HK1074881B - Arrays of microparticles and methods of preparation thereof - Google Patents

Description

Microparticle arrays and methods of making the same

This application claims priority to U.S. provisional application 60/343621 filed on 12/28/2001. U.S. provisional application 60/343621 is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to high unit density microparticle arrays and methods of making the same. The invention also relates to a multi-chip array and a preparation method thereof. The invention further provides methods of performing bioassays using high unit density arrays and multichip arrays.

Background

Array formats for biological and chemical analysis can provide accurate results quickly and with reduced effort. (Nature Genetics, 1999 Vol.21(1) supplement pp.3-4) arrays of biological probes such as DNA, RNA or protein molecules are typically formed by deposition and immobilization or by in situ synthesis on an inert substrate. In these prior art methods, array formation typically requires direct attachment of probe molecules to a substrate, which may be composed of organic (e.g., polymers such as nitrocellulose) or inorganic (e.g., glass or silicon) materials.

The use of silicon as a base layer provides certain advantages of methods involving the complete set-up of semiconductor wafer and chip processing. In semiconductor processing, wafers are modified and transformed in a series of various processing steps to produce desired features. Typically, a variety of identical features are fabricated simultaneously on each wafer by parallel processing to form individual segments on the wafer. Manufacturing time can be greatly saved by using parallel or batch processing to manufacture the same features. In addition, batch processing produces a high degree of chip uniformity, and very small ("sub-micron") features can be precisely fabricated using certain photolithography and etching methods. Thus, structures with a high feature density can be fabricated on very small chips. After processing is complete, individual segments are cut from the wafer, a process known as singulation (division) to obtain a multiplicity of chips. (Peter Van Zant, "Microchip failure", 3^rd edition，McGraw Hill 1998)。

Semiconductor wafers containing different functional chips may be assembled in a final packaging process by interconnecting the different chips or by merely bonding two wafers having different functional chips, and then cutting a stack of wafer laminations (stack of wafers). Efficient semiconductor manufacturing processes can significantly enable rapid industrial production. Highly complex systems are developed for chip production, packaging and quality control.

Biochips are arrays of different biomolecules ("probes") that can bind to specific targets bound to a solid support (solid support). There are two basic methods for preparing biochips.

The first method involves placing an equal volume of a solution containing presynthesized probe molecules of interest on a planar substrate and then immobilizing the probe molecules at the designated locations. For example, probe solutions are applied to a substrate ("spot") to form probe arrays of custom compositions that are one-dimensional position coded (Kricka, Larry J., "Immunoassay", Chapter 18, pages 389-. The molecular probes may be attached directly to the surface of the substrate or may be attached to a solid support and then deposited or attached to the substrate to form an array. Microparticles ("beads") represent one type of such carriers. Beads have the advantage of separating the process of preparing and testing the substrate from the process of preparing, applying and testing probes and analytical chemistry (US patent No. 6251691). Beads of various sizes and compositions are widely used in chemical and biological assays as well as in combinatorial synthesis.

The deposition, printing, and spotting methods of the probe array production have some undesirable characteristics. First, state of the art deposition and printing techniques can only produce arrays with low feature density (low feature density) because the typical spot size is 100 microns and the spot-to-spot separation is 300 microns. Second, probe deposition methods to date have not been able to produce consistent spots, with significant point-to-point differences. Third, the spotting methods, including variations such as electrophoretic deposition onto patterned electrodes (U.S. patent No.5605662), require extensive equipment and logistical support to perform array production at any meaningful scale. In particular, the spotting method cannot perform batch fabrication of probe arrays. Thus, when batch processing formats are used to efficiently produce substrates, the subsequent step of "biofunctionalizing" these substrates by applying chemical or biological probes is inefficient because it is not suitable for batch processing formats and requires many separate spotting steps. This method of manufacturing a large number of identically functionalized chips is therefore very time consuming and expensive compared to methods using parallel processing methods.

A second method of preparing probe arrays involves in situ photochemical synthesis of linear probe molecules such as oligonucleotides and peptides using methods similar to standard procedures for semiconductor processing, photolithography. These methods have been widely used in recent years to synthesize oligonucleotides at specified positions on a glass or similar substrate in a parallel multi-step photochemical reaction (U.S. Pat. No. 5143854; Proc. Nat. Acad. Sci. USA, 1996, 93: 13555-.

Although parallel processing, which produces a large number of probe arrays directly on one wafer at a time, has the advantages of batch processing yielding scalability and inherently improved uniformity, there are serious drawbacks to the fabrication of probe arrays. First, in a series of one-step reactions, only simple, relatively short linear molecules are suitable for synthesis, and in practice, only arrays of short oligonucleotides are prepared by this method. Second, the reaction often does not proceed to completion, resulting in significant compositional inhomogeneity. Third, all semiconductor processing must be completed before introduction of the biomolecules, as the biomolecules may not be compatible with the harsh environment of certain semiconductor processing steps. This limitation may preclude various advantages in semiconductor manufacturing techniques. Fourth, if the functionalization is done on a batch fabrication format, the fabrication process defines the chemical or biochemical composition ("content") of each chip on the wafer. That is, introducing a change in the probe design requires a corresponding change in the overall manufacturing process. Customization, while theoretically possible, requires a change in the necessary masking step required to photochemically synthesize the desired probe molecule. The cost and time delay of this approach makes customization impractical.

Brief Description of Drawings

FIGS. 1a and 1b illustrate the process of the present invention.

FIG. 2 shows an example of a chip containing an array of beads. The chip comprises three layers (L1, L2, L3). L1 is a silicon based layer (A1) with a micromachined array of containment beads; l2, patterned SiO₂(100nm thick); l3 is Si₃N₄Layer (5-10nm thick).

Fig. 3 shows an example wafer design.

FIG. 4 shows an example of designing a chip containing an array of beads. The base layer is silicon (Si). 12 contact Star pattern (12-tip star pattern) in 100nm thick SiO₂On the layer. The star interior is not coated with SiO₂Covered, while the star exterior is SiO₂And (6) covering. There is an array of closely packed hexagonal recesses (recess) in the center. The total number of recesses is 4012.

Figure 5 provides an example of a surface structure for a protective bead. H1 is a well with straight side walls that retains (retain) beads. H2 is a pyramid recess that can receive a bead. H3 is a column set that confines one bead. Hx is a recess that can accommodate multiple posts.

Fig. 6 shows an example of an array structure. A1 and a2 are arrays of rectangular recesses. A3 is an array of hexagonal recesses.

Fig. 7 shows an example of chip grouping.

Fig. 8 illustrates a chip packaging method. A, B, C and D are chips with different functions. The wafer may be divided into chips according to the scribed lines. Individual chips with different functional groups separated from different wafers may be put together. A 4-chip package consists of 4 different functionalized interconnected chips for biological applications. The 4 chips may be arranged in a variety of ways, non-limiting examples include square or linear.

Fig. 9 illustrates a method of assembling chips by moving free chips in rows and columns.

FIG. 10 shows an example of a chip design in which one probe array is placed at a corner of each chip. By combining such 4 chips in this manner in the figure, a larger array can be formed.

FIG. 11 illustrates a method of manufacturing a chip comprising an array of beads of the invention.

Fig. 12 is a photograph of a hydrogel formed on a silicon wafer.

FIG. 13a illustrates fluorescence images of bead arrays on a chip before hydrogel formation and after gel exfoliation. The number of beads and bead positions are the same. FIG. 13b illustrates the results of the chip reactions with and without hydrogel treatment.

FIG. 14 illustrates a removable chip carrier and its application and reaction chamber.

FIG. 15 illustrates an example of a random coded array; (b) chip library (c) random assembly of chips in a chip library, (d) random tiling (tilling) of arrays.

FIG. 16 illustrates the design of an array with beads of different sizes assembled simultaneously or sequentially.

Fig. 17 illustrates the design of a multi-chip carrier.

Disclosure of Invention

The present invention provides a parallel processing method having advantages of a semiconductor manufacturing method. In addition, the method of the invention is flexible and can meet the requirements of different quantities and different analyses. The present invention combines the flexibility of being able to select array contents with high feature density (high feature density) with the economies of scale of parallel (batch) array assembly. The present invention provides a method of assembling randomly coded, carrier-displayed probe arrays of selectable composition at designated locations in spaces delineated on a base layer, which can then be divided into a plurality of chips having carrier-displayed probe arrays. In another embodiment, the singulated chips (without carrier-displayed probes) from one or more base layers are contacted with carrier-displayed probes of a desired species to form chips having a desired array. The present invention provides a multi-chip array by combining chips prepared from substrates having different kinds of carrier display probes. The invention also describes designing chips of substrates and display carriers such as chemically labeled microparticles to optimize the chip display microparticle arrays in biological tests and analysis for a variety of target analytes, including biomolecules such as nucleic acids, proteins, cells, and the like.

The method of producing the biochip of the invention comprises patterning (pattern) a substrate to form a plurality of chip areas, scribing (delicateate) the separation boundaries between the chip areas, assembling at least one bead array comprising biofunctionalized, optionally encoded beads on the surface of the substrate, and dividing the chip areas to form individual biochips. As described above, the segmentation may be done prior to assembly of the bead array on the chip surface. (As used herein, the term "biochip" refers to a chip having biomolecules attached to its surface, such as is used in biological assays.) non-limiting examples of biomolecules include oligonucleotides, nucleic acid fragments, proteins, polypeptides, ligands, receptors, antigens, antibodies, and individual members of biological binding pairs. Furthermore, the term "division" (or segmentation) refers to a method of obtaining a chip by breaking the connection between the individual chip regions on the base layer or a base layer subunit containing a plurality of chips. Furthermore, the terms "functionalized" and "biofunctionalized" refer to binding substrates for biomolecules (e.g., molecular probes), including methods of attachment to the surface of beads.

The invention also provides methods of making analytical devices comprising a plurality of molecular probes. The method comprises selecting molecular probes from a probe library and attaching them to a plurality of beads to form a bead sub-population. The bead subpopulations are attached to a major surface of a substrate containing a chip having decodable indicia identifying the source wafer. The wafer is then diced to produce a variety of biochips. The process is repeated for at least one other bead subpopulation comprising different molecular probes. The resulting biochips are then assembled to form a bio-array.

In another aspect of the invention there is provided an assay device prepared according to the method of the invention. The device of the present invention includes a base layer that is segmented to define separate chip regions. Such a substrate optionally may be further patterned and segmented to define sub-regions of confinement (restrain) one or more carriers, such as beads.

In another embodiment, the invention comprises a partitioned and optionally patterned substrate further comprising one or more species of target analyte-detecting carrier probes.

The segmented (fractional) wafer described above, with or without carrier probe arrays, is also an embodiment of the present invention. Preferably, the chip comprises an array of carrier probes.

