Industrial Agricultural Biotech – Michael Seul, Chiu Wo Chau, Bioarray Solutions Ltd
Abstract for “System and Method for Programmable Illumination Pattern Generation”
“An apparatus for programming illumination patterns to manipulate colloidal particulates or biomolecules suspended between electrodes is disclosed. The apparatus uses LEAPS (Light controlled electrokinetic assembly particles near surfaces) and relies on: AC electrical field-induced assembly particles: The patterning of the electrolyte/silicon dioxide/silicon interface to exert spatial controls over the assembly process; and external illumination for real-time control. The apparatus projects patterns of illumination onto planar surfaces. A LEAPS electrode, This allows the creation of patterns with graphical design software or drawing software on a personal computers and the projection or sequence of those patterns (?time-varying pattern?). The interface is created using a liquid crystal panel (LCD panel) and an optical design that images the LCD panel onto the surface. This allows for the assembly and arrangement of particles in such patterns.
Background for “System and Method for Programmable Illumination Pattern Generation”
“I?Ions Electric Fields and Fluid Flow: Field Induced Formation of Planar Bead Arrays”
“Electrokinesis” refers to the phenomenon of electromagnetic fields causing a mobile ions around charged objects in an electrolyte. A diffuse ion cloud is formed when an object with a given surface charge is immersed into a solution containingions to screen it. The arrangement of two layers of immobile charges and a screening cloud of mobile counter-ions in the solution is called a “double layer”. The fluid is not electroneutral in this area of finite thickness. The region is not electroneutral because of the electric fields that act on it. These ions will cause the fluid to entrain in the diffuse layer. The fluid’s spatial distribution will determine the flow fields. The simplest form of electrokinetic phenomena is electroosmosis. This happens when an electric field is applied parallelly to the surface of a sample vessel or electrode with fixed surface charges. For example, a silicon oxide electrode (in neutral pH range). The electric field accelerates counter-ions in an electrode double layer, causing them to drag along solvent molecules and resulting in bulk fluid flow. This effect may be significant in narrow capillaries, and can be used to your advantage when designing fluid pumping systems.
“Electrophoresis” is a similar phenomenon. It refers to field-induced transports of charged particles in an electrolyte. An electric field accelerates the mobile ions within the particle’s double layer, just like electroosmosis. Contrary to the previous case, if the particle is mobile, it will compensate the field-induced motion (and the resulting Ionic Current) by moving in an opposite direction. The role of electrophoresis is important in industrial coating processes. It is also of interest, along with electroosis, in connection with the development capillary electrophoresis to be a majorstay in modern bioanalytical seperation technology.
“In restricted geometries such as the one of a shallow experiment chamber, such as a?sandwich’, it is possible to observe the effects of electroosmotic flow in confined spaces. Two planar electrodes. The surface charge distribution and topography on the bounding electrode surfaces are crucial in determining the nature of electroosmotic flow. This is a “sandwich”. An electrochemical cell can be made by two electrodes separated by a small gap. The oxide-capped silicon wafer will form the bottom electrode, while the optically transparent, conducting ITO will form the other electrode. “The silicon (Si) wafer is a thin slice from a single crystal silicon. It has been doped to achieve electrical conductivity at suitable levels and then insulated from electrolyte solution with a thin layer silicon oxide (SiOx).
A (DC or AC electric field applied to the electrode surface can induce the reversible aggregation beads into planar aggregates. The phenomenon was previously observed in a cell made from two conductive ITO electrodes. (Richetti Prost, Barois, J. Physique Lettr. 45, L-1137 to L-1143 (1984), which the contents are incorporated in this document by reference). However, it was previously demonstrated that electrokinetic flow is responsible for the attractive interaction between beads (Yeh Seul and Shraiman?Assembly Of Ordered Colloidal Aggregates By Electric Field Induced Fluid Flu?, Nature 386 57?59 (1997)), whose contents are also incorporated in this document by reference). This flow is due to the effect of non-uniformities in spatial distribution of current near the electrode. These non-uniformities can be explained by the presence of colloidal beads near the electrode. Each bead blocks the movement of ions within the electrolyte. It has been shown that a single bead can generate a toroidal fluid flow centered at the bead when it is placed close to the electrode’s surface. Multiple methods can be used to create fluid flow lateral toward low impedance regions by intentionally creating spatial non-uniformities. These methods will be described in the following sections.
“Particles embedded within the electrokinetic flow can be advected without regard to their particular chemical or biological nature while simultaneously altering flow field. The electric field-induced assembly planar aggregates or arrays can be applied to a variety of colloidal particles, including: beaded polymer resins (beads) ), lipids vesicles and whole chromosomes. Cells and biomolecules such as DNA and proteins, as well metal or semiconductor clusters and colloids.
