Patent Application
20120231453
The present invention relates to the art of microstructures, and more particularly to encoded microparticles and biological assays which can be performed employing the encoded microparticles.
Microparticles or nanoparticles are often referred to as structures whose characteristic dimensions are on the order of micrometers or less, such as those with volumes of 1 mm3 or less. Due to their unique properties arising from their small characteristic dimensions, microparticles have found distinguishable applications in laboratory research and many industrial fields. Encoded microparticles possess a means of identification and are an important subclass of the general field of microparticles. Because encoded particles carry information and can be physically tracked in space and time, they greatly extend the capabilities of non-encoded particles. A particularly important application for encoded microparticles is multiplexed bioassays, including those involving DNA and proteins. Other important fields for encoded microparticles include combinatorial chemistry, tagging, etc. Many biochemical and non-biochemical applications as will be discussed herein below.
For many applications, one more desirable attributes include: a large number of identifiable codes (i.e. a high codespace), accurate and reliable identification of the encoded particles, material compatibility for a particular application, low cost manufacturing of the microparticles (on a per batch, per particle, and per code set basis), and flexibility in the detection systems.
Several approaches to produce encoded microparticles have been developed in the past, such as fragmented colored laminates, colored polystyrene beads, quantum dot loaded polymer beads, rare-earth doped glass microbarcodes, electroplated metal nano rods, diffraction grating based fiber particles, and pattern bars and disks, and other types of microparticles. These technologies however suffer from any of a number of limitations, such as, insufficient codespace, high cost, inadequate precision, poor performance in applications, problematic clumping incapability of large scale manufacture, and complicated preprocessing or assay procedures.
Therefore, what is desired is an encoded microparticle or a set of encoded microparticles carrying coded information, methods of making the same, methods for providing the codes for microparticles, methods for fabricating the microparticles, methods and systems for detecting microparticle, and methods and systems for using.
As an example of the invention, an encoded microparticle and methods for using and making are provided. In one example, a method for detecting an analyte in a test fluid includes providing a set of biochemically active microparticles, each microparticle comprising a spatial code; wherein a layer of the microparticles is arranged on a surface during analysis, detecting electromagnetic radiation from the microparticles in order to detect the spatial codes of the individual microparticles; and wherein the microparticles undergo Brownian motion during the detection of the spatial codes.
Embodiments wherein the encoded microparticles are employed in many different biological assays are contemplated herein. For instance, while the concept of polymerase chain reaction (PCR) has been in the field for several decades and has now grown to include quantitative PCR (qPCR). Disclosed are methods of multiple qPCR utilizing the encoded microparticles.
Embodiments are also disclosed which employ the present encoded microparticles in multiplex chromosome immunoprecipitation (ChIP) assays. The technique of ChIP is often used to investigate interactions between proteins and DNA in the cell, especially for instance, the binding of transcription factors to specialized binding elements or canonical DNA binding sequences, i.e. cross-linked ChIP (XChIP). In a typical ChIP assay, one may isolate the protein-bound oligonucleotides and then determine the isolated oligonucleotides sequences by microarray analysis, i.e. ChIP on chip, or on PCR, qPCR or other sequencing technique. The multiplex-ability of the present encoded microparticles, combined with new signal amplification techniques, allows detection of these isolated oligonucleotides on a single molecule level using encoded microparticles and bDNA technology.
While the appended claims set forth the features of the present invention with particularity, the invention, together with its objects and advantages, may be best understood from the following detailed description taken in conjunction with the accompanying drawings of which:
b is a side view cross-section of the microparticle in
a schematically illustrates another example encoded microparticle of the invention;
b schematically illustrates an another example encoded microparticle of the invention;
a and
d is a cross-sectional view of another exemplary microparticle during an exemplary fabrication of the invention;
a to
a to
a and
a and
a to
a shows a montage of 12 dense reflectance images of encoded microparticles;
b shows a transmission fluorescence microscope image of example microparticles of the invention;
a to 27c show schematic diagrams of encoded microparticles of the present invention with surface indentations that form a spatial code;
d shows an example of encoded microparticles comprising indentations;
a to 28c show the non-uniform aerial density measured normal to the particle surface for corresponding particles in
a to
a to
a illustrates an exemplary assay in which the microparticles of the invention can be used;
b illustrates another exemplary assay in which the microparticles of the invention can be used;
An encoded microparticle is provided carrying a code, and a set of encoded microparticles are provided with distinguishable codes, wherein the codes comply with a pre-determined coding scheme. Preferably, the microparticles in the examples below have a volume of 1 mm3 or less. The microparticle of the invention enables fast, precise and less complicated detection of the code. Methods for providing the codes on microparticles, methods for fabricating the microparticles, methods and systems for detecting the microparticle, and methods and systems for using the microparticles are also disclosed.
In the following, the invention will be discussed with reference to specific examples. It will be appreciated by those skilled in art that the following discussion is for demonstration purposes, and should not be interpreted as a limitation. Instead, other variations without departing from the spirit of the invention are also applicable.
As an example,
The microparticle in this particular example has a set of segments (e.g. segment 102) and gaps (e.g. gap 104) intervening the segments. Specifically, segments with different lengths (the dimension along the length of the microparticle, e.g. along the Y direction) represent different coding elements; whereas gaps preferably have the same length for differentiating the segments during detection of the microparticles. The segments of the microparticle in this example are fully enclosed within the microparticle, for example within body 106. As an alternative feature, the segments can be arranged such that the geometric centers of the segments are aligned to the geometric central axis of the elongated microparticle. A particular sequence of segments and gaps represents a code. The codes are derived from a pre-determined coding scheme.
Segments of the microparticle can be any suitable form. In an example of the invention, each segment of the microparticle has a substantially square cross-section (i.e. the cross-section in the X-Z plane of a Cartesian coordinate as shown in
The microparticle example of
The code elements, i.e. the segments and gaps, may take any desired dimensions. As an example of the invention, each coding structure has a characteristic dimension that is 5 μm (microns) or less, such as 3 microns or less, and more preferably 1 micron or less, such as 0.8 or 0.5 microns or less. In particular, when gaps are kept substantially the same dimension while the segments vary in dimension, each gap preferably has a characteristic dimension that is 1.5 microns or less, such as 0.8 or 0.5 microns or less.
As one example, if forming the microparticles on a 12-inch silicon wafer with 0.13 line widths, the gap areas can be made to have 0.13 μm minimum widths, with the less transparent segments having widths of from 0.13 μm to much larger (depending upon the desired length of the particle and the encoding scheme and code space desired). Minimum gap widths, as well as minimum segment widths, of from 0.13 to 1.85 μm (e.g. from 0.25 to 0.85 μm) are possible depending upon the wafer fabrication used. Of course larger minimum gap and segment lengths (e.g. 1.85 to 5.0 μm, or more) are also possible. Other sized wafers (4 inch, 6 inch, 8 inch etc.) can of course be used, as well as wafers other than silicon (e.g. glass), as well as other substrates other than silicon (larger glass panels, for example).
Though the microparticle may have the same length in the X, Y and/or Z directions, preferably the encoded microparticle has a ratio of the length to width of from 2:1 to 50:1, e.g. from 4:1 to 20:1. In an example of the invention, the microparticle has a length (e.g. the dimension along the Y direction) of 70 microns or less, 50 microns or less, 30 microns or less, such as 20 microns or less, 16 microns or less, or even 10 microns or less. The width (e.g. the dimension along the X direction), as well as the height (the dimension along the Z direction), of the microparticle can be 15 microns or less, 10 microns or less, 8 microns or less, 4 microns or less, or even 1 microns or less, such as 0.13 micron. Widths as small as from 0.5 to 2 microns are also possible. Other than the shape as shown in
The coding structures and gaps of the microparticles can take any suitable form as long as the coding structures and gaps together represent detectable codes. As mentioned above, the cross-section of the microparticles, as taken perpendicular to the length of the particle, can be square, rectangular, circular, elliptical, or any desired shape such as jagged or curved shapes or other profiles. When the cross-section is rectangular, the rectangle preferably has an aspect ratio (the ratio of the length to the width or height) of 2:1 or higher, such as 4:1 or higher, 10:1 or higher, 20:1 or higher, or even 100:1 or higher, but preferably less than 500:1. The ratio of the width to height can be around 1:1 (square cross section), or have a ratio of from 1:4 to 1:1 preferably a ratio that allows the particle to rest on either the sides defining the width or height of the particle such that the code of the microparticle can be detected regardless of which of the elongated sides the particle rests.
To facilitate fast, cost-effective, reliable, and easy detection of the code represented by the coding structures and gaps, it is preferred that each coding structure is as omni-directional as possible to the detection means. That is—each coding structure exhibits substantially the same geometric appearance or detectable properties when observed from at least two directions, more preferably from four (or all, if not four-sided in cross section) directions perpendicular to the length of the microparticle. Accordingly, the coding structures preferably possess rotational symmetry along the length of the microparticle, such as 2-folded or 4-folded rotational symmetry.
A microparticle of the invention can have any suitable number of coding structures depending upon the shape or length of the particle, and the code space desired. Specifically, the total number of coding structures of a microparticle can be from 1 to 20, or more typically from 3 to 15, and more typically from 3 to 8.
