Classifications

• • 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
  • G01N21/21—Polarisation-affecting properties
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
• • 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
  • G01N21/75—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

• the present invention relates to methods and apparatus for detecting, qualitatively and quantitatively, molecular interaction.
• a “probe” is intended to mean a known entity which may either be immobilized or free floating.
• a “target” or “ligand” is an "unknown” entity which has a known, specific complementary or reactive relationship with a specific probe. Probe- target combinations may be referred to as “complexes” or “pairs”.
• a “primaiy amplifier” or “mass enhancing unit” is a “bulky” entity which has an interaction or bonding with a pair, or with a linking element that bonds to the pair.
• a “secondary amplifier” interacts with the primary amplifier or with the combination of primary amplifier-probe-target pair complex to form a new combination.
• a “tertiary amplifier” is any successive mass enhancer which can interact with a primary amplifier-probe-target pair complex or amplifier.
• Bin factor is the ratio of the molecular weight of the amplifier to that of the probe-target pair. However, bulk or mass is not always the primary parameter in enhancing recognition of a probe-target pair. Other parameters, such as optical or electrical properties, may be important to a detection device.
• a reaction takes place on a surface in the evanescent field of a total internal reflecting (TIR) surface.
• TIR total internal reflecting
• the resultant beam is directed to a detector, for example, a CCD chip/camera, which can visualize the entire surface of interest.
• a detector for example, a CCD chip/camera
• the reacted area then provides a feature which differs from the non-reacted areas.
• the specific location of the feature identifies the reaction which produced the feature.
• Detecting the unique feature can present a challenge if the reagents, and the reacting and non-reacting areas are sufficiently small so as to require extremely sensitive detection systems.
• the prior art has relied upon tagging or marking to enable the identification of a reaction.
• the marking or tagging step was unnecessary.
• highly sensitive techniques were required to. detect the reaction and identify the reactants.
• a complex product which can be the result of any reaction (e.g., an antibody in the case of bioactive materials), is used to develop, enhance or "amplify" a probe-target reaction so that the resultant compound can be detected with less sensitive equipment or the detection limit of sensitive equipment may be effectively lowered so that smaller quantities of the target may be detected.
• any reaction e.g., an antibody in the case of bioactive materials
• probe molecules that have an exclusive or specific affinity for a "target” molecule resulting in the formation of a probe-target pair.
• amplifier molecules or compounds that have a specific attraction or affinity for a class of probe-target pairs, or for a particular subset of a class of probe-target pairs.
• Nucleic acid probes can react with complementary RNA or DNA sequences to hybridize, forming a double stranded sequence. Once hybridization has taken place, an antibody specific to lengths of double stranded nucleic acid material is added and incubated. Generally, the antibody will have a molecular weight and bulk many times that of the probe and target, resulting in an enlarged and enhanced probe-target combination that is more readily detected, even with less sensitive optical or other systems.
• bioassays or immunoassays are based on the reaction of a bioactive substance (or "antigen") with a specific complex conjugate of that bioactive substance (or "antibody”). It is believed that the present invention can be extremely useful in the performance of bioassays or immuiioassays. Furthermore, U.S. Pat. No.
• ssDNA single stranded DNA
• a surface can be prepared with a matrix of probes affixed thereto, each with a different sequence. The x-y location in the matrix is known for each probe element.
• the unknown target compound is prepared as ssDNA and is reacted with the probe material. If the probe and target have complementary sequences, there will be a reaction at the site and the two single strand DNA samples will hybridize into a double stranded DNA (“dsDNA”) chain.
• dsDNA double stranded DNA
• the amplifying antibodies are now introduced to the matrix surface.
• the amplifying antibodies are selected for their specificity for dsDNA as determined by the structure 'of their variable region.
• the amplifying antibodies are generally an order of magnitude more massive than the hybridized pair. All antibodies of a particular class, i.e. IgA, IgM, IgG, etc., have different molecular structures and mass and all are relatively bulky.
• the amplifying antibodies will bind to the dsDNA. Under certain conditions, the amplifying antibodies will aggregate or agglomerate, nucleating upon the antibody bound to the probe-target pair, resulting in a structure of massive size and bulk.
• the active Fab fragment or F(ab") 2 fragments may be separated from the immunoglobulin by papain or pepsin cleavage, respectively, providing a smaller, sterically and kinetically favorable recognition unit.
• biotinylation of the antibody fragments would provide for linkage to avidin or avidin-containing bulky compounds.
• secondary amplifying antibodies specific for the primary amplifying antibodies, could be added which will bind to the primary antibodies to create, at a particular x-y location, a larger object, detectable with less sensitive equipment, such as relatively insensitive optics and easily recognized by using a camera which can visualize the entire matrix.
• Such an aggregation or agglomeration could be detected by an atomic force microscope, among other surface techniques, which can determine height profile changes.