The flow-mediated attraction between beads can travel a great distance. This is important for the applications that will be described. Planar aggregates form when an electric field is applied externally and then disassemble once the field is removed. The array assembly process is determined by the strength of the applied field. This determines the arrangement of the beads within the array. As a function, the applied voltage increases and beads form planar aggregates. These are composed of particles that are mobile and loosely packed. Then, they adopt a tighter packing and then exhibit a spatial arrangement resembling a raft or bubbles. Reversible transitions are possible between states of increasing internal ordeal, and complete disassembly is possible when applied voltage is removed. Another arrangement is that beads at low initial concentration form small clusters, which then assume positions within an orderly?superstructure?.
“II?”Patterning Silicon Oxide Electrode Surfaces
“Electrode patterning in accordance with a predetermined design facilitates the quasi-permanent modification of the electrical impedance of the EIS (Electrolyte-Insulator-Semiconductor) structure of interest here. Electrode-patterning modifies the EIS impedance spatially to determine the ionic current near the electrode. Beads can either seek out or avoid high ionic current regions depending on the frequency of the electric field. Spatial patterning allows for explicit external control over the shape and placement of beads arrays.
There are many methods for patterning, but two techniques offer the most advantages. The first is UV-mediated regrowth of a thin layer of oxide on a properly prepared silicon substrate. This method avoids photolithographic resist patterning or etching. The UV illumination mediates the transformation of exposed silicon to oxide in the presence of oxygen. The thickness of the oxide layer is dependent on its exposure time. This can be spatially modulated through the use of patterned masks in the UV illumination path. This thickness modulation, which is typically around 10 Angstroms in size, results in spatial modulations in the impedance at the Si/SiOx interface. However, the top surface remains flat and chemically homogeneous to the electrolyte solution. A second method that can produce spatial modulations in electrode surface charge distribution is UV-mediated photochemical oxygenation of a suitable chemical substance. This first occurs on the SiOx surface as a monolayer film. This allows fine control of local features and electrokinetic flow.
“A variation on this photochemical modulation involves the creation of lateral variations in the EIS impedance, and thus in the current generated by the applied electric field. This can be achieved by controlling UV exposure to cause a slow lateral variation of the oxide thickness and/or the surface charge density. Controlling lateral gradients is key to inducing lateral bead transportation. It also facilitates fundamental operations such as the capture and channeling beads to a predetermined destination using conduits made of impedance elements embedded in Si/SiOx interface. Photochemical patterning of functionalized chemicals overlayers can also be applied to other types electrode surfaces, including ITO.
“III?Light-controlled Modulation of the Interfacial Impedance”
The basis for controlling the electrokinetic forces that mediate the aggregation of beads is the spatial and temporal modulation in the EIS-impedance according to a pattern or external illumination. Remote control of the formation, placement, and rearrangement bead arrays is possible via light-modulated electrokinetic assembly. This allows for a wide variety of interactive manipulations of colloidal beads as well as biomolecules.
It will help to review the photoelectric properties of semiconductors in order to understand the basic principle of this method. This includes the EIS structure, the Insulating SiOx and Semiconductor structures (I), E (Electrolyte solution) and S (Semiconductor). The photoelectric characteristics of this structure are closely related to those of a standard Metal-Insulator-Semiconductor (MIS) or Metal-Oxide-Semiconductor (MOS) devices which are described in S. M. Sze, ?The Physics of Semiconductors?, 2nd Edition, Chapt. 7 (Wiley Interscience 1981), whose contents are incorporated by reference.
“The interface between semiconductor and insulating oxide layers deserves special attention. Understanding the MOS structure’s electrical response to light is crucial. This concept refers to a small, but finite space charge region that forms at the Si/SiOx Interface in the presence of an applied bias potential. An effective bias in the form a junction potential is present for the EIS structure under all conditions. Space charge regions form due to distortions of the semiconductor’s conduction and valence bands (?bandbending?). The interface is in its vicinity. This is due to the fact that the interface has a bias potential. However, the insulating oxide prevents charge transfer. In electrochemical terms, this means that the EIS structure eliminates Faradaic effect. Instead, opposite sign charges accumulate on either side the insulating oxide layer to generate a finite degree of polarization.
“In the presence a reverse bias, the conduction and valence band edges of an N-doped semiconductor bend upwards near the Si/SiOx Interface and electrons flow out from the interfacial area in response to the corresponding voltage gradient. A majority carrier depletion layer forms near the Si/SiOx Interface. This region is able to produce electron-hole pairs through light absorption. If they don’t recombine instantaneously, electron-hole pairs can be split by the local acting electric field and a corresponding current flows. This latter effect allows for control over the electrokinetic assembly beads in an electrolyte solution.
Two aspects of an equivalent circuit that represents the EIS structure can be used to better understand the frequency dependence of light-induced modulation. The first is a close analogy between the detailed electrical characteristics at the electrolyteoxide interface and the depletion layer at interface between semiconductor and insulator. The depletion layer has similar electrical characteristics to the double layer. It also exhibits a voltage-dependent capacitance. The depletion layer’s impedance can be reduced by illumination, as we have already discussed. The second reason is that the oxide layer’s capacitive electrical response means that it will only pass current above a threshold (?threshold?) frequency. If the applied voltage frequency exceeds the threshold, illumination may lower the effective impedance for the entire EIS structure.
