WO2000068419A2 - Appareil et procede permettant de controler et de detecter les interactions entre une petite molecule et une biomolecule - Google Patents

Appareil et procede permettant de controler et de detecter les interactions entre une petite molecule et une biomolecule Download PDF

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Publication number
WO2000068419A2
WO2000068419A2 PCT/CA2000/000504 CA0000504W WO0068419A2 WO 2000068419 A2 WO2000068419 A2 WO 2000068419A2 CA 0000504 W CA0000504 W CA 0000504W WO 0068419 A2 WO0068419 A2 WO 0068419A2
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biomolecule
acoustic wave
small molecule
wave device
liquid
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PCT/CA2000/000504
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English (en)
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WO2000068419A3 (fr
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Mark Mcgovern
Michael Thompson
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Sensorchem International Corporation
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Priority to CA002368315A priority Critical patent/CA2368315A1/fr
Priority to AU43880/00A priority patent/AU4388000A/en
Priority to EP00924999A priority patent/EP1190093A2/fr
Publication of WO2000068419A2 publication Critical patent/WO2000068419A2/fr
Publication of WO2000068419A3 publication Critical patent/WO2000068419A3/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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
    • 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
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
    • 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
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • 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
    • G01N2291/0422—Shear waves, transverse waves, horizontally polarised waves
    • 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
    • G01N2291/0423—Surface waves, e.g. Rayleigh waves, Love waves
    • 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
    • G01N2291/0426—Bulk waves, e.g. quartz crystal microbalance, torsional waves
    • 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

  • This invention relates to methods of and apparatus for qualitative and quantitative analysis of biomolecule interactions using viscosity measurements.
  • a biosensor comprises a biochemical component attached to a form of electronic transducer.
  • the biochemical component usually comprising proteins or nucleic acids, is exposed to a particular chemical compound, and any resulting chemical interaction is electronically detected by the transducer.
  • Transducer technology for use in biosensors can be based on piezoelectric effects, which are changes in shape or conformation of certain solid crystals when an electric voltage is applied to them, or, conversely, the production of an electric voltage when such a solid crystal is mechanically deformed. If the crystal is used as one of the components of an "oscillating circuit", the crystal will determine the oscillation frequency of the whole circuit. The crystal itself vibrates at a "resonant frequency" which is determined by the physical shape and size of the crystal, among other factors. Quartz is the most commonly used piezoelectric crystal, although many others exist. Under suitable conditions, the circuit will oscillate very accurately at the same frequency, which is measured in Hertz (Hz).
  • Hz Hertz
  • Piezoelectric crystals can be used as the basis of biosensor transducer platform technologies. If any material is allowed to contact a clean piezoelectric crystal surface, as the device oscillates, it will change the resonant frequency of the device. The size of the observed frequency change can be used to measure the quantity of material which adhered to the crystal surface.
  • a biosensor transducer platform comprising a platform-like quartz crystal, a first electrode on its lower surface and a second electrode on its upper surface, with the immobilized biomolecule on the second, upper electrode, is used.
  • the resulting bonding or hybridization of the nucleic acid in the test solution (analyte) to the immobilized nucleic acid on the electrode causes a change in the vibrational frequency of the circuit, as compared with that of the circuit involving the immobilized nucleic acid itself.
  • the existence and magnitude of the change of frequency is a measure of the presence and quantity of the nucleic acid under test, and can be electronically translated into detection of the presence and measurements of the quantity of the nucleic acid under test, in the analyte solution.
  • United States patent No.5, 595, 908 to Fawcett teaches a method for detecting polynucleotide hybridization using a piezoelectric crystal.
  • a polynucleotide is immobilized on a surface of a piezoelectric crystal. After washing and drying the crystal, the resonance frequency of the piezoelectric crystal is measured through means for determining the resonant frequency of a piezoelectric crystal.
  • a separate source of polynucleotide is exposed to the polynucleotide-coated piezoelectric crystal for a sufficient length of time and under conditions suitable for hybridization. After washing and drying the crystal, the resonance frequency of the crystal is then again measured, and the difference between the resonance frequency before and after the incubation period indicates the extent of hybridization.
  • the washing and drying steps required by Fawcett's method render it expensive and time consuming.
  • Fawcett nor Gizeli et al. teach a method for monitoring the binding of small molecules to biomolecules.
  • the invention provides a method for monitoring small molecule-biomolecule interactions, comprising the steps of: (a) binding a biomolecule to a substrate;
  • the substrate is a part of an acoustic wave device, and the acoustic wave device is selected from the group consisting of a piezoelectric device, a magnetic acoustic resonator sensor, a surface acoustic wave device, a thin rod acoustic wave device, a shear horizontal acoustic wave device, and a plate mode with acoustic sensor.