The invention also includes an assay device for detecting one or more target analytes. Such assay devices of the invention comprise one or more biochips comprising a functionalized bead array suitable for detecting one or more desired target analytes. In a preferred embodiment, a plurality of different biochips are attached to a single support to provide the ability to detect different target analytes.

In another aspect, the invention provides a method of performing a biological assay comprising contacting a plurality of biochips bound to a support with a solution comprising at least one target analyte and detecting, directly or indirectly, said analyte. The plurality of biochips can comprise at least one subpopulation of biochips having a biofunctionalized array. Optionally, the plurality of biochips can comprise at least two subsets of biochips, wherein different subsets of biochips have different sizes or different bead array geometries.

In another aspect of the present invention, there is provided a method of performing an assay using the above assay device. The method comprises exposing a biochip array of the assay device to a solution containing at least one target analyte and detecting the reaction product.

Another aspect of the invention provides a method of making a biochip carrier comprising covering a solid substrate having at least one hydrophilic major surface with a patterned hydrophobic layer for spatially defining an array of biochips.

The invention also provides a method of assembling an array of beads on a surface of a semiconductor substrate comprising placing a patterned dielectric film on the surface of the semiconductor substrate, wherein the dielectric film forms a boundary on the surface of the substrate, and incorporating the beads in solution into a region of the substrate designated as an array of beads, wherein the region is defined by the boundary.

Another aspect of the invention provides a method of depositing beads directly onto a surface of a semiconductor substrate to form an array of beads, the method comprising adding a solution of beads to a surface of a patterned semiconductor substrate, the substrate containing a structure to receive the beads and mechanically agitating the solution to induce the beads to settle in the structure.

Another aspect of the invention provides a bead array comprising a bead array on a biochip protected with a removable coating. Wherein the bead array comprises a plurality of beads having surface-attached molecular probes, and the coating has the property of being non-reactive with the molecular probes on the surface of the beads.

The present invention also provides a method for quality control in the process of manufacturing a biochip. The method comprises optionally encoding biofunctionalized beads, exposing the recessed, patterned substrate containing the containment beads to a solution containing the beads, and optionally imaging the beads to ensure that the recesses are sufficiently occupied (occupy).

Detailed Description

The present invention provides compositions and methods for designing and producing arrays of desired compositions and patterns comprising chemical or biological entities, such as biomolecules, e.g., nucleic acids and proteins. In particular, the method of the present invention combines the flexibility of real-time (real-time) selection of array contents and high feature density with the economies of scale of parallel processing to assemble randomly coded, carrier-displayed probe array diversity of selectable compositions at designated locations of delineated wafer spacings ("chips"). The invention also includes methods of forming location and composition encoded arrays of such chips. Further, the present invention provides wafer and chip designs for solid supports such as labeled microparticles ("beads") and labeled chips ("tiles") optimized in biological tests and analyses involving biomolecules and cells.

The present invention provides methods and processes for making high unit density arrays of microparticles that are biologically or chemically functionalized. Such arrays can be produced in adjustable quantities, in flexible formats and with preselected compositions. The methods and processes of the present invention can be performed in batch and parallel formats. In particular, the invention relates to the fabrication of such particle arrays on one or more wafers so that a portion or all of a particular wafer exhibits one or more such particle arrays having a preselected composition and functionality. The invention also relates to packaging the resulting array of microparticles in a multi-chip format.

The present invention provides arrays having compositions that depend on the end use of the array. Arrays containing from about one bead to several million beads can be fabricated. Typically, the array will comprise one to one billion or more, depending on the size of the beads and substrate and the end use of the array. The preferred range for high feature density arrays is from about 1000000000 (billion) to 1 bead/mm²More preferably 1000000 to 100 beads/mm²Most preferably 100000 to 1000 beads/mm²。

The microparticles of the present invention are functionalized to include chemical or biological entities such as DNA, RNA, and proteins. These entities may be selected to provide flexibility in selecting the contents of the array depending on the application of interest. In addition, because such microparticle arrays have a high feature density, they can be designed to optimize the array for performing biological assays of interest. Such arrays are disclosed, for example, in PCT/US01/20179 and US Patent No.6251691, which are incorporated herein by reference in their entirety.

The methods of the overall process of the present invention can be divided into 4 general categories, referred to as pre-assembly, post-assembly, and packaging. Such classification is not some way of attempting to limit a certain class. As further described below, fig. 1a and 1b particularly illustrate the process of the present invention.

I. Before assembly

The pre-assembly method involves performing a chip layout, fabricating a wafer according to this layout, optionally scribing (scribes) the wafer, and then cleaning and inspecting if necessary. As described in fig. 1a and 1b, the singulation (as described below) may follow the method of assembly (fig. 1a) or follow the method of wafer fabrication (fig. 1 b). If individual chips are derived from a wafer, the resulting chips are sorted and each chip may be labeled as described below to identify such chips based on their functionality.

Chip layout

One chip layout is shown in fig. 2. It is understood that a "chip" may be any three-dimensional shape. Each chip comprises a substrate (e.g., layer 1(L1)) in which biofunctionalized beads are assembled to form a microparticle array. Many types of substances can be used as the substrate. Suitable materials have certain desirable properties. These properties can be divided into mechanical (e.g. strength), electronic (e.g. with boundary impedance (interface impedance) that can be modified), optical (e.g. flatness, transparency, well-defined optical absorption spectra, minimal autofluorescence, high reflectivity) and chemical (e.g. processes that are easy to perform for precise features or for surface reactivity of deposited dielectric layers that can be covalently linked) properties. Is suitable forNon-limiting examples of substrates include semiconductors (e.g., silicon), insulators (e.g., sapphire, mica, and ruby), ceramic substances, and polymers (e.g., Mylar @)^TM，Kapton^TMAnd Lucite^TM)。

In certain embodiments, the base layer can be a semiconductor wafer, such as a monocrystalline semiconductor wafer, which is commonly used in the semiconductor device industry. In other embodiments, the substrate may be any patternable solid substrate that is inert to reagents used in chip fabrication and bioanalysis. Non-limiting examples of such substrates include glass, plastic, and polymers.

Fig. 2 illustrates a chip with rectangular cross-linked regions and is not intended to limit other chip geometries. In fig. 2, L1 represents an intermediate layer (although layers on both sides of L1 are not required). The recessed array a1 of L1 was where the bead array was built. The shape of the recess a1 need not be square. Non-limiting examples of other suitable shapes include triangular, rectangular, pentagonal, hexagonal, and circular. One of the functions of the recessed array a1 is to aid in aligning and protecting the beads by creating a rectangular structure on the wafer or chip to limit the movement of the beads on the surface.

Optionally, the chip may also contain a second layer (L2). Layer L2 comprises a patterned insulating dielectric layer (e.g., silicon dioxide). An example of one possible pattern is given by fig. 3, which shows a star pattern in the middle of the chip. The shaded areas are dielectric material and the white areas are areas where the dielectric is removed. The thickness of the dielectric layer is typically, but not limited to, 100 nm. If an electric field is applied perpendicularly to the chip, an inhomogeneous potential is formed near the chip surface due to the L2 pattern. The electric field may be applied to the surface as described in US Patent No.6251691 (referred to as "LEAPS") incorporated herein by reference. Using LEAPS, beads in a liquid solution applied to the surface of a substrate are placed in a varying transverse electric field gradient when an AC potential is applied to the substrate. This electric field gradient drives the beads in solution so the beads will accumulate on the a1 region where surface structure is established on L1. Thus, the pattern of L2 may be any pattern that can cause beads to accumulate at a particular location on the substrate, although it should be recognized by those patterns that are more effective than other patterns in causing bead accumulation.

The patterning and predetermined design of dielectric layer L2 facilitates the quasi-permanent modification of the electrical resistance of the bead solution-dielectric-semiconductor formed electrolyte-insulator-semiconductor (EIS) structure. By spatially adjusting the EIS impedance, the electrode pattern determines the ion current in the vicinity of the electrode. Depending on the applied electric field frequency, the beads are either picked out or high ion current regions are avoided. Spatial patterning thus allows for explicit external control over the placement and shape of the bead array.

Optionally, the chip containing the bead array may contain a protective, usually surface-covering, passivation layer. The function of the L3 layer is to act as an interface between the chip and the chemical liquid medium comprising the bead suspension, the biological assay sample or the wash chip. Therefore, the L3 layer should be relatively stable to chemical attack and the surrounding environment (robust). It should also protect the functional probes attached to the beads from electrostatic forces during assembly of the bead array. In some embodiments, the L3 layer also minimizes bead adhesion to the chip surface during bead array assembly. The L3 layer is preferably inert to biological samples and is preferably non-fluorescent in the same wavelength range as used for fluorescence detection in biological assays. In addition, its presence should not cause a change in the electric field distribution near the chip surface, which would prevent the use of LEAPS for bead assembly. By way of example, the L3 layer may be a thin layer of LPCVD (low pressure chemical vapor deposition) silicon nitride having a thickness of about 40 to about 100A.

The L3 layer may also be engineered by chemical treatment to change surface properties. For example, a silicon nitride surface may be oxidized to produce SiO_x(i.e., SiO)₂And/or substoichiometric silicon oxide) or silicon oxynitride (SiO)_xN_y) Both of which are hydrophilic and facilitate dispersion of an aqueous sample. In other embodiments, the surface SiO_xOr SiO_xN_yCan advance oneStep (ii) functionalisation with silanol groups to create a hydrophobic surface.

Finally, the backside of each chip may be coated with a metal or metal alloy for electrical contact (preferably ohmic contact). For example, if the chip is made of a silicon-based layer, the backside of the chip may be coated with a thin chromium adhesion layer and a thicker gold layer, using methods conventional in the semiconductor industry. Although gold coatings are inert to most chemicals and have high conductivity, other ohmic contact coatings may be used if chemically compatible with other fabrication methods. Non-limiting examples include titanium nitride/tungsten and titanium tungsten/tungsten. The chips may be coated with a metal or metal alloy before or after singulation.

Optionally, one side of the chip and/or its parallel opposing side (parallel side) may be coated with a magnetically reactive substance. This can be accomplished by assembling the beads on one or both sides of the chip beads using conventional assembly methods. The method described in US Serial No.10/032657, applied on 28.12.2001, which is incorporated by reference, may be used. Prior to assembly, the magnetically reactive species may be functionalized to provide additional chemical and biological functionality. Alternatively, all sides of the chip may be encoded by random-absorbing beads on the chip carrier by using methods known in the art. The array configuration provides a miniaturized tag ("chip ID") to identify the chip and the wafer source ("wafer ID"). Each chip ID is derived from the number S, which represents a distinguishable configuration of a randomly encoded array of L sites, given by the following way, where r (k) n (disordered) samples of (indistinguishable) particles, 1. ltoreq. k.ltoreq.n, can be distributed in the L sites:

s (L; n; r (k), 1. ltoreq. k. ltoreq.n) | L! /[ r (1)! r (2)! .. r (k)! .. r (n)! An array indicating a large number of possible combinations is L16, consisting of 4 distinguishable bead types, each occurring 4 times (r (1) · r (4) ═ 4), can exhibit S (16; 4; r (k) ≦ 4) | 16 |, and! [ (4!) (4!) (4!) ], or about sixty-three million distinguishable configurations.