“Effective reduction of EIS impedance is also dependent on light intensity, which affects the rate at which electron-hole pairs are generated. The majority of photogenerated electrons are free to flow out of the depletion area and contribute to the photocurrent, even if there is no significant recombination. The rest of the hole charge builds up near the Si/SiOx Interface and blocks the electric field from the depletion area. The rate of recombination decreases and electron-hole separation efficiency (and hence the photocurrent) decreases. Given the values of frequency, amplitude, and voltage applied, it is expected that the current will increase in intensity as the illumination intensifies. Then, it will decrease. The impedance decreases initially to a minimum (at maximum current), and then it decreases.”
This intensity dependence can be used to inducing the lateral displacement beads between partially covered and fully masked regions. The fully exposed areas will correspond to regions of interface with the lowest impedance and therefore the highest current. Beads will be drawn into these areas as the illumination intensities increase. The effective EIS impedance of fully exposed regions will decrease as the photocurrent increases. This can lead to an inversion of current’s lateral gradient. The fully exposed areas will be used to draw beads. You can also use time-varying variations in the illumination pattern to influence bead movement.
“IV?Integration in Biochemical Analysis in a Miniaturized and Planar Format”
“Planar array assays are a good option for biomolecular screening and medical diagnostics. They have the advantage of high parallelity and automation, which allows them to achieve high throughput in multi-step, complex analytical protocols. Miniaturization will reduce pertinent mixing times due to the small spatial scale. It will also result in reduced sample and reagent volumes, as well a reduction in power requirements. Integration of biochemical analytical methods into a miniaturized system surfaced on a planar substrate (chip?) would be significant. This would result in significant improvements in performance and cost reductions in diagnostic and analytical procedures.
“In the context of DNA manipulation, and analysis, initial steps were taken (i.e. miniaturization) by combining on glass substrate, the restriction enzyme treat of DNA, and the subsequent separation enzyme digests using capillary electrophoresis. See, for instance, Ramsey, PCT Publication Number. WO 96/04547 is incorporated by reference. Alternatively, the amplifying of DNA sequences using the polymerase chain react (PCR) and subsequent electrophoretic separation are described in U.S. Pat. Nos. Nos.
These standard laboratory processes were demonstrated in a miniature format but have not been applied to create a complete system. Additional manipulations will be required to create a complete system, such as binding and functional assays, front-end sample processing and small signal detection followed by information processing. Complete functional integration is the real challenge. This is where system architecture and design limitations on individual components will become apparent. A fluidic process is needed to concatenate analytic steps that require spatial separation and transport to new places of sets of analytes. There are many options, including electroosmotic pumps and droplet transport by temperature-induced local surface tension gradients. These techniques are possible in demonstration experiments but require a lot of system design to manage the large DC voltages needed for electroosmotic mixing.
The present invention combines three functional elements to provide an apparatus and method that allow for the interactive spatial manipulation of colloidal particle (?beads?) in real-time. The interface between an electrolyte solution and a light sensitive electrode is used to allow for the interaction of molecules and colloidal particles. These three functional elements include the electric field-induced assembly planar particle arrays at the interface between an insulating electrode or a conductor electrode and an electrolyte. The spatial modulation and control of interfacial resistance by UV-mediated oxide growth or surface-chemical patterning. Finally, the interactive, real-time control of interfacial Impedance by light. External intervention is possible to adjust the spatial distribution of ionic electrons and the fluid flow mediating array assembly. This invention has many advantages. It is important to introduce spatial non-uniformities within the EIS structure. These inhomogeneities can be either permanent or temporary as a result of taking advantage of the EIS structures’ physical and chemical properties.
“The invention concerns the realization of an integrated, functionally complete system for performing biochemical analysis on a silicon wafer surface or other substrate. The present invention also allows for the fabrication of surface-mounted optical elements, such as lens arrays, that can be fabricated using the method and apparatus.
The combination of these three functional elements gives the invention the ability to manipulate beads and bead arrays with planar geometry. This allows for the application of biochemical analytical methods. These fundamental operations are applicable to aggregates or arrays of colloidal particles, including beaded polymer resins (also referred to by latices), whole chromosomes cells, biomolecules such as proteins and DNA as well as metal and semiconductor colloids and clusters.
“Sets of colloidal particles can be captured and arrays formed on designated areas of the electrode surface (FIGS. 1 a, 1 band FIGS. 2 a?d). The particles and their arrays formed in response to the applied fields can be channeled along conduits in any configuration. These conduits may either be embedded in Si/SiOx by UV-oxide patterning, or delineated using an external pattern. This channeling (FIGS. 1 c, 1 d, 1 e, FIGS. 3 c, 3 D), in a direction that is normal to the applied electric field. This relies on lateral gradients of the EIS structure’s impedance and, therefore, in the field-induced field. FIGS. These gradients can be introduced using appropriate patterns of illumination. This allows for the implementation of a gated version (FIG. 1 e). For the alignment of long-lived particles such as DNA near the electrode’s surface, the electrokinetic flow that mediates the array assembly process can also be used. The present invention also allows for methods of sorting and separating particles.
