WO1994013835A1 - Detection de macromolecules - Google Patents

Detection de macromolecules Download PDF

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Publication number
WO1994013835A1
WO1994013835A1 PCT/US1993/012307 US9312307W WO9413835A1 WO 1994013835 A1 WO1994013835 A1 WO 1994013835A1 US 9312307 W US9312307 W US 9312307W WO 9413835 A1 WO9413835 A1 WO 9413835A1
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WIPO (PCT)
Prior art keywords
macromolecules
probe
active surface
former
molecules
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Application number
PCT/US1993/012307
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English (en)
Inventor
Stanley L. Bogdanski
Stefan Nowinski
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Granta Laboratories Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Granta Laboratories Limited filed Critical Granta Laboratories Limited
Priority to AU58512/94A priority Critical patent/AU5851294A/en
Publication of WO1994013835A1 publication Critical patent/WO1994013835A1/fr

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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
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4788—Diffraction
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N2021/757—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated using immobilised reagents

Definitions

  • This invention relates to the detection of macromolecules.
  • the selectivity of the method is based on the complementary structure which exists between certain pairs of molecules, such as: a DNA or RNA single strand molecule and its complementary single strand, an antibody and its antigen, and an enzyme and its substrate (or coenzyme) .
  • the reagent used comprises the molecular fragment which associates itself to the analyte.
  • Enhanced sensitivity and an improved limit of detection is often achieved by use of a reagent labelled with a molecular tag.
  • the label makes the reagent and, therefore, the analyte, highly detectable by rendering it fluorescent, bioluminescent, chemiluminescent, radioluminescent, colourimetric, etc.
  • a further technique for labelling is by attaching an enzyme to the reagent and measuring the changes brought on by the introduction of the appropriate substrate.
  • Amplification techniques often result in multiplying the interfering substances along with the analyte.
  • Assay formats are classed as either heterogeneous or homogeneous, depending on whether a reagent is immobilized on a solid or whether it remains in a liquid, respectively. Assays are also classified as to whether they are competitive or non-competitive.
  • Rl is selective towards the analyte, A, and is immobilized say on the surface of a microtitre plate.
  • the sample containing the analyte is introduced and then washed away, leaving some of the Rl occupied by the analyte.
  • a second reagent, R2 which is labelled and selective for the first reagent, Rl, is added and washed away.
  • the response of the detector towards the labelled reagent is indirectly proportional to the amount of analyte originally present.
  • two reagents are used. The first is immobilized and selective to the analyte. The analyte is captured, as described above. The second regent is labelled and selective to the analyte. The following detector signal is directly proportional to the concentration of the analyte.
  • Homogeneous techniques basically are the same as the above but rely on a measurable change occurring to labelled reagent when it becomes associated to an analyte.
  • Homogeneous assays are mostly of the competitive type and are usually relatively simple to perform and automate but have limited sensitivity, i.e. 10 "9 mol/l.
  • the present invention is relatively simple to implement, has great sensitivity and retains the advantage of a direct analytical measurement.
  • a heterogeneous method in which the unlabelled reagent molecule is attached to a surface at the edge of a small former which is configured so as to create a diffraction pattern when placed into a specially designed detector. Changes in this diffraction pattern indicate whether the reagent is associated with an analyte or whether it is unassociated, i.e., no analyte is present.
  • the former can be configured in several ways to achieve an amplified diffraction pattern and allow an analyte measurement, e.g., as a slit, as a wire, as a ball or as multiples of these discrete elements.
  • Our method is based on the spatial redistribution of the diffracted light leaving a discrete element or former of small dimension which results from the apparent change in dimension of that former when it becomes associated with analyte molecules.
  • the active surface where the molecular association takes place lies along the path of the light used to illuminate the former and forms the edge of the former thus defining the dimension producing the diffraction pattern.
  • both the spatial distribution of the diffraction pattern and the change in it which occurs due to the presence of the analyte are well defined and are used to advantage in a system which is highly selective for the analyte and minimizes interferences.