The desired code can be incorporated in and represented by the microparticle in many ways. As an example, the coding elements of the pre-determined coding scheme can be represented by the segment(s)—e.g. segments of different lengths represent different coding elements of the coding scheme. Different spatial arrangements of the segments with the different (or the same) lengths and intervened by gaps represent different codes. In this code-incorporation method, the intervening gaps preferably have substantially the same dimension, especially the length in the direction to which the segments are aligned. As another example, the codes are incorporated in the microparticle by arranging gaps that vary in lengths; while the segments have substantially the same dimension and are disposed between adjacent gaps. In another example, the both segments and gaps vary in their dimensions so as to represent a code. In fact, the code can also be represented in many other alternative ways using the segments, gaps, and the combination thereof.
For representing a code derived from the predetermined coding scheme, the segments and gaps are arranged along the length (the Y direction) of the elongated microparticle (2D, or even 3D, arrangements however are also possible). Specifically, the segments and gaps are alternately aligned along the length with the each segment being separated (possibly fully separated and isolated) by adjacent gaps; and each gap is separated (possibly fully separated and isolated) by adjacent segments, which is better illustrated in a cross-sectional view in
In an example of the invention, any suitable number of segments can be used—e.g. from 2 to 20, or more typically from 3 to 15 segments (more typically from 3 to 8 segments) of less transparent material (as compared to the intervening gaps between the segments) are provided within the encoded microparticle. To form the code, it is possible that the segments of less transparent material are varying lengths. Alternatively, the segments of less transparent material could each have substantially the same length whereas the intermediate segments of more transparent material could have varying lengths. Of course, the segments of more transparent material and the intermediate segments of less transparent material could both have varying lengths in order to represent the code.
Referring to
In order to enable detection of codes incorporated in microparticles, the segments and gaps in each microparticle can be composed of materials of different optical, electrical, magnetic, fluid dynamic, or other desired properties that are compatible with the desired detection methods. In one example the segments and gaps are directly spatially distinguishable under transmitted and/or reflected light in the visible spectrum. For example, when the code detection relies upon optical imaging, the distinguishable property (segments vs. gaps) can be a difference in transmissivity to the particular light used for imaging (which can be any desired electromagnetic radiation—e.g. visible and near-visible light, IR, and ultra-violet light. The segments can be made to be more light absorbing (or light reflecting) than the intervening spacing material (or vice versa). When the code detection relies upon the electrical property measurements, the property can be resistance and conductance. When the code detection involves magnetic methods, the properties can be inductance and electro-inductance. When the code detection involves fluid dynamic methods, the property can be viscosity to the specific fluid used in the code detection. Regardless of which specific property is relied upon, the segments and gaps are preferred to exhibit sufficient difference in the specific property such that the difference is detectable using the corresponding code detection method. In particular, when the code is to be detected by means of optical imaging, the segments and gaps are composed of materials exhibiting different transmissivity (in an optical transmittance mode) or reflectivity (in optical reflectance mode) to the specific light used in imaging the microparticles. For example, the segments of the microparticle of the less transparent material can block and/or reflect 30% or more, preferably 50% or more, or e.g. 80% or more, of the visible light or near visible light incident thereon.
Given the fact that transmissivity of electromagnetic radiation through an object varies with the thickness of the object, it is preferred that the segments that are capable of blocking and/or reflecting 30% or more, preferably 50% or more, or e.g. 80% or more (or even 90% or more), of the detection light; while the gaps between the coding structures are provided from materials and at dimensions that are capable of transmitting 50% or more, 70% or more, 80% or more, or even 90% or more of the detecting light. Alternatively, the segments and gaps are composed of different materials such that the ratio of the transmissivity difference is sufficient to detect the codeγ, e.g. is 5% or more, 10% or more, 20% or more, 50% or more, and 70% or more. The transmissivity is defined as the ratio of the light intensities of the passed light to the incident light.
The microstructure can be made of organic and/or inorganic materials or a hybrid of organic and inorganic material. Specifically, the gaps (which are preferably more transmissive to visible or near-visible light) and segments (which are preferably less transmissive to visible or near-visible light as compared to gaps) each can be composed organic or inorganic materials, or a hybrid organic-inorganic material. The segments can be composed of a metal (e.g. aluminum), an early transition metal (e.g. tungsten, chromium, titanium, tantalum or molybdenum), or a metalloid (e.g. silicon or germanium), or combinations (or nitrides, oxides and/or carbides) thereof. In particular, the segments can be composed of a ceramic compound, such as a compound that comprises an oxide of a metalloid or early transition metal, a nitride of a metalloid or early transition metal, or a carbide of a metalloid or early transition metal. Early transition metals are those from columns 3b (Sc, Y, Lu, Lr), 4b (Ti, Zr, Hf, Rf), 5b (V, Nb, Ta, Db), 6b (Cr, Mo, W, Sg) and 7b (Mn, Tc, Re, Bh) of the periodic table. However, preferred are early transition metals in columns 4b to 6b, in particular tungsten, titanium, zirconium, hafnium, niobium, tantalum, vanadium and chromium.
The gaps which are in this example more transparent, can comprise any suitable material that is more transparent than the segments. The spacing material can be a siloxane, siloxene or silsesquioxane material, among others, if a hybrid material is selected. The spacing material, if inorganic, can be a glass material. Thin film deposited silicon dioxide is a suitable material, with or without boron or phosphorous doping/alloying agents. Other inorganic glass materials are also suitable such as silicon nitride, silicon oxynitride, germanium oxide, germanium oxynitride, germanium-silicon-oxynitride, or various transition metal oxides for example. A spin on glass (SOG) could also be used. If an organic material is used for the gap material, a plastic (e.g. polystyrene or latex for example) could be used.
Both the segments and the gaps can be deposited by any suitable methods such as CVD (chemical vapor deposition), PVD (physical vapor deposition), spin-on, sol gel, etc. If a CVD deposition method is used, the CVD could be LPCVD (low pressure chemical vapor deposition), PECVD (plasma enhanced chemical vapor deposition), APCVD (atmospheric pressure chemical vapor deposition), SACVD (sub atmospheric chemical vapor deposition), etc. If a PVD method is used, sputtering or reactive sputtering are possible depending upon the desired final material. Spin on material (SOG or hybrid organic-inorganic siloxane materials
As a more specific example, the segments can be comprised of a any suitable silicon material such as CVD (chemical vapor deposition) deposited amorphous silicon. Polysilicon or single crystal silicon area also suitable as are a wide range of other materials as mentioned above. It is preferred, but not necessary, that the material selected for the segments has a high degree of deposition thickness control, low surface roughness, control of etching—both patterning and release (e.g. using a dry plasma etch for patterning and a wet or dry chemical etch for release), and CMOS process compatibility. The gap material can be CVD deposited silicon dioxide. The silicon dioxide may include doping/alloying materials such as phosphorous or boron. Temperature considerations may be taken into account in choosing a combination of more and less transparent materials for the segments and gaps.
a schematically illustrates another example encoded microparticle of the invention. Referring to
b schematically illustrates an another example encoded microparticle of the invention. Referring to
In the examples as discussed above, the microparticle is composed of materials of selected distinguishable properties, such as distinguishable optical properties. In the example above, one material has a greater transparency or optical transmissivity than the other material, which difference is detectable under magnification. A specific example of the above is where one material is a light absorbing material, and the other material is a translucent or transparent material with greater light transmittance in the visible spectrum (or in another spectrum should a different detection system be used—e.g. UV, IR etc). In another example, one material is a light reflecting material whereas the other material is either light absorbing or light transmitting. A detectable difference where one material is more opaque and the other material is less opaque, or where one material is more reflective and the other material is less reflective, are within the scope of this example. As mentioned above, the alternating portions of opaque and transparent materials can be made of silicon and glass among other materials. Given the fact that transmissivity (and reflectivity) of almost all materials exhibit dependencies from the thickness of the material, the microparticle may be formed such that the coding structures (i.e. the structures representing coding elements of a code) are derived from a single material.
Referring to
For facilitating the application of the microparticles, especially biological/biochemical/biomedical/biotechnology applications wherein the sample bio-molecules are to be attached to the surfaces of the microparticles, an immobilization layer may be desired to be coated on the surfaces of the microstructures.
b schematically illustrates a transmissive-mode image of the microparticle in
The microparticle of
A coding structure layer is deposited and patterned so as to form the coding structures, such as structures 218, 222, 220, 224. After forming the coding structures, surrounding layer 224 is deposited on the formed coding structures. Because the surrounding layer will be exposed to the target sample in the assay, it is desired that layer 224 is composed of a material that is resistant to chemical components in the assay solution wherein the microparticles are to be dispensed. Moreover, for holding the probe molecules, such as nucleic acids (e.g. DNA or RNA), proteins, antibodies, enzymes, drugs, receptors, or ligands, molecules on the surface of the layer, layer 224 is desired to be capable of immobilizing the probe molecules.
The following exemplary fabrication processes will be discussed in reference to microparticles with segments and gaps, however it should be noted that the following methods are applicable to many other types of code elements.