“Colloidal particles can be placed in designated areas, and kept there until they are released or disassembled. You can define the overall shape of an array by UV-oxide patterning, or by changing the pattern of illumination. This allows the creation of functionally distinct compartments on the electrode surface, whether permanent or temporary. The arrays can be modified in real-time and may be combined with other arrays (FIG. 1 f) Or split into multiple subarrays, or clusters (FIG. 1 g, FIGS. 4 a, 4 b). You can also adjust the local order of the array and the lateral particle densities by using the external electric field, or by adding a second, chemically inert component to the array.
“The invention allows the combination of fundamental operations to create increasingly complex products and process. The following examples illustrate how analytical procedures can be used to solve a variety of problems in materials science and pharmaceutical drug discovery. These and other functionalities can be integrated in a planar geometries. The present invention provides the ability to create temporary or permanent compartmentalization to spatially isolate concurrent processes and subsequentail steps in a protocol. It also allows for the manipulation of sets of particles in such a way that they can be concatenated in different areas of the substrate surfaces.
“This invention is for a system for programming illumination patterns. The present invention discloses a novel method and apparatus to generate patterns of illumination and project them onto planar surfaces or onto planar interfaces such as the interface formed by an electrolyte-insulator-semiconductor (EIS), e.g., as described herein. The present invention allows the creation of patterns or sequences thereof using graphical design software or drawing software on a personal computers and the projection of those patterns or sequences (?time-varying pattern?). The LCD panel is projected onto the interface by an optical design that images the LCD panel onto the surface. LCD technology allows flexibility and control over spatial layout, time sequences and intensities. You can also create illumination patterns. This allows you to create patterns with rapidly changing light intensities, or patterns with slowly changing intensity profiles.
“The present invention uses patterns of illumination to control assembly and lateral motion of colloidal particle within an enclosed fluid environment. Particles can be inducible to move between the two electrode surfaces that are planar and bound by the liquid with the help of an electric field. This field is time-varying and can be applied between the electrode’s planar electrode surfaces. It can also induce movement into and out of the illuminated areas of the electrode depending upon the layout of the patterns, the frequency and intensity of the transmitted light, the electric field strength, the junction gap separation, and the semiconductor doping levels.
“In conjunction the present invention disclosing an programmable illumination generator, advanced operations in array reconfiguration, segmentation, and (spatial encoding) are possible which, in turn, lead to a variety advanced operations and applications.”
“Applications according to the invention are described where patterns are created by projecting fixed masks that define bright and dark areas of illumination on the substrate. The present invention describes a programmable generator that allows for flexibility and control over the placement and direction of colloidal particles. Particles can be?dragged? to form dense planar layers. You can also?drop them? Interactively by?dragging? You can also?drop? The graphical design displayed on a computer monitor using a mouse. Alternately, you can program a sequence of patterns or a transformation pattern to manipulate arrays in a planned manner. Multiple ?sub-assemblies? Under illumination, multiple particles can be controlled simultaneously in different areas of the substrate.
“BRIEF DESCRIPTION DES DRAWINGS”
“Other objects features and advantages of this invention will be easier to understand when combined with the detailed description of an embodiment. This will be understood only as an illustration and the accompanying drawings will reflect aspects of that embodiment.
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“FIGS. 6 a.c are side views of a layout-preserving process to transfer a microtiter plate from a planar cells to a microtiter plates;
“FIG. 7 shows the inclusion of spacer particles in bead clusters.
“FIG. “FIG.8 is an example of binding assay variations;
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“FIGS. 14 a.d are photos of different forms of light-induced arrays;
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“FIG. 16 is a photo showing a “drag and drop?” operation applied to particles
“FIG. 17 shows how an illumination profile can be used to create a subarray border;
“FIGS. 18 a and 18b are photos that illustrate the creation and maintenance of particle confinement patterns.
“FIG. 19 is a photo that illustrates the preferential collection only of one type of particle in the mixture into an illuminated space under conditions that ensure the exclusion of all other particles.
“FIGS. “FIGS.
“FIGS. 21 a and 21b are photos taken in succession while sweeping an illumination pattern across an sample containing small colloidal particles (2.8?m in diameter), which were deposited at random locations on a planar substrate.
“FIGS. 22 a and 22b show examples of the methods and procedures for chemical and spatial encoding arrays and decoding arrays using selective anchoring individual beads to substrates, segmentation and fractionation.
“FIG. 23 is an example of random sequential injection.