• non-human IgG and human immunoglobulins are the probe-target pair, as a means to detect antibodies in human serum, thereby determining if a person has been exposed to a particular antigen. If IgM that is specific for the non-human anti-human- immunoglobulin complex, or even specific just for the human immunoglobulin, is used as the primary amplifying antibody, the bulk of the pair can be increased two-and-a half fold. Yet other probe-target pairs can be identified, with the amplifying antibodies that can be used with such pairs.
• yet other, secondary amplifying antibodies can be employed, with or without markers, that are reactive with either the hybridized pair or with the primary amplifying aiitibodies to increase the detectability of the reacted pair through techniques which utilize separating processes that are based upon mass differentiation.
• the present invention can be utilized with other analytes of interest.
• results can be observed with electron or conventional microscopes, colorimetry, gravimetric analysis, chromatography and spectroscopy. If it is desired to use tags or markers, yet other techniques of the prior art could be employed at much lower levels of sensitivity.
• It is still another object of invention utilize secondary amplifying compounds that can bind to the primary amplifying compounds that are bound to the specific probe-target reactions, or, ideally, the probe-target-primary- amplifier complex.
• FIG. 1 is a schematic representation of a prior art detection scheme relying on a radioactive or fluorescent label
• FIG. 2 is a schematic representation of an exemplary detection technique in accordance with the present invention
• FIG. 3 is a chart correlating size with the various stages taught with reference to an exemplary embodiment of a method and apparatus in accordance with the present invention
• FIGS. 4 AND 5 depict the optical observation via total internal reflection as an aspect of an exemplary embodiment of a method and apparatus in accordance with the present invention
• FIG. 6 is a chart associating layer thickness with various probe- target pairs as an aspect of an exemplary embodiment of a method and apparatus in accordance with the present invention
• FIG. 7 is a table associating layer thickness with various probe-target pairs as an aspect of an exemplary embodiment of a method and apparatus in accordance with the present invention.
• FIG. 8 is a table summarizing exemplary approaches to using the method and apparatus in accordance with the present invention.
• FIG. 1 shows the prevailing prior art sequence in which a target 11 hybridizes with the probe 13 which has been bound to a substrate 15 to form a probe-target pair 17 whose identity is to be ascertained is made detectable. It has been a common practice in the prior art to add a specific linker 19 to which an amplifier 21 which has a tag or label 23 can bind. The presence of the tag or label 23 is usually the result of a separate processing step which may include the addition of the linking ligand upon the target. It is the presence of the tag or label, however, that makes the probe target pair 17 detectable. Equipment which is sensitive to the tag or label 23 allows the target to be uniquely identified.
• FIG. 2 shows for an exemplary embodiment of the method and apparatus in accordance with the present invention a probe element 12 which can be a ssDNA fragment bound to a substrate 10.
• the substrate 10 may be a glass slide.
• dsDNA pair 16 there exist antibodies or primary amplifying bodies 30. These primary amplifying bodies 30 can only bind to the hybridized dsDNA pairs 16 but not to the ssDNA probe 12 or target elements 14.
• FIG. 3 is a progression showing relative size of the various resultant combinations starting with the substrate 10 with an immobilized probe 12.
• the probe-target pair 16 can be measured in fractions of nanometers of film thickness on the surface of the substrate 10.
• the scale increases to the 30 nanometer range, detectable by sensitive systems such as atomic force microscopes, or the techniques of the copending applications of the common assignee. With these values, other "fine" detection systems ' such as spectrometers or gravimetric devices, can find evidence of the probe-target reaction.
• the techniques of the present invention are useful with any probe-target pair for which specific amplifier compounds can be identified. It is believed that the, technique can be applied in any situation involving an unknown material or target at the molecular stage which can react with a probe of comparable size. Amplifying materials can be identified which can bind to the probe-target combination.
• EXAMPLE 1 Oligonucleotide Probe and Target; IgM Primary Amplifying Body
• One area in which the present invention is useful is with the dry state surface scanning detection of an oligonucleotide probe-target pair on an aldehyde derivatized glass substrate.
• the process entails the following steps:
• Tris-EDTA-NaCl buffer (“TE-NaCl buffer”), spot a 30 mer oligonucleotide modified with C6 amino at 3' end to the slide surface to act as a probe. Dry the probe for 12 hours at room temperature (25° C) and ⁇ 30% relative humidity.
• the prepared substrate can then be "read” by an atomic force microscope which will detect the hybridized pair and the associated amplifier.
• Polystyrene microspheres may be obtained from Bangs Laboratories, Inc. (9025 Technology Drive, Fishers, IN 46038-2886; www.bangslabs.com) in derivatized form in sizes from 25nm to lOOOnm, the IgG being supplied by the customer. The IgG is attached via the Fc portion, leaving the variable region exposed and available for coupling to antigens. lOOn spheres are a preferable first choice.