  • the acoustic wave device is selected from the group consisting of a piezoelectric device, a magnetic acoustic resonator sensor, a surface acoustic wave device, a thin rod acoustic wave device, a shear horizontal acoustic wave device, and a plate mode with acoustic sensor.
  • the biomolecule is bound to the electrode, and the electrode is bound to the acoustic wave device.
  • the biomolecule may be bound to the electrode by physiosorption or by binding using neutravin-biotin, thiol-gold or TTU silane.
  • the biomolecule may be a polynucleotide, a polypeptide, and it may be any biological molecule or compound which undergoes a conformational change upon binding to a small molecule
  • the liquid is aqueous
  • the liquid may contain a physiological buffer
  • the method of the invention may further compnse the subsequent steps of (h) removing the liquid by introducing new liquid not containing the small molecule, (l) measuring frequency of oscillation of the acoustic wave device to obtain a third value, and
  • the method of the invention may also further compnse the subsequent steps of (k) introducing into the new liquid a second small molecule, (l) measu ⁇ ng frequency of oscillation of the acoustic wave device to obtain a fourth value,
  • the small molecule is less than 2800 daltons In another embodiment, the molecule is less than 1000 daltons In a further embodiment, the small molecule is less than 500 daltons
  • the small molecule may be a pharmaceutical agent
  • the method of the invention further comprises the step of contacting a side of the acoustic wave device with a gas. The gas may flow across a side of the substrate. The gas side may be maintained separate from the liquid side.
  • the invention also provides an apparatus (22) for monitoring small molecule- biomolecule interactions, comprising:
  • a wet surface (46) attached to the substrate for contact with a liquid and for binding with a biomolecule (iii) a dry surface (48) attached to the substrate; and (iv) a detection apparatus (42, 44) for determining the resonance frequency of the substrate.
  • the apparatus may further comprise a liquid flow chamber (28) for flowing liquid over the wet surface.
  • the apparatus further comprises a gas flow chamber (30) for flowing gas over the dry surface.
  • the substrate and the detection apparatus together are an acoustic wave device selected from the group consisting of a piezoelectric sensor, a magnetic acoustic resonator sensor, a surface acoustic wave device, a thin rod acoustic wave device, a shear horizontal acoustic wave device, and a plate mode with acoustic sensor.
  • acoustic wave device selected from the group consisting of a piezoelectric sensor, a magnetic acoustic resonator sensor, a surface acoustic wave device, a thin rod acoustic wave device, a shear horizontal acoustic wave device, and a plate mode with acoustic sensor.
  • the biomolecule is bound to an electrode (12), and the electrode is bound to the detection apparatus.
  • the biomolecule may be bound to the electrode by physiosorption or by binding using neutravin-biotin, thiol-gold or TTU silane.
  • the wet surface is located on a face of the substrate opposing the dry surface.
  • a process for detecting the interaction of small molecules with biomolecules which comprises contacting a liquid solution suspected of containing a small molecule of interest with the biomolecules in a biosensor, the biosensor comprising a piezoelectric material, an electrode electrically connected to the piezoelectric material, the biomolecules immobilized on the electrode, and an electrical circuit involving the electrode and the piezoelectric material and having characteristic, measurable electrical output signals, and monitoring change in at least one of the electrical output signals caused by interaction of the small molecule of interest with the immobilized biomolecules.
  • FIGURE 1 is a diagrammatic top view of a piezoelectric sensor platform for use m the invention
  • FIGURE 2 is a diagrammatic side view thereof
  • FIGURE 3 is a diagrammatic side view of the top electrode of the device with biomolecules immobilized thereon
  • FIGURE 4 is a similar vtew of the biosensor mounted in a flow cell
  • FIGURE 5 is a schematic diagram of a DNA sequence bound to a biosensor
  • FIGURE 6 is a graph showing frequency modulation in response to the addition of small molecules to the flow cell
  • FIGURE 7 is a graph showing frequency modulation m response to the addition of small molecules to the flow cell (DNA-antibiotic interaction)
  • FIGURE 8 is a graph showing frequency modulation in response to the addition of small molecules to the flow cell (RNA-antibiotic interaction)
  • FIGURE 9 is a graph showing frequency modulation in response to the addition of small molecules to the flow cell (RNA-peptide interaction)
  • the present invention provides a process whereby biosensors based upon viscosity effects and resonance measurements as descnbed herein may be used to detect, to quantify and to monitor the chemical and biochemical reactivity and properties of small molecules, l e those of less than about 2,800 Da moleculai weight
  • biosensors based upon viscosity effects and resonance measurements as descnbed herein may be used to detect, to quantify and to monitor the chemical and biochemical reactivity and properties of small molecules, l e those of less than about 2,800 Da moleculai weight
  • frequency changes caused by binding of small molecules to biomolecules such as nucleic acids and proteins immobilized on oscillating circuits much larger than was expected can be produced, and, in fact, in the opposite direction from that predicted.