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“FIG. 26 a?b shows a method for producing a composite particle arrangement exhibiting a concentric collection of discrete compositional bands;
“FIG. “FIG. 27 illustrates the principle that imposing conditions favoring expulsion particles from sub-regions illuminated with high intensities;
“FIG. 29 shows the light-induced fluid flow at the boundary of illuminated and unilluminated areas of a substrate.
“The functional elements of the invention can be combined to create a set fundamental operations for interactive spatial manipulation of colloidal molecules and particles, as well as planar aggregates near an electrode surface. The following describes the fundamental operations of this “toolset”. These operations are listed in ascending order of complexity. It is helpful to use a classification scheme that takes into account the number of inputs or outputs involved in any given operation. A?three-terminal’ would be the combination of two different arrays or sets of particles into one. operation that requires two inputs as well as one output. Three-terminal reverse operation involves one input and two outputs. It is the division of an array into two subarrays.
“Capture-and-hold is the fundamental one-terminal operation. operation (FIG. 1a), which creates an array of particles in a defined area of arbitrary outline on a surface, delineated either by UV-mediated Oxid patterning or a corresponding pattern projected onto a Si/SiOx substrate. FIGS. FIGS. FIG. FIG. FIG. FIG. 2 b shows an electric field (10Vp-p source at 1 kHz), and bead capture takes place within the thin oxide zone 22. The array will grow in less than one second. It will continue to grow for approximately 10 seconds after that, as beads arrive at greater distances to increase the region 22’s outward growth perimeter. The array stops growing when it reaches the outer limit of its target area. This is the area that is defined by the thin oxide with a low impedance. The applied voltage determines the internal state of the beads. Higher values encourage denser packing and eventually form ordered arrays with hexagonal crystalline configurations in the form of bubble rafts. The array will remain in place as long the voltage is applied. The array will be disassembled if the voltage is removed.
“The ?capture-and-hold? “Capture-and-hold” operation can also be performed under visible or infrared illumination. For example, a mask with the desired layout may be projected onto the Si/SiOx electro. On a Zeiss UEM microscope, a 100 W quartz microscope light source was used. Affixing masks or apertures in the intermediate image plane provided the desired shape in the electrode’s plane (when the microscope is properly focused under Koehler illumination) has been possible. An IR laser diode of 3 mW output at 650-680 nm has also been used. External illumination is preferred to oxide patterning in order to modify the spatial confinement pattern of particles.
“Related To?capture and-hold?” “Related to?capture-and-hold?” (FIG. 1 b), which removes particles from a specified area of the surface. An inversion occurs when the frequency of the applied voltage is increased to around 100 kHz. This results in particles that assemble in the thin oxide portion of the surface (e.g. region 22 FIG. 2 b) instead of forming structures that surround the perimeter of the target area. This effect is not sufficient. The exclusion of particles from desired areas can also be achieved, analogously to the original “capture-and-hold” method. Operations by simply embedding a corresponding structure into the Si/SiOx interface via UV-mediated oxide regrowth. FIG. 2 c, 2 d are achieved under conditions that are identical to those in FIGS. 2 a, 2 b are achieved by applying 20V (pp), at 10 kHz. The oxide thickness in non-designated areas 24 is about 30 Angstroms. However, the value for the designated square areas 26 are approximately 40 Angstroms. This indicates a higher impedance at the applied frequency.
“The ?capture-and-hold? “Capture-and-hold” allows the spatial compartmentalization and separation of functionally distinct areas on the substrate surface. This operation allows for the spatial isolation of particles of different chemical types, which can be introduced to the electrochemical cells at different times and injected at different locations.
Translocation (FIG.) is the fundamental two-terminal operation. 1 c, or the controlled transporting of a set particles from location O on the surface to location F; O and F are the target areas to which one-terminal operations can be applied. Translocation uses a one-dimensional, lateral beam transport. This is done by applying a current along a conduit connecting O and F. FIGS. 3. a and 3. b, or projecting a linear pattern of illumination. This channeling operation causes beads to move in the direction that has lower impedance than the arrow in FIGS. 3. a and 3. b are in line with the electrokinetic flow.
To create a lateral current at the Si/SiOx interface, Oxide patterning can be used in two ways. FIG. 3 c shows the simplest way to do this. FIG. 3 c shows the simplest method. It depicts a large holding area 32 that is fed by three narrow conduits 34. These conduits are formed by thermal oxide etching. To form a bead array, beads move along narrow conduits 34 to reach the holding area 32. FIG. FIG. 3 d shows a larger view of FIG. 3 c. This is a large-scale view of FIG. 3 a. FIGS. FIGS. 3 c, 3 d show that the voltage applied was 10V (pp), at 10 kHz. Alternate methods for creating bead transportation, such as UV-mediated oxide growth, include controlling the thickness of the oxide along the conduit. You can achieve this by UV exposure using a graduated filter. To cause lateral transport, there are only 5-10 Angstroms difference in oxide thickness between O & F. This situation does not require that the aspect ratios of the conduit or holding areas be changed. FIG. 3 b.”