The microstructure of the invention can be fabricated with a method that fall into the broad field of micro-machining, such as MEMS fabrication methods. MEMS use the techniques of the semiconductor industry to form microscale structures for a wide variety of applications. MEMS techniques typically, but not in all circumstances, include the deposition of thin films, etching using dry and/or wet methods, and lithography for pattern formation. Because MEMS is an offshoot of the semiconductor industry, a vast worldwide manufacturing infrastructure is in place for cost-effective, high volume, precision production. Generally speaking, the more similar the full MEMS process is to existing integrated circuit processes, e.g. CMOS compatible, the more accessible this infrastructure is.
The microstructure of the invention can be fabricated in many ways, such as fabrication methods used for integrated circuits (e.g. interconnects) or MEMS. In the following, an exemplary fabrication method compatible with the MEMS fabrication for making a microparticle will be discussed with reference to
Referring to
Referring to
The patterning of the layers can be done in many methods, one of which is photolithography that is widely used in standard fabrication for semiconductor integrated circuits and MEMS devices. The most common form of photolithography used in the MEMS industry is contact photolithography. A reticle (aka mask) is typically composed of a binary chrome pattern on a glass plate. The reticle is placed very near or in contact with a photoresist covered wafer (or other substrate). UV light is shone through the mask, exposing the photoresist. The wafer is then developed, removing the photoresist in the exposed regions (for positive-tone photoresist). The pattern on the reticle is thus transferred to the photoresist where it serves as a mask for a subsequent etching step.
Projection photolithography is another type of photolithography that is used exclusively in modern integrated circuit manufacturing. Instead of bringing the mask into physical contact, projection photolithography uses a system of lenses to focus the mask pattern onto the wafer. The primary advantage of this system is the ability to shrink the mask pattern through the projection optics. A typical system has a five times reduction factor. In general, much smaller feature sizes can be printed with projection as compared to contact lithography. A projection photolithography system is also known as a step-and-repeat system (or stepper for short). The maximum pattern or field size on the mask is significantly smaller than the wafer diameter. The mask pattern is repeatedly exposed (“stepped”) on the wafer forming an array of “dies”. The stepping distance is the distance the wafer stage travels in X and Y between exposures and is usually equal to the die size. This typical scheme produces a non-overlapping array of identical dies, allowing for subsequent parallel processing of the dies on the wafer.
The hard mask layer (150) is further patterned so as to form discrete areas, as shown in
In the example above, the patterning of the hard mask layer is performed in two separate lithography steps. In an alternative example, the reticle may comprise a pattern such that the patterning of the hard mask can be accomplished with a single lithography step. As a further alternative, the hard mask can be omitted and either a two step or single step lithography process used.
After patterning the top hard mask layer, silicon layer 148 is etched so as to form corresponding discrete silicon areas on the substrate, such as silicon segments 168 and 172, with areas there between for material of greater transparency (e.g. gap areas 170 and 172, as shown in
After patterning silicon layer 148, transmissive layer 168 is then deposited as shown in
The microparticles are then separated from each other, while still attached to the underlying substrate, as shown in
The indentations are as a result of the particular fabrication method; and can remain in the final product, or can be removed by, for example, planarization—e.g. chemical-mechanical-polishing (CMP) techniques. In fact, the indentations in some situations can be beneficial for code detection and/or fluorescence quantitation using fluorescent methods because the binding of a fluorescently tagged material to the surface of the microbarcode is greater in the indentation areas (per unit length of the microbarcode), the so called indentation signal enhancement, fluorescence can be greater in the indentation areas and can be used to determine the code (with or without other transmissive or reflective techniques discussed herein below). The same indentation signal enhancement would be applicable with reporter systems other than fluorescence, e.g. radioactive reporters, etc.
Though a silicon wafer was mentioned as the substrate in the example given above, a glass substrate, such as a glass wafer or larger glass sheet or panel (e.g. like those used in the flat panel display industry) could be used. Glass (or silicon) wafers can be of any suitable size e.g. 4 in., 6 in., 8 in. or 12 in. When a glass wafer is used, typically an additional sacrificial layer will first be deposited (for later removal during the release step). The sacrificial layer can be semiconductor material, such as silicon, an early transition metal, such as titanium, chromium, tungsten, molybdenum, etc. or a polymer, such as photoresist, as mentioned earlier herein.
A scanning-electron-microscopy (SEM) image of a segment (e.g. segment 102) in
An SEM image of a multiplicity of microparticles fabricated with the exemplary fabrication method as discussed above is presented in
The microparticles of the invention can be fabricated at the wafer-level, and released either at the wafer level or die level. Specifically, a plurality of dies each comprising a set of microparticles can be formed on a wafer. The microparticles on each die may or may not be the same—that is the microparticles on each die may or may not have the same code. After forming the microparticles, the dies can be separated from the wafer; and the wafer(s) on the singulated dies can be then removed. An exemplary wafer-level fabrication method is demonstrated in
Referring to
After the formation of the microparticles, the wafer is then broken into dies as shown in
The releasing step can be performed in many ways, such as dry etch, wet etch, and downstream plasma etch. In an exemplary bulk wet etch, shown schematically in
After pelleting the particles through centrifugation or lapse of time, the liquid (so called supernatant) is removed and the particles are washed several times in water or a solvent. “Washing” refers to the successive replacement of the supernatant with a new liquid, usually one involved in the next chemical processing step. After detaching the microparticles from the substrate (or wafer), the substrate can be removed from etchant—leaving the microparticles in tubes. The released microparticles can then be transferred to containers for use.
The microparticles can be fabricated on the wafer level, as shown in
The tendency of the microparticles to form a monolayer is not trivial. Monolayer formation involves many factors, such as the surface charge state (or zeta potential) of the microparticles, the density of microparticles in a specific solution, the fluid in which microparticles are contained, and the surface onto which the microparticles are disposed. Accordingly, the microparticles of the invention are comprised of materials and are constructed in a form that favors the maintenance of a charged state sufficient to substantially overcome stiction forces; and thus microparticles are capable of undergoing Brownian motion which facilitates the formation of a reasonably dense monolayer of particles.
In biological applications, the microparticles are often used to carry biochemical probe molecules. For immobilizing such probe molecules, the microstructure preferably comprises a surface layer, such as a silicon dioxide layer, which can be chemically modified to attach to the probe molecules. In accordance with an example of the invention, the microparticles are constructed such that the microparticles are capable of forming a monolayer, for example, at the bottom of a well containing a liquid; and the monolayer comprises 500 or more particles per square millimeter, more preferably 1,000 or more, 2,000 or more, or 3,000 or more microparticles per square millimeter. In an alternative example, the microparticles can form a monolayer that such that the detectable particles occupy 30% or more, 50% or more, or 70% or more of the total image area (i.e. the image field of view). In connection with the example mechanism of self-assembled monolayer formation, it is preferred that the 2D diffusion coefficient of the microparticles of the invention is greater than 1×10−12 cm2/s. For accommodating the monolayer of the microparticles, the container for holding the microparticles in detection preferably has a substantially flat bottom portion.
The system depicted in
a shows a montage of 12 dense reflectance images of encoded microparticles. Approximately 6,000 particles are in the images. The particles are a small fraction of the approximately 200,000 particles total in a well of a 384 wellplate. The total particles are approximately 10% of a set that contains 1035 codes (batches). The set was formed by combining approximately 2,000 particles from each of the 1035 batches where each batch contained approximately 2 million particles of a single code. These images are a subset of a larger image set from which data regarding identification accuracy is presented below.
b shows a transmission fluorescence microscope image of example microparticles of the invention. Shown are here, in addition, small, elongated, encoded microparticles with an outer surface that is entirely glass. Shown are a multiplicity of non-spherical encoded particles with a silica (e.g. glass or silicon dioxide) outer surface and a length less than 70 μm (e.g. less than 50 um.). The length of the example particles in this particular example is 15 um.
In this image, the particles are in a solution that contains suspended fluorescent molecules. The fluorescent molecules, when excited by the microscope light source, provide illumination from above (i.e. behind with respect to the collection optics, see
For successfully identifying the microparticles, e.g. reading the codes incorporated therein, the images of the microparticles may be processed. Such image processing can be performed with the aid of software programs. According to exemplary examples of software programs and algorithms, pairs of raw and processed image are presented in
Referring to
Table 3 shows identification data for image sets that include those images shown in
The microparticles included in Table 3 have a codespace of 30,069, wherein the codespace is defined as the total number of possible codes with the particular particle design, i.e. with the chosen coding scheme and coding scheme parameters. A pre-determined identification method assigns one of the 30,069 possible codes based on the analysis of the particle segment information. 1035 codes were randomly selected, manufactured, and mixed to form the collection. When analyzing the identification of the collection, if the software assigned code is one of the 1035, it is assumed to be correct. The number of “correctly” identified particles divided by the total is called the “ID %”. This assumption underestimates the error rate (1−ID %) by the probability that a random error falls within the 1035 present codes, or 1035 divided by 30,069=about 3%. The assumption therefore ignores this 3% deviation and provides a close approximation to the true identification accuracy.