When using a randomly coded array to generate many tags T, where T < S, for many practical applications a large constellation space of size S is sampled to reduce the chance of duplication. One particular advantage of using randomly encoded bead arrays to construct labels is that they are readily, inexpensively produced in large quantities in a miniaturized form and in a one-step process by the methods of the present invention. The randomly coded bead array format of chip IDs is easily constructed to share a common subdomain (subfield) or subcode (subcode) that can be used to determine whether two or more chips are from the same wafer. For example, if a total of N bead types are used to generate chip IDs on N wafers, p types may be reserved, where p < N, and p is selected such that 2p > N. Thus, a wafer-specific subcode was constructed that contained only the remaining n-p bead types. For example, given an N-16 bead type to construct a chip ID containing a chip subcode identifying each chip from one of the N-100 wafers, a p-7 bead type may be reserved to construct a 7-digit binary to identify each of the 100 wafers by the absence of up to 7 reserved bead types. For example, one wafer in the group lacks the 7 reservation types used, and the other 7 lacks one reservation type. The encoding beads are functionalized and carry probe molecules on their surface. The encoded magnetic particles may also be magnetized and may exhibit chemical and biological functionality.

An example of a fabricated chip is shown in fig. 4. The base layer is Si (100), an n-type phosphorus-doped wafer having a resistivity of 1.5-4 ohm-cm. The chip is a square with a side length of 1.75mm and a thickness of 0.5 mm. The L2 layer was 1000A of thermally grown silicon dioxide with a 12-contact star opening in the middle. The dimensions of the star are shown in figure 4. The center of the chip has a tightly packed hexagonal array of recesses containing 68 rows and 59 columns. The dimensions of the hexagonal recess are shown in fig. 4. The L3 layer was a 60A thick LPCVD silicon nitride layer that covered the entire chip except for the sidewalls and bottom of the hexagonal recess where there was only bare silicon with native silicon oxide.

Figures 5a and 5b illustrate non-limiting examples of other structures suitable for limiting the movement of beads. H1 is a recess or cavity with straight side walls that retains a single bead. H2 is an inverted pyramidal recess that can receive a bead. H3 is a set of columns that confine one bead. Hx is a recess that can accommodate multiple beads. The upper diagram (fig. 5a) shows a top view of the structure, while the lower diagram (fig. 5b) shows a cross-sectional view. In one embodiment, a straight sidewall spacer (component) H1 that holds only one bead is used. This structure serves to confine the beads in a liquid medium. The shaded areas of fig. 5 are the underlying material and the white areas are empty spaces. The space shape is not limited to a square; for example, a pyramidal recess H2 may be used to accommodate a bead spacer. Further, while the bottom of the recess is preferably flat, this is not required in certain embodiments.

Fig. 6 shows an example of an array structure. A1 and a2 are rectangular arrays of recesses. A3 is a hexagonal array of recesses. Various structures may be fabricated on a chip or wafer to form an array or multiple arrays on the surface of the chip or wafer. These structures may be the same or different types of structures and/or structures of different sizes may coexist. 3 illustrative embodiments are set forth in FIG. 6. In the figure, the hatched area is a recess. The unshaded area is the original substrate surface (which is covered with a thin film). The arrangement a1 is a square concave regular cartesian array (regular cartesian array). Array A2 is a square recessed alternating checkerboard array (alternating checkerbioard array). Arrangement a3 is an array of hexagonal recesses. Although the array of the present invention is not limited to a regular array, a regular array facilitates elucidation of the reaction results.

Of course, the location of the array in the chip is not limited to the center. For example, the array may be located at one corner, as shown in FIG. 10. In addition, more than one array may be present on a chip. For example, 4 arrays can be fabricated on one chip as shown in FIG. 10. In some embodiments, a different set of beads is added to each of the 4 arrays on the chip. One way to accomplish this bead distribution in a large scale process is to use masks that expose only one array per chip at a time. Different sets of beads were added to the exposed array. The mask is then moved to expose another array on the chip and the process is repeated. After 4 replicates, each chip had 4 arrays of 4 different sets of beads.

1.2 wafer fabrication

Several methods can be used to pattern selected chips on a wafer using wafer techniques such as, for example, photolithography or material etching. The method of choice depends on the wafer design requirements. Depending on the different requirements, the wafer is subjected to one or more manufacturing cycles to produce a fully processed wafer. Each wafer cycle in this process includes, but is not limited to, 3 steps: (i) material growth and/or deposition; (ii) printing (lithograph); and (iii) etching. Each cycle typically produces a structural layer. One or more layers may be cycled depending on the target configuration on the wafer.

For example, the species growth or deposition may be through SiO₂Growth on silicon or dielectric substances such as SiO₂、Si₃N₄Or chemical vapor deposition of other substances, or deposition of metals such as aluminum, chromium, gold, titanium, or other metals. The printing step may comprise photolithography, electron beam printing (e-beam lithography), x-ray lithography or imprint lithography (imprints). The etching step may include removing an amount of material within certain areas defined by the masking layer, such as but not limited to photoresist. Non-limiting examples of etching methods include anisotropic etching, such as reactive ion etching, biased crystal plane (crystal-biased) wet chemical etching, or isotropic etching, such as isotropic wet chemical etching, vapor etching, or plasma etching.

1.3 scribing

To effectively perform the functionalization, the wafer processing is followed by chip area scribing so that the chip area is suitable for batch parallel processing. To this end, the present invention preferably scores the wafer to score the regions so that subsequent singulation can separate them into individual chips. In other embodiments of the invention, the chips are separated using techniques that do not require dicing. The purpose of the scribe lines is to create break lines to facilitate separation of individual chips in the wafer dicing step without damaging or destroying the individual chips. It should be noted that although the scribe line is to be broken, it is strong enough not to damage the chip during subsequent processing steps. For example, scribe lines may be created using a dicing machine (e.g., DISCO, Dynatex, or Loomis Industry) such that the scribe lines are only a fraction of the wafer thickness. A roller is then applied to the scribe line in the vertical direction to further scribe the individual chips. Scribing may be done using a stone-drilling scriber; the inter-chip channel (trench) on a silicon wafer can be created by chemical etching using wet chemistry, such as for example potassium hydroxide/water solution at high temperature. The wafer may also be dry etched by deep reactive ion etching to create well-defined channels between the chips.

1.4 wafer cleaning and inspection

In the dicing step, dust or particles may be generated. In order to protect the wafer surface, a protective layer is applied to the wafer surface in one embodiment of the invention. For example, the layer may be an adhesive tape (if it does not damage the wafer surface), a photoresist coating, or other organic coating. After scribing, the protective layer is removed by peeling it off the wafer (e.g., if it is a tape) or dissolving it in a suitable solvent. For example, the photoresist layer may be removed by dissolving it in acetone, and then rinsing the wafer with isopropanol. If trace amounts of protective coating remain on the wafer, a more aggressive cleaning process may be used. In one embodiment, the wafer is cleaned with an oxygen plasma to remove trace organics. In another embodiment, the wafer is cleaned by an RCA cleaning method, which is a standard cleaning method in the semiconductor industry, which includes an ammonium hydroxide/hydrogen peroxide mixture heated to about 75 ℃. In another embodiment, the wafer is cleaned at elevated temperatures (about 60℃.) with a mixture of concentrated sulfuric acid and hydrogen peroxide.

1.5 chip grouping (grouping)

Fig. 7 shows an example of chip (C3) grouping. C1 is a wafer or any base unit that facilitates batch fabrication as used in the semiconductor industry; c2 is a subunit of C1 (could be the entire C1) which consists of the required number of chips; c3 is a chip, which is the minimum unit of biochip. Typically, C2 is the complete unit for chip functionalization. After functionalization, C2 was separated into individual C3.

1.6 bead functionalization and combination (pooling)

As used herein, the terms "microsphere," "microparticle," "bead," and "particle" are used interchangeably. Compositions of beads include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic substances, thoria gel, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as sepharose, cellulose, nylon, cross-linked micelles, and teflon (teflon). (see "Microsphere Detection Guide", Bangs Laboratories, Fishers, IN.) the particles need not be spherical and may be porous. The bead size may be in the range of nanometers (e.g., 100nm) to millimeters (e.g., 1mm), with beads in the range of about 0.2 microns to about 200 microns being preferred, and in the range of about 0.5 microns to about 5 microns being more preferred.

In some embodiments of the invention, the beads are functionalized prior to distribution to the surface of the wafer so that each bead has a specific type of biological probe attached to its surface. Various methods of functionalizing beads are suitable for use in the present invention. Suitable methods depend in part on the nature of the material used to make the beads. For example, beads are functionalized by attaching binding agent molecules, including nucleic acids, including DNA (oligonucleotides) or RNA fragments, using methods known in the art, such as using one of several conjugation reactions known in the art; a peptide or protein; aptamers (aptamers) and small organic molecules (G.T. Hermanson, Bioconjugate technologies (academic Press, 1996); L.Illum, P.D.E.Jones, Methods in Enzymology 112, 67-84 (1985)). In certain embodiments of the invention, functionalized beads have a binding agent molecule (e.g., DNA, RNA, or protein) covalently attached to the bead. The beads may be stored in a bulk suspension containing a buffer for later use. Functionalization typically requires a one-or two-step reaction that can be performed in parallel using standard hydraulically controlled robots to covalently attach any number of desired functions to a given bead. Beads of core-shell architecture may be used, the shell consisting of a thin polymeric barrier layer, the preferred composition of which is selected; and functionalized according to the application of the target assay.

In some embodiments of the invention, the beads are color-coded with a fluorescent dye. For different analytical applications, the beads may comprise additional dye-labeled biological substances on their surface. To detect the beads and the signal analyzed, fluorescence microscopy imaging can be used.

The bead library was created by preparing a subset of different sets of beads. Each bead subpopulation is prepared by attaching one type of molecular probe from a probe library to a plurality of beads to form a subpopulation. Each bead subpopulation is distinguished by having fluorescent dye color coding or other methods.

Assembly of

Bead arrays are assembled by protecting the beads on the surface of the wafer or part of the wafer. Prior to protecting the beads on the wafer surface, a bead library is formed by chemically encoding or staining the beads with optically distinguishable labels containing one or more fluorophore dyes that are spectrally distinguishable by excitation wavelength, emission wavelength, excited state time, or emission intensity. The optically distinguishable labels can be used to stain beads at a specific rate, as disclosed, for example, by Fulwyler, U.S. Pat. No. 4717655. Staining may also be carried out by swelling the Particles by methods known to those skilled in the art (Molday, Dreyer, Rembaum & Yen, J.mol.biol 64, 75-88 (1975); L.Bangs, "Uniform Latex Particles", Seragen Diagnostics, 1984). For example, up to 12 distinguishable bead populations can be encoded by swelling with two colors (waving) and bulk staining, each with 4 intensity levels, and mixed in 4 molar ratios. International application No. PCT/US/98/10719, which is incorporated herein by reference in its entirety, discloses a combined color coding of the outer and inner surfaces. Color coding is also discussed in United States patent No.6327410, which is incorporated herein by reference in its entirety.