  • the piezoelectric crystal-based device when operated with the piezoelectric crystal in contact with liquid is effectively much more sensitive than the Sauerbrey equation suggests.
  • AWDs acoustic wave devices
  • the liquid closest to the AWD surface can "slip" along the vibrating surface, so that it is the viscosity and "stiffness" of the liquid which is being probed by the ultrasonic or acoustic waves emitted by the vibrating AWD.
  • the transmitted acoustic waves may couple back to the AWD, and thereby detect the presence of, and hence changes in the characteristics of, nearby molecules.
  • the molecules in fact change the frequency of the acoustic waves. Binding of or interaction of small molecules with biomolecules immobilized on or near the AWD causes further changes, as compared with the immobilized biomolecules themselves.
  • acoustic wave biosensors or acoustic wave devices, AWDs, and this term is sometimes used herein to denote such devices.
  • MMARS magnetic acoustic resonator sensors
  • surface acoustic wave devices thin rod acoustic wave devices
  • shear horizontal acoustic wave devices shear horizontal acoustic wave devices
  • plate modes with acoustic sensor examples include quartz, lithium tantalate, Rochelle salt, and lead titanate zirconate ceramics
  • means for detecting the resonance frequency of a crystal can be provided in a variety of ways.
  • a crystal will be interposed between electrode material, the leads of which are cormected to an oscillator circuit.
  • a frequency meter or the like attached to the output of the oscillator circuit is used to measure the resonance of the crystal.
  • the electrode material need not be in physical contact with the crystal.
  • an efficient way of preparing a piezoelectric crystal for use in the present invention is to deposit electrode material on opposite surfaces of the crystal.
  • the electrode material can be deposited on the central region of opposing crystal surfaces.
  • a band of electrode material can be deposited on the crystal surface to form a pathway from the electrode material deposited on the central region of crystal surface to the edge of the crystal where leads for connecting to an oscillator circuit can be attached.
  • Any suitable electrode material can be used in the practice of this invention. Such materials include, but are not limited to, gold, silver, aluminum, nickel, chromium, titanium tantalum and the like.
  • AWD is a magnetic acoustic resonator sensor
  • the glass wafer upon which the biomolecule is bound will be in contact with a frequency counter.
  • Biomolecules can be chemically bonded directly to a piezoelectric crystal surface or indirectly via the electrode or via a material previously deposited on the crystal surface. Such other material can be electrode material or a thin layer of polymeric or other bonding material, such as intermediate molecule links.
  • biomolecules may be bound to the electrode by physiosorption or by binding using neutravin-biotin, thiol-gold or TTU silane.
  • a synthetic polynucleotide can be bonded chemically to the crystal surface to which a second, naturally occurring, polynucleotide can be attached by enzymatic ligation.
  • a polynucleotide can be ligated by enzymatic reaction (DNA or RNA ligase) to a different polynucleotide which is covalently bonded by chemical reaction to a functional group which is in turn chemically bonded to a substrate, for example, a polymeric substrate.
  • enzymatic reaction DNA or RNA ligase
  • Effective polymeric substrates include those polymers characterized by hydrolytically stable, hydrophobic backbones substituted with reactive pendant groups.
  • such polymers include, but are not limited to, copolymers of ethylene or propylene and N-(6-x-hexyl)-acrylamide, copolymers of styrene and p-x-methyl styrene, copolymers of alkylated siloxanes and 6-x-hexyl and alkyl substituted siloxanes, and similar polymers where x is a reactive group such as amino, sulfhydryl, iodo, bromo, chloro, carboxyl, hydroxy, chloro carbonyl, dimethylsilyl and similar groups which are capable of combining with polynucleotides or derivatized polynucleotides.
  • Other polymers useful in the present invention include, but are not limited to, poly(butyl methacrylate), polyurethane and the like.
  • One of the most sensitive areas for immobilizing the polynucleotides is the central portion of the crystal surface.
  • the liquid solution suspected of containing the small molecule of interest is flowed continuously across the immobilized biomolecules, and measurements of chosen electrical signals are made continuously as the solution flows.
