WO2002060575A2 - Biocapteur a puits permettant de detecter des evenements moleculaires ou cellulaires - Google Patents

Biocapteur a puits permettant de detecter des evenements moleculaires ou cellulaires Download PDF

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Publication number
WO2002060575A2
WO2002060575A2 PCT/US2002/003181 US0203181W WO02060575A2 WO 2002060575 A2 WO2002060575 A2 WO 2002060575A2 US 0203181 W US0203181 W US 0203181W WO 02060575 A2 WO02060575 A2 WO 02060575A2
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WIPO (PCT)
Prior art keywords
well
signal
signal transmission
transmission structure
interior surface
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PCT/US2002/003181
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English (en)
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WO2002060575A3 (fr
Inventor
Mark A. Rhodes
Barrett J. Bartell
Kurt D. Kramer
Hong Peng
Henry H. Liu
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Signature Bioscience, Inc.
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Priority to AU2002250009A priority Critical patent/AU2002250009A1/en
Publication of WO2002060575A2 publication Critical patent/WO2002060575A2/fr
Publication of WO2002060575A3 publication Critical patent/WO2002060575A3/fr

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    • 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
    • G01N33/5438—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to bioelectrical sensors, more particularly to a well-based bioelectric sensor for detecting molecular events.
  • Bioelectric sensors are well known in the biotechnical arts and use electronic signals to detect and identify the structure and interactions of molecules, as well as cellular function and activity.
  • Bioelectric sensors (“biosensors”) are typically constructed in a parallel-plate capacitor device in which layers of probes are immobilized on of the opposing surfaces between the capacitor plates. The probes and sought targets have a high binding affinity and when bound, operate to change the capacitance of the biosensor. The change in capacitance can be measured by passing an electronic signal (typically a time-varying ac signal) between the two capacitor plates before and after binding and comparing the respective responses. Examples of these types of biosensors are disclosed in U.S. Pat. No. 5,653,939 to Hollis et al., as well as U.S. Patent No. 5,187,096 to Giaever et al which discloses a similar structure for cell-based measurements.
  • the capacitor-type biosensor suffers from some disadvantages, one being the relatively low frequency range over which the biosensor can be used.
  • the measured cells or molecules situated between the plates will reside in an aqueous environment which significantly attenuates signals propagating between the two plates.
  • the highest frequency signal measurements are typically in the high KHz region to low MHz.
  • an improved biosensor is sought.
  • the biosensor includes a well structure and a signal transmission structure.
  • the well structure has an interior surface which defines an interior cavity.
  • the signal transmission structure is formed on at least a portion of the interior surface of the well structure and is operable to support the propagation of an electromagnetic signal above 10 MHz.
  • the signal transmission structure includes a signal line formed on at least a portion of the interior surface of the well structure and configured to conduct a time-varying voltage.
  • the signal transmission structure also includes one or more ground elements formed on the at least a portion of the interior surface of the well structure and configured to maintain a time-invariant voltage, the one or more ground elements spaced apart from and extending generally parallel to the signal line.
  • FIG. 1 illustrates a first embodiment well-based coplanar biosensor in accordance with the present invention.
  • Fig. 2 A illustrates a second embodiment of the well-based biosensor in accordance with the present invention.
  • Fig. 2B illustrates a micro-miniature version of a signal transmission structure in accordance with one embodiment of the present invention.
  • Fig. 3 illustrates a first embodiment of an array of well-based biosensors in accordance with the present invention.
  • Fig. 4 A illustrates a second embodiment of an array of well-based biosensors 400 in accordance with the present invention.
  • Fig. 4B illustrates a top view of the one of the substrate layers illustrated in Fig. 4A.
  • Fig. 4C illustrates a top view of a third embodiment of an array of well- based biosensors in accordance with the present invention.
  • Fig. 5 illustrates a switch connection schematic for a well-based biosensor array in accordance with one embodiment of the present invention.