  • Our method allows detection of the analyte directly, without the necessity of further amplification, e.g., by PCR (polymerase chain reaction), or the use of labelled reagents. This results in a direct analysis, less prone to interferences and contamination, optimized for selectivity, and offering a relatively simple analytical procedure.
  • the technique is amenable to the use of reagent labels and additional reagents to further improve the sensitivity and limit of detection.
  • the probe concentrates the analyte from a relatively large volume of solution to a known specific small area, depending on its binding affinity between captured reagent on the probe and the analyte. This enhances sensitivity and lowers the level of detection as compared to optical techniques that depend on the analyte concentration on a larger surface area or in a larger volume, such as those depending on the reduction of light being transmitted.
  • detection systems other than diffraction, which take advantage of the enhanced signal resulting from this concentration step and localization of the analyte.
  • the detection technique includes multiple analyte detection, blank readings and tests for false positives and false negatives by incorporating the appropriate sections into the probe during the fabrication. This is accomplished without increasing the complexity of the analytical procedure.
  • the probe is configured to perform as a continuous detector, say on the eluent of a chromatographic column, or to serve as a consumable, for a preliminary, quick test to be performed by a doctor in his surgery.
  • the probe giving a positive test result and which is presumed to contain the analyte can be sent to a clinical laboratory for further tests, including amplification, and final confirmation.
  • the invention features method and a system for detection of macromolecules that employs a probe comprising a discrete former of predetermined small size and selected geometry capable of diffracting light of a selected wavelength to produce a diffraction pattern, the former having an active surface which lies along the path of the light with reagent molecules exposed for binding the macromolecules to the active surface; a light source adapted to emit light of the selected wavelength, the radiation being directed to and extending along the active surface; the wavelength of the light and the small size of probe being so related that the macromolecules when bound to the active surface are non-transparent and produce a diffraction pattern change which is detectable and greatly amplified in dimension relative to the size of the macromolecule that produces the change; a detector arranged to detect at least a portion the diffraction pattern; and a system control adapted to determine the presence of the macromolecules on the basis of the change of the diffraction pattern.
  • Preferred embodiments of this aspect of the invention may have one or more of the following features.
  • the limited size of the active surface of the probe is effective to concentrate the macromolecules of interest on its surface for detection.
  • the reagent molecules are either DNA or RNA molecules of a sequence complementary to the sequence of DNA or RNA molecules of interest, or protein molecules.
  • the protein molecules are either enzymes adapted to detect substrates or co-enzymes, or antibodies associated to an antigen of interest.
  • the critical dimension of the probes' active surface is of the order of 0.1 to 20 microns and the macromolecules being detected have dimension of the order of fractions of a nanometer to nanometers.
  • the probe has a wire former that includes an intermediate layer with attached reagent molecules.
  • the probe comprises a former that defines a slit further including an intermediate layer with attached reagent molecules at the edges of the slit.
  • the probe comprises a 0.1 to 20 micron size sphere former that includes an intermediate layer with attached reagent molecules.
  • the probe comprises a former that defines an aperture including an intermediate layer with attached reagent molecules at the edge of the aperture.
  • the reagent molecules are arranged in a manner to form detection and reference surfaces.
  • the detection and reference surfaces enable quantitative or qualitative detection of the macromolecules.
  • the invention features an apparatus for the detection of macromolecules comprising a light source, a probe comprising a former of predetermined dimensions and a detector, all arranged in a geometry such that the detector observes the diffraction pattern of the probe, the former having an active surface of such dimension that its diffraction pattern at the detection plane is of the order of centimeters from 1st to 2nd order peaks, whereby changes on the active surface of the order of nanometers in the dimension of the probe result in movements of the order of millimeters in the position of the diffraction peaks allowing the attachment of macromolecules to the active surface of the probe to be detected.
  • Preferred embodiments of this aspect of the invention may have one or more of the following features.
  • the radiation is modified (such as being polarized) , to enhance the interaction of the radiation with the analyte macromolecules.