In an alternate example, a flow-cell enabling the microparticles flowing in a fluid can be provided for detection by continuous imaging, as shown in
Another alternative microparticle of the invention is schematically illustrated in
In another example, the fluorescent layer may be comprised of fluorophores, or other luminescent materials. The fluorescent layer may interact with molecular species in an assay, for example with fluorescently labeled nucleic acids or protein samples via Fluorescence Resonant Energy Transfer processes. In yet another example, the microparticles may have a non-fluorescent layer, wherein incorporated in or on the layer are molecules, for example quenchers that interact with luminescent emitter molecules.
a to 27c show schematic diagrams of encoded microparticles of the present invention with surface indentations that form a spatial code. The microparticle may be fabricated by many methods including the aforementioned examples.
In exemplary examples of encoded microparticles comprising indentations, the surface of the particles have fluorescent, or otherwise emitting, molecules attached to or in the surface, as shown in
The invented general method of generating the codes on microparticles consists of the use of multiple lithographic printing steps of a single code element per particle region. The multiple printing steps create multiple code elements per particle region. The code elements taken together form the code for the microparticle. In a preferred example, the printing steps are performed on many particles in parallel using a master pattern. A master pattern comprises an array of single code elements per particle region. A code element may represent more than one physical feature, such as holes, stripes, or gaps. The master pattern is printed multiple times such that a multiplicity of microparticles with complete codes is formed, wherein the multiplicity of microparticles comprises identical particles (e.g. all particles have the same code). Variations upon this theme, for example wherein the multiplicity of microparticles are not identical, are anticipated and will be described in detail below. Between multiple print steps, a component of the overall printing system changes to translate the code element within the particle region. In a most preferred example, this change is a movement of the substrate on which the particles are formed. In another preferred example, this change is the movement of the master pattern. In yet other examples this change is the movement of an optical element such as a mirror.
An exemplary example of the general method of generating code using multiple print steps involves photolithography as the printing mechanism, e.g. contact photolithography and projection photolithography. An exemplary example of projection photolithographic utilizes a step and repeat system (aka stepper). A reticle contains a code pattern that has a single code element per particle. Through multiple exposures of this code pattern at different lateral offsets, a multiplicity of code elements (per particle) is created. Combined, these code elements form a complete code. The lateral offsets define the code and are programmed into the stepper software. The offsets, and therefore the code, can be changed on a per die or per wafer basis. The codes printed on different dies on a wafer and/or different wafers in a lot are thus controlled by software and can be arbitrarily changed. This enables a powerful flexibility in the manufacture of large sets of codes. A single mask set, having one to a few masks, can be used to generate an arbitrary number of codes, numbering into the 105 range and beyond.
The code pattern, shown in
An exemplary example of the invented method for producing codes uses photolithography and positive-tone photoresit. Positive-tone means that the areas exposed to light are developed away. For a negative-tone resist, exposed regions are what remain after development. The photocurable epoxy SU-8 is an example of a negative-tone resist. In an alternate example using a negative-tone resist such as SU-8, the regions that are to be segments are exposed to light instead of the regions that are to be gaps.
An alternate example of the general method of generating code using multiple print steps utilizes stamping (aka imprint lithography) as the printing mechanism, and is schematically depicted in
In addition to the microparticle as illustrated in
Referring to
Another example of a system for organizing the multi print method using a stepper is to exposure all of the code elements within a single die before moving on to the next die. Of course, a number of offsets other than four could be used. Though this and other examples of the general method of producing codes on microparticles has been described with respect to using a projection photolithography and a stepper, contact lithography and other patterning methods may also be used.
The invented method of producing codes, for example the use of a photolithographic step and repeat system to form a complete code through multiple exposure steps of a single reticle field, may be used to apply unique codes to many types of components, e.g. MEMS and IC devices.
The microparticles as discussed above have incorporated therein codes derived from any desired coding scheme, such as binary or non-binary coding.
By way of example,
In an exemplary example, the coding scheme utilizes code elements placed at locations spanned by interval lengths smaller than the code element size itself. This deviates from the standard binary coding where the code consists of the absence or presence of a feature at discrete, evenly spaced locations. In the preferred embodiment of this coding scheme, naturally applicable to the above structure manufactured using the multiple print technique, the code element is the gap in the segmented inner opaque material. The gap size is chosen to be one that is reliably defined by the stepper and photolithography process and also resolvable by the microscope (working at the desired magnification). The gap size, interval length, and particle length determine the codespace (number of codes possible). The determination of a codespace involves tradeoffs between particle density on the wafer, identification accuracy, optical detection system complexity, and particle number per microscope image. Codespaces of over a million can be produced and accurately identified using practical parameter combinations.
In the example of a standard binary coding scheme, the particle would be divided into units of equal length. Each unit could then be black or white, 0 or 1. Because the particle is symmetric, there are two codes that are the same when one is reversed (so called “degenerate” codes). When counting the codes, one from each of the pair of degenerate codes is preferably discarded. Without the degeneracy, there would be 2N possible codes, where N is the number of bits (units). With the degeneracy, there are about half that number. Exactly, the number of possible codes with the standard binary format is [2N+2floor[(N+1)/2]]/2. In the example of the high contrast encoded microparticle structures of the present invention, previously shown in
The non-binary coding scheme mentioned above has many advantages in the fabrication and detection of microparticles, including providing for high codespaces and robust code identification. In the example of the coding scheme, the reliability of the microparticle fabrication process is improved by permitting optimization of patterning and etch conditions for features, of a single size, e.g. gaps in the segments having a single width.
In the exemplary examples of encoded microparticles and methods of determining codes therein, e.g. as shown in
In general, a high codespace is desirable. In the field of genomics, having a codespace in the tens of thousands is especially important because it enables full genomes of complex organisms, such as the human genome, to be placed on a single particle set. The top portion of Table 1 shows the effect of varying the delta parameter, d, on the codespace. Shrinking d gives many more codes but places increased demand on the optical system. The need to resolve a smaller d means that a more expensive objective would typically be used. Practically, the lower limit of the gap interval distance is set by the resolution by the optical system (manifested as the pixel size of the digital image captured using a CCD camera). Using a 60× objective and 6.2 mm 1024×1024 CCD chip, an interval distance of d=0.4 μm equals approximately 4 pixels. If the interval distance is reduced to 0.3 μm (3 pixels), there are 105,154 codes. The codespace can be extended into the millions for longer particle lengths, L, and/or smaller gap widths, w.
The lower portion of Table 1 shows the effect of varying the length of the particle at fixed w and d. The length L is inversely proportional to the density of particles on the die (number of particles per unit area). The length also affects the number of particles in an image and thus throughput (particles detected per second). Tradeoffs exist between codespace, density, identification, and throughput. Optimization of the coding scheme parameters will determine the selected coding scheme for a particular application.
In the upper right photograph of
The encoded microparticles, systems, and methods of using the encoded microparticles have a wide range of applications in the fields of biology, chemistry, genetics and medicine, as well as in security and commercial fields involving the tagging of monetary bills, identification cards and passports, commercial products, and the like. In one example, the encoded microparticles can be used in for molecular detection, such for as analyzing, quantifying and sequencing DNA, RNA, and proteins. In other examples, combinatorial chemistry or drug screening assays are performed as known in the art.
Referring to the flowchart shown in
The binding of the encoded microparticles and molecules produces a “pooled probe set” through step 414. The pooled probe set is a mixture of encoded microparticles where each code has a unique probe sequence attached to the particle surface. The pooled probe set can then be used to determine the amount of individual targets present in a mixture of targets. The mixture of targets is referred to as the sample and is typically derived from a biological specimen. The sample is then labeled, typically with a fluorophore at step 416. When the sample is mixed with the pooled probe set, the probes and targets find each other and bind in solution. With nucleic acids, this reaction, step 418, is called hybridization and is very selective. After binding, the particles are imaged to read the codes and quantify the fluorescence at step 420. Referring to the lookup table, the amounts of the different target species, or the number of different species and their identities, in the mixed sample can now be determined at step 422.
The samples exposed to the encoded microparticles may be a purified biological extract or a non-purified sample, including but not limited to whole blood, serum, cell lysates, swabs, nucleic acid samples, such as genomic samples, or tissue extracts. The samples may be produced by culturing, cloning, dissection, or microdissection. Cells may serve as either the sample or probe in a bioassay utilizing the microparticles and other aforementioned inventions.
It is noted that multiple different samples may be identified in a single bioassay as discussed above. Before the detection and after the hybridization, the encoded microparticles can be placed into wells of a well plate or other container for detection. In one detection example, the encoded microparticles settle by gravity to the bottom surface of the well plate. The encoded microparticles in the well can be subjected to centrifugation, sonication, or other physical or chemical processes (multiple washing steps, etc.) to assist in preparing the encoded microparticles for detection. In another example, the encoded microparticles can be placed onto a glass slide or other specially prepared substrate for detection. In yet other examples, the encoded microparticles are present in a flow stream during detection, or present in a suspended solution. In other words, the code of the encoded microparticles may be read by flow cytometry using a flow cytometer.
The term “conjugation” is used to refer to the process by which substantially each encoded microparticle has one or more probe molecules attached to its surface. Methods of conjugation are well known in the art, for example in Bioconjugate Techniques, First Edition, Greg T. Hermanson, Academic Press, 1996: Part I (Review of the major chemical groups that can be used in modification or crosslinking reactions), Part II (a detailed overview of the major modification and conjugation chemicals in common use today), and Part III (discussion on how to prepare unique conjugates and labeled molecules for use in applications).