When forming an array of beads, there are many possible ways to protect the beads on the chip surface. The recesses formed during the wafer fabrication step provide spacing that retains the beads on the surface of the substrate. The effectiveness of protecting (or immobilizing) the beads depends on the size of the recess relative to the size of the bead. The size of the recess for this purpose is such that the depth of the recess is about 0.5 to 1.5 times the diameter of the bead used. More preferably, the recess is sized such that when the bead is stabilized in the recess by gravity, its highest point is below the top of the rim and there is only enough space to accommodate the volume of the other bead 1/3. In addition, although it is preferred that the size of the recesses be larger than the size of the beads, in this preferred embodiment, each recess should not be able to accommodate more than one bead. Also, the opening of the recess is slightly larger than the bead. For example, the hexagonal array shown in fig. 4 conforms to a bead having a diameter of 3.2 microns.

It may not be necessary to use recesses in the substrate to accommodate the beads. For example, a plurality of posts may be arranged on the surface of the substrate to confine the beads. One possible configuration is shown in the upper (top) and lower (perspective) views of fig. 5 c. Here, each bead is bounded by 6 pillars surrounding it. The number of columns is not limited to 6, but may be 3 or more. Further, any other raised or recessed surface structure may be used, including bumps, posts, bumps, and indentations. In other embodiments, a large recess may be used that can accommodate more than one bead. For example, fig. 5d shows a large recess with straight side walls. The top view of the upper figure shows that the horizontal dimension of the large recess is more than twice the diameter of one bead.

As noted above, the geometry and size of the recesses used to assemble the microparticle array can be varied. In some embodiments, the geometry and size are altered by depositing a layer of silicon oxide or polymer after the pores are formed by etching. For example, a recess having a reentrant sidewall profile may be formed by this deposition method. As used herein, the term "re-entrant sidewall profile" refers to a recess having a surface that is recessed to an opening having a diameter that is less than the diameter of the bottom of the recess. Recesses with re-entrant sidewall profiles formed by this method have higher bead retention rates during processing and analysis.

The beads may be attached to the surface by covalent or van der waals forces, electrostatic forces, gravitational forces, magnetic or other forces. These combination methods may also be used in combination. In one embodiment, the bead array may be generated by picking a sample of the designated encoded beads (aliquot) from each pool in the bead array composition described. Samples of the "assembled" (dispensed) suspension are dispensed onto selected substrates, such as scribe streets of a wafer.

In other embodiments, the bead arrays can be prepared using LEAPS. In these embodiments, a first planar electrode is provided substantially parallel to a second planar electrode ("sandwich" configuration), the two electrodes being separated by an aperture containing an electrolyte. The surface or the inside of the first planar electrode is patterned by an interfacial (interfacial) patterning method, as described below. The encoded and functionalized beads are introduced into the gap. When an AC voltage is applied in the gap, the beads form a randomly encoded bead array on a first electrode (e.g., a chip or wafer). Alternatively, an array of beads can be formed on one photo-sensing electrode (e.g., a chip or wafer) using LEAPS. Preferably, the above-described sandwich configuration is also used for the planar photo-sensitive electrode and the further planar electrode. Furthermore, the two electrodes are separated by a gap containing an electrolyte. Functionalized and encoded beads are introduced into the gap, which form an array on the photosensitive electrode when an AC voltage and light are applied in combination.

The substrate (e.g., chip or wafer) used in the present invention can be patterned using the LEAPS interface patterning method, by patterned growth of, for example, oxide or other dielectric substances to create the desired impedance gradient configuration under application of an AC electric field. Alternatively, the patterned base layer may be obtained by selectively doping the inner regions of the base layer with an impurity. The pattern can be designed to produce the desired configuration of AC field induced fluid and corresponding particle transport. A base layer may be patterned on a wafer using semiconductor processing techniques. In addition, the substrate is spaced by depositing a thin film of UV-patternable, light-transmissive polymer, such that the desired pattern of fluid channels and spaces is attached to the substrate to confine fluid in one or more discrete spaces, thereby accommodating multiple samples on a given substrate.

For example, spatial encoding can be accomplished in a single liquid phase during array assembly, e.g., using LEAPS to assemble a planar bead array in any desired configuration in response to a changing electric field and/or light pattern projected onto a substrate. LEAPS creates a lateral gradient of interfacial impedance between the silicon chip and the solution that modulates the electrohydrodynamic forces that mediate array assembly. The electrical requirements are moderate: a low AC voltage of below 10Vpp is typically applied between the two planar electrodes in a fluid gap of typically 100 m. This assembly method is fast and optically programmed: under an electric field, an array containing thousands of beads was formed within a few seconds. Multiple sub-arrays can also be created in a multi-fluid phase maintained at spaced chip surfaces. Alternatively, spatial encoding is accomplished by assembling separate chips, each carrying at least one random coded array from a specific library, into a designated multi-chip configuration.

In one embodiment, the method disclosed in PCT/US01/20179 (referred to as "READ"), incorporated by reference in its entirety, can be used to prepare custom bead arrays for performing multiplex biomolecule analyses of the present invention. Using READ, arrays are prepared by applying a separate batch process to produce application specific substrates (e.g., wafer-level chips) and to produce chemically encoded and biologically functionalized beads (e.g., -10)⁸Bead/1001 suspension). Preferably, bead Quality Control (QC) is performed separately prior to array assembly, such as determining morphological and electrical properties, examples of the latter include surface ("Z") potential and surface conductivity. In addition, the beads in suspension are analyzed prior to introduction into the substrate. This is to optimize the array conditions, usually with the goal of maximizing array sensitivity and specificity and minimizing bead-to-bead variation. For substrates, the QC step may include optical inspection, ellipsometry, and electrical transfer (electrical transfer) measurements.

Once the chemically-encoded and biofunctionalized beads are combined with a substrate (e.g., a chip or wafer), the LEAPS or another active deposition method can allow for rapid assembly of dense arrays at designated areas of the substrate. By assembling in the same fluid phase, problems such as point-to-point or chip-to-chip variability can be avoided without the need for rework (retool) or design methods. Furthermore, the consistency of these methods can lead to optimization of beads independent of the nature of the chip and the array conditions. In addition, multi-bead arrays can be formed simultaneously in discrete fluidic spaces maintained on the same chip or wafer. Once formed, these multi-bead arrays can be used for parallel processing of multiple samples. The integration of LEAPS and microfluidics creates a separate, miniaturized, optically programmable platform for parallel analysis of proteins and nucleic acids.

Once the functionalized and encoded beads are prepared and bound to the substrate, binding of the analyte to the binding reagent on the beads can be performed before or after assembly of the randomly encoded array on the substrate. For example, a bead array may be formed after analysis, and an analysis image and a decoding image of the array may be subsequently obtained. Alternatively, beads may be assembled and immobilized on an array by physical or chemical means to produce a randomly encoded array. The DC voltage can be used to produce a random coded array. A DC voltage, typically 5-7V (for beads in the range of 2-6 μm and a gap size of 100-150 μm) and applied for < 30s in a "reverse bias" configuration so that the n-doped silicon base layer forms the anode, causes the array to compress, facilitating contact of adjacent beads in the array and simultaneously causing the beads to move to regions of high electric field in close proximity to the electrode surface. Beads can be anchored to the surface by van der waals forces or "tethers" (tethers) that extend from the bead surface, such as polylysine and streptavidin.

After assembly of the beads, the chip or wafer is inspected and imaged by fluorescence microscopy to obtain a decoding map. Decoding can be used later to identify the location and functionality of each individual bead.

The percentage of array positions that are filled is preferably higher than 50%, more preferably higher than 90%. To test the effectiveness of the recesses to retain the beads on the surface of the substrate, the chip containing the bead array was placed in an aqueous solution and shaken continuously for 3 days. Image comparison before and after this test showed that more than 99% of the beads remained in the recesses on all test chips.

After assembly

During the post-assembly process, the bead array is covered with a protective surface. Before and after covering the beads, the wafer containing the bead array is divided into one or more bead chips.

3.1 protective particles

In certain embodiments of the invention, a gel covering the bead array region can be used to prevent bead migration. In other embodiments, recessed bottom and/or side wall chemical functional groups can be used to attach the beads to a surface. Charged polymers may also be used to coat the chip prior to bead deposition. The charge of the overlying polymer is selected to be opposite to that of the beads so that the beads can be attached to the polymer by electrostatic forces. When a bead is positioned in a charged polymer-coated recess, the coulomb attraction between the bead and the recess sidewalls and bottom holds the bead in the recess. In this way, the bead retention rate during processing and analysis is increased. In some embodiments, a second charged polymer is deposited on the surface of the chip after the beads are placed in the recesses. The second polymer is selected to have the same charge as the beads so that no polymer is deposited on the beads, but the surface charge of the chip is neutralized. Several variations of this technique can be applied to minimize core process changes. For example, a single polyelectrolyte layer may be used, or a multi-layer structure (with alternating positive and negative electropolymer layers) may be constructed to produce a coating with a more uniform and controlled thickness. Also, instead of polymers, charged polymer nanoparticles may be used alone or in combination with charged polymers. Uncharged but low T's may also be used_g(glass transition temperature) polymer and/or nanoparticle coatings enhance the adhesion of the beads to the chip surface.

3.2 protection of assembled arrays

Removable coatings may be used to protect the biofunctionalized beads on the wafer or segmented biochip array prior to segmentation into biochips. There is therefore a need for a method of protecting bead arrays from environmental dust, dirt and other contaminants during storage of biochips or wafers. The present invention provides a coating for protecting a biochip and a wafer and a method for preparing the coating. In a preferred embodiment, the coating protects the beads of the bead array from environmental contaminants and prevents degradation of biomolecules at the bead surface. Furthermore, the coating can be easily removed from the surface of the biochip before the bioanalysis is performed.

In certain embodiments, the coating comprises an inert, non-reducing sugar, such as trehalose, which does not interact with reactive chemical moieties such as amino groups in peptides and proteins, and thus prevents the general degradation or aggregation upon drying with other excipients.

In other embodiments, a hydrogel (e.g., agarose hydrogel) can be used to prevent contamination, dehydration, and physical damage during storage. The hydrogel may be peeled off the substrate surface prior to performing the biological assay. The exfoliation action not only removes the hydrogel but also cleans the surface of any excess beads that are not located in the array locations defined by the recesses or other retaining structures. These excess beads embedded in the hydrogel can be recovered for further use.