External illumination is used to define conduits. The illumination intensity along the conduit can be varied to create the required impedance gradient. This has the advantage of making the conduit a temporary structure. Also, the direction of motion can be reversed or modified if necessary. The present invention provides mechanisms for light-mediated active linear transport (light-mediated) of planar aggregates made of beads. This can be controlled interactively. This can be achieved by moving an external pattern of illumination across a substrate surface to entrain the array of beads or electronically changing the shape of that pattern to incite particles to move.
“Two modes light-mediated, active transportation arc:
“i) Direct Translocation? Tractor beam? This is a method for translocating arrays and delineating their overall shapes by setting parameters to favor particle assembly in illuminated areas of the surface. The pattern is simply followed by arrays. The fluid’s mobility limits the rate of motion and so depends on the particle diameter and fluid viscosity.
“ii] Transverse Array Contraction is a bead transport system that allows fluids to flow through flexible tubing. This very general idea can be implemented using the light-control component. Multi-component planar beads are confined to a rectangular channel by UV-patterning, if desired, or by simply using light. The channel is free of beads by diffusion. A transverse illumination pattern is created that matches the channel’s dimension. The time is then varied to create a transverse constriction, which travels in one direction. This constriction wave can be created in many ways. One method to create a constriction wave is to project a mask onto the sample. The mask pattern can then be moved in the desired manner. This technique can also be applied electronically by controlling the illumination patterns of suitable light sources.
“The control of lateral beam transport through changing or moving patterns in illumination has the advantage of being able to apply it whenever and wherever (on any given substrate surface) is required. It does not require the imposition of gradients in impedance using predefined UV patterning. A pre-defined impedance pattern, on the other hand can offer additional capabilities when used in conjunction with light control. It may be possible to transport beads using a substrate-embedded, impedance gradient in order to separate the beads according to mobility.
“Conduits connecting O to F do not have to be straight. As tracks direct the movement of trains, conduits can be bent in any way you like (FIG. 1 d). 1 d. 1 e permits transport of particles between O and F after the conduit has been opened (or made in real-time) by a gated version of translocation (FIG. This operation uses UV oxide patterning to create two holding areas O and F. Light control is used to temporarily establish O and F. The conduit is lit with sufficient intensity to block the passage. The conduit can be opened by reducing the intensity or removing the illumination. The former allows beads to be transported by light, while the latter prevents them from being transported.
“The three fundamental operations that are essential to be successful are the merging or splitting of sets of beads (FIGS). 1 f, 1 g FIG. 1 f involves the two previous fundamental operations of?capture and-hold? applied to two spatially separated sets of beads at locations O1 and O2, their respective channeling along merging channels into a common target zone, and their eventual channeling into a final destination, a third area, F.
“The division of an array into subarrays (FIG. 1 g is an exception to a more complicated sorting operation. Sorting is the process of dividing beads from a set or array into two subsets. This can be done by analyzing their fluorescence intensity. The simpler case is that an array held in O must be divided into two subarrays following a demarcation line. Subarrays must be moved to F1 or F2 as the target areas. This is done according to the above conditions. operation to the array in O. Conduits link O to F1 or F2. The array is divided by high intensity illumination along a narrowly defined line. This again relies on gated translocation to control transport away from the holding space O. Another version, called indiscriminate split, randomly assigns particles to F1 or F2 through gated translocation of O’s array into F1 or F2 after conduits have been opened as previously described.
“FIGS. 4 a and 4b depict a variant where beads are located in the region O (FIG. 4 a) can be divided into multiple regions F1, F2, and. . . Fn (FIG. 4 b). For carboxylated polystyrenespheres with 2 micron diameter, this is achieved by increasing the voltage, typically from 5V (pp), to 20V (20 pp). The field-induced particle Polarization causes the array to fragment into smaller clusters. Splitting is used to distribute particles over a larger substrate area for presentation to potential analytes in solutions and subsequent scanning of individual clusters using analytical instruments to make individual readings.
The three functional elements of this invention may be combined to produce additional fundamental operations that control the orientation anisotropic objects embedded within the electroosmotic flow. This is created by the applied electrical field at the electrode’s surface. Gradients in the impedance are used to control the direction of flow in the substrate’s plane. These gradients can be shaped in accordance with the channeling operation. This is used to controllably align anisotropic items as shown in FIG. 1 h and can be used to align biomolecules such as DNA.
“Permanently anchoring an array on a substrate is another fundamental operation. Anchoring chemistries that are analogous to those using heterobifunctional crosslinking agents to anchor proteins via the formation of amide bonds can be used to accomplish this best. Another class of coupling chemicals for permanent anchoring is molecular recognition. This can be done, for instance, between biotinylated and surface-anchored streptavidin.