The probes attached to the surface of the encoded microparticles typically have known attributes or properties. In an example, the probes can be derived from biological specimens or samples and used in the screening, including but not limited to genetic sequencing, of large populations where typically, the derivatives from one member of the population is applied to a single code, typically a multiplicity of particles of a single code. Preferably, microparticles having the same code have attached substantially the same probe molecules; whereas microparticles having different codes likewise have different probe molecules.
One of the most powerful features of a multiplexed assay using solution arrays of encoded microparticles as the platform instead of planar microarrays is the flexibility to add functionality to the assay by simply adding new encoded microparticles. With standard microarrays, once the arrays are printed or synthesized, the microarray typically cannot be changed. If the researcher wants to change the probes on the array or add probes for new genes, typically entirely new arrays would then be produced. With pooled probe sets of encoded microparticles, new probe and encoded microparticle conjugates can easily be added to the existing pooled probe set. In practice the new probes could be different probes for an already represented gene, probes for alternative splicing variants of genes, or tiling probes for genes.
The present encoded microparticles of the invention can be used as major functional members of biochemical (or chemical) analysis systems, including but not limited to solution based microarrays, biochips, DNA microarrays, protein microarrays, lab-on-a-chip systems, lateral flow devices (immune-chromatographic test strips) and the like. Applications include, but are not limited to, gDNA and protein sequencing, gene expression profiling, genotyping, polymorphism analysis, comparative genomic hybridization (CGH), chromatin immune-precipitation (ChIP), gene copy number determination, single nucleotide polymorphism (SNP) determination, methylation detection, as well as discovering disease mechanisms, studying gene function, investigating biological pathways, and a variety of other biochemical and biomolecular related applications such as inspection and analyses of proteins, peptides, polypeptide, and related biochemical applications.
Assay architectures may include those well known in the art, including but not limited to DNA/DNA hybridization, DNA/RNA or RNA/RNA hybridization, enzymatic assays such as polyemerase extension, ligation, and qPCR. The encoded microparticles can also be used in microfluidic or lab-on-a-chip systems or any flow based cytometric systems, including but not limited to those systems wherein sample preparation, biochemical reaction, and bio-analyses are integrated.
For example, fluorescent tags can be employed when an optical imaging method based on the presence of fluorescence is used. Radioactive labels can be used when the microparticles are utilized to expose or develop relevant photographic films. Alternatively, enzymatic tags can be used when the detection involves detection of the product of the enzyme tag that is released when the sample molecules bind to or react with the probe molecules on the microparticles. Other tagging methods are also possible, as set forth in Schena et al., “Quantitative monitoring of gene expression patterns with a complementary DNA microarray”, Science, 1995, 270-467, the subject matter of which is incorporated herein by reference in its entirety. Other labeling techniques include, but are not limited to, chemiluminescence, colorimetric assays, phosphorescence, quantum dot analysis, fluorescence resonance energy transfer (FRET) labels, biosensors, and other known labels in the art.
Samples without labels can also be reacted with the microparticles. For example, molecular beacon probes can be applied to the microparticle. Molecular beacon probes typically contains a hairpin structure that, upon binding the labelless, or in some examples labeled, sample molecules unfold, thus producing a signal indicative of the binding events. Such molecular beacon probes, as well as other probes, may be used in assays involving FRET, where for example fluorophores or quenchers are placed on or in the surface of the microparticles.
For facilitating fast, reliable, and efficient bioassay for large number of sample molecules, it is preferred, but not necessary, that the encoded microparticles are capable of arranging themselves substantially in a monolayer on a surface, such as the bottom surface of the well in which the encoded microparticles are contained. The encoded microparticles may optionally exhibit Brownian motion in the liquid in which the optical detection is performed. Given the specific liquid in which the microparticles are hybridized and detected, it is preferred that the 2D diffusion coefficient of the microparticles is equal to or greater than 1×10−12 cm2/s and/or 10% or more, such as 15% or more, or even 20% or more, and 50% or more of the microparticles are measured to undergo a lateral displacement of 20 nm or greater, such as 30 nm or greater, or even 50 nm or greater—in a time interval of 1 second or less, or preferably 3 seconds or less, or five seconds or less.
The detectable encoded microparticles, which are referred to as those that are able to be accurately detected by the desired detection means, such as optical imaging using visible light, are capable of occupying 30% or more, 40% or more, and typically 50% or more of the surface area on which the microparticles are collected together, such as a portion of the bottom surface of the container in which the microparticles are contained. Defining an area in which at least 90% of all the microparticles are disposed (typically at least 95% or more typically at least 99%, and often 100%), the microparticles can be seen to have a density of 1000 particles/mm2 or more, such as 1500 particles/mm2 or more, 2000 particles/mm2 or more, and typically 3000 particles/mm2 or more (e.g. 5000 particles/mm2 or more). The detection rate within the above-mentioned area, which rate is defined as the ratio of the total number of detected microparticles (microparticles with spatial codes detected) of a collection of microparticles under detection to the total number of the collection of microparticles, is preferably 80% or more, typically 90% or more, or more typically 99% or more.
Another preferred example of the invention is a kit comprising biochemically active encoded microparticles that contains 200 or more, more preferably 500 or more, 1000 or more, or even 10,000 or more different codes within the kit (due to the large codespace enabled by the invention, even larger numbers of codes). Due to statistical sample requirements of convenient liquid pipetting and a desired redundancy of particular codes within the kit, more than 10 particles of the same code are typically provided (20 or more, or even 30 or more microparticles of the same code) within the kit, as in some example applications the redundancy improves the overall assay performance. The term “biochemically active encoded microparticles” is refers to microparticles that have biological or chemical moieties on surfaces and thus can be used in assays; and the term “moieties” are referred to as molecular species; including but are not limited to nucleic acids, synthetic nucleic acids, oligonucleotides, single stranded nucleic acids, double stranded nucleic acids, proteins, polypeptides, antibodies, antigens, enzymes, receptors, ligands, and drug molecules, cells, and complex biologically derived samples.
Universal adapter schemes may be employed in assays to provide a set of non-interacting synthetic sequences that are complementary to oligonucleotide probe sequences of the probes. Genotyping can be performed using common probes and allele-specific reporters or allele-specific probes and common reporters. Amplification assays such as those involving PCR, qPCR, padlock probes, or Molecular Inversion Probes (MIPs) can be performed using the particles of the current invention. Examples of two of these assays are provided in
Protein based assays are also applicable. These include but are not limited to sandwich immunoassays, antibody-protein binding assays, receptor-ligand binding assays, or protein-protein interaction assays. Examples of these assays are shown in
A single type of protein can be chemically attached to encoded microparticles of possessing a single code and thereby act as the probe. Upon mixing of the encoded microparticle-protein conjugates and reaction with a sample, proteins that interact and bind to one another are determined by the presence of detectable label adjacent to encoded microparticles.
The square cross section of the encoded microparticle structures of the present invention provides an improvement over the prior art by providing an increased area of contact in the shape of a flat, rectangular surface. Prior art particles that are spherical or cylindrical in shape limit the contact areas to single points or lines respectively. This invention is not limited to proteins: any interacting molecules may be used with this assay architecture. Also, the omni-directional encoded microparticles of the present invention may be used in conjunction with any other encoded particles including but not limited to fluorophores, quantum dots, latex or glass beads, colloidal metal particles, spectroscopically active particles, SERS particles, or semiconductor nanorods.
The encoded microparticles may be used in conjunction with a 2D planar array of molecules. Interaction between molecules on the surface of the particles and those contained in spots on the 2D planar array are determined by the binding of the particles to the spots. The presence of the encoded microparticles in the predetermined spot locations, preferably after washing steps, indicates a binding interaction between the molecules on the encoded microparticles and the molecules on the 2D planar array. The assay result can be determined by identifying 1) the particle code, and 2) the spot location. This is shown in
The encoded microparticles of the invention have many other applications. For example, by placing protein-detection molecules (e.g., ligands, dyes which change color, fluoresce, or generate an electronic signal upon contact with specific protein molecules) onto the encoded microparticles as probes, bioassay analyses can be performed, i.e., evaluation of the protein and/or gene expression levels in a biological sample. As another example, by placing (cellular) receptors, nucleic acids/probes, oligonucleotides, adhesion molecules, messenger RNA (specific to which gene is “turned on” in a given disease state), cDNA (complementary to mRNA coded-for by each gene that is “turned on”), oligosaccharides & other relevant carbohydrate molecules, or cells (indicating which cellular pathway is “turned on”, etc.) onto the encoded microparticles as probes, the encoded microparticles can be used to screen for proteins or other chemical compounds that are implicated in disease (i.e., therapeutic target); as indicated by (the relevant component from biological sample) adhesion or hybridization to specific spot (location) on the microarray where a specific (target molecule) was earlier placed/attached. In fact, the microparticles of the invention can be applied to many other biochemical or biomolecular fields, such as those set forth in the appendix attached herewith, the subject matter of each is incorporated herein by reference.