  • a variety of different solutions can be flowed across the biomolecules of the device successively, in a continuous operation, and measurements correlated to the different solutions.
  • biomolecules includes proteins and nucleic acids, and other polypeptides and polynucleotides, as well as any biological molecule or compound which may undergo a change in size, shape, or conformation upon binding with a small molecule.
  • the AWD is not completely immersed in the liquid.
  • the vibration of the AWD is less damped and produces better data, and the risk of shorting out the electrode is reduced.
  • the electrical signal used as the basis of measurement of changes caused by the interaction can be any detectable output which changes as a result of the interaction.
  • it can be the frequency of oscillation of the piezoelectric crystal, as detected by the circuitry.
  • changes in impedance of the crystal are used as the basis of measurement.
  • pulsed electrical power is supplied to the crystal, and the resolution of the impedance measurements can be improved by using a selection of different frequencies of the power input.
  • a specific type of piezoelectric-based AWD for use in the present invention is a thickness shear mode device, TSM.
  • One process of the present invention accordingly uses an acoustic wave device (AWD), nucleic acids immobilized on the AWD, a flow cell which contains the AWD and which permits the flow of liquids across the surface of the AWD to which the biomolecules are attached, a means of sending and receiving electronic signals to and from the AWD to determine changes in acoustic frequency associated with small molecule interaction with nucleic acid or protein targets, a means of storing and processing the electronic signals collected from the AWD, and a method to interpret the data and correlate it to the determination of small molecule interaction affinity for biomolecules.
  • AWD acoustic wave device
  • the AWD is a type of biosensor used as a means of detecting the presence of molecules dissolved in a liquid medium.
  • An AWD produces and propagates acoustic waves into a liquid medium.
  • the present invention provides a process, and suitable apparatus, for detecting or monitoring the interaction of small molecules, especially those of molecular weight 2800 Da or less, with biomolecules such as nucleic acids, which comprises immersing immobilized biomolecules under test in a solution containing the small molecule of interest, generating acoustic waves in the solution by use of an AWD, and detecting and analyzing frequency changes in the acoustic waves attributable to interaction of the small molecules of interest with the immobilized biomolecules under test.
  • the AWD is made of a piezoelectric material, such as quartz, and is shaped into planar form, often circular.
  • the device should also be shaped in such a way as to allow the surfaces to vibrate parallel to the plane of each face.
  • metal electrodes are affixed to allow intimate electrical contact, so that the piezoelectric effect can occur.
  • the AWD is made useful for biosensor applications by attaching or immobilizing biomolecules onto the AWD surface. It is well known that biomolecules interact very selectively with other molecules to form aggregate compounds. By immobilizing a particular biomolecule species onto the AWD, a very selective biosensing device can be made.
  • silane adhesion agents have been used to attach biomolecules to biosensors, including piezoelectric AWD's.
  • One specific and highly effective method is disclosed in International Patent Application PCT/CA/00969.
  • the AWD is housed in a flow cell which performs several functions. It protects the AWD from damage. It allows electrical contact to be made with the AWD. and allows the electronic signals to pass from the AWD, through the flow cell, and to the outside of the flow cell, where the contacts terminate. It also allows liquid or gas to flow over one or both sides of the AWD.
  • Each face of the AWD is suitably positioned over a separate chamber of a pre-determined volume. The liquid flows into one of the chambers through one port, through the chamber, and out of the chamber through a second port. The other chamber is not connected to the liquid supply, and may be kept sealed or purged with gas.
  • the faces of the AWD are sealed, typically by using "o-rings".
  • Liquids can be introduced into the flow cell by means of a suitable pump, such as peristaltic, syringe, or piston, so that a continuous flow of liquid passes through the flow cell.
  • a suitable pump such as peristaltic, syringe, or piston
  • Water is the most commonly used liquid for this purpose; however, many additives may be dissolved in the water so as to provide an environment suitable for measuring biomolecular interactions.
  • Cations such as lithium, sodium, potassium, magnesium, calcium, ammonium, alkyl ammonium, quaternary ammonium, guanidinium
  • anions such as chloride, phosphate, carboxylate, sulfate, sulfonate, carbonate, borate), buffers (to regulate pH), solubilizing agents (detergents, surfactants, organic solvents), chelators (such as EDTA), and anti-bacterial/anti-microbial substances may be present in the water.
  • Gases can be introduced through the flow cell, if desired, by means of a pressurized tank, so that continuous flow of gas passes through the flow cell. Air may be used for this purpose, however, almost any inert gas will be suitable.
  • the gas used is relatively dry, in order to maintain a constant humidity in the gas chamber of the flow cell.