  • the radiation is parallel and coherent to generate the optimum diffraction pattern.
  • the macromolecules are modified by staining or other process.
  • the macromolecules are labeled by a radioactive, fluorescent, bioluminescent, chemiluminescent, electroluminescent, chromophoric or conductive element, or an enzyme.
  • the radiation making up the diffraction pattern is monochromatic, achieved, e.g., by use of a monochromatic source or a narrow band filter anywhere in the beam path prior to the detector.
  • the probe is of a "positive" shape such as a wire or a sphere, or a "negative” shape such as a slit or a pin-hole having at least one dimension of a suitably small magnitude so as to create a suitably large diffraction effect that results in magnification of surface changes.
  • the invention features a system and method of detection of macromolecules (as in diagnosis of disease when the macromolecules are biological molecules of clinical significance) whereby a suitable reagent (biological examples include: single strand DNA, RNA antibodies, or other reagent molecule) is attached to an active surface of small dimensions and the probe is presented to a suitably prepared sample, the presence of analyte molecules in the sample resulting in binding of those molecules to the reagent molecules and producing a small dimensional change on the active surface and thereafter observing the probe, the presence of analyte molecules being thereby detected directly, without the use of complicated labelling procedures as used in some other diagnostic techniques.
  • a suitable reagent biological examples include: single strand DNA, RNA antibodies, or other reagent molecule
  • Fig. 1 is a diagrammatic view of an embodiment of analyzer system in accordance with the present invention.
  • Fig. 2 is one preferred embodiment of an analyte selective probe with active surface located at the edges of a slit;
  • Fig. 2A is the analyte selective probe of Fig. 2 adapted for in situ detection
  • Figs. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, and 31 show schematically a method of fabrication of the analyte selective probe of Fig. 2;
  • Fig. 4 is another preferred embodiment of the analyte selective probe with the active surface on a wire held within a tube which provides a flow through cell configuration for introducing a sample;
  • Figs. 5A, 5B, 5C, and 5D show fabrication of the analyte selective probe of Fig. 4;
  • Figs. 6, 6A, and 6B show a transmission mode embodiment of the analyzer system of Fig. 1;
  • Fig. 7 shows a diffraction pattern monitored by a split detector arrangement of the analyzer system
  • Fig. 8 shows multiple diffraction patterns formed by several beams and monitored by multiple detectors of the analyzer system
  • Figs. 9 and 9A show a diffraction pattern obtained by a random orientation of the active surface, and an annular detector used to detect this pattern, respectively;
  • Figs. 10 and 10A show the reflection mode embodiments using a slit probe and a wire probe, respectively.
  • a system for detecting macromolecules includes a source 10 emitting electromagnetic radiation 12 that is directed to a probe 20, containing the sample and held in a holder 19 in line with a detector 100.
  • An active surface 28 of probe 20 is aligned and interacts with the radiation.
  • the substances present on active surface 28 alter the outgoing radiation 16 in a predictable manner which is detected by detector 100.
  • the entire operation is controlled by a process control unit 120 that specifies the type of radiation to be emitted from source 10, controls location of probe 20 and defines the mode of operation and position of detector 100.
  • Detection system 100 has several detectors that can be used simultaneously or interchangeably depending on the type of the probe used, wavelength of the detection beam and reference beams, the overall source-probe-detector arrangement and the mode of operation. Depending on the mode of operation and the wavelength of the source, the selected detector is a photomultiplier, photodiode, CCD array, or photoconductive detector.
  • the invention uses several types of probe 20. Referring in particular to Fig. 2, analyte- selective probe 20 includes a rigid transparent substrate 22, a thin Si0 2 layer 24, and a nontransparent layer 26.
  • a thin section of layers 24 and 26 is removed to form a transparent slit which is 0.1 ⁇ m to 20 ⁇ m wide and about 0.02 mm to 20 mm long.
  • the exposed silicon dioxide surfaces 28 of the slit are chemically treated to introduce reactive sites (-NH 2 ) which are used to immobilize single strands of DNA having a sequence of nucleic acids which form matching base pairs with the complementary nucleic acids found on the targeted analyte DNA molecule.
