * 1982-11-27 1984-05-30 Canon K.K., Tokio/Tokyo Geraet zur informations-aufzeichnung und/oder -wiedergabe
US6306598B1 (en) * 1992-11-13 2001-10-23 Regents Of The University Of California Nucleic acid-coupled colorimetric analyte detectors
WO2002014867A2 (fr) * 2000-08-11 2002-02-21 Agilix Corporation Systemes de detection ultrasensibles
CA2496777A1 (fr) * 2002-08-29 2004-05-06 Bioscale, Inc. Capteur resonnant et systeme de detection
US20050079548A1 (en) * 2003-07-07 2005-04-14 Plexxikon, Inc. Ligand development using PDE4B crystal structures
US7759134B2 (en) * 2003-09-10 2010-07-20 Auburn University Magnetostrictive ligand sensor

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7691583B2 (en) 2000-07-12 2010-04-06 Cornell Research Foundation, Inc. High sensitivity mechanical resonant sensor
US7939273B2 (en) 2000-07-12 2011-05-10 Cornell Research Foundation, Inc. High sensitivity mechanical resonant sensor
WO2008106469A1 (fr) * 2007-02-26 2008-09-04 University Of Florida Research Foundation, Inc. Capteurs de force de cellule vivante et procédés d'utilisation de ceux-ci
US8826724B2 (en) 2010-12-24 2014-09-09 Honeywell International Inc. Carbon dioxide sensor

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