Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-26
2001-09-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C250S559050, C359S368000, C356S300000, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06294327
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of imaging. In particular, the present invention provides a method and apparatus for high resolution imaging of a sample which has been labeled with a material having strong light scattering and/or reflection properties, for example, metal colloid markers. According to the present invention, specific binding analytes are detected by performing imaging using light scattering and reflection illumination.
The present invention is useful in a variety of applications where detection is required. One useful application includes, for example, use with biopolymer arrays. For example, a pioneering technique for creating high density nucleic acid arrays is set forth in U.S. Pat. No. 5,445,934. The present invention is useful in any application in which one can attach a label having strong light scattering and reflection properties to a molecule of interest.
BACKGROUND OF THE INVENTION
The present invention provides a method and instrument for analyzing a sample, such as polymer assays. Examples of such a polymer assay include nucleic acid arrays, protein or polypeptide arrays, carbohydrate arrays, and the like. In addition, the present invention can be used both with samples that are immobilized and in solution. Any number of possible samples can be used with the present invention. Various types of scanners have been used to extract information from a sample. For example, previous instruments for reading samples have employed detection schemes that are responsive to fluorescence in order to reveal specific interactions or hybridizations.
Rather than using fluorescent labeling, it is known to use a solution of particles which scatter light effectively to label nucleic acid arrays. For example, a solution of metal particles, called a metal colloid, could be used. Any other particle which scatters light can also be used to label a sample. More specifically, it is known to detect one or more components of the reaction between a specific binding protein and the corresponding bindable substance, in which one or more labeled components are used, that are obtained by coupling particles of a dispersion of a metal, metal compound or polymer nuclei, as disclosed in U.S. Pat. No. 4,313,734 entitled “Metal Sol Particle Immunoassay.”
Further, it is possible to employ a detection method using a two dimensional optical wave guide which allows measurement of real time binding or melting of a light scattering and reflection label at capture zones on a DNA array, as described in “Real Time Detection of DNA Hybridization and Melting on Oligonucleotide Arrays using Optical Wave Guides” by Don I. Stimpson, Joanell V. Hoijer, WangTing Hsieh, Cynthia Jou, Julian Gordon, Tom Theriault, Ron Gamble and John Baldeschwieler.
The above-described document employs a technique for detecting specific binding analytes typically employing a scanning technique that relies on total internal reflectance. This technique is also known in the art as evanescent wave detection. For example, referring to
FIG. 1
, a cross-section of a transparent array substrate surface of the base of a nucleic acid array is shown. Accordingly, to achieve total internal reflection from the interface of the glass and an aqueous buffer used in the nucleic acid array, the internal incidence angle of light from the scanner must approach 90 degrees. Because the illuminating rays bend toward normal incidence when entering the dense glass chip from air, it is not possible to achieve such a shallow internal incidence angle by simply illuminating nearly parallel to the plane of the transparent array substrate.
With total internal reflectance technology, it is possible to illuminate the sample through the edge of the transparent array substrate. However, this approach is cumbersome and expensive. Moreover, although it may be possible to illuminate the edge of the transparent array substrate with a sample residing in a plastic cartridge, such an arrangement would require that one edge face of the substrate be of fairly high optical quality. This would result in higher packaging costs.
Another possible solution which will allow the use of total internal reflection techniques for reading genetic information from nucleic acid arrays involves the use of a coupling prism which is affixed near the edge of the planar surface. Such a coupling prism allows the illumination to enter the dense transparent array substrate at an angle nearer to normal incidence. Total internal reflection techniques employing a coupling prism require that space be provided for the coupling prism thereby precluding space for probes.
Although total internal reflection techniques may be used with samples in which washing reduces the concentration of residual labels to practically undetectable levels, in such applications, total internal reflection techniques generate undesired background scattering from both the glass/aqueous interface and the glass/air interface.
In addition, other known techniques for labeling with scattering labels tend to bind or react at inappropriate places on the nucleic acid array. For example, metal colloids have been used in blot assays, for example, home pregnancy test kits. Generally, such kits use a colorimetric assay in which colloid agglutination occurs on a white substrate. Test results are determined by light attenuation by the metal colloid which introduces a color.
There exists a need for an apparatus and method for imaging samples which have been labeled with a scattering label having a high scattering signature.
SUMMARY OF THE INVENTION
The present invention is directed to a scanner instrument and method for scanning a sample such as a nucleic acid array by using a novel light scattering and reflection technique. In particular, the combined use of reflection imaging and diffuse scatter imaging has been found to maximize dynamic range and detection limits for samples labeled with scattering labels and bound to nucleic acid arrays. The novel light scattering and reflection technique may be used successfully in various applications because such applications employ a transparent array substrate which exhibits particular optical characteristics, as described below. The sample according to the present invention has been labeled with a scattering label having a strong light scattering and reflection properties. One example of such a scattering label is a metal colloid but the present invention is not limited to the use of a metal colloid and in fact any material with strong light scattering and reflection properties may be used. The present invention does not rely on evanescent wave or total internal reflection techniques. The light scattering and reflection illumination technique of the present invention provides superior optical results to previous methods but at significantly lower cost.
According to a preferred embodiment of the present invention, a light scattering and reflection illumination technique is used for detecting genetic information on a sample in which only reflection mode imaging is used. In another embodiment of the present invention, a sample is imaged through a novel combination of reflection mode imaging and scatter mode imaging. The external incidence angle of light used is typically in a range of angles from near zero to over 45 degrees as measured with respect to the surface normal but any angles may be used.
In addition, an instrument is disclosed which provides imaging according to the novel illumination and collection technique described above.
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EPO Search Authority International Search Report dated Sep. 1997 for PCT/US98/18541.
Rava Richard P.
Trulson Mark O.
Walton Ian D.
Affymetrix Inc.
Banner & Witcoff , Ltd.
Horlick Kenneth R.
Siew Jeffrey
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