  • Fig. 6 illustrates a method for manufacturing a well-based biosensor in accordance with one embodiment of the present invention.
  • molecular event refers to the interaction of a molecule of interest with another molecule (e.g., molecular binding) and to all structural properties of molecules of interest.
  • Structural molecular properties include the presence of specific molecular substructures (such as alpha helix regions, beta sheets, immunoglobulin domains, and other types of molecular substructures), as well as how the molecule changes its overall physical structure via interaction with other molecules (such as by bending or folding motions), including the molecule's interaction with its own solvation shell while in solution.
  • the simple presence of a molecule of interest in the region where detection/analysis is taking place is not considered to be a "molecular event,” but is referred to as a "presence.”
  • binding events are (1) simple, non-covalent binding, such as occurs between a ligand and its antiligand, and (2) temporary covalent bond formation, such as often occurs when an enzyme is reacting with its substrate. More specific examples of binding events of interest include, but are not limited to, ligand/receptor, antigen/antibody, enzyme/substrate, DNA/DNA, DNA/RNA, RNA/RNA, nucleic acid mismatches, complementary nucleic acids and nucleic acid/proteins. Binding events can occur as primary, secondary, or higher order binding events.
  • a primary binding event is defined as a first molecule binding (specifically or non-specifically) to an entity of any type, whether an independent molecule or a material that is part of a first surface, typically a surface within the detection region, to form a first molecular interaction complex.
  • a secondary binding event is defined as a second molecule binding (specifically or non- specifically) to the first molecular interaction complex.
  • a tertiary binding event is defined as a third molecule binding (specifically or non-specifically) to the second molecular interaction complex, and so on for higher order binding events.
  • Examples of relevant molecular structures are the presence of a physical substructure (e.g., presence of an alpha helix, a beta sheet, a catalytic active site, a binding region, or a seven-trans-membrane protein structure in a molecule) or a structure relating to some functional capability (e.g., ability to function as an antibody, to transport a particular ligand, to function as an ion channel (or component thereof), or to function as a signal transducer).
  • Molecular structure is typically detected by comparing the signal obtained from a molecule of unknown structure and/or function to the signal obtained from a molecule of known structure and/or function.
  • Molecular binding events are typically detected by comparing the signal obtained from a sample containing one of the potential binding partners (or the signals from two individual samples, each containing one of the potential binding partners) to the signal obtained from a sample containing both potential binding partners.
  • cellular event refers in a similar manner to reactions and structural rearrangements occurring as a result of the activity of a living cell (which includes cell death). Examples of cellular events include opening and closing of ion channels, leakage of cell contents, passage of material across a membrane (whether by passive or active transport), activation and inactivation of cellular processes, as well as all other functions of living cells. Cellular events are commonly detected by comparing modulated signals obtained from two cells (or collection of cells) that differ in some fashion, for example by being in different environments (e.g., the effect of heat or an added cell stimulant) or that have different genetic structures (e.g., a normal versus a mutated or genetically modified cell). Morpholic changes are also cellular events. Other examples of cellular events are illustrated in applicant's concurrently filed application entitled "Methods for Analyzing Cellular Events," (Atty. Docket No. 23 US) herein incorporated by reference in its entirety for all purposes.
  • a molecular event e.g., binding of a potential drug with a receptor
  • a biological sample capable of undergoing biological functions e.g., a cell or a cell-free enzyme system
  • the molecular event can be amplified by the biological function and, if desired to increase sensitivity, the change resulting from the function can be detected rather than the molecular event itself.
  • detectable amplified signals include the permittivity change of a cell resulting from the opening or closing of an ion channel when a molecular binding event occurs and a physiological reaction (e.g., synthesis of a protein) of a cell when a drug interacts with a cellular receptor.
  • binding event detection can be referred to as detection of a "cellular molecular event” (as opposed to a “non-cellular molecular event,” which is one that occurs in the absence of cells). Similar language can be used to describe cell-free enzyme-system molecular events.