• the microspheres may be dyed to absorb the wavelength of interest if an optical method of probing is desired.
• the microspheres may be coupled to goat anti-mouse-IgM serve as a secondary amplifier for a primary amplifying antibody.
• a preferred method is as follows: Prepare the hybridized surface and add the IgM as set forth in EXAMPLE 1.
• SEQUENCE SEEKERTM commercial sequence-specific polyamide product is used to bind to a double- stranded probe-target pair, streptavidin-coated polystyrene microspheres are preferred (dyed or un-dyed, as needed) (SEQUENCE SEEKERTM, Prolinx, Inc., Bothell, WA; www.prolinx.com) . Prepare the hybridized surface
• polyamides which are commercially known as SEQUENCE SEEKERTM (Prolinx, Inc., Bothell, WA; www.prolinx.com) and which can be conjugated with specific compounds, for example biotin.
• SEQUENCE SEEKERTM is drawn to strands of hybridized DNA and if the sequence contains the right sequence of five base pairs, the polyamide will bind itself to the minor groove of the dsDNA.
• sequence seeker is not the amplifier, but serves as a "recognition unit" similar to the specific peptide structure in the variable region of the antibody, which is responsible for specificity.
• a biotin is bound to the sequence seeker to act as a linking compound with streptavidin or streptavidin-coupled compounds, which may serve as the mass amplifier.
• Some primary amplifying bodies 30 can bind or agglomerate to primary amplifying bodies that have been bound to a probe-target pair.
• secondary amplifying bodies 32 which can bind to the primary amplifying bodies 30 that are bound to a hybridized probe-target pair 16. It is then possible to aggregate or agglomerate a rather massive structure at the site of the probe-target reaction so that there is a single, unique, massive structure in the matrix that can be more readily detectable by less sensitive detectors including, but not limited to, optical, gravimetric, topographical or other mass discriminating techniques and others.
• Adding the secondary amplifying bodies 32 increases the size by a factor of approximately 2.5.
• a preferred method is as follows:
• the streptavidin itself is more massive (68 kilodaltons) than the dsDNA probe-target pair, providing a primary mass amplifying effect.
• Examples of a non immunoglobulin antigens as a targets can include protein arrays having importance to agricultural industry in certifying that processed foods are free of contaminating allergens, GMOs.
• Police and national security agencies may wish to use the present invention to improve existing arrays that are used to detect explosives such as TNT.
• the probe element 12 is part of a matrix of other, different probes (or ssDNA fragments).
• Known matrix arrangements are capable of providing a match to an unknown target probe or ssDNA, the identity of which is to be found by a test.
• a procedure for this exemplary embodiment includes the steps recited for EXAMPLE 1 above for oligo probes, except that there are numerous different probes placed in a multi-well plate (for example, a 396-well plate).
• the plate is placed in a spotting robot, which prints the oligo probes on the slide in an arrayed pattern determined by the software and experimenter.
• the substrate can have total internal reflection (TIR) as taught in the prior patent applications of the common assignee, placing the probe element in the evanescent field.
• TIR total internal reflection
• tertiary amplifiers 34 which bind to the secondary amplifying bodies 32, creating even more massive combinations at the site of the probe-target reaction, lowering the threshold of detection in many detecting schemes.
• the ability to bind a tertiary amplifier 34 to the site would provide an additional twenty-fold size increase from the initial probe-target thickness value.
• the addition of secondary and tertiary amplifying bodies or amplifiers permits the use of "gross" detection systems such as microscopy, filtration or other size exclusion techniques and any mass based separation system.
• Streptavidin is readily available coupled to a variety of materials, such as polystyrene, polyaniline, or metallic microspheres, which are even more massive. Additionally, streptavidin has four binding sites for biotin, allowing biotinylated antibodies or biotinylated microspheres to be attached, serving as the secondary amplifying body, and the streptavidin itself also serves as the specific target for antibodies, which in that case would be the secondary amplifying body.
• FIG. 5 is a bar chart showing relative heights expressed in nanometers (nm). As seen in the chart, the bare aldehyde slide surface has a height of 2.2 nm. With attachment of a 1 ⁇ M DNA probe, the height increases to 2.4 nm. The probe-target pair of the 1 ⁇ M DNA probe and 1 ⁇ M DNA target has a height of 3.9 nm. With the addition of a primary amplifier, 0.1 ⁇ l of antibody, the height increases to 7.2 nm. However, if excess antibody is added as the primary amplifier, the height is increased tenfold to 72 nm. For confirmation of the experiment, the height of a l ⁇ M DNA probe with
• Fig. 4 shows an exemplary embodiment of the present invention in which a total internal reflection (TIR) is used to observe the result of amplification.