It will be appreciated by those of skill in the art that a new and useful microparticle and a method of making the same have been described herein. The large sets of encoded microparticles produced by this invention can be a fundamental technology that will have far reaching applications, especially in the field of biotechnology and more specifically genomics. It has the potential to dramatically reduce the cost of highly multiplexed bioassays. Moreover, enables researchers to easily design custom content solution arrays. The researcher can also easily add new particle types to the pooled set, for instance including new found genes of interest with the microparticles of the invention.
In view of the many possible embodiments to which the principles of this invention may be applied, however, it should be recognized that the embodiments described herein with respect to the drawing figures are meant to be illustrative only and should not be taken as limiting the scope of invention. Those of skill in the art will recognize that the illustrated embodiments can be modified in arrangement and detail without departing from the spirit of the invention.
For example, the encoded microparticle may have a six sided shape with four elongated sides and two end sides. The encoded microparticle can be configured such that the code of the encoded microparticle can be detectable regardless of which of the four elongated sides the barcode is disposed on. The encoded microparticle may have a ratio of the length to width from 2:1 to 50:1, or anywhere from 4:1 to 20:1. The length of the encoded microparticle is preferably from 5 to 100 μm and more preferably less than 50 μm. The width of the microparticle can be from 0.5 to 10 μm. In other examples, the length of the microparticle can be less than 10 μm, less than 25 μm, less than 25 μm; less than 5 μm, less than 27 μm; and the width of the microparticle can be less than 3 μm. The ratio of width to height of the microparticle can be from 0.5 to 2.0. The ratio of the length to width of the microparticle can be from 2:1 to 50:1. The cross section taken along the length of the microparticle is substantially rectangular with a length at least twice the width.
The microparticle may have a glass body with segments embedded therein. The difference of the transmissivity of the glass body and segments can be 10% or more. The glass body may have a length of less than 50 μm and a width of less than 10 μm with the glass body having a volume of from 5 to 500 μm3. The encoded microparticle may have 2 to 15, 3 to 10, or 4 to 8 portions of less transparent material within the encoded microparticle. The code incorporated in the microparticle can be binary or non-binary or any other desired codes. The microparticle may have biochemical molecules attached to one or more surfaces of the microparticle, such as DNA and RNA probes with a density of from 102 to 106 μm2. When fabricated on the wafer-level, the wafer may have a surface area of from 12.5 in2 to 120 in2, and wherein there are at least 3 million microparticles per in2 of the wafer. The wafer may have at least one million codes are formed on the substrate, or at least two hundred different codes are present within the one million codes, or at least 3000 different codes are present within the one million codes. When placed in a liquid buffer, for example in a bioassay, the microparticles can form a single monolayer with a 2 dimensional diffusion coefficient of the microparticles greater than 1×10−12 cm2/s and more preferably greater than 1×10−11 cm2/s.
The encoded microparticles of the present invention possess a barcode which enables differentiation of each different microparticle. The encoded microparticles may have a size of about 2 μm×2 μm×15 μm. As already discussed above, various oligonucleotides can be conjugated to the encoded microparticles such that each code corresponds to a unique oligonucleotide sequence.
Oligonucleotides (alternatively referred to as a polynucleotide or polynucleotides) may include, but are not limited to, for instance, any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids (PNAs), modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2′-O-methylated oligonucleotides), and the like. The nucleotides of the polynucleotide can be deoxyribonucleotides, ribonucleotides or nucleotide analogs, can be natural or non-natural, and can be unsubstituted, unmodified, substituted or modified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like. The polynucleotide can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The polynucleotide can be, e.g., single-stranded or double-stranded.
The term “analog” in the context of nucleic acid analog is meant to denote any of a number of known nucleic acid analogs such as, but not limited to, LNA, PNA, etc. For instance, it has been reported that LNA, when incorporated into oligonucleotides, exhibit an increase in the duplex melting temperature of 2° C. to 8° C. per analog incorporated into a single strand of the duplex. The melting temperature effect of incorporated analogs may vary depending on the chemical structure of the analog, e.g. the structure of the atoms present in the bridge between the 2′-O atom and the 4′-C atom of the ribose ring of a nucleic acid.
Various bicyclic nucleic acid analogs have been prepared and reported. (See, for example, Singh et al., Chem. Commun., 1998, 4:455-456; Koshkin et al., Tetrahedron, 1998, 54:3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97:5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8:2219-2222; Wengel et al., PCT International Application Number PCT/DK98/00303 which published as WO 99/14226 on Mar. 25, 1999; Singh et al., J. Org. Chem., 1998, 63:10035-10039, the text of each is incorporated by reference herein, in their entirety). Examples of issued US patents and Published U.S. patent applications disclosing various bicyclic nucleic acids include, for example, U.S. Pat. Nos. 6,770,748, 6,268,490 and 6,794,499 and U.S. Patent Application Publication Nos. 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114, 20030087230 and 20030082807, the text of each of which is incorporated by reference herein, in their entirety.
Various 5′-modified nucleosides have also been reported. (See, for example: Mikhailov et al., Nucleosides and Nucleotides, 1991, 10:393-343; Saha et al., J. Org. Chem., 1995, 60:788-789; Beigleman et al., Nucleosides and Nucleotides, 1995, 14:901-905; Wang, et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9:885-890; and PCT Internation Application Number WO94/22890 which was published Oct. 13, 1994, the text of each of which is incorporated by reference herein, in their entirety).
Oligonucleotides in solution as single stranded species rotate and move in space in various energy-minimized conformations. Upon binding and ultimately hybridizing to a complementary sequence, an oligonucleotide is known to undergo a conformational transition from the relatively random coil structure of the single stranded state to the ordered structure of the duplex state. With these physical-chemical dynamics in mind, a number of conformationally-restricted oligonucleotides analogs, including bicyclic and tricyclic nucleoside analogues, have been synthesized, incorporated into oligonucleotides and tested for their ability to hybridize. It has been found that various nucleic acid analogs, such as the common “Locked Nucleic Acid” or LNA, exhibit a very low energy-minimized state upon hybridizing to the complementary oligonucleotide, even when the complementary oligonucleotide is wholly comprised of the native or natural nucleic acids A, T, C, U and G.
Other examples of issued US patents and published applications include, but are not limited to: U.S. Pat. Nos. 7,053,207, 6,770,748, 6,268,490 and 6,794,499 and published U.S. applications 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114 and 20030082807; the text of each is incorporated by reference herein, in their entirety.
Additionally, bicyclo[3.3.0] nucleosides (bcDNA) with an additional C-3′,C-5′-ethano-bridge have been reported for all five of the native or natural nucleobases (G, A, T, C and U) whereas (C) has been synthesised only with T and A nucleobases. (See, Tarkoy et al., Helv. Chim. Acta, 1993, 76:481; Tarkoy and C. Leumann, Angew. Chem. Int. Ed. Engl., 1993, 32:1432; Egli et al., J. Am. Chem. Soc., 1993, 115:5855; Tarkoy et al., Helv. Chim. Acta, 1994, 77:716; M. Bolli and C. Leumann, Angew. Chem., Int. Ed. Engl., 1995, 34:694; Bolli et al., Helv. Chim. Acta, 1995, 78:2077; Litten et al., Bioorg. Med. Chem. Lett., 1995, 5:1231; J. C. Litten and C. Leumann, Helv. Chim. Acta, 1996, 79:1129; Bolli et al., Chem. Biol., 1996, 3:197; Bolli et al., Nucleic Acids Res., 1996, 24:4660). Oligonucleotides containing these analogues have been found to form Watson-Crick bonded duplexes with complementary DNA and RNA oligonucleotides. The thermostability of the resulting duplexes, however, is varied and not always improved over comparable native hybridized oligonucleotide sequences. All bcDNA oligomers exhibited an increase in sensitivity to the ionic strength of the hybridization media compared to natural counterparts.
A bicyclo[3.3.0] nucleoside dimer containing an additional C-2′,C-3′-dioxalane ring has been reported in the literature having an unmodified nucleoside where the additional ring is part of the internucleoside linkage replacing a natural phosphodiester linkage. As either thymine-thymine or thymine-5-methylcytosine blocks, a 15-mer polypyrimidine sequence containing seven dimeric blocks and having alternating phosphodiester- and riboacetal-linkages exhibited a substantially decreased Tm in hybridization with complementary ssRNA as compared to a control sequence with exclusively natural phosphordiester internucleoside linkages. (See, Jones et al., J. Am. Chem. Soc., 1993, 115:9816).
Other U.S. patents have disclosed various modifications of these analogs that exhibit the desired properties of being stably integrated into oligonucleotide sequences and increasing the melting temperature at which hybridization occurs, thus producing a very stable, energy-minimized duplex with oligonucleotides comprising even native nucleic acids. (See, for instance, U.S. Pat. Nos. 7,572,582, 7,399,845, 7,034,133, 6,794,499 and 6,670,461, all of which are incorporated herein by reference in their entirety for all purposes).