  • test sample refers to the material being investigated (the biological sample, defined below) and the medium/buffer in which the analyte is found.
  • the medium or buffer can included solid, liquid or gaseous phase materials; the principal component of most physiological media/buffers is water.
  • Solid phase media can be comprised of naturally occurring or synthetic molecules including carbohydrates, proteins, oligonucleotides, Si0 2 , GaAs, Au, or alternatively, any organic polymeric material, such as Nylon ® , Rayon ® , Dacryon ® , polypropylene, Teflon ® , neoprene, delrin or the like.
  • Liquid phase media include those containing an aqueous, organic or other primary components, gels, gases, and emulsions.
  • Exemplary media include celluloses, dextran derivatives, aqueous solution of d-PBS, Tris, deionized water, blood, cerebrospinal fluid, urine, saliva, water, and organic solvents.
  • a biological sample is a sample of biological tissue or fluid that, in a healthy and/or pathological state, is to be assayed for the structure(s) or event(s) of interest.
  • biological samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, pleural fluid, and cells from any of these sources.
  • Biological samples also include cells grown in cultures, both mammalian and others.
  • Biological samples further include sections of tissues such as frozen sections taken for histological purposes. Although a biological sample is often taken from a human patient, the meaning is not so limited.
  • the same assays can be used to detect a molecular event of interest in samples from any mammal, such as dogs, cats, sheep, cattle, and pigs, as well as samples from other animal species (e.g., birds, such as chickens or turkey) and plants (e.g., ornamental plants and plants used as foods, such as corn or wheat).
  • the biological sample can be pretreated as necessary by dilution in an appropriate transporting medium solution or concentrated, if desired, and is still referred to as a "biological sample.” Any of a number of standard aqueous transporting medium solutions, employing one of a variety of transporting media, such as phosphate, Tris, or the like, preferably at physiological pH can be used.
  • pretreatment of a more general sample by dilution, extraction, etc.) once it is obtained from a source material do not prevent the material from being referred to as a sample.
  • the term "signal transmission structure” refers to a two conductor transmission structure operable to support the propagation of an electromagnetic signal.
  • the two conductor transmission structure includes a signal line and one ⁇ r more ground elements, the signal line and ground elements attached together by a dielectric substrate.
  • the signal line is configured to conduct a time-varying voltage
  • the ground elements are configured to maintain a time-invariant voltage only which is typically ground (zero volts) potential.
  • Exemplary embodiments of the signal transmission structure include coplanar waveguide signal lines, slot lines, and the like.
  • the term “electromagnetically coupled” refers to the transfer of electromagnetic energy between two objects, e.g., the signal transmission structure and molecular events occurring within the test sample.
  • the two objects can be electromagnetically coupled when the objects are in direct or indirect physical contact, (e.g., molecular events attached along the surface of the planar signal line or on a physical intervening layer or structure), or when the objects are physically separated from each other (e.g., molecular events suspended within solution flowing through a flow tube, the flow tube positioned within the detection region).
  • the term “electromagnetically couples” will indicate the interaction of an electromagnetic signal (e.g., the incident test signal) with an object (e.g., molecular events occurring within the test sample).
  • the term "well structure” refers to a vessel which has an open, covered, or pierceable top and which is operable to retain a test sample solution therein.
  • the well structure has integrally formed side and bottom surfaces.
  • the side and bottom interior surfaces are constructed from separated pieces.
  • the material composition, shape, and size of the well structure will vary depending upon a number of factors, such as the aforementioned bottom and side wall construction.
  • the well structure's interior surface, defined by the side wall and bottom interior surfaces (and a top surface if the well opening is covered) may be diverse, the bottom interior surface in some embodiments consisting of a flat, concave, conical, or other surface geometry.
  • Fig. 1 illustrates a well-based biosensor 100 in accordance with one embodiment of the present invention.