  • Surface 28 has a well defined depth, surface roughness, and geometry and specific orientation with respect to incoming beam 12.
  • the O.l ⁇ m to 20 ⁇ m wide slit is fabricated by a series of photolithographic steps.
  • probe 20 is formed by depositing thin layer 24 of Si0 2 by chemical vapor deposition (CVD) , sputtering or other technique onto transparent substrate 22 (e.g. , microscope slide lightly coated with thin gold layer to make inactive) .
  • transparent substrate 22 e.g. , microscope slide lightly coated with thin gold layer to make inactive
  • the thickness of Si0 2 layer 24 is selected depending on the desired depth of active surface area 28.
  • a photoresist 25 is deposited on top of the silicon dioxide layer 24.
  • the first photolithographic (Fig. 3B) step defines the dimensions of the slit on the probe and starts the process of removing the desired section of Si0 2 layer 24 which exposes active surface 28.
  • Photoresist 25 is exposed and developed to define the slit.
  • Si0 2 layer 24 is removed by anisotropic etching (or by ion milling) through the Si0 2 layer (Fig. 3C) .
  • photoresist 25 is removed.
  • the nontransmitting layer 26 that is opaque to radiation of selected wavelengths (e.g., a reflecting gold layer) is deposited onto layer 24.
  • photoresist 25 is again deposited, exposed and developed to define a slit that coincides with the removed section in the Si0 2 layer (Fig. 3E) .
  • Etching is used again to create a transparent slit in nontransmitting layer 26 (Fig. 3F) .
  • a photolithographic step is used after deposition of both layers 24 and 26.
  • a photoresist is deposited on layer 26, exposed and developed to define the slit.
  • the slit is created by ion milling or by combination of ion milling and anisotropic etching through layers 24 and 26.
  • the silicon dioxide surface 28 is activated for the attachment of a reagent molecule.
  • the silicon dioxide surface is activated using a silylating agent to form activated ester of silica 29, i.e., N- hydroxysuccinimidyl-silica located on surface 28 (Fig. 3G) .
  • the desired single strand DNA fragments are synthesized by standard techniques (e.g., using an Applied Biosystems Synthesizer) .
  • the fragments have an additional section with an aminoalkyl group at the 5'- end.
  • the 5 , -aminoalkyl-DNA 30 is then coupled to an N- hydroxysuccinimidyl ester silica located on the activated edge surface 28 (Fig. 3H) .
  • This reaction gives a stable amide linkage between the DNA and the silica, as described by Solomon, et al. in Analytical Biochemistry 203, 58-69 (1992).
  • Probe 20 contains a long, thin slit that has the interior Si0 2 edges coated with analyte selective reagent e.g., single strand DNA fragments 30, Fig. 3H and Fig. 2, of a particular, known sequence.
  • the sequence is chosen to be complementary to the sequence of the DNA material, analyte of interest, the particular probe is designed to detect.
  • the probe uses different nucleic acid sequences for detection of different analytes of DNA.
  • active surface 28 of probe 20 is contacted with the analyte solution by immersion or introducing the test solution to the probe to bind the analyte DNA material 32, Fig. 31.
  • Fig. 31 depicts the probe after it has captured the analyte from the sample and the slit has been reduced in width by the buildup of analyte on the active surfaces of the sides of the slit.
  • the analyte solution is prepared by lysing cells to release DNA material.
  • the double strand DNA is dissociated into single strands by adjusting the conditions of temperature, salt concentration and/or pH.
  • the strands are cut into suitable lengths using restriction enzymes.
  • the analyte DNA molecule, the target may be considerably larger than the associated DNA immobilized on the surface of the probe.
  • the analyte DNA material is bound to the immobilized strands by controlling the conditions in the micro-environment of the slit.
  • a washing step is used to remove potential interference molecules which are less strongly bound.