“General Experimental Conditions”
Experimental studies have demonstrated that the functional elements of the invention, which include the electric-field induced assembly planar particle arrays, spatial modulation of interfacial resistance by UV-mediated oxide and surface-chemical patterning, and finally the control over the interfacial impingance by light, were successfully demonstrated. These experiments used n-doped silicon wafers with resistivities in the range 0.01 Ohm cm. They were capped with thermally grown oxide layers varying in thickness from several thousand Angstrom to thin oxide layers that were regrown after the original “native” was removed. Under UV illumination from a source of deuterium in the presence oxygen, oxide was grown in HF to thicknesses ranging between 10 and 50 Angstroms. To produce features within the range of several microns, thermally grown oxide was lithographed using standard techniques.
“Surfaces were cleaned according to industry standards RCA and Piranha cleaning protocol. Before use, substrates were kept in Millipore water purification water. The contact angle of a 20 microliter droplet placed on the surface was measured and then viewed through a telescope. The contact angle refers to the angle that is subtended by the surface and the angle that is tangent (in side view), at the point of contact. A contact angle of 90 degrees would be for a droplet that is perfectly hemispherical. Surface chemical derivatization with mercapto-propyl-trimethoxysilanc (2% in dry toluene) produced surfaces giving typical contact angles of 70 degrees. The contact angle was reduced to zero by oxidation of terminal thiol functionality in the presence UV radiation. This occurred in less than 10 minutes of exposure to the UV source. Similar silane reagents could also be used to create hydrophobic surfaces. These surfaces had contact angles exceeding 110 degrees.
“Simple ?sandwich? Electrochemical cells were made by using kapton film to act as a spacer between Si/SiOx (ITO) and conductive indium Tin oxide (SiOx), which was deposited on a thin layer of glass. Silver epoxy was used to make contacts to platinum leads. It was applied directly to the ITO electrode’s top and (oxide-stripped to) backside of Si electrode. AC fields were generated by a function generator in this two-electrode arrangement. The applied voltages ranged from 20V to 1 MHZ and the frequencies varied from DC to 1 Mhz. High frequencies favour the formation of particle chains linking the electrodes. The currents were displayed on an oscilloscope and monitored by a potentiostat. Laser illumination was used for epi-fluorescence and reflection differential interference contrast microscopy. A simple microscope illuminator of 100 W was used to produce light-induced modulations in EIS impenetration. The laser diode emitting light at 680?680 nm also worked well.
“Colloidal beads were used, anionic and cationic, as well as nominally neutre, with a diameter ranging from several hundred Angstroms up to 20 microns. They were stored in NaN2 solutions.”
To avoid any non-specific interactions between particles or between particles and electrode surfaces, colloidal stability was carefully considered. Colloid suspensions were carefully avoided from bacteria contamination.
“Typical operating conditions that produced, except where otherwise noted, most of these results were: 0.2mM NaN2 solution (sodium azide), containing particles at a concentration such as to produce not more then a monolayer of particles when deposited onto the electrode; DC potentials in excess of 1?4V and AC potentials between 500 Hz.10 kHz and 500 Hz.10 kHz; electrode gap of 50 microns; anionic beads (carboxylated polystyrene beads) of 2 micron in diameter as well as nominally neutral polystyrene beads with a 2?20 micron in diameter;
“The present invention’s method and apparatus can be used in many different areas. We will discuss some of these in detail. Each example contains background information, followed by an application of the invention to the particular application.
“EXAMPLE I”
“Fabrication Surfaces and Coatings with Designed Properties.”
The present invention can be used to create planar surfaces or coatings with desired properties. The functional elements of this invention allow the formation of arrays of particles with a wide variety of sizes (approximately 100 Angstrom up to 10 microns), chemical composition, or surface functionality. These arrays can be placed on designated substrate areas and delineated. The interparticle spacing or internal order of the array can be controlled by changing the applied field before anchoring it to the substrate. These newly formed surfaces have pre-designed optical, mechanical and chemical characteristics that can be modified by subsequent treatments such as chemical cross-linking.
“The mechanical or chemical modification of surfaces or coatings principally determines how materials interact in a wide variety of applications that depend upon low adhesion (e.g. the familiar?nonstick?). surfaces that are important for housewares, low friction (e.g. to reduce wear in computer hard drives), hydrophobicity (the ability to repel water), catalytic activation or chemical functionality to either suppress or promote molecular interactions with surfaces. This last area is crucial for the development of biosensors and other bioelectronic devices. A large number of applications rely on surfaces with defined topography and/or chemical function to act as templates for controlling the growth morphology or command surfaces. Controlling the alignment of optically active molecules in thin organic films deposited, such as liquid crystal display applications.
“It has been extensively researched how thin organic films can be absorbed from liquids or gases using a variety of methods. Despite their simplicity and widespread use, these methods can prove difficult to manage in order to produce reliable and reproducible results. Molecular films cannot be used to create surfaces with a consistent topography.