  • the biosensor 100 is one of a multitude of biosensors formed in a multi-well tray structure 120, for instance, a 96, 384 or 1536-well tray as know in the art.
  • the tray structure 120 is constructed from materials such as polycarbonate, polyethylene, polypropylene, polystyrene, or such similar materials, although in other embodiments materials such as polytetrafluoroethylene, glass, quartz, fused silica, polyimide, silicon dioxide, gallium arsenide, or other dielectric materials typically used in the construction of high frequency circuits may be used in alternative embodiments.
  • the biosensor 100 may be formed in a conventionally-sized test tube or any other type of sample container and may be constructed from the aforementioned or similar materials.
  • a cover (not shown) is used to enclose the well structure, thereby insulating it from environmental effects and evaporation. Suitable cover materials include silicon, polyurethane, latex, polycarbonate, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene.
  • a gas permeable material such as polydimethylsiloxane (PDMS) may be used.
  • the biosensor 100 includes a signal transmission structure 150, which, in one embodiment comprises a coplanar waveguide signal line having a center signal line 152 and two laterally located and parallel extending ground elements 154.
  • the center signal line 152 and ground elements 154 are metallized strips composed of 1.5 um gold deposited on top of 100 A titanium adhesion layer.
  • the width of the center signal line 152 and lateral separation from the ground elements 154 is usually determined by the desired characteristic impedance of the structure 150 and the dielectric constant of the material 120 onto which the transmission structure 150 is formed.
  • the desired characteristic impedance of the transmission structure 150 is 50 ohms and the well structure 120 is composed of borosilicate glass ( ⁇ r ⁇ 4.6), thereby setting the width of the center signal line 152 to 0310 mm and the lateral separation between the center signal line 152 and the ground elements 154 to 0.038mm.
  • borosilicate glass ⁇ r ⁇ 4.6
  • the signal transmission structure 150 consists of a signal line and single ground element, forming a conventional slot line transmission structure known in the art of high frequency circuit design. The separation distance between the transmission and ground element in this embodiment can be determined based upon the desired characteristic impedance and dielectric constant of the interconnect substrate, as known in the art.
  • Other signal transmission structures may also be used in embodiments under the present invention.
  • the eenter signal line and ground elements 152 and 154 extend to well rim 162, down the well side wall 164, and along the bottom interior surface 166, extending about halfway into the cavity 1 8.
  • This transmission structure 150 forms a one-port transmission structure in which an input test signal is supplied to, and the modulated signal is recovered from the same signal port 156. Modifications of this embodiment include terminating the signal line and ground elements 152 and 154 along the side wall 162, or extending them across the entire bottom interior surface 166, and terminating partially up the opposing face of the side wall 164. In another embodiment, the signal transmission structure 150 forms a two-port measurement structure in which the center signal line and ground elements 152 and 154 extend across the entire bottom surface 166, up the opposing face of the side wall 164, existing the opposing side of the rim 162, to form an output port (not shown) from which the modulated signal is recovered.
  • portions of the signal transmission structure 150 may be passivated or otherwise rendered inactive to external effects over a particular length.
  • the side wall portion(s) of the signal transmission structure 150 may be coated with glass, a polymer, or similar material to insulate it from the contained test sample, and to otherwise maintain the desired characteristic impedance of the signal transmission structure 160 while in the presence of the test sample.
  • the passivation layer may include an outer metallic layer to further electrically insulate this portion of the signal transmission structure from the contained test sample.
  • the well-based biosensor of the present invention is operable to detect molecular events (as defined above) in solution, suspension, and on the surface of the signal transmission structure 150.
  • Solution and suspension-phase detection is accomplished by propagating an incident test signal (at one or more frequencies) along the signal transmission structure which is exposed to the contained test sample.
  • a portion of the incident test signal electromagnetically couples to the molecular event occurring in solution or suspension, the event modulating one or more characteristics (the amplitude, phase, frequency, group delay, etc.) of the incident test signal.