  • the selectivity of the probe is related to the number of associated base pairs being formed between the probe DNA and the analyte DNA, whereas the limit of detection is more closely related to the size of analyte molecule and depth of the active surface layer containing the probe DNA.
  • Wire probe 40 is mounted in a capillary tube 50 using mounting ends 54A and 54B.
  • the sample and subsequent wash solutions are introduced to the probe via ports 52A and 52B.
  • wire probe 40 comprises a former 42 of 0.1 to 20 microns in diameter (e.g., tungsten wire) .
  • the former is coated with a thin layer of silicon dioxide 43 introduced by chemical vapor deposition (Fig. 5B) .
  • Fig. 5B silicon dioxide 43 introduced by chemical vapor deposition
  • Fig. 5C selected areas of silicon dioxide layer are then activated using a silylating compound to form an activated ester such as N- hydroxysuccinimidyl-silica 44.
  • the desired single strand DNA segments with a DNA sequence complementary to the sequence of a particular analyte are then immobilized on activated surfaces 44 creating active surfaces 46A and 46B (Fig. 5D) in the same way as described for the slit probe.
  • slit probe 20 designed for the multiple analyte detection includes similar sections as described above for the wire probe.
  • the light source 10 (e.g., an excimer laser, Helium-Neon laser, tunable YAG laser, etc.) emits continuous or pulsed monochromatic, collimated electromagnetic radiation of a suitable wavelength. Depending on the reagent and analyte being detected, the source is selected on the basis of wavelength from a range of ultra-violet, visible or infra-red lasers. Alternatively, light source 10 includes a source of white light. Suitable optics and filters are used to scan the wavelengths and direct the beam. The radiation is split into one or more detection beams and one or more reference beams. The geometry of the beam:active-surface interaction is controlled. Material 26 surrounding the active surface of probe 20 is opaque to the radiation so that the plane of the active surface lies in the direction along the path of the radiation.
  • the wavelengths are selected on the basis of the active surface and probe configuration, spectral absorbance, reflectance or scatter characteristics of the reagent and analyte (suitable stains or other absorption enhancing techniques may be used) and the dimensional change as related to the presence of the analyte.
  • the dimensional change from fractions of a nm to few microns, is measured by monitoring the corresponding change in the diffraction pattern of the active surface with and without the analyte.
  • the analyzer system can scan the active surfaces at different wavelengths and angles to obtain diffraction patterns with optimal signal-to-noise conditions.
  • Fig. 6 is a diagrammatic view of the transmission mode embodiment of the slit probe.
  • Source 10 emits collimated monochromatic light 12.
  • Probe 20 is aligned to have plane of the active surface 28 oriented along the path of the radiation.
  • the diffracted light 16 is imaged by a spherical lens 18 onto an image plane 80 at a predetermined distance from the lens.
  • a detector 102 of detection system 100 placed in image plane 80 detects the far field (Fraunhofer) diffraction pattern 90 formed by the beam passing over the slit aperture containing active surface 28.
  • ⁇ is the angle from the axis of the straight through beam.
  • Pattern 90 has a series of maxima and minima at distances from the axis of the straight through light which are inversely related to the width of the slit.
  • the output of the detector is digitized and processed in the data analyzer of computer 122.
  • Fig. 6A shows diagrammatically wire probe 40 placed in the analyzer system operating in the transmission mode embodiment.
  • Fig. 6B shows diagrammatically an embodiment wherein wire probe 40 is used in a flow cell arrangement. In the flow cell arrangement, the test solution is recirculated or added to a flowing stream of cleansing solution.
  • multiple element detector 104 formed by several light detecting elements (e.g. photodiodes) , is located at a fixed position. As shown in Fig. 7, detector 104 determines changes in the radiation intensity at a fixed point of a selected peak of diffraction pattern 90. From the change in the detected intensity, (i.e., movement of the pattern relative to the detector) presence and concentration of the analyte is determined.
  • the size of the photosensitive surface area of the detector is optimized according to probe dimensions, distance between the detector and probe, and further optical considerations to give the best analyte resolution.