3.3 chip segmentation

After functionalization, the chip set (wafer or sub-unit of a wafer) is segmented. If the wafer has been scribed, the separation is performed by breaking the connections between the chips. This can be accomplished by rolling a roller across the backside of the wafer in a vertical direction along the scribe line, in accordance with the method described in U.S. patent No. 3790051. Alternatively, by other methods, e.g. using Dynatex International^TMThe manufactured GST Scriber/Breaker completed the segmentation. The individual chips obtained by this method are ready for packaging. In addition to the singulation methods described herein, any other singulation method, such as laser cutting, may be used to achieve the objectives of the present invention.

In some embodiments, the wafer or subunit is singulated prior to biofunctionalization. Each chip is then either biofunctionalized identically or by exposing subsets of the chip to different bioactive groups.

IV. encapsulation

Using the method of the present invention, a variety of chips can be produced by assembling randomly encoded arrays of probe molecules displayed on encoded beads. Each chip cut from a uniquely identified wafer may contain one or more arrays of randomly encoded beads. This method of random assembly of the invention encompasses an embodiment wherein the bead displayed probes on a chip containing a randomly encoded array are members of a large probe library displayed on a label chip selected from a variety of wafers. The chips of different wafers may be selected and assembled to form a combined (bumped) chipset. Preferably, the chip displays a decoding mark that identifies the origin of the wafer. An array of coded chips can be formed by random assembly on a planar surface, a method also known as random tiling, as described in FIG. 15 d. Random tiling refers to a method of assembling a set of coded chips into a planar arrangement or array so that each chip or portion of each chip in the assembly or array can be optically inspected.

The hierarchical scale of random assembly from bead array level to chip array level provides the flexibility to rapidly generate large custom compositions and arrays of probe sets with high feature density. Such arrays can be used to display a large set of probes for gene expression profiling (gene expression profiling) or for methylation profiling of DNA by analytical methods known in the art. In addition, random tiling methods of attaching multiple chips to a single support, as described below, provide a novel, rapid and flexible method of performing combinatorial (posing) and deconvolution strategies known in the art when it is desired to expose multiple probe arrays to different reactions. For example, arrays displaying partially overlapping probe sets are readily produced by appropriate construction and chip selection.

After assembling the bead arrays of the present invention, the wafer is diced to allow manipulation of individual marker chips. In random tiling, a marked chip selected from one or more wafers is placed on a surface (preferably the surface of a flat substrate) on which the chip can be moved to form a multi-chip assembly corresponding to a desired layout. To facilitate tight packaging, the chip may be designed to exhibit a convex symmetric shape such as a square, triangle, or hexagon. To reduce the distance between bead arrays on adjacent chips, the chips can be designed to exhibit interlocking shapes (FIG. 14 c).

Sliding assembly

In this embodiment, multiple singulated wafers are placed on a common large substrate having sides showing the probe arrays facing downward. In a preferred embodiment, the probe array is recessed to prevent direct contact with the substrate. One or more chips are randomly selected from each wafer, and a die (coins) is slid over a face in a similar manner to form an array of chips by sliding the chips to designated assembly areas. Generalizing this approach to the rank steering as shown in FIG. 8. In this embodiment, the tiling process can be monitored and recorded by standard optical and machine vision instruments used for semiconductor inspection. This instrument can be used to track individual wafers from a chip source to their respective final positions, and can directly perform position encoding and decoding of the assembled chip array. After the assembly process is completed, a multi-chip carrier (as described) is aligned with one or more chip arrays arranged in the assembly area, and the chips are then laid down and bonded to form a multi-chip assembly. To facilitate bonding, the carrier may be pre-coated with an adhesive or may be coated with a magnetic substance if the chip is magnetized by the method.

This slide assembly method preferably uses a mechanical contact, such as a suction device known in the art that can lift and manipulate individual chips. Alternatively, the magnetizable chips are manipulated using a magnetic pen that can select one or more chips from each wafer. The wafer (and the chips contained thereon) is magnetized by depositing a magnetic substance such as nickel or nickel-iron alloy ("permalloy"), as is known in the art for example for semiconductors and ceramics, by means of electroplating or electroless deposition. Alternatively, paramagnetic particles may be introduced as part of an array of randomly encoded microparticles displaying probe molecules or as a separate feature, for example in the form of an array assembled at a designated portion of each chip. The array of magnetizable particles may be located on one side or the other of the chip containing the array of randomly encoded probes.

Slide assembly typically involves handling individual chips and becomes quite cumbersome as the number of constituent chips of the chip array increases. The situation is even more pronounced if the chip is small, exhibiting linear dimensions of, for example, 100 μm or less. For example, the ceramic substrate may form a chiplet in the shape of a cube or near-cube in this dimension. In this case, the individual chips are preferably manipulated by methods known in the art for manipulating glass or polymer particles of similar size.

Collective Assembly (Collective Assembly)

In this embodiment, chips cut from individual wafers are stored in a bulk suspension using an inert storage buffer such as high purity water containing trace amounts of azide. The chips are suspended by mechanical or magnetic agitation. Chip assemblies (pools) are formed by dispersing and mixing selected suspensions. Optionally, traces of glucose or other highly soluble molecular weight components may be added to the suspension to increase viscosity and thus improve fluid properties. Random deposition is accomplished by loading and depositing the dispersed suspension onto a planar substrate using a syringe, pipette or capillary or by using continuous methods known in the art to produce arrays including those colloidal particles that cause flow and capillary forces (Adachi, E et al, Langmuir, Vol.11, 1057-.

In random deposition, a template may be provided to the substrate to guide the positioning of the individual chips and to contain the individual chips at designated locations on the substrate. In one embodiment, the chips are collected from the mixed suspension by inserting a net into the suspension and withdrawing it so that each chip is lifted (lifted) or scooped out (scoop out). Preferably, a chip, particularly a chip placed on a flat, featureless base layer, is sufficiently separated from other chips to prevent partial overlap and stacking before "continued increase" into a close-packed configuration. Separation, for example by sliding tissue (see above) or by mechanical agitation, has the advantage of inducing "drum modes" on flexible substrates such as polymer substrates, as practiced in the art.

In a preferred embodiment, the chip is "continuously added" mechanically, for example, by placing the chip in a fluid that flows parallel to the surface of the substrate in a sandwich flow cell (sandwich flow cell) wherein the chip is pushed against an obstruction at the distal end of the cell.

Preferably, the randomly assembled chip is oriented to expose the active side displaying the array of randomly coded probes. When the active side is not exposed, the chip must be flipped. Flipping the chip into the preferred orientation is accomplished by mechanical agitation and cycling the bonded correctly oriented chip (coated with a heat or light activated bonding adhesive). Alternatively, inversion during mechanical agitation is aided by moving the centroid to the undesired side of the chip, such as by metallization. The metal-plated chips can be deposited in the presence of a magnetic field gradient perpendicular to the surface of the base layer to provide slow enough settling in a high viscosity medium to allow the chips to adopt the correct orientation as they approach the surface.

Inversion can also be facilitated by creating three-dimensional shapes, such as pyramidal shapes, with the pyramidal tips facing away from the active surface, as produced by standard semiconductor etching methods.

The chip may be packaged in a single or multiple chip packages. In a multi-chip package, chips containing different arrays of biological probes are placed on the same carrier. Fig. 8 shows 4 chips packaged together to form a square bonded chip or a linear bonded chip. The 4 chips may be adhered to a common carrier such as a glass slide, or they may be attached to the carrier by other means such as adhering a magnetic substance to the back of the chips so that they adhere to the magnetic carrier. Chip processing is not limited to the use of pick-and-place equipment. After singulation, the chips may be grouped in rows or columns. These rows and columns of chips can be moved by restriction bars (confinements bars). Fig. 9 shows different combinations of chips obtained by selectively arranging different rows of chips.

Fig. 10 shows another package design. The 4 chips with arrays at the corners combine to form one chip with a larger array in the middle. If the arrays on 4 chips contain different functional probes, a large array will contain 4 times the amount of information that a single chip contains.

A variety of chips can be produced by assembling randomly coded arrays of probes displayed on beads. Each chip may include one or more arrays of randomly encoded beads and may be diced from a uniquely identified wafer. In another embodiment, a chip containing a randomly encoded array of probes on beads may be a member of a probe library. In this embodiment, each chip of the multi-chip array exhibits a decodable indicia that identifies the source wafer.

Glass surfaces or other similar surfaces on the glass slide can be used to make multi-chip carriers. For preparing the slide as a carrier, a coating, such as Teflon, can then be applied^TMTo create an annular opening or hole (i.e., a glass region without any teflon coverage). Each hole is a circle with a diameter of 6.5 mm. One or more chips may be bonded to the glass surface in one well. A typical slide is 25X 75mm and 1mm thick, with a 2X 5 array of wells. Typical chip sizes are 1.75 × 1.75mm, up to 4 chips can be bonded to the glass surface in each well. Each chip in the same well may have a different set of assembly beads prior to bonding to the carrier. For example, if each chip has an array containing 39 bead sets, a well with 4 different chips will have a total of 4 × 39-156 beads. On the other hand, for larger chips (e.g., 4.5 x 4.5mm square), one well is completely occupied by one chip. For the pore sizes described herein, each pore can retain up to 40 μ l of liquid (typically an aqueous solution). Typically, a 20. mu.l volume of sample solution is added to each well for performing the biological reaction so that each chip is completely covered with the sample solution. Since the Teflon coating outside the wells is hydrophobic, it is water solubleThe liquid sample will not spill. The form of the carrier slide can be designed to suit certain applications. For example, a row of 8 wells on a slide can be used to analyze 8 samples. Furthermore, a 4 × 8 well array can be used to analyze 32 samples. Also, more wells (e.g., 96384 and 1536) arranged on a single slide can be used to analyze more samples.

In certain embodiments of a removable chip carrier, the chip is bonded to a substrate such as glass, stainless steel, plastic, silicon, or ceramic. The entire carrier unit is movable and can be transferred during processing to expose the chip to different reaction media, such as reaction chambers, elution chambers and signal reading stages.

In other embodiments, the removable chip carrier comprises a slot and a slot on which the chip is protected. By covering the chip in the removable chip carrier, contamination during transfer can be minimized. In some embodiments, the slot of the removable chip carrier also serves as a processing environment. If desired, a reaction gas or liquid solution for a different purpose such as performing biological analysis or cleaning the chip may be added to the tank and then removed (evacuated). In addition, the removable chip carrier may have means (means) to change the thermodynamic properties of the wells, such as the well pressure or temperature.