  • the resulting modulated signal can be recovered either through a reflection (known as "Su” or "S 2 ") measurement or an insertion loss (“S 2 ⁇ " or "Si 2 ”) measurement, known in the art of high frequency circuit analysis.
  • the imparted modulation is indicative of the presence and identity of the molecular event occurring within the test sample.
  • the distance over which the test signal can travel between the signal transmission structure 150 and the event will typically range from 10 "10 m to 10 "2 m (or anywhere therebetween), depending upon such factors as the composition of the test sample, event concentration within the test sample, amplitude and frequency of the test signal, length of the exposed signal transmission structure 160, sensitivity of the signal detector, and the desired detection time.
  • the molecular event is physically attached (directly, or indirectly through intervening layers or linkers) to the signal transmission structure.
  • the incident test signal travels along and electromagnetically couples from the signal transmission structure to the directly or indirectly attached molecular event.
  • the event modulates a property (as described above) of the incident test signal, the modulation being recoverable by either a reflected or insertion loss measurement.
  • the modulation can be used as an identifier for each molecular event, as each imparts a unique modulation to the test signal at one or more frequencies.
  • Fig. 2 A illustrates a second embodiment of the well-based biosensor 200 in accordance with the present invention.
  • the biosensor 200 includes an upper substrate 220 and a lower substrate 250.
  • the upper substrate 220 includes a cavity 222 extending through the depth of the upper substrate 220.
  • the lower substrate 250 includes a top surface 252 on to which a center signal line 255a and two lateral ground elements 255b are formed.
  • an annular ring is used instead of the upper substrate 220.
  • the coplanar and slot structures do not typically employ a non-planar ground plane, one may be additionally employed in alternative embodiments, for instance ground plane metallization may be deposited on the bottom surface of the lower substrate 250.
  • the signal transmission structure 255 may comprise a slot line structure in an alternative embodiment.
  • the upper substrate 220 is positioned on top of the lower substrate 250 and the two are aligned so that at least a portion of the signal transmission structure (the center signal line 255a and the ground elements 255b) extends into the cavity 222.
  • the top and lower substrates 220 and 250 are attached, thermally bonded by raising the glass near its softening point in one embodiment, to retain a predefined volume of a test sample within the cavity 222.
  • the upper substrate 220 and the lower substrate 250 are composed of borosilicate glass are attached by thermal bonding.
  • the signal transmission structure 255 is formed by metal deposition using titanium or chrome adhesion layer (lOOA - 200 A) followed by a 1-2 um gold film and patterning using standard UN photolithography.
  • Fig. 2B illustrates a micro-miniature version ⁇ f the signal transmission structure 255 in accordance with one embodiment of the present invention.
  • the microminiature version of the signal transmission structure 255 permits the construction of higher density biosensor arrays, as further illustrated below.
  • the micro-miniature signal transmission structure 255 includes the center signal line 255a and spaced apart ground elements 255b which taper from wider sections to narrow sections within the cavity 222 of the upper substrate.
  • the upper substrate and cavity 222 are similarly miniaturized and attached over the top of the signal transmission structure 255.
  • the center signal line 255a tapers to a width of 0.1-0.5 um. Separation between the center signal line 255a and the ground elements 255b tapers from 20-50 um to 0.3 um within the cavity 222.
  • the center signal line 255a and ground elements 255b are composed of O.lum - 1.5 um gold deposited on a sapphire dielectric layer 260 using semiconductor processing techniques or similar precision photolithography.
  • the upper substrate consists of polydimethylsiloxane (PDMS) having a well diameter on the order of 1 mm.
  • Fig. 3 illustrates a first embodiment of an array of well-based biosensors 300 in accordance with the present invention.
  • the illustrated array 300 includes four substrate layers 310 ⁇ -310 .
  • the upper substrate 310 ⁇ functions as a upper substrate (described above) having an array of well structures 320, extending through the depth of the substrate 310 ⁇ .