For instance, U.S. Pat. No. 7,399,845 provides 6-modified bicyclic nucleosides, oligomeric compounds and compositions prepared therefrom, including novel synthetic intermediates, and methods of preparing the nucleosides, oligomeric compounds, compositions, and novel synthetic intermediates. The '845 patent discloses nucleosides having a bridge between the 4′ and 2′-positions of the ribose portion having the formula: 2′-O—C(H)(Z)-4′ and oligomers and compositions prepared therefrom. In a preferred embodiment, Z is in a particular configuration providing either the (R) or (S) isomer, e.g. 2′-O,4′-methanoribonucleoside. It was shown that this nucleic acid analog exists as the strictly constrained N-conformer 2′-exo-3′-endo conformation. Oligonucleotides of 12 nucleic acids in length have been shown, when comprised completely or partially of the Imanishi et al. analogs, to have substantially increased melting temperatures, showing that the corresponding duplexes with complementary native oligonucleotides are very stable. (See, Imanishi et al., “Synthesis and property of novel conformationally constrained nucleoside and oligonucleotide analogs,” The Sixteenth International Congress of Heterocyclic Chemistry, Aug. 10-15, 1997, incorporated herein by reference in its entirety for all purposes).
A “polynucleotide sequence” or “nucleotide sequence” is a polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.
Two polynucleotides “hybridize” when they associate to form a stable duplex, e.g., under relevant assay conditions. Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays” (Elsevier, N.Y.), as well as in Ausubel, infra.
The “Tm” (melting temperature) of a nucleic acid duplex under specified conditions (e.g., relevant assay conditions) is the temperature at which half of the base pairs in a population of the duplex are disassociated and half are associated. The Tm for a particular duplex can be calculated and/or measured, e.g., by obtaining a thermal denaturation curve for the duplex (where the Tm is the temperature corresponding to the midpoint in the observed transition from double-stranded to single-stranded form).
The term “complementary” refers to a polynucleotide that forms a stable duplex with its “complement,” e.g., under relevant assay conditions. Typically, two polynucleotide sequences that are complementary to each other have mismatches at less than about 20% of the bases, at less than about 10% of the bases, preferably at less than about 5% of the bases, and more preferably have no mismatches.
While PCR techniques have been practiced in the art for decades, the concept of performing quantitative PCR has only recently emerged as a reproducible and useful technique used for quantitating the number of messages of RNA present in a sample, for instance, or the relative amounts of a specific gene transcript or mtDNA, or even the presence of specific miRNA or other types of RNA species, for example. (See, for instance, Fernandez-Jimenez et al., “Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA,” PLoS One, 6(12):e28910, 2011, Alcoser et al., “Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts,” BMC Biotech., 11:124, 2011, all of which are incorporated herein by reference in their entireties for all purposes). Often qPCR techniques are combined with other techniques to accomplish an investigational study, as in Hannemann et al., “Quantitative high-resolution genomic analysis of single cancer cells,” PLoS One, 6(11):e26362, 2011, where qPCR is coupled with array-CGH tiling to analyze the genetics of single cells, which are isolated using a microscope and microdissection techniques. (See also, VanGuilder et al., “Twenty-five years of quantitative PCR for gene expression analysis,” Biotechniques, 44(5):619-626, 2008, incorporated herein by reference in its entirety for all purposes).
As can be seen in the art, qPCR is a powerful tool which can be easily manipulated in many ways to detect many different genetic events and parameters. A PCR reaction can be performed on the present encoded microparticles since they are of a size that would allow binding and enzymatic activity of polymerase enzymes. Therefore, it is also possible to perform multiplex real-time PCR (qPCR) the encoded microparticles. Such a technology may be used to investigate many genetic questions such as gene expression, gene copy number, SNP genotyping, etc.
Each code of group of encoded microparticles may correspond to a unique oligonucleotide sequence attached as a probe to that encoded microparticle (or encoded microparticles). Each of the different encoded microparticles may then be placed into a reaction vessel with the appropriate amount of polymerase and primers and control genes to allow quantitative real time PCR of a sample. The encoded microparticles may be visualized, for instance at the bottom of a well plate as described above, and their position and code determined. Either before or after this the PCR reactions may be initiated. Alternatively, the PCR reactions may be initiated simultaneously while reading the codes. Label signals may then be detected in real time as the reactions proceed to perform real-time PCR or qPCR on the encoded microparticles. Label signals may be dissected within the microwell plate so that a particular quantity and rate of label appearance or disappearance, as the case may be depending on which labeling system is utilized, may be directly associated with a particular encoded microparticle and a particular code. Using the lookup table, the code may then be correlated with the unique probe sequence and the amount and/or copy number, etc. of that target directly determined in real time.
Other variations of this assay will be clear to one of skill in the art, wherein various targets are desired to be quantitated, such as the aforementioned miRNA, mtDNA, mRNA, ESTs, doped synthetic targets, and other nucleotide species.
The identity and presence of various SNPs may also be determined through the use of DNA analogs, such as the constrained-ethyl nucleotide analog family. Placing such analogs, such as cEt, at the SNP location allows the probe to distinguish between different SNPs and thereby enables detection and quantitation (determination of copy number as well) of various SNP-containing genes. (See, Latorra et al., “Design considerations and effects of LNA in PCR primers,” Mol. Cell. Probes, 17:253-259, 2003). Additionally, various label dye methodologies have been developed specifically for SNP determination in qPCR. (See, Kostrikis et al., “Spectral genotyping of human alleles,” Science, 279:1228-1229, 1998).
As reported in the literature, various labeling techniques may be employed in conducting qPCR reactions. For instance, a label-quencher pair may be incorporated into a primer, according to standard practice, said primer being complimentary to a sequence in the target. These are sometimes referred to as molecular beacons. As the polymerase reaction proceeds, the 5-prime to 3-prime exonuclease activity of the polymerase will release the quencher of the label-quencher primer pair as the polymerase reaches the primer hybridized to the target, thereby allowing the fluorescent, or other, half of the pair to emit a signal to be detected. Other hybridization probe-based and amplicon-based labels are known, such as those which provide a donor and acceptor FRET pair with a donor on one probe/primer and acceptor on another which when hybridized to the PCR product produce and in close proximity to one another a FRET signal. Another class of such labels includes “light-up probes” which are made of peptide nucleic acid (PNA) and possess an asymmetric cyanine dye which becomes intensively fluorescent upon binding and hybridizing to its target sequence. (See, Svanvik et al., “Detection of PCR Products in Real Time Using Light-Up Probes,” Anal. Bioch., 287:179-182, 2000, available from LightUp Technologies, AB., Huddinge, Sweden).
Other methods employ the use of single labels which intercalate into double-stranded DNA, such as SYBR® green I (see Giglio et al., “Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR,” Nuc. Acids Res., 31(22):e136, 2003). Other newer intercalating dyes are being developed, such as The minor groove binding asymmetric cyanine dye 4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridinium iodide (BEBO) and (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) (BOXTO) and its positive divalent derivative BOXTO-PRO, (4-[3-methyl-6-(benzoxazole-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-(3-trimethylammonium-propyl)-quinolinium dibromide). (See, Bengtsson et al., “A new minor groove binding asymmetric cyanine reporter dye for real-time PCR,” Nuc. Acids Res., 31(8):e45, 2003, and Ahmad, Ashraf “BOXTO as a real-time thermal cycling reporter dye,” J. Biosci., 32(2):229-239, 2007). Thus, only when the probe is hybridized to the target and a polymerase reaction begins will the label intercalate into the double-stranded DNA and emit a detectable signal. Furthermore, these different types of labels and strategies may be combined. (See, Stahlberg et al., “Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis,” BioTechniques, 40:315-319, 2006). These and other similar labeling and detection techniques are well described in the art and known to one of skill in the art.
It is a unique advantage of the present encoded microparticles in this system that only one type of label need be used in a qPCR experiment employing the present encoded microparticles, though multiple labels may be used if needed. That is, because there is a single unique code associated with each unique probe on each particle, only one label need be used to detect and quantitate the qPCR reaction on each microparticle. Thus, one may assay as many as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100 or even 200 different targets in a single assay using a single label type because each unique target will correspond to a unique probe attached to one or more unique encoded microparticles correlated with one or more unique codes in the assay.
The advantages provided by such a streamlined and efficient assay include the requirement of less starting material and fewer laboratory consumables and resources because fewer assays need be run, better-controlled experiments because a large number of controls may be run simultaneously in each assay, lower cost, quicker and more simplified assays may be run using the present encoded microparticles in a multiplex qPCR assay. Other advantages of this assay, as described above, will be apparent to the investigator designing experiments based on the principles disclosed herein.
Chromatin immunoprecipitation (ChIP) is a powerful tool for identifying specific regions of the genome which are associated with, or bound by, proteins such as histones, transcription factors, and gene regulators, etc. Antibodies are employed that recognize the specific protein of interest for study or a specific modification of a protein. The initial step of a ChIP assay is chemical cross-linking of protein-protein and protein-DNA in live cells with formaldehyde or similar reagents, such as paraformaldehyde, EDC, etc. This step allows cross-linking and covalent attachment of any proteins associated with the genomic DNA. After cross-linking, the cells are lysed and crude extracts are sonicated to shear the genomic DNA. Proteins, together with cross-linked DNA, are subsequently immune-precipitated by incubation of the sheared lysates with antibodies specific for the proteins the user wishes to study. (See, for instance, Southall et al., “Chromatin profiling in model organisms,” Brief Funct. Genomic Proteomic., 6(2):133-140, 2007, and Carter et al., “Applications of genomic microarrays to explore human chromosome structure and function,” Hum. Mol. Gen., 13(2):R297-R302, 2004, and references cited therein).