  • the analyzer system determines, in real time, changes in the diffraction pattern that correspond to the active surface changes due to increasing presence of the analyte.
  • the above described configurations observe changes on the scale of sub-nanometers to nanometers using a slit of few microns. This resolution, which is dependent on the chemical and optical nature of the species present on active surfaces 28, is sufficient to detect the single strands DNA.
  • Further reagents may be used to enhance the presence of the analyte and give a large detector response, e.g., by adding to the dimension of the analyte layer or making the analyte less transparent to the radiation from the source. Referring to Fig.
  • Slit probe 20 may comprise several slits with active surfaces 28, each adapted to detect different species of analyte.
  • the high sensitivity is obtained from the small area of the probe onto which the analyte is concentrated, a high affinity of binding between analyte and immobilized reagent, and a detector based on diffraction, which increases in amplification as the probe size is reduced.
  • a detector based on diffraction which increases in amplification as the probe size is reduced.
  • the applications of the probe/detector govern the operating conditions and the design of the probe.
  • a simple "yes/no" qualitative test requiring maximum sensitivity benefits from using a probe with a high captive reagent concentration immobilized on a probe containing a minimum active surface area.
  • the same conditions should be optimized to obtain maximum analyte/reagent affinity.
  • a continuous detector may benefit from a larger active surface area, lower reagent concentration and sub-optimal conditions in order to obtain a quicker response time and avoid interferences accumulating over time.
  • the analyzer system utilizes one or more detection beams and one or more reference beams arranged to probe different active areas of the probe sequentially or simultaneously.
  • the concentration of the attached single strands of DNA reagent is varied along the slit length periodically or continually.
  • the detection beam may scan the active surface with varying concentration of the analyte bound to the reagent while the reference beam scans a corresponding active surface with varying concentration of the reagent.
  • the signals obtained from the diffraction pattern are subtracted.
  • the examination sequences are chosen to calibrate the probe, to eliminate false positive or negative readings, to determine concentration of one or more analytes in test solution.
  • a closed system is achieved by adding a transparent layer 27 onto nontransmitting layer 26 to form a closed space. Then, a suitable micro-flow fluid handling system is attached to circulate fluid through the closed volume. The closed system enhances the control over temperature and pH parameters required to control the binding steps.
  • the wire probe of Fig. 4 is used in the same way, wherein the closed system is achieved by connection of the micro- flow system to ports 52A and 52B.
  • binding of the analyte is detected by changes to the time base profile of the detected light.
  • the technique uses either a disposable probe or a probe that can be regenerated for multiple uses depending on the application.
  • the multiple use probe is adapted to release the analyte from the active surface (e.g., by thermally cycling the probe or by pH cleansing) .
  • the active surface of slit probe 20 (or wire probe 40) includes multiplicity of surfaces with single strands of DNA arranged in a selected geometry that allow only molecules of a certain length to adhere. For example, there may be two active surfaces separated by a selected distance. Each surface has an immobilized reagent, and the selection of analyte is based on two different molecular associations having a defined distance (molecular size) between them.
  • the source- probe-detector alignment can be simplified by using several randomly positioned wires or micro-spheres as a former.
  • the diffraction pattern is circular in nature.
  • beam 12 irradiates a suspension of spherical probes 60 (e.g., former is a 3 to 5 micron silica ball similar to the ones used in the solid phase chromatography and creates diffraction pattern 95 that is monitored by a detector 112.
  • Detector 112 has the detector elements arranged in one or more annular rings centered on the straight through beam (Fig. 9A) . Detector 112 is positioned at a distance from the axis that enables proper resolution of the diffraction pattern.
  • Figs. 10, and 10A show diagrammatically the reflecting mode embodiment of the analyzer system, wherein the diffraction pattern is observed by detecting the reflected radiation from the probe.
  • the reflecting embodiment of slit probe 20 requires flat transparent substrate 22 made reflective by depositing an additional reflective layer 23 onto layer 22 prior to the slit formation.