• a polarized light source assembly 112 has a light source 126, a beam forming member 128 (if the nature of the light source is such as to make beam forming useful or necessary), a polarizer 130 and an optical retarder 132.
• the total internal light reflection assembly 114 has an optical element 134 which has an optical surface 136. Also shown is a specimen slide 138 on the optical surface 136, and between them an index matching substance 140. Because of the index matching a total internal reflection surface (TIR surface) is defined as the upper surface 139 of the specimen slide 138.
• a specimen 142 is on the TIR surface 139 of the slide 138.
• the optical element 134 is a prism configured along with the index matched slide 138 in relationship to the incoming light beam 120 and the exiting light beam 122 such that the beam reflects only a single time at the TIR surface 139 and then exits the prism. If the specimen is placed directly on the optical surface 136, then the optical surface 136 would be the TIR surface. But this is not the usual application as the specimen (such as a biochip) is usually prepared more conveniently on a specimen slide 138 and placed in the apparatus.
• the post-reflection detector assembly 116 has a polarizer 144 and a two- dimensional array detector 146, preferably a camera of the CCD type.
• the processor 118 is a specially programmed computer and output means for processing the image into a representation of film thickness variations spatially resolved over the area imaged.
• a image is acquired by detecting changes spatially distributed in the local polarization state in the beam's cross-section caused by the total internal reflection. This provides information about the presence and composition in the array of substances on the substrate surface for each resolvable point on the surface. Different polarization state changes are included in the cross-section of the reflected beam indicative of the substances on the specimen in the location in the specimen array corresponding to a position in the detector.
• the processor 118 receives the data as an electrical signal 124 and characterizes the change of polarization state spatially over the two-dimensional array.
• the analysis and processing is done in one embodiment by comparing the known polarization state of the incoming light from the light processing assembly 112 with the changed polarization state of the reflected light 122, spatially resolved two-dimensionally within the beam which provides a map of spatially distributed points of spots in the specimen array.
• the polarization shift is then analyzed by the processor 118 to provide information of the presence and properties of elements in the chemical specimen.
• Other known techniques such as null processing or phase modulation can be used to determine the change in polarization state.
• the light source ember 126 may be a light emitting diode (LED), a superluminescent diode (SLD), an incandescent light source, or a laser. If an LED or SLD is used, the set-up shown in FIG. 4 is appropriate, where the beam forming member 128 is a collimator. if an incandescent light source is used, an optical filter is also used.
• the light source 126 for the apparatus is a quasi- monochromatic light sources of moderate bandwidth.
• the light source 126 is preferably an LED of moderate bandwidth.
• the bandwidth is a full width half maximum wavelength in the range of about 10 nanometers to 50 nanometers, and more preferably a full width half maximum wavelength in the range of about 30 nanometers to 50 nanometers.
• the optical retarder could be placed instead in the exiting beam path 122 before the polarizer 144.
• the optical retarder could be placed instead in the exiting beam path 122 before the polarizer 144.
• FIG. 5 an alternative embodiment is shown,
• the light source is a laser 150
• a moving diffuser 152 is adapted to produce speckle offsetting fluctuation of the minima and maxima in the speckle pattern caused by the laser.
• the moving diffuser 152 is attached to a mechanical actuator 154 which is preferably a moter and servoapparatus for providing the speckle offsetting fluctuations.
• the beam 120 then proceeds through the beam forming element 128, the polarizer 130 and the optical retarder 132, exiting the light source assembly 120.
• metal nanospheres coupled to recognition units, primary amplifiers, or secondary amplifiers could be used.
• streptavidin-coupled gold nanospheres which are in turn coated with colloidal silver are used to amplify biotinylated biomolecules. If biotinylated DNA, RNA, or protein targets are used, enhancement with streptavidin-gold nanoparticles, with or without silver colloid enhancement, will amplify signals when probed by ellipsometry, reflectometry, evanescent .techniques, light microscopy, high resolution scanning, scanning electrochemical probe microscopy, and AFM.
• a general procedure, adaptable to the particular probe-target system b those skilled in the art, is as follows:
• Solution A Dissolve 80 mg silver acetate (code 85140; Fluka, Buchs, Switzerland) in 40 mL of glass double- distilled water. (Silver acetate crystals can be dissolved by continuous stirring within about 15 min.)
• Solution B Dissolve 200 mg hydroquinone in 40 mL citrate buffer.
• a mass amplifier is added to a probe-target pair (with or without a linking element).
• the presence of the mass amplifier permits detection of the probe-target pair.
• Additional mass amplifiers such as secondary and tertiary amplifiers, create opportunities to use less sensitive detecting equipment and/or lower concentrations of the target material.

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• 2003
  • 2003-05-28 CN CNA038268639A patent/CN1823085A/zh active Pending
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