Current ChIP assays require the critical step of reversing protein-DNA cross-links in the immunoprecipitated material, followed by purification of the now uncross-linked DNA fragments and PCR amplification of those fragments. These steps are very time consuming and labor intensive. Further they only allow detection of a single fragment sequence at a time. The presently disclosed ChIP on encoded microparticles method adapts the QuantiGene® technology into a encoded microparticle multiplex platform.
Label amplification technologies exist which is coupled with encoded microparticle use to provide additional functionality to the traditional ChIP assays. For instance, the QuantiGene® technology (Affymetrix, Inc., Santa Clara, Calif.) provides a simple and efficient means of significantly amplifying a single analyte capture event. Based on a branched-DNA (bDNA) methodology, this amplification technique has been highlighted in, for instance, U.S. Pat. Nos. 7,803,541, 7,927,798, 7,968,327, 7,615,351, and 7,951,539, and related US patents and patent applications (incorporated herein by reference in their entirety for all purposes). See also, Flagella et al., “A multiplex branched DNA assay for parallel quantitative gene expression profiling,” Anal. Bioch., 352(1):50-60, 2006.
The ChIP on encoded microparticles method can be used for two general types of ChIP experiments: Cross-linking ChIP (X-ChIP or XChIP) which uses chromatin fixed with formaldehyde and fragmented by sonication and native chromatin ChIP (N-ChIP or NChIP) which uses native chromatin prepared by nuclease digestion of cell nuclei.
One of the unique capabilities of the present ChIP method is that it allows multiplex detection of various regions of genome which may be associated with proteins of interest. Another unique and especially helpful feature of the present method is that it can be performed directly from crude cell extracts without reverse-linking, DNA isolation and PCR. These labor-intensive and time-consuming steps may be skipped due to the unique advantages provided by the addition of the QuantiGene® label amplification technology with the present encoded microparticles.
It is a unique advantage of the present encoded microparticles in this system that only one type of label need be used in a ChIP experiment employing the present encoded microparticles, though multiple labels may be used if needed. That is, because there is a single unique code associated with each unique probe on each particle, only one label need be used to detect and quantitate the various targets on each microparticle. Thus, one may assay as many as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100 or even 200 different targets in a single assay using a single label type because each unique target will correspond to a unique probe attached to one or more unique encoded microparticles correlated with one or more unique codes in the assay.
Chromatin immunoprecipitation (ChIP) is a popular method used to identify regions of the genome associated with specific proteins, some of which are crucial for vital cellular functions including gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. The ChIP assay has been used to study both histones and non-histone proteins, such as transcription factors, within the context of the cell. Compared with histone proteins, transcription factors and other DNA binding proteins have a weaker affinity. Therefore, it is critical to incorporate a cross-link step such as formaldehyde to cross-link proteins to DNA. After cross-linking, the chromatin are released from nuclei and sheared into 200-500 bp fragments, which are then immunoprecipitated with specific antibodies. DNA sequences cross-linked to the protein of interest, such as transcription factors, co-precipitate as part of the chromatin complex, which is then isolated using magnetic protein G beads, i.e. magnetic beads having attached to them proteins which bind immunoglobulins (commercially available from many vendors). After reverse cross-linking (in a traditional ChIP assay), the associated DNA is released from the complex and is subjected to PCR analysis.
ChIP-on-chip or ChIP-on-Glass, also known as genome-wide location analysis, is a technique for isolation and identification of the DNA sequences bound by specific DNA binding proteins in cells. These binding sites may indicate functions of various transcriptional regulators and help identify their target genes during development and disease progression. The identified binding sites may also be used as a basis for annotating functional elements in genomes. The types of functional elements that one can identify using ChIP-on-chip include promoters, enhancers, repressor and silencing elements, insulators, boundary elements, and sequences that control DNA replication. For instance, the ChIP-on-chip is a technological platform used by the ENCODE consortium to map functional elements in the human genome.
Traditional ChIP assays, including ChIP-on-chip and ChIP-on-glass, are time consuming, cumbersome and/or limited in ability to quantitate since they require reverse cross-linking and DNA isolation before PCR-based amplification and detection. This may lead to variation in quantitation and the requirement of performing numerous replicates to obtain accurate data. Furthermore, the exponential nature of PCR amplification, together with the small quantities of target molecules, means that trivial variations in reaction components and thermal cycling conditions and mis-priming events during the early stages of the PCR can greatly influence the final yield of the amplified product. Finally, qPCR is limited in the number of genes that can be analyzed in a multiplexed expression assay by the maximum number of dyes available to the investigator. The cost increases substantially when four or more dyes are used in a single assay.
DNA sequences cross-linked to the protein of interest, such as transcription factors, precipitate with the bound chromatin sequences. The genetic material is isolated after the DNA binding proteins are precipitated using commonly available magnetic protein G beads. The magnetic protein G beads may be washed in a filter plate with a magnetic holder. At this point, without reverse cross-linking and DNA isolation, the bound DNA in the complex may be released by addition of a quantity of protease K. The gene sequences may then be directly detected with the present encoded microparticle assay. No PCR-based amplification is necessary since the label-amplification system (QuantiGene®) is able to detect single molecule quantities of genetic material.
In the present ChiP-on-bead method, different classes of capture microparticles may be used; with each class of microparticle conjugated with a unique capture probe (CP) oligonucleotide that confers DNA specificity to the encoded microparticle. This multiple ChIP method is accomplished through the use of encoded microparticles. However, it is noted that other particles, or beads, may also be used that are compatible with the QuantiGene® system, such as fluorescent microspheres (beads) (available from Luminex, Corp., Austin, Tex.) which may also act as unique solid support structures for the hybridization of specially designed oligonucleotide probe sets.
Each probe set in the QuantiGene® system contains capture extenders (CE, usually has 5 CEs), label extenders (LE, usually has 10 LEs) and optionally blockers (BL, usually has 6 BLs), whose sequences are selected based on the sequence of the target DNA in order to recognize the particular DNA. However, the tails of multiplex CEs are designed to discriminate between different encoded microparticles (or fluorescent microspheres within the bead array) while quantitatively capturing the associated target DNA fragment. The CEs are typically optimally approximately 40 nucleotides in length, but can range in length from 20-60 bases. Roughly half of the sequence of the CE is complementary to the target DNA, and the other half is complementary to the capture probes that are immobilized on the encoded microparticles. The Les are also approximately 40 nucleotides in length, but can range in length from 20-60 bases. Again, roughly half of the LE sequence is typically complementary to sections of the target DNA fragment, and the other half is complementary to the amplifier oligonucleotides. The BLs hybridize across any regions of the target DNA sequences that are not covered by CEs and Les, i.e. non-overlapping target sequences.
Signal amplification in the multiplex ChiP-on-bead assay occurs when a bDNA molecule hybridizes to the LE. These bDNA molecules contain hybridization sites for as many as 45 biotinylated label probes (but may be as few as 20, 25, 30, 35, 40, or as many as 50, 60, 70, 80 or even 100), which are oligonucleotides that hybridize to amplifier molecule branched DNA. Upon addition streptavidin-conjugated phycoerythrin, a 45-fold amplification of fluorescence signal is achieved for each amplifier molecule associated with the target DNA fragment attached on the bead.
It should be noted that, as disclosed in the above-mentioned references describing the QuantiGene® system, many other types of labels may be used, including, but not limited to, such labels as phosphorescence, radioactivity, chemiluminescence, colorimetry, quantum dots, biosensors, molecular beacons, and the like. Signals (MFI, Mean Fluorescence Intensity) generated from each bead are proportional to the amount of each DNA fragment captured on the surface of each DNA-specific probe set. In this fashion, this multiplex ChIP-on-bead/microparticle assay allows amplification of the label rather than the target. In this method, signal amplification occurs without the need of purification and amplification of the target DNA.
As a further optional embodiment, the encoded microparticles, after having bound thereto the target DNA or RNA and the label amplification system, may be analyzed by use of a flow cytometry instrument. That is, the labeled encoded microparticles may be passed through a flow cell and the code determined and fluorescence or other label detected simultaneously, or nearly simultaneously, as the particles are pass through the flow cell. The encoded microparticles may optionally exhibit magnetic properties and thereby be pre-aligned in a proper orientation to allow detection of the spatial code as they pass through the flow cell. This, combined with robotic liquid handling instrumentation which is commercially available, will allow complete automation of this process for high throughput analysis of a multitude of samples, i.e. hundreds or more samples, without the need for labor-intensive pipetting and other manipulation of the solutions comprising the sample and the encoded microparticles and antibodies and such.
Therefore, the invention as described herein contemplates all such embodiments as may come within the scope of the following claims and equivalents thereof.
This U.S. patent application is a continuation-in-part of Ser. No. 11/521,153 filed Sep. 13, 2006 which claims priority from co-pending U.S. provisional application Ser. No. 60/762,238 filed Jan. 25, 2006 and U.S. provisional application Ser. No. 60/716,694 filed Sep. 13, 2005, the subject matter of each being incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60762238 | Jan 2006 | US | |
| 60716694 | Sep 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11521153 | Sep 2006 | US |
| Child | 13336699 | US |