  • the gas chamber may be kept at a constant humidity by means of effective sealing of the chamber.
  • the present invention may be used in conjunction with multiple samples of small molecules passing into the flow cell to evaluate their affinities for the biomolecule immobilized onto the AWD.
  • the small molecules are normally stored in separate containers, or can also be stored as mixtures.
  • the sample concentrations can be either known or unknown.
  • a known volume of sample is injected into the flow cell for analysis by means, for example, of a Rheodyne sample injection valve.
  • a Rheodyne sample injection valve One method is to use a commercially-available "autoinjector" device which possesses such a valve, and is capable of injecting known volumes of sample into the continuously flowing liquid, which then travels through the appropriate tubing to the flow cell. Each sample is injected sequentially.
  • the autoinjector method allows multiple samples to be processed in a predetermined order automatically.
  • the autosampler "XXL 232" supplied by Gilson is suitable for this purpose.
  • the electrical contacts which terminate on the outside of the flow cell, are connected to an appropriate electronic measurement device which is capable of reading the particular frequency that the AWD is operating, at a given interval of time.
  • the electronic measurement device should be capable of transmitting electrical power to the AWD, as well as being capable of reading frequency.
  • One such method is known as the "network analysis method", in which a Hewlett-Packard 4395 A network/' spectrum/impedance analyzer is used to characterize the AWD primarily by what is known as “series resonant frequency”. Many other parameters such as parallel resonant frequency, phase, impedance, resistances, capacitances, and inductances may be used, in an analogous manner.
  • the Hewlett-Packard 4395 A is controlled by a computer program, which may be installed on a separate computer system, that allows the measurements to be started at a predetermined time and date, carried out at predetermined intervals of time throughout the course of an experiment, and stopped at a predetermined time and date.
  • Hewlett-Packard 4395 A are also stored as a data file in an appropriate format which allows the data to be graphed as time vs frequency, or time vs some other electronic parameter. This allows the magnitude and/or the area of the peaks present in the data graph to be determined.
  • the magnitude and/or the area of the peaks over time correspond to the relative strength of the small molecule interaction. If one small molecule sample generates a greater peak height, and/or peak area signal compared to a signal generated by a different small molecule, then the first small molecule can be interpreted as having a greater affinity for the biomolecule than does the second small molecule. If both small molecule samples generate the same signal intensity over time, but the first sample was known to be more dilute than the second sample, then the first sample can be interpreted as having a greater affinity for the biomolecule than does the second small molecule.
  • Figs. 1 and 2 show a quartz substrate 10 having a top electrode 12 on its top surface and a similar bottom electrode 14 on its bottom surface, both in electrical contact with the substrate.
  • the arrows on Fig. 2 indicate the ability of the substrate to oscillate in the plane of its surfaces on application of electric power of appropriate frequencies.
  • Fig. 3 shows biomolecules 16, e.g. nucleic acids or proteins, immobilized to the upper surface of the top electrode 12 through the intermediary of a chemical immobilizing agent 18, e.g. a cross-linked silane optionally including linkers and tethers as disclosed in aforementioned International Patent Application PCT/CA98/00969, the disclosure of which is inco ⁇ orated herein by reference.
  • a chemical immobilizing agent 18 e.g. a cross-linked silane optionally including linkers and tethers as disclosed in aforementioned International Patent Application PCT/CA98/00969, the disclosure of which is inco ⁇ orated herein by reference.
  • Fig. 4 shows the biosensor 20, including the substrate 10, electrodes 12, 14 and immobilized biomolecules 16 inside a flow cell 22 and ready for operation in the process of the invention.
  • the flow cell 22 has an outer housing 24 inside which is mounted a cell 26 having an upper chamber 28 and a lower chamber 30.
  • the biosensor 20 is mounted on a seal 32 separating the chambers 28 and 30, with the top electrode
  • the biosensor has a wet surface 46 attached to the substrate 10 for contact with a liquid and for binding with a biomolecule.
  • Liquid containing the small molecule of interest fills the upper chamber 28 and flows continuously therethrough, from liquid inlet 34 to liquid outlet 36, both protruding outside the housing 24.
  • Gas such as nitrogen fills the lower chamber 30, and flows continuously therethrough from gas inlet 38 to gas outlet 40, similarly protruding outside the housing 24.
  • inert gas permits free oscillation of the piezoelectric substrate, and maintains an inert environment, of controlled humidity (preferably dry) in contact with the bottom surface and bottom electrode 14, for increased reliability of results.
  • liquid inlet 34 is connected via suitable pumping arrangements to a multiwell plate containing a plurality of different liquid solutions for analysis.