  • the lower substrate 310 4 operates as described above having a signal transmission structure formed on the top surface.
  • Each of the intermediate substrates 310 2 and 310 3 include one or more cavities extending therethrough, and a signal transmission structure formed on its top surface.
  • the well structures 320 are staggered and are of different depths.
  • the shallow wells (320 1 and 320 6 ) are located at the outermost ends, and bottom out at the top surface of substrate 310 2 .
  • the deepest wells (320 3 and 320 ) are located in the center, and bottom out at the top surface of the lower substrate 310 4 .
  • the intermediate depth wells (320 2 and 320 5 ) are located between the shallow and deepest wells, and bottom out at the top surface of subsfrate 310 3 . This well pattern extends in into the plane of the figure, forming the entire array.
  • the array consists of a 6x16 structure, forming a 96-well array, the wells 320, having a diameter of 8 mm and a depth of 50 mm.
  • the size, depth, or number of wells vary is also possible in the present invention. For instance, arrays emulating conventional 384 or 1536 well trays are just a few of the possible embodiments under the present invention.
  • each of the signal transmission structures 330j is formed along the bottom interior surface of each of the well structures 320j.
  • the signal transmission structures 330 extend midway into the well structures 320j, although the structures 330, may extend a shorter or longer distance in alternative embodiments.
  • the substrates 31 ⁇ ! -310 4 are made successively wider (left to right) in order to provide connectivity to the signal transmission structures 330; extending from the array 300.
  • Materials such as glass, quartz, fused silica, polyimide, fired ceramic paste, silicon dioxide, gallium arsenide, or other dielectric materials typically used in the construction of high frequency circuits may be used in alternative embodiments.
  • Adjacently stacked substrates 310j are attached, preferably water-tight at least around the bottom interior surface to contain aqueous-based test samples.
  • FIG. 4 A illustrates a second embodiment of an array of well-based biosensors 400 in accordance with the present invention.
  • This array 400 is similar in construction to the first embodiment 300, having four substrates 410 ⁇ - 410 4 comprising a 6x16, 96-well structure.
  • signal transmission structures 432, 434, 436, and 438 are additional patterns which extend at least partially into well structures 420 2 , 420 3 , 420 4 , and 420s, respectively.
  • Fig. 4B illustrates a top, left half view of the second substrate 410 2 illustrated in Fig. 4A.
  • the top left half surface includes a signal transmission structure 330j, the left end of which extends to the left edge of the array 400 and the right end of which extends (at least partially) into the into well 420 1 .
  • the top surface also includes a first set of three plated-through vias 440 1 which extend vertically between, and electrically connect respective lines of signal transmission structures 330 2 and 432. Plated-through vias are well known in the art of miniature circuit design and may be formed using conventional photolithographic techniques or semiconductor processing.
  • the signal fransmission structure 432 extends at least partially into the well 420 2 , functioning as the well's biosensor.
  • miniature wires are fed through the vias 440] to provide electrically connectivity between respective lines of signal transmission structures 330 2 and 432.
  • a second set of vias 440 2 (or alternatively conductive wires) extend between, and electrically connect respective conductors of signal transmission structures 330 3 and 434 in the aforementioned manner.
  • the right side of signal transmission structure 434 extends (at least partially) into well 420 3 , functioning as that well's biosensor.
  • the top right surface of the second substrate 410 2 is a mirror image of Fig. 4B.
  • Fig. 4C illustrates a top view of a third embodiment of an array of well- based biosensors in accordance with the present invention.
  • the array 450 includes one lxN switch 451 and one Nxl switch 452, N being the number of signal transmission structures 453 connected between switches 451 and 452.
  • the signal transmission structures 453 (coplanar waveguide lines, slot lines, and the like) are formed on the top surface of substrate 456, suitable materials being glass, alumina, polyimide, or other similar materials listed herein or conventionally known.