  • Reflective layer 23 reflects the diffracted radiation which is separated from the incoming beam by a half silvered mirror 17 at 45° or a similar optical element suitable for the selected wavelength.
  • Detector system 100 is positioned to observe the reflected diffraction pattern in the same way as discussed above.
  • the reflecting embodiment of wire probe 40 (Fig. 10A) includes a mirror surface 41 positioned behind the wire. The incoming radiation, diffracted by the active surface of probe 40, is reflected back onto the wire and then detected by detector 100.
  • Optimization of detection may be achieved by altering the incidence angle of the radiation, by polarizing the radiation and rotating the polarized plane angle relative to the active surface, by adjusting the pulse parameters of radiation, or by modulating the frequency.
  • the light source can operate in a pulse mode to prevent thermal effects on the active surface or to scan different regions of the active surface interchangingly.
  • Each of these techniques provides a means of internal noise and drift reduction of the detector as well as that of the environment (e.g., vacuum, gas or liquid) wherein the measurement is conducted.
  • probe embodiments use only a former with the immobilized selective reagent bound directly to the former.
  • a number of reactive intermediates selectively paired with reactive moieties can be used.
  • Reagent molecules can be arranged so as to coil and grow more dense when affected by the analyte molecule, effectively changing the active surface. Immobilization occurring at several points between the reagent and active surface can ensure optimal positioning of molecules for their subsequent detection.
  • the reagent molecules so arranged would respond to the angle of the plane using polarized radiation. This arrangement reduces noise and enhances the detection.
  • the Si0 2 layer activated by a silylating agent is replaced by other "active" layers to which the preferred type DNA strands are bound, for example, polystyrene, nitro-cellulose, nylon, polycarbonate or polyurethane.
  • the detection surfaces are again fabricated by photolithography; however, other methods such as micromachining, laser ablation or molding may also be used.
  • probes with different shapes of active surfaces arranged to selectively concentrate the analyte An aperture with a 0.1 to 20 micron diameter resolves features down to 0.1 nm layers of analyte.
  • the slit type apertures were described in detail; however, other geometries such as circular or square hole apertures may be also used.
  • the active surfaces may be located on structures that are complementary to the described apertures.
  • the probe is an opaque long, thin bar or a dot with the activated surfaces located on the outside edges.
  • An equivalent of a circular aperture is a small sphere.
  • the diffraction signal can be augmented by enlarging the analyte layer or making it less transparent. This is accomplished by using reagents which would react or combine with the now immobilized analyte. These reagents could merely add to the size of the analyte layer, stain the analyte, or attach an enzyme (or reactive molecule) which would subsequently deposit a precipitate in this immediate vicinity.
  • the probe can subsequently be also reacted with reagents which fluoresce, luminesce, etc.

Abstract

Procédé et système de détection de macromolécules comprenant une source de lumière, une sonde comportant un gabarit ayant des dimensions prédéterminées et un détecteur, tous ces éléments étant placés selon une géométrie telle que le détecteur observe l'image diffractée de la sonde. Le gabarit comporte une surface active dont la dimension est telle que son image diffractée au niveau du plan de détection est de l'ordre de plusieurs centimètres entre les première et deuxième crêtes d'ordre, de sorte que des modification de l'ordre de plusieurs nanomètres des dimensions de la surface active provoquent des déplacements de l'ordre de plusieurs millimètres de la position des crêtes de diffraction qui permettent à des macromolécules recherchées de se fixer sur la surface active de la sonde soumise au procédé de détection. Cette invention est également utile pour détecter des macromolécules biologiques qui sont importantes du point de vue clinique pour diagnostiquer des maladies, et ce directement sans employer de procédures de marquage complexes telles que celles qu'on utilise dans d'autres techniques de diagnostic. La sonde peut également être utilisée en association avec d'autres techniques connues étant donné que la surface active est capable de concentrer fortement les macromolécules par rapport à leur concentration dans la solution.