  • the solutions are pumped sequentially through the upper chamber 28 of the flow cell, while the electrodes of the substrate are connected to suitable circuitry via electrical connections 42, 44. Readings of output from the electrodes are made and suitably displayed continuously, in real time, as the solutions are flowed through, and appropriately recorded for analysis.
  • Small molecules of molecular weight up to about 2,800 Da are monitored for biomolecule interactions according to the present invention.
  • One specific example of such a molecule is the Tat-20 peptide, which interacts with RNA (TAR) which can be immobilized as the biomolecule in the present invention. This provides a monitor of HIV infection.
  • TAR RNA
  • the method of the invention can be used to screen the activity of small molecules of up to about 2800 Da molecular weight, for their interaction with various nucleic acids immobilized on the substrate.
  • small molecule refers to a molecule with a molecular weight of less than 2800 daltons
  • small molecule refers to a molecule with a molecular weight of less than 1000 daltons
  • small molecule refers to a molecule with a molecular weight of less than 500 daltons
  • the small molecule is a potential pharmaceutical agent, such as a potential diagnostic agent or drug
  • Piezoelectric crystals were rinsed with ACS-grade acetone, ACS-grade ethanol and copious amount of ddH 2 O, followed by drying with a stream of dry helium, prior to any immobilization. All buffers / solutions were degassed with dry helium prior to use.
  • Tris buffer pH 7.5: lO mM Tris-HCl
  • PBS buffer pH 7.4: 10 mM phosphate
  • Tris buffer pH 7.5 and all strands are 25 base-pairs long.
  • a 9 MHz gold (Au) piezoelectric quartz crystal was washed, dried and mounted in the flow cell.
  • Tris buffer was passed through the flow cell at pump speed #1 ( ⁇ 0.065 mL/min) and the baseline is monitored until stable.
  • the change m frequency was allowed to stabilize prior to the next injection and this same allowance is made for all subsequent steps.
  • a 9 MHz chromium (Cr) piezoelectric quartz crystal was washed and placed m a humidifying chamber overnight. Silamsation was performed in a glovebox, via immersion in a 1 mM solution of TTU in dried toluene, for a period of 2 hrs. The crystal was then rinsed with dried toluene, ACS-grade chloroform and dried with a stream of nitrogen. A 20 uM solution of thiol-terminated ssDNA m PBS (pH 7 4). was reacted with the linker bis-bromomethyl benzene-2-sulfonate (BMBS) for 1 hr.
  • BMBS linker bis-bromomethyl benzene-2-sulfonate
  • the resulting complex was then purified m a NAP column, conditioned with PBS pH 3 to minimize analyte retention.
  • the purified aliquot was pH-adjusted to 7.5, introduced onto the TTU surface and allowed to incubate for 1 hr.
  • the crystal was then rinsed thoroughly with ddH-,0. dried with a stream of helium and mounted m the flow cell Tris buffer was passed through the flow cell at pump speed #1 ( ⁇ 0.065 mL/min) and the baseline was monitored until stable.
  • FIG. 6 shows a schematic diagram of a DNA sequence, SCDNA102, which has known binding sites for various antibiotics, bound to a biosensor.
  • Crystals prepared using the methods set out above were mounted in the flow cell and the appropriate buffer is passed through to establish a baseline. Crystals could be modified just prior to assay and kept in the flow cell under Tris until the appropriate buffer switch is performed.
  • the analytes were injected at various volumes (475 uL - 200 uL) and flow rates (pump speed #1 - #5). according to the nature of the interaction and the desired throughput.
  • Figure 6 is a graph showing frequency modulation in response to the addition of small molecules to the flow cell to assess the interaction of the small molecules with single stranded SCDNA102, shown in Figure 5
  • various antibiotics were added to the flow-through system, using the following pattern small molecule A, wash, small molecule A, wash, small molecule B, wash, small molecule B, wash
  • actmomycin, daunomycm, ethidmm bromide, and echinomycm all produced marked, reproducible peaks or valleys in the frequency data
  • distamycm and tetracyclme failed to produce any noticeable effect This is to be expected as SCDNA102 does not contain distamycm and tetracyclme binding sites (see Figure 5)
  • Example 5 Drug small molecules interaction with prepared surfaces using RNA biomolecule
  • Example 7 Peptide small molecules interaction with prepared surface using RNA biomolecule

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Abstract

L'invention concerne un appareil (22) permettant de contrôler les interactions entre une petite molécule et une biomolécule. Un substrat (10) oscillant comprend une surface humide (46) permettant le contact avec un liquide et la liaison avec une biomolécule ; et une surface sèche (48). Un appareil de détection (42, 44) détermine la fréquence de résonance dudit substrat avant et après l'interaction. Ainsi des petites molécules sont contrôlées, en vue de l'interaction avec des biomolécules ; ce contrôle est effectué par contact d'une solution contenant lesdites petites molécules avec les biomolécules immobilisées soumises à un test ; et par création d'ondes sonores à l'aide d'un dispositif à ondes sonores. L'interaction entre les petites molécules et les biomolécules immobilisées provoque un changement de fréquence, lequel changement peut être détecté puis analysé de manière électronique afin de contrôler ladite interaction.