  • An annular ring 454 is positioned over each of the signal transmission structures 453 and attached to the substrate 456, thereby forming a well structure.
  • Feed lines 457 supply signals between switches 451 and 452 and connectors 458, examples of which include SMA, BNC, N, 3.5 mm, 2.4 mm, or other connectors suitable for the test frequency.
  • the switch state of switches 451 and 452 are controlled via control lines 459.
  • Fig. 5 illustrates one embodiment of a switch schematic 500 for a 96-well array in accordance with the present invention.
  • the switch schematic 500 includes a signal source/detector assembly 510, and a switch bank 520 including one 1x2 switch 522, two 1x3 switches 524, and six 1x16 switches 526 connected as shown.
  • Each of the switches includes a control line 555 for controlling which one of the plurality of output (left) ports is connected to the input (right) port.
  • the control lines 555 are connected to a microprocessor 550 which, responsive to an input digital word, activates the control lines 550 to connect the signal source/detector 410 to a specific well structure.
  • the switches 522, 524, and 526 are preferably bi-directional to propagate signals in both the forward direction (during test signal launch) and the reverse direction (during recovery of the modulated signal in a one-port measurement configuration).
  • Signal amplifiers may be included between one or more of the switches to provide gain (in one or both directions) for compensating for signal loss.
  • the signal source/detector assembly 510 may consist of a vector or scalar network analyzer, a signal generator in combination with a vector voltmeter or similar measurement instrument, or a time domain reflectometers test set.
  • Switches 522, 524 and 526 preferably are low NSWR (voltage standing wave ratio), low insertion loss devices, and may be either discrete components or monolithically formed with the substrates and transmission structures, for instance, as a part of a semiconductor fabrication process.
  • Control lines 555 may consist of external wires or monolithically formed signal lines integrated into the array.
  • Microprocessor 550 is typically located externally to the array, but in some embodiments may be integrally formed as a part of the array.
  • Fig. 5 illustrates a one-port test setup in which an incident test signal is supplied to, and a reflected modulated signal recovered from the well-based biosensors.
  • a two-port measurement system in which the signal transmission structure extends through the well structure to a signal detector may be employed.
  • a portion of the incident test signal continues propagating in the forward direction and is recoverable by the signal detector.
  • the test signal insertion loss (difference in amplitude between the incident and recovered signals) may be measured and used to detect and identify molecular events occurring within the biosensor well.
  • Fig. 6 illustrates a method for manufacturing a well-based biosensor in accordance with one embodiment of the present invention.
  • a upper substrate having a cavity extending therethrough is provided.
  • the upper substrate can be provided without the cavity, and the cavity formed subsequently using standard photolithographic or semiconductor processing techniques, by laser, chemical etch, ultrasonic, waterjet, or other conventionally known techniques.
  • a lower substrate is provided.
  • the upper and lower substrates are composed of glass, although other materials such as polytefrafluoroethylene, glass, quartz, fused silica, polyimide, silicon dioxide, gallium arsenide, or other dielectric materials typically used in the construction of high frequency circuits may be used in alternative embodiments.
  • a coplanar waveguide fransmission line in one embodiment, is deposited onto the top surface of the lower substrate.
  • the coplanar waveguide line consists of a center signal line of width 0.310 mm separated by 0.038 mm from two adjacent ground elements, all of which are composed of 2 um gold sputtered over 100 A titanium.
  • the upper substrate is positioned ⁇ n top of the lower substrate, such the top surface of the lower substrate forms the bottom interior surface of the cavity.
  • the upper and lower substrates are aligned (using, for instance, an optical microscope) such that the signal transmission structure deposited on the top surface of the lower substrate extends at least partially into the cavity along the bottom interior surface of the well structure. In a specific embodiment, the signal transmission structure extends approximately halfway into the cavity.
  • the upper substrate is attached to the top surface of the lower subsfrate.
  • the attachment preferably provides a water-tight seal around the bottom of the cavity.