PCT/US1993/012307 1992-12-16 1993-12-16 Detection de macromolecules WO1994013835A1 (fr)

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GB9226229A GB2273772A (en) 1992-12-16 1992-12-16 Detection of macromolecules utilising light diffraction

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US7815854B2 (en) 2004-04-30 2010-10-19 Kimberly-Clark Worldwide, Inc. Electroluminescent illumination source for optical detection systems
US7829328B2 (en) 2003-04-03 2010-11-09 Kimberly-Clark Worldwide, Inc. Assay devices that utilize hollow particles
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CN102033054A (zh) * 2001-12-11 2011-04-27 金伯利-克拉克环球有限公司 对基于衍射的诊断的结果进行观察和分析的方法
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US7384598B2 (en) 2001-12-21 2008-06-10 Kimberly-Clark Worldwide, Inc. Diagnostic device
US7244393B2 (en) 2001-12-21 2007-07-17 Kimberly-Clark Worldwide, Inc. Diagnostic device and system
US7651841B2 (en) 2001-12-24 2010-01-26 Kimberly-Clark Worldwide, Inc. Polyelectrolytic internal calibration system of a flow-through assay
US7695979B2 (en) 2002-05-03 2010-04-13 Kimberly-Clark Worldwide, Inc. Biomolecule diagnostic devices
US8110349B2 (en) 2002-05-03 2012-02-07 Kimberly-Clark Worldwide, Inc. Method for producing biomolecule diagnostic devices
US7771922B2 (en) 2002-05-03 2010-08-10 Kimberly-Clark Worldwide, Inc. Biomolecule diagnostic device
US7670786B2 (en) 2002-08-27 2010-03-02 Kimberly-Clark Worldwide, Inc. Membrane-based assay devices
US7662643B2 (en) 2002-12-19 2010-02-16 Kimberly-Clark Worldwide, Inc. Reduction of the hook effect in membrane-based assay devices
US8034397B2 (en) 2003-04-03 2011-10-11 Kimberly-Clark Worldwide, Inc. Methods of making assay devices utilizing hollow particles
US7829328B2 (en) 2003-04-03 2010-11-09 Kimberly-Clark Worldwide, Inc. Assay devices that utilize hollow particles
US7851209B2 (en) 2003-04-03 2010-12-14 Kimberly-Clark Worldwide, Inc. Reduction of the hook effect in assay devices
US7943395B2 (en) 2003-11-21 2011-05-17 Kimberly-Clark Worldwide, Inc. Extension of the dynamic detection range of assay devices
US7713748B2 (en) 2003-11-21 2010-05-11 Kimberly-Clark Worldwide, Inc. Method of reducing the sensitivity of assay devices
US7781172B2 (en) 2003-11-21 2010-08-24 Kimberly-Clark Worldwide, Inc. Method for extending the dynamic detection range of assay devices
US7943089B2 (en) 2003-12-19 2011-05-17 Kimberly-Clark Worldwide, Inc. Laminated assay devices
US7815854B2 (en) 2004-04-30 2010-10-19 Kimberly-Clark Worldwide, Inc. Electroluminescent illumination source for optical detection systems
US7796266B2 (en) 2004-04-30 2010-09-14 Kimberly-Clark Worldwide, Inc. Optical detection system using electromagnetic radiation to detect presence or quantity of analyte
US7964340B2 (en) 2004-06-30 2011-06-21 Kimberly-Clark Worldwide, Inc. One-step enzymatic and amine detection technique
US20110136165A1 (en) * 2007-01-22 2011-06-09 Borivoj Vojnovic Detecting objects
US9212985B2 (en) * 2007-01-22 2015-12-15 Isis Innovation Limited Detecting objects
US20160153959A1 (en) * 2007-01-22 2016-06-02 Isis Innovation Limited Method of detecting objects
US9546993B2 (en) * 2007-01-22 2017-01-17 Oxford University Innovation Limited Method of detecting objects
US8883094B2 (en) 2008-01-03 2014-11-11 University Of Central Florida Research Foundation, Inc. Detection of analtyes using metal nanoparticle probes and dynamic light scattering
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