PCT/CA2000/000504 1999-05-05 2000-05-05 Appareil et procede permettant de controler et de detecter les interactions entre une petite molecule et une biomolecule WO2000068419A2 (fr)

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CA002368315A CA2368315A1 (fr) 1999-05-05 2000-05-05 Appareil et procede permettant de controler et de detecter les interactions entre une petite molecule et une biomolecule
AU43880/00A AU4388000A (en) 1999-05-05 2000-05-05 Apparatus and process for monitoring and detecting small molecule-biomolecule interactions
EP00924999A EP1190093A2 (fr) 1999-05-05 2000-05-05 Appareil et procede permettant de controler et de detecter les interactions entre une petite molecule et une biomolecule

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CA 2271179 CA2271179A1 (fr) 1999-05-05 1999-05-05 Procede pour surveiller et detecter des interactions entre de petites molecules et des biomolecules

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US6803205B2 (en) 2000-11-08 2004-10-12 Surface Logix, Inc. Methods of measuring enzyme activity using peelable and resealable devices
US6967074B2 (en) 2000-11-08 2005-11-22 Surface Logix, Inc. Methods of detecting immobilized biomolecules
US7001740B2 (en) 2000-11-08 2006-02-21 Surface Logix, Inc. Methods of arraying biological materials using peelable and resealable devices
US7101669B2 (en) 2000-04-12 2006-09-05 Sensorchem International Corporation Enzyme-based regeneration of surface-attached nucleic acids
US7351575B2 (en) 2000-11-08 2008-04-01 Surface Logix, Inc. Methods for processing biological materials using peelable and resealable devices
WO2008048222A2 (fr) 2005-08-19 2008-04-24 Intel Corporation Procédé et dispositif fondé sur une technologie cmos pour analyser des molécules et des nanomatériaux sur la base de l'affichage électrique d'évènements de liaison spécifiques sur des électrodes fonctionnalisées
US7371563B2 (en) 2000-11-08 2008-05-13 Surface Logix, Inc. Peelable and resealable devices for biochemical assays
WO2008073042A1 (fr) * 2006-12-13 2008-06-19 Biosensor Applications Sweden Ab (Publ) Procédé répétable en continu de détection d'antigènes dans un volume d'essai
US7439056B2 (en) 2000-11-08 2008-10-21 Surface Logix Inc. Peelable and resealable devices for arraying materials
WO2010127122A1 (fr) * 2009-04-29 2010-11-04 The Trustees Of Columbia University In The City Of New York Structure fbar-cmos monolithique telle que pour une détection de masse
CN101595387B (zh) * 2006-12-13 2014-01-29 生物传感器应用国际有限公司 在测试体积中测试抗原的连续可重复方法
US9255912B2 (en) 2009-04-29 2016-02-09 The Trustees Of Columbia University In The City Of New York Monolithic FBAR-CMOS structure such as for mass sensing
US9741870B2 (en) 2012-10-17 2017-08-22 The Trustees Of Columbia University In The City Of New York Systems and methods for CMOS-integrated junction field effect transistors for dense and low-noise bioelectronic platforms
EP3222993A4 (fr) * 2014-11-21 2018-08-01 Centre National De La Recherche Scientifique Système de détection de molécule
US10122345B2 (en) 2013-06-26 2018-11-06 The Trustees Of Columbia University In The City Of New York Co-integrated bulk acoustic wave resonators
US10600952B2 (en) * 2016-05-20 2020-03-24 Pulmostics Limited Surface acoustic wave sensor coating
US11674928B2 (en) * 2017-06-16 2023-06-13 Foundation For Research And Technology Hellas Detecting nucleic acids in impure samples with an acoustic wave sensor

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US5880552A (en) * 1997-05-27 1999-03-09 The United States Of America As Represented By The Secretary Of The Navy Diamond or diamond like carbon coated chemical sensors and a method of making same
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US4999284A (en) * 1988-04-06 1991-03-12 E. I. Du Pont De Nemours And Company Enzymatically amplified piezoelectric specific binding assay