  • the attachment process is a fusion process in which the substrates are subjected to a ramped temperature profile in a quartz tube furnace. The highest temperature depends on the choice of material, but is near the softening point of the glass.
  • Alternate bonding methods include medical grade UV-cured acrylate compounds and amorphous fluoropolymer. which can be applied via spin coating and patterned using standard lithographic techniques and plasma etching. The last two techniques may be performed at ⁇ 120° C and allow attachment of the upper and lower substrates made from dissimilar materials.

Abstract

L'invention concerne un biocapteur à puits permettant de détecter des événements moléculaires ou cellulaires qui comprend une structure de puits et une structure de transmission de signal. Ladite structure de puits comprend une surface intérieure définissant une cavité intérieure. Ladite structure de transmission de signal est formée sur au moins une partie de la surface intérieure de la structure de puits, et fonctionne de façon à supporter la propagation d'un signal électromagnétique supérieur à 10MHz. Selon un mode de réalisation spécifique, la structure de transmission de signal comprend un circuit de transmission formé sur au moins une partie de la surface intérieure de la structure de puits, et est configurée de façon à acheminer une tension à variation temporelle. La structure de transmission de signal comprend également au moins un élément à la terre formé sur au moins une partie de la surface intérieure de la structure de puits, et est configurée de façon à maintenir une tension temporellement invariable, l'élément à la terre étant espacé du circuit de transmission et s'étendant généralement parallèlement à ce circuit de transmission.
PCT/US2002/003181 2001-02-01 2002-01-30 Biocapteur a puits permettant de detecter des evenements moleculaires ou cellulaires WO2002060575A2 (fr)

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US77571801A 2001-02-01 2001-02-01
US09/775,718 2001-02-01
US92952001A 2001-08-13 2001-08-13
US09/929,520 2001-08-13

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WO2003016894A2 (fr) * 2001-08-13 2003-02-27 Signature Bioscience, Inc. Biocapteur de guide d'ondes coplanaire conçu pour detecter des evenements moleculaires ou cellulaires
WO2003101618A1 (fr) * 2002-05-31 2003-12-11 Cancer Research Technology Ltd Substrat destine a supporter un ensemble d'echantillons experimentaux
WO2007106402A2 (fr) * 2006-03-10 2007-09-20 President And Fellows Of Harvard College Procedes et appareil pour irradiation en champ proche
WO2009138939A1 (fr) * 2008-05-13 2009-11-19 Nxp B.V. Réseau de capteurs et son procédé de fabrication
JP2015224920A (ja) * 2014-05-27 2015-12-14 株式会社エンプラス 流体取扱装置

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016894A2 (fr) * 2001-08-13 2003-02-27 Signature Bioscience, Inc. Biocapteur de guide d'ondes coplanaire conçu pour detecter des evenements moleculaires ou cellulaires
WO2003016894A3 (fr) * 2001-08-13 2004-04-08 Signature Bioscience Inc Biocapteur de guide d'ondes coplanaire conçu pour detecter des evenements moleculaires ou cellulaires
WO2003101618A1 (fr) * 2002-05-31 2003-12-11 Cancer Research Technology Ltd Substrat destine a supporter un ensemble d'echantillons experimentaux
WO2007106402A2 (fr) * 2006-03-10 2007-09-20 President And Fellows Of Harvard College Procedes et appareil pour irradiation en champ proche
WO2007106402A3 (fr) * 2006-03-10 2008-02-21 Harvard College Procedes et appareil pour irradiation en champ proche
WO2009138939A1 (fr) * 2008-05-13 2009-11-19 Nxp B.V. Réseau de capteurs et son procédé de fabrication
CN102026724B (zh) * 2008-05-13 2014-03-12 Nxp股份有限公司 传感器阵列和制造传感器阵列的方法
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JP2015224920A (ja) * 2014-05-27 2015-12-14 株式会社エンプラス 流体取扱装置

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