V. analysis

The biochip comprising the bead array of the present invention can be used to perform various bioassays and chemical analyses. Once assembled, the bead arrays on the biochips of the invention can be imaged to record analytical signals and can be decoded to identify probe-bound target analytes associated with individual beads in the array. Bead arrays provide a system that can be used to read the results of a multi-step biological or chemical analysis sequence. In addition, because there are multiple probes involved with an array comprising different target analytes, multiple target analytes can be detected simultaneously. In addition to providing the ability to detect the presence or absence of specific target analytes, the bead arrays of the present invention have also been found to be useful in determining the affinity constant of probe-bound target analytes. Thus, biochips have a wide range of applications for detecting, for example, biomolecules such as TNF- α and IL-6. Other non-limiting applications include genotyping by polymorphic analysis; analyzing gene expression; quantitative multiplexed profiling of cytokine expression, analysis of genes and gene products in the same fluid sample; analyzing affinity fingerprints; and multiplex (multiplexed) analysis of reaction kinetics. Other analyses and analytical determinations can be performed using the biochips of the invention, as described in U.S. Pat. No.6327410, which is incorporated by reference.

Examples

The invention will be better understood from the following examples. However, one skilled in the art will readily appreciate that the specific methods and results discussed herein are merely illustrative of the invention as set forth in the claims below.

Example 1: wafer fabrication and design of chips containing bead arrays

The manufacturing process of a chip containing an array of beads as shown in FIG. 4 is described in FIG. 11. The base layer was a100 mm diameter, 0.5mm thick silicon wafer with a (100) n-type phosphorus doped crystal orientation. Suitable impedance ranges for these wafers are 1.5-4 ohm-cm. Typically wafers are batch manufactured in as many as 25. The first step comprises SiO₂And (5) growing. Firstly, the wafer is cleaned by an RCA cleaning method, comprising the following steps: (1) the wafer is placed in NH with a volume ratio of 1: 5₄OH∶H₂O₂(30％)∶H₂Soaking the mixture of O in 75 deg.c for 10 min; (2) rinsing with cascade water batch cleaning (cascade water batch cleaning) using 18M Ω -cm of water; (3) the wafers were placed in a volume ratio of 1: 5 HCI (36%): H₂O₂(30％)∶H₂Soaking the mixture in the O mixed solution for 10 minutes at 75 ℃; and (4) rinsing with cascade flow water batch cleaning until the water in the batch is at least 16M Ω -cm. The wafer is dried by rotation and then put in a horizontal furnace for SiO₂And (5) growing. The wafers were placed vertically on a quartz boat (quartz boat) and introduced at 1050 deg.C and 760torr pressure with O₂+ HCl (4%) oxidation furnace. The oxidation time was 34 minutes. By this method, a SiO of 1000A is obtained₂Layer, as confirmed using ellipsometry (refractive index: n ═ 1.46, thickness variation: < 5%).

With SiO₂The wafer of (1) was spin coated with photoresist (Shipley 1813) at 4000rpm (30 seconds of spinning) and then baked at 115 ℃ for 60 seconds on a hot plate to remove the solvent. The wafer was then exposed to UV light (365-. After UV exposure, the wafers were developed by an AZ300MIF developer (developer) for 60 seconds, rinsed in DI water and blown dry with a stream of compressed dry nitrogen. The wafer was then left without a buffered oxide etchant (6: 1 mixture of ammonium fluoride and 50% aqueous hydrogen fluoride) for 2 minutes to etch the SiO in the exposed areas₂(asterisks in FIG. 11). The wafer was then rinsed with DI water and then soaked in 1165Microposit photoresist remover at 60 ℃ for 60 minutes to remove the photoresist. The wafers were then rinsed in DI water and blown dry with dry compressed nitrogen. The entire process produces a wafer with a patterned oxide layer.

After the oxide patterning step, the wafer was cleaned by RCA method and then placed on a horizontal furnace for silicon nitride (Si)₃N₄) And (6) depositing. Two silicon nitrides may be used: standard and low stress (low stress) nitrides. The deposition conditions were as follows: LPCVD nitride (standard), pressure 200mtorr, temperature 800 ℃, SiCl₂H₂＝30sccm，NH₃90 sccm; LPCVD nitride (low stress): pressure 150mtorr, temperature 850 deg.C, SiCl₂H₂＝47sccm，NH₃10 sccm. After 2 to 3 minutes of deposition, Si₃N₄The film thickness is between 60 and 90A.

The next step is to fabricate the array structure. The wafers were spin coated with photoresist OCG 12i at 4000rpm (spin time 30 seconds) and then baked on a hot plate at 90 deg.CFor 60 seconds to remove the solvent. The wafer was exposed to UV light (365nm) and repeated photo-etching was performed using a GCA-630010 xi-line Stepper (Stepper). After exposure, the wafers were baked on a hot plate at 115 ℃ for 90 seconds, then developed by an AZ300MIF developer for 60 seconds, rinsed in DI water, and blown dry with a stream of compressed dry nitrogen. The wafers were baked in a 90 c oven for 20 minutes. Wafers were etched in a Plasma Therm 72 etcher using CF₄Gas reactive ion etching etches to remove the silicon nitride in the exposed areas (hexagonal features in the array). Oxygen reactive ion etching may then be used to remove residual polymer on the hexagonal features. The hexagonal recesses were fabricated by Deep Reactive Ion Etching (DRIE) using an Unaxis SLR 770 ICP deep silicon etcher (strained Bosch fluoride process). The process was adjusted to etch a 3.8 micron deep recess in 2-3 minutes. The depth is controlled within 0.3 micron. After etching, the wafer was immersed in 1165Microposit photoresist remover at 65 ℃ for 60 minutes to remove the photoresist. The wafers were rinsed in DI water and blown dry with dry compressed nitrogen. The wafer was then processed by oxygen plasma in a GaSonics, Aura1000, Downflow photoresist stripper to remove any residual polymer in the hexagonal recesses created during DRIE. The wafer was then spin coated with a protective photoresist coating (Shipley1813, rotation 4000rpm, spin time 30 seconds) and then baked on a hot plate at 115 ℃ for 60 seconds to remove the solvent. The gold of 500A was coated on the back with 100A of chromium as an adhesion layer to sell the wafer. The native silicon oxide layer on the back of the wafer was stripped prior to the coating process using argon ion sputtering.

The fabricated wafer was diced from the surface to define each chip (size of each chip, 1.75 x 1.75mm square). The depth of the wound is 2/3 the wafer thickness. After cutting, the photoresist was removed by soaking it in 1165Microposit photoresist remover at 60 ℃ for 60 minutes, then rinsed in DI water and blown dry with a stream of compressed dry nitrogen. The wafers are then typically soaked in NanoStrip (a mixture of concentrated sulfuric acid and hydrogen peroxide) at 60 ℃ for 2 hours, then rinsed in DI water and blown dry with a stream of compressed dry nitrogen. After these steps, the wafer is ready for the bead assembly step.

After assembly of the beads, additional beads that are not protected in the recess may be removed. One method of removing unprotected beads is to wipe the chip or wafer surface with a wet cotton swab. Another method is to wash away the unprotected beads with a stream of water almost parallel to the surface of the chip or wafer. Another method involves growing a gel on the surface and then peeling the gel.

Example 2: functionalized beads and formation of bead arrays

Color-coded, tosyl-functionalized 3.2 μm diameter beads were used as solid phase carriers (solid phase carriers). Several sets of distinguishable color codes were generated by Particles stained using standard methods (bangs. l.b., "united Latex Particles", seragen diagnostics inc., p.40). The stained beads were functionalized with neutral streptavidin (Pierce, Rockford, IL), a biotin-binding protein to mediate immobilization of biotinylated probes or primers. In a typical small-scale coupling reaction, 200. mu.l of a suspension containing 1% beads are washed three times with 500. mu.l of 100mM phosphate buffer/pH 7.4 (buffer A) and resuspended in 500. mu.l of this buffer. After 20. mu.l of 5mg/ml neutravidin was added to the bead suspension, the reaction was carried out overnight at 37 ℃. The coupled beads were then washed once with 500. mu.l PBS/pH7.4 (buffer B) containing 10mg/ml BSA, resuspended in 500. mu.l of this buffer and reacted at 37 ℃ for 1 hour to block unreacted sites on the bead surface. After blocking, the beads were washed 3 times with buffer B and stored in 200. mu.l of this buffer.

Biotinylation at the 5' end of the probe (for detection of the target molecule) and primer (which can be used as a template for subsequent catalytic reaction extension of the hybridized DNA target to identify the reaction) coupled to the bead; a 15 carbon triethylene glycol linker was inserted between biotin and oligonucleotide to minimize the destructive effects of surface immobilization in subsequent reactions. For each primer, a binding reaction was performed using 50. mu.l of the bead suspension. The beads were washed once with 500. mu.l of 20mM Tris/pH7.4, 0.5M NaCl (buffer C) and resuspended in 300. mu.l of this buffer. A primer solution (2.5. mu.l of a 100. mu.M solution) was added to the bead suspension and reacted at room temperature for 30 minutes. The beads were washed three times with 20mM Tris/pH7.4, 150mM NaCl, 0.01% triton and stored in 20mM Tris/pH7.4, 150mM NaCl.

An example bead array is assembled as follows. The bead suspension obtained by the above procedure was washed 5 times with deionized water (all water used was highly pure and sterile with an impedance of 18 M.OMEGA. -cm or more), and then suspended in 0.01mM TRIS Base + 0.01% Triton x-100 aqueous solution. The bead content in the suspension was 0.5%. Mu.l of the bead suspension was added to the surface of a 4.5mm square chip containing an array of beads via a micropipette (micro-pipe). The chip was then subjected to the LEAPS method. Counter electrode (counter electrode) is a piece of glass coated with a layer of Indium Tin Oxide (ITO). The gap between the chip surface and the ITO-coated glass was 100 microns. The AC power was applied in the order shown in table 1.

Table 1AC application sequence. The voltage is half the peak-to-peak amplitude.

Step (ii) of	Time (minutes)	Frequency (Hz)	Voltage (+/-volt)	Function(s)
					1	2	2000	3	AC on
2	2	1000	3	AC on
					3	2	500	3	AC on
4	2	2000	3	AC on
					5	2	500	3	AC on
6	2	2000	3	AC on
					7	2	200	3	AC on
8	2	2000	3	AC on
					9	2	200	3	AC on
10	0	200	0	AC stop

After completion of the sequence, the beads within the area of the star pattern are concentrated in the array area. The concentration of the beads is aided by the presence of a star pattern induced fluid (flow) pattern. After the beads settled for 15 minutes, the apparatus was slowly immersed in pure water. The ITO glass coating was slowly lifted up and the water was slowly drained off so that the surface of the chip was visualized. At this time, the surface may be dried by leaving the chip at room temperature for a certain period of time or by baking the chip in an oven at 55 ℃ for 5 minutes. The dried chip was soaked in pure water for 15 minutes, and then the surface of the chip was gently wiped several times with a wet cotton swab to remove beads that were not in the array. The chip was then rinsed 3 times with pure water and then dried by blowing compressed nitrogen gas to the surface thereof. Finally, the chip was examined by fluorescence microscopy to ensure that there were no additional beads outside the array.