“An alternative solution to the problem is modification of conductive surface by electrophoretic deposit of suspended particulates. This technique is widely used in industry to coat metal parts with paint and deposit phosphor on display screens. In practical applications, active deposition significantly increases the kinetics for formation. This is in contrast to passive adsorption organic films from solution. Electrophoretic deposition is a process that uses high DC electric fields to create layers where particles are permanently attracted to the surface. Although particles found in monolayers so-deposited are often randomly distributed, it is possible to form monolayers of polycrystalline gold colloids (150 Angstrom) on carbon-coated copper grids. In most cases, however, carbon-coated copper grids are not recommended as substrates.
Summary for “System and Method for Programmable Illumination Pattern Generation”
“I?Ions Electric Fields and Fluid Flow: Field Induced Formation of Planar Bead Arrays”
“Electrokinesis” refers to the phenomenon of electromagnetic fields causing a mobile ions around charged objects in an electrolyte. A diffuse ion cloud is formed when an object with a given surface charge is immersed into a solution containingions to screen it. The arrangement of two layers of immobile charges and a screening cloud of mobile counter-ions in the solution is called a “double layer”. The fluid is not electroneutral in this area of finite thickness. The region is not electroneutral because of the electric fields that act on it. These ions will cause the fluid to entrain in the diffuse layer. The fluid’s spatial distribution will determine the flow fields. The simplest form of electrokinetic phenomena is electroosmosis. This happens when an electric field is applied parallelly to the surface of a sample vessel or electrode with fixed surface charges. For example, a silicon oxide electrode (in neutral pH range). The electric field accelerates counter-ions in an electrode double layer, causing them to drag along solvent molecules and resulting in bulk fluid flow. This effect may be significant in narrow capillaries, and can be used to your advantage when designing fluid pumping systems.
“Electrophoresis” is a similar phenomenon. It refers to field-induced transports of charged particles in an electrolyte. An electric field accelerates the mobile ions within the particle’s double layer, just like electroosmosis. Contrary to the previous case, if the particle is mobile, it will compensate the field-induced motion (and the resulting Ionic Current) by moving in an opposite direction. The role of electrophoresis is important in industrial coating processes. It is also of interest, along with electroosis, in connection with the development capillary electrophoresis to be a majorstay in modern bioanalytical seperation technology.
“In restricted geometries such as the one of a shallow experiment chamber, such as a?sandwich’, it is possible to observe the effects of electroosmotic flow in confined spaces. Two planar electrodes. The surface charge distribution and topography on the bounding electrode surfaces are crucial in determining the nature of electroosmotic flow. This is a “sandwich”. An electrochemical cell can be made by two electrodes separated by a small gap. The oxide-capped silicon wafer will form the bottom electrode, while the optically transparent, conducting ITO will form the other electrode. “The silicon (Si) wafer is a thin slice from a single crystal silicon. It has been doped to achieve electrical conductivity at suitable levels and then insulated from electrolyte solution with a thin layer silicon oxide (SiOx).
A (DC or AC electric field applied to the electrode surface can induce the reversible aggregation beads into planar aggregates. The phenomenon was previously observed in a cell made from two conductive ITO electrodes. (Richetti Prost, Barois, J. Physique Lettr. 45, L-1137 to L-1143 (1984), which the contents are incorporated in this document by reference). However, it was previously demonstrated that electrokinetic flow is responsible for the attractive interaction between beads (Yeh Seul and Shraiman?Assembly Of Ordered Colloidal Aggregates By Electric Field Induced Fluid Flu?, Nature 386 57?59 (1997)), whose contents are also incorporated in this document by reference). This flow is due to the effect of non-uniformities in spatial distribution of current near the electrode. These non-uniformities can be explained by the presence of colloidal beads near the electrode. Each bead blocks the movement of ions within the electrolyte. It has been shown that a single bead can generate a toroidal fluid flow centered at the bead when it is placed close to the electrode’s surface. Multiple methods can be used to create fluid flow lateral toward low impedance regions by intentionally creating spatial non-uniformities. These methods will be described in the following sections.
“Particles embedded within the electrokinetic flow can be advected without regard to their particular chemical or biological nature while simultaneously altering flow field. The electric field-induced assembly planar aggregates or arrays can be applied to a variety of colloidal particles, including: beaded polymer resins (beads) ), lipids vesicles and whole chromosomes. Cells and biomolecules such as DNA and proteins, as well metal or semiconductor clusters and colloids.
The flow-mediated attraction between beads can travel a great distance. This is important for the applications that will be described. Planar aggregates form when an electric field is applied externally and then disassemble once the field is removed. The array assembly process is determined by the strength of the applied field. This determines the arrangement of the beads within the array. As a function, the applied voltage increases and beads form planar aggregates. These are composed of particles that are mobile and loosely packed. Then, they adopt a tighter packing and then exhibit a spatial arrangement resembling a raft or bubbles. Reversible transitions are possible between states of increasing internal ordeal, and complete disassembly is possible when applied voltage is removed. Another arrangement is that beads at low initial concentration form small clusters, which then assume positions within an orderly?superstructure?.
“II?”Patterning Silicon Oxide Electrode Surfaces
“