US5501986A (en) * 1988-04-06 1996-03-26 E. I. Du Pont De Nemours And Company Piezoelectric specific binding assay with mass amplified reagents
US5374521A (en) * 1991-09-17 1994-12-20 Kipling; Arlin L. Acoustic reflection process for molecular sensing using a bulk acoustic wave quartz sensor
US5880552A (en) * 1997-05-27 1999-03-09 The United States Of America As Represented By The Secretary Of The Navy Diamond or diamond like carbon coated chemical sensors and a method of making same
WO1999020640A2 (fr) * 1997-10-16 1999-04-29 Sensorchem International Corporation Immobilisation de monocouches oligonucleotidiques par liaison covalente a densite superficielle elevee

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7101669B2 (en) 2000-04-12 2006-09-05 Sensorchem International Corporation Enzyme-based regeneration of surface-attached nucleic acids
US7439056B2 (en) 2000-11-08 2008-10-21 Surface Logix Inc. Peelable and resealable devices for arraying materials
US6967074B2 (en) 2000-11-08 2005-11-22 Surface Logix, Inc. Methods of detecting immobilized biomolecules
US7001740B2 (en) 2000-11-08 2006-02-21 Surface Logix, Inc. Methods of arraying biological materials using peelable and resealable devices
US7351575B2 (en) 2000-11-08 2008-04-01 Surface Logix, Inc. Methods for processing biological materials using peelable and resealable devices
US6803205B2 (en) 2000-11-08 2004-10-12 Surface Logix, Inc. Methods of measuring enzyme activity using peelable and resealable devices
US7371563B2 (en) 2000-11-08 2008-05-13 Surface Logix, Inc. Peelable and resealable devices for biochemical assays
JP2009509175A (ja) * 2005-08-19 2009-03-05 インテル・コーポレーション 官能性電極上の選択的結合事象を電気的に読み取ることにより分子及びナノ材料を分析する方法及びｃｍｏｓ型デバイス
WO2008048222A2 (fr) 2005-08-19 2008-04-24 Intel Corporation Procédé et dispositif fondé sur une technologie cmos pour analyser des molécules et des nanomatériaux sur la base de l'affichage électrique d'évènements de liaison spécifiques sur des électrodes fonctionnalisées
WO2008048222A3 (fr) * 2005-08-19 2008-07-24 Intel Corp Procédé et dispositif fondé sur une technologie cmos pour analyser des molécules et des nanomatériaux sur la base de l'affichage électrique d'évènements de liaison spécifiques sur des électrodes fonctionnalisées
CN101595387B (zh) * 2006-12-13 2014-01-29 生物传感器应用国际有限公司 在测试体积中测试抗原的连续可重复方法
WO2008073042A1 (fr) * 2006-12-13 2008-06-19 Biosensor Applications Sweden Ab (Publ) Procédé répétable en continu de détection d'antigènes dans un volume d'essai
US9255912B2 (en) 2009-04-29 2016-02-09 The Trustees Of Columbia University In The City Of New York Monolithic FBAR-CMOS structure such as for mass sensing
CN102414855A (zh) * 2009-04-29 2012-04-11 纽约市哥伦比亚大学信托人 如用于质量感测的单块fbar-cmos结构
WO2010127122A1 (fr) * 2009-04-29 2010-11-04 The Trustees Of Columbia University In The City Of New York Structure fbar-cmos monolithique telle que pour une détection de masse
US9741870B2 (en) 2012-10-17 2017-08-22 The Trustees Of Columbia University In The City Of New York Systems and methods for CMOS-integrated junction field effect transistors for dense and low-noise bioelectronic platforms
US10122345B2 (en) 2013-06-26 2018-11-06 The Trustees Of Columbia University In The City Of New York Co-integrated bulk acoustic wave resonators
EP3222993A4 (fr) * 2014-11-21 2018-08-01 Centre National De La Recherche Scientifique Système de détection de molécule
US10843191B2 (en) 2014-11-21 2020-11-24 Centre National De La Recherche Scientifique (Cnrs) Molecule detecting system
US10600952B2 (en) * 2016-05-20 2020-03-24 Pulmostics Limited Surface acoustic wave sensor coating
US11674928B2 (en) * 2017-06-16 2023-06-13 Foundation For Research And Technology Hellas Detecting nucleic acids in impure samples with an acoustic wave sensor

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WO2000068419A3 (fr) 2001-12-27
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EP1190093A2 (fr) 2002-03-27

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