Example 3: forming an array of beads

The bead slurry was spread directly over the array area on the chip. The bead slurry on the array surface was gently agitated with a wet cotton swab (K1). The motion of the K1 may be circular, linear, or other meaningful means, and is generally parallel to the chip surface. After agitating the slurry several times, the beads were moved to the array. The chip surface was then cleaned by using K1 to erase additional beads not in the array. This method can be scaled in a multi-chip assembly from single chip to wafer scale and can be automated.

An example of one processing step to form an array of beads is as follows. 2 microliters of 1% microparticles (approximately 3.2 microns in diameter) in 100 microliters of phosphate buffered saline (also known as PBS: 150mM, NaCl; 100mM, NaP, pH7.2) were used to assemble an array of 8 microparticles on a silicon chip (2.5X 2.5mM) with 4000 microwells per chip. The following steps were used:

(1) microparticles in PBS were collected by centrifugation (14000g, 1 min) in 1.5ml centrifuge tubes. Other collection means may be used.

(2) The supernatant was discarded by aspiration using a pipette (transfer pipette).

(3) Resuspend the microparticles in 5. mu.l of 5% 10mM glycerol in Tris pH 7.5.

(4) The microparticles in the glycerol solution were collected by centrifugation. Other collection means may be used.

(5) The glycerol solution was aspirated from the pellet.

(6) Resuspend pellet in 1 μ l 5% glycerol, 10mM Tris, pH 7.5.

(7) The 8 silicon chips were placed on double-sided tape affixed to a microscope slide.

(8) A volume of 0.1 microliter of the particle suspension was added to each chip in the area with 4000 wells.

(9) The swab was washed with water from the wash bottle.

(10) The wet swab was blow dried for 30 seconds by using compressed air. The air flow removes excess moisture from the cotton swab. In addition, air also blows some of the fibers from the surface, making the tampon more fluffy.

(11) After completion of steps 9 and 10 (about 2 minutes) the water content of the suspension of step 8 reached equilibrium due to the evaporation of the bead suspension in air and the deliquescent nature of the glycerol in solution. The droplets are more slurry-like because of the increased viscosity. To assemble the microparticle array, the bead slurry was gently agitated several times in a circular motion with the tip of a wet cotton swab. The loose fibers of the cotton ball send the beads into the micropores on the surface (fig. 3).

(12) The particle occupancy of the micropores was checked by using fluorescence microscopy. If the occupancy is not satisfactory, step 11 may be repeated.

(13) Excess particles were gently wiped from the chip by using a cotton swab. To eliminate excess moisture on the surface, the swab does not squeeze the chip.

(14) The chip was dried by blowing compressed nitrogen gas to the chip surface.

(15) The assembled microparticles prepared by this method can be used for analysis or stored in a solution at 4 ℃ for later use.

In this example, to deposit an array of microparticles directly on a silicon chip, the microparticles are suspended in a small volume of 5% glycerol, 10mM Tris pH 7.5. However, high solution viscosity or ion concentration may interfere with LEAPS when the particles are suspended in other solutions (e.g., assembling the particles on a substrate such as a designated area of a patterned or illustrated electrode) if LEAPS is used to assemble the beads. Thus, it is preferred that the ionic concentration of the suspension be about 1.0mM or less, preferably between about 0.1mM and 1.0 mM. Further, it is recommended that the viscosity of the suspension is about 100cp or less.

In addition, certain salts, such as sodium phosphate and sodium chloride, may form crystals at elevated concentrations during step 11. Such crystals may interfere with biomolecules at the surface of the beads. Thus, their use in bead suspensions is not recommended.

Example 4: direct deposition

The direct deposition method described herein is a simple method for efficiently assembling arrays of microparticles on solid surfaces. For example, a 0.25 microliter volume of a 1% solution of microparticles (10mg/ml, which corresponds to 168000 beads) is sufficient to assemble an array at an occupancy of greater than 95% on a silicon chip containing 4000 microwells. In other words, 2% of the beads in the suspension are filled in the micropores of the surface. In addition, the assembly process is performed in an aqueous solution at room temperature with a neutral pH. These mild conditions ensure that the reactivity of molecules such as DNA, RNA, peptides and proteins remains unchanged during assembly when immobilized on particles. Thus, microparticle arrays assembled using this method are compatible with a variety of biochemical assays. Moreover, this assembly method can be scaled in assembly from single chip to wafer scale and can be automated to produce large numbers of microparticle arrays.

The direct deposition process is further illustrated by the following examples. 2 microliters of a 1% microparticle solution (microparticles approximately 3.5 microns in diameter) was added to 100 microliters of phosphate buffered saline (also known as PBS: 150mM, NaCl; 100mM, NaP, pH7.2) to form 8 microparticle arrays on silicon chips (2.5X 2.5mM) having 4000 microwells per chip. The method comprises the following steps:

(1) microparticles in PBS were collected by centrifugation (14000g, 1 min) in plastic centrifuge tubes (eppendorf tubes). Other collection means may be used.

(2) Removal of supernatant by aspiration using pipette

(3) Resuspend the pellet in 5. mu.l of 5% glycerol in 10mM Tris pH 7.5.

(4) The particles in the glycerol solution were collected by centrifugation. Other collection means may be used.

(5) The glycerol solution was extracted from the pellet.

(6) Resuspend pellet in 1 μ l of 5% glycerol, 10mM Tris, pH 7.5.

(7) The 8 silicon chips were placed on double-sided tape affixed to a microscope slide.

(8) 0.1 microliter of the particle suspension was added to each chip over an area having 4000 microwells.

(9) The swab was washed with water from the wash bottle.

(10) The wet swab was dried by blowing with compressed air for 30 seconds. The air flow removes excess moisture from the swab. In addition, the gas also blows some fibers away from the surface, making the tampon more fluffy.

(11) Due to the evaporation of the bead suspension in air and the deliquescent nature of the glycerol in solution, the water content of the suspension of step 8 reaches equilibrium after steps 9 and 10 are completed (1-2 minutes). The droplets become a slurry because of the increased viscosity. To assemble the microparticle array, the bead slurry was gently agitated several times with a wet cotton swab in a circular motion. The loose fibers of the cotton ball send the beads into the micropores on the surface.

(12) The particle occupancy of the microwells was checked by using a fluorescence microscope. If the occupancy is not satisfactory, step 11 is repeated.

(13) Excess particles were gently wiped off the chip by using a cotton swab. To eliminate excess water on the surface, the swab does not squeeze the chip.

(14) Following step 13, the assembled microparticle array is ready for analysis or stored in a solution at 4 ℃ for later use.

For assembly of arrays using direct deposition, microparticles suspended in a small amount of 5% glycerol, 10mM trisph7.5 solution were used. The use of concentrated glycerol (i.e. above 5%) increases the viscosity of the bead slurry and the specific gravity of the solution in the droplets on the chip (step 11). Also, this may include assembly effectiveness. Although the solution used in the direct deposition method is not limited to 10mM Tris, pH7.5, it should be noted that certain salts, such as sodium phosphate and sodium chloride, tend to form crystals at high concentrations, as in step 11. Salt crystals not only reduce the occupancy of particulates in the pores, but may also damage molecules on the surface during assembly.

It is also recommended to store the chip or wafer containing the chip in a wet tank for a short period of time (e.g. 30 minutes) before the analysis application to allow the beads to settle by gravity in the recess. Centrifugation of the assembled array bound to a glass slide can facilitate the sedimentation process. The recommended centrifugation settings are as follows:

a centrifuge: sorvall centrifuge model RT6000B

A rotor: sorvall swing bucket model H1000B

Speed: 2000RPM

Time: 5 minutes

The operation items are as follows: setting up a centrifuge in a10 ℃ cooling mode

Setting brake to off mode

The speed was slowly increased from 0to 2000 at the first 2 minutes, and then

Centrifuge again at 2000RPM for 5 minutes.

Equivalent equipment and arrangements may be used for this purpose.

For microscopic examination, a variety of immersion media can be used for placing the chip on the slide. One example is the use of a deposition medium containing 2.25M tetraethylammonium hydrochloride (tetra butylammoniumchloride), 37.5mM Tris, pH8.0, 25% glycerol.

Example 5: parallel assembly of biochip arrays

The present invention provides a method for assembling biochip arrays in parallel. In this embodiment, chips from different wafers form an array of biochips. One non-limiting example is illustrated in fig. 9, which shows that 4 different wafers produce 4 chips: A. b, C and D. The rows or columns of chips may be combined in any geometry to form an intermediate chip matrix. In a preferred embodiment, the chips have a regular geometric shape (e.g., square or rectangular) and the corresponding intermediate chip matrix also has a regular set shape. The rows or columns are then lifted from the intermediate chip matrix so that the rows or columns contain different types of chips. Depending on the application, a mixed row or column may contain more than one copy of a certain type of biochip. The mixed rows and/or columns formed in these embodiments can be integrated into a biochip array for biological analysis. In a preferred embodiment, the semiconductor chip handling apparatus is used to assemble an intermediate chip matrix and extract mixed rows or columns. By forming the intermediate chip matrix using long rows or columns of chips, it is possible to produce many mixed rows or columns simultaneously. In this way, a mixed row or column may be generated in large numbers.

Example 6: protection of biochips by sugar coating

The functionalized beads are assembled on the chip using standard methods. After assembly, the chip surface was washed, 2-4. mu.l of a 1% trehalose solution (α -D-glucopyranosyl- α -D-glucopyranoside, a naturally occurring, glass-forming disaccharide) in DI water was spread over the chip (surface size: 1.75X 1.75mm) and dried under ambient conditions. When dried, a glass film forms on the substrate and encapsulates the assembled beads. Although the film is stable even under high humidity conditions, the film dissolves immediately upon exposure to liquid water.

To evaluate the effect of film formation on the activity of functionalized particles, neutravidin-functionalized particles were assembled on a biochip. Some biochips are passivated with trehalose solution and are in normal ambient conditions as described above, while others are not coated with trehalose solution and are stored at 4 ℃ for 2 weeks. The biological activity of the biocoated chips was found to be similar to that of the uncoated biochips stored at 4 ℃.

Example 7: hydrogel as a multifunctional reagent in wafer cleaning, storage and particle recovery

Agarose hydrogel can be used as an exfoliating agent to remove particles from the chip and allow for later recovery of the particles. Hydrogels can also be used as a storage substance to prevent wafer and particle degradation and dust.

The functionalized particles were assembled on a 6 inch wafer containing the chips. To clean the particles remaining on the surface, a 1% agarose solution (melting point 95 ℃, gelation temperature 50 ℃) was poured onto the wafer at 55 ℃ and held at ambient conditions or at 4 ℃ until gelation occurred. Gels of different thickness, from microns to millimeters, can be produced by using strakes (spacers) of different thickness. The edge strips provide a barrier at the edge of the wafer to prevent the agarose solution from running off the edge. Beads located on the wafer surface rather than in the recesses are embedded in the gel. When the solution is fully cured, the gel film and embedded beads can easily peel off. A small amount of water remaining on the surface was then blown dry immediately using a stream of compressed nitrogen. In this way, the wafer surface is kept clean.

To analyze the effect of the exfoliation process on occupancy and the effect of the agarose gel membrane on the activity of the functionalized particles, the particles were assembled on a chip, which was then subjected to decoding analysis and extension (extension) analysis. Fig. 12 shows that the exfoliation process did not reduce the occupancy (i.e., no particles were exfoliated from the recesses). It is believed that the viscosity of the gel solution plays a role in retaining the particles in the pores. The higher the viscosity of the gel solution, the lower the tendency of the solution to go into a recess before gelation, so the occupancy is less likely to be affected. SSP in chip analysis showed comparable signal and CV (fig. 13ab), indicating that gel did not affect assay sensitivity.

Agarose gels are thermally reversible, physically cross-linked hydrogels. For particle recovery purposes, agarose should be used which has a very low melting point (m.p. < 50 ℃, gelation temperature, 8-17 ℃). Subsequently, the agarose gel can be remelted at 50-55 ℃ and the embedded particles can be recovered. The biological activity of the biomolecules on the particles is maintained under these conditions.

Such hydrophilic hydrogels can be used not only as exfoliation agents, but also as storage agents to prevent degradation, dust and physical damage to the particles/wafers during storage and transportation.

Example 8: polymer coating

Small batches of cleaned individual chips (approximately 5 to 20) were placed in small Teflon containers (volume 5ml) containing 1ml of 1% (1mg/ml) polyallylamine hydrochloride (Mw-15000) or 0.1% polylysine solution (Sigma Aldrich). Incubate the chip at room temperature for 1-2 hours and gently shake. Then, it was taken out from the polymer solution and dried at a temperature ranging from 50 to 70 ℃ for 1 hour. This process typically leaves a thick, non-uniform polymer coating film on the surface of the chip. These modified chips were used to assemble beads using standard methods. The final surface cleaning step of the assembly process removes most of the excess polymer and excess beads. The presence of the polymer coating improves the adhesion of the beads to the chip surface and, after such treatment, the retention of the beads in the recesses is significantly improved.

Example 9: packaging biochips to form multi-chip carriers for biological analysis and methods of adding bead arrays and making same

The choice of the type of packaging for a particular biochip depends on the application. Usually, for convenience, one or more biochips are attached to a chip carrier. The carrier may be as simple as a glass slide, or may be a complex device (cartridge) with fluidic manipulation, temperature control, signal recording and other functions. The biochip can be permanently bonded to the carrier by glue or reversibly bonded by various means such as magnetic or mechanical force.

Example 9A-manufacture of multi-chip carriers from glass slides: to prepare the slides as carriers, teflon coatings are then applied to create circular openings or wells (i.e., areas of glass without teflon coverage). Each hole is circular with a diameter of 6.5 mm. One or more chips may be bonded to the glass surface in one hole. A typical glass slide is 25X 75mm and 1mm thick with a 2X 5 array of wells. Chips with a typical size of 1.75 x 1.75mm, up to 4 chips can be bonded to the glass surface of each well. Each chip in the same well may have a different set of assembled beads prior to binding to the carrier. For example, if each chip has an array containing 39 bead sets, wells with 4 different chips will have a total of 4 × 39 ═ 156 beads. On the other hand, for larger chips (e.g., 4.5 x 4.5mm square), one chip can occupy one hole. For the well sizes described herein, each well can hold up to 40 μ l of liquid (typically an aqueous solution). Typically, a volume of 20. mu.l of sample solution is added to each well to perform a biological reaction, so each chip is completely covered with sample solution. Since the teflon coating outside the well is hydrophobic, the aqueous sample does not spill out. The form of the carrier slide can be designed to suit certain applications. For example, a single row of 8 wells on a slide can be used to analyze 8 samples. Also, a 4 × 8 array of wells can be used to analyze 32 samples. Also, more wells (e.g., 96384 and 1536) can be arrayed on a single slide to analyze more samples.

Example 9B-removable chip carrier: in certain embodiments of the removable chip carrier, the chip is bonded to a substrate such as glass, stainless steel, plastic, semiconductor, or ceramic. The entire carrier unit is movable and can be transferred during processing to expose the chip to different reaction media, such as reaction chambers, washing chambers, and signal readers. (see FIG. 14 for one embodiment).

In other embodiments, the removable chip carrier comprises a well or a well for attaching a chip. By covering the chips in the removable chip carrier, contamination during transfer can be minimized. In some embodiments, the slots of the removable chip carrier also serve as a processing environment. If desired, reactive gases or liquid solutions for different purposes, such as performing biological analyses or cleaning the chip, can be applied to the removable chip carrier and subsequently evacuated. In addition, the removable chip carrier may have means to change the thermodynamic properties of the cell, such as cell pressure or temperature.

Claims (36)

1. A method of producing a biochip comprising the steps of:

patterning a wafer substrate to form a plurality of chip regions;

assembling at least one bead array comprising functionalized beads on a wafer-based surface of at least one chip region, said beads being encoded with an optical signal detectable at the bead surface without separating said beads; and

the wafer is divided to form a plurality of individual biochips.

2. The method of claim 1, wherein the method further comprises scribing the wafer substrate in accordance with the scribed chip regions.

3. The method of claim 1, further comprising the step of cutting channels on said patterned wafer along boundaries between said chip regions.

4. The method of claim 3, wherein the cleaving is accomplished by using wet chemicals.

5. The method of claim 3, wherein the cutting is accomplished by using dry etching.

6. The method of claim 1, wherein the bead array is protected by a removable protective coating prior to segmentation.

7. The method for producing a biochip of claim 1, further comprising fabricating a bead-limiting structure in the chip region, the bead-limiting structure comprising a bead-movement-limiting recess.

8. The method of claim 7, wherein the recess is defined by photolithography and formed by reactive ion etching.

9. The method of claim 7, wherein each of said recesses has straight sidewalls and a predetermined depth.

10. The method of claim 9, wherein the recess has a reentrant sidewall profile formed by modifying a recess having straight sidewalls by subsequently depositing a layer of a substance adhering the sidewalls to produce the desired reentrant sidewall profile.

11. The method of claim 10, wherein the substance is silica.

12. The method of claim 1, further comprising the step of evaluating and controlling biochip quality by optically imaging beads to ensure that recesses formed in the chip area are occupied by beads.

13. A method of packaging biochips produced according to the method of claim 1, comprising selecting biochips from a plurality of wafer substrates and assembling them on a planar surface to form a combined biochip kit.

14. A method of packaging biochips produced according to the method of claim 1, wherein said combined biochip sets form an assay device.

15. The method of claim 13, wherein the biochips are assembled at an assembly area by sliding the biochips from a plurality of wafer substrates present on a common substrate.

16. The method of claim 15, wherein the biochip is labeled to identify the source of the wafer substrate.

17. The method of claim 16, wherein the biochip is randomly assembled.

18. The method of claim 16, wherein biochips from a plurality of wafer substrates are present in a suspension and wherein the suspension is deposited on a planar substrate.

19. The method of claim 13, wherein biochips from a plurality of wafer substrates are selected and placed on a common plane.

20. A method of packaging biochips produced according to the method of claim 1, comprising selecting at least one biochip from at least one wafer substrate and placing the at least one biochip in a liquid confinement region on a carrier substrate.

21. The method of claim 20, wherein said carrier substrate comprises a hydrophilic major surface and a patterned hydrophobic layer.

22. The method of claim 21, wherein the patterned hydrophobic layer on the carrier substrate defines a liquid confinement region.

23. A biochip prepared by a method comprising the steps of:

patterning the wafer substrate to form a plurality of chip regions;

assembling at least one bead array comprising functionalized beads on a surface of the wafer substrate in at least one chip region, the beads being encoded with an optical signal detectable at the bead surface without separating the beads; and

the wafer is divided to form the individual biochips.

24. The biochip of claim 23, wherein the wafer substrate further comprises a scribe-line according to the chip area.

25. The biochip of claim 23, wherein the wafer substrate further comprises channels along the boundaries of the chip regions that cut the patterned wafer.

26. A bead array comprising a removable coating that protects the bead array on a biochip, the bead array comprising a plurality of functionalized beads having surfaces to which molecular probes are attached, the beads being encoded with an optical signal that is detectable at the bead surfaces without isolating the beads, and the coating having the property of being non-reactive to the molecular probes on the bead surfaces.

27. A semiconductor wafer substrate comprising at least one chip region, the chip region comprising:

an array of recesses in the substrate that limit movement of the beads;

a patterned insulating dielectric layer for adjusting the impedance of an electrolyte-insulator semiconductor structure when the base layer is in contact with a bead solution, wherein the patterned dielectric layer is positioned such that beads in the bead solution tend to accumulate in the recessed array when another electric field is applied; and

a protective passivation layer covering a surface of a substrate.

28. The semiconductor wafer substrate of claim 27, further comprising a plurality of chip regions.

29. The semiconductor wafer substrate of claim 27, wherein said patterned insulating dielectric layer defines an area having a star-shaped perimeter.

30. The semiconductor wafer substrate of claim 29, wherein said array of recesses comprises recesses having a shape selected from the group consisting of: triangular, pentagonal, hexagonal, and circular.

31. A method of assembling an array of beads, the method comprising:

providing a semiconductor wafer substrate of claim 27, wherein said patterned dielectric layer defines regions on said substrate with and without a dielectric layer, and wherein said array of recesses is located in the regions without the dielectric layer;

applying a bead solution to a surface of a substrate, the surface comprising the patterned dielectric layer; and

bead arrays are assembled using LEAPS, wherein an electric field of a first frequency is applied to induce bead migration to a region having a dielectric and an electric field of a second frequency is applied to align beads in the recessed array.

32. A wafer substrate comprising a plurality of chip regions defined by patterning and scribing said wafer substrate, wherein said chip regions comprise regions of at least one constraining bead and wherein said scribing can break said chip regions to form a biochip comprising one or more chip regions.

33. The wafer substrate of claim 32, further comprising at least one subpopulation of beads attached to a surface of at least one chip region of said substrate and wherein said bead subpopulation is formed from at least one bio-functionalized molecular probe selected from a probe library and attached to a plurality of beads.

34. A biochip produced by breaking at least one wafer substrate and separating the chip regions to form the plurality of biochips according to claim 33.

35. The biochip of claim 34, wherein the biochip is obtained by breaking two or more wafer substrates comprising different bead subpopulations.

36. A method of retaining a set of charged beads at designated locations on a surface of a semiconductor wafer substrate comprising depositing a first charged polymer on the surface of the semiconductor wafer substrate, the first charged polymer having a charge opposite to that of the charged beads, depositing the beads onto the surface of the semiconductor wafer substrate, and depositing a second charged polymer on the surface of the semiconductor wafer substrate, the second charged polymer having a charge opposite to that of the first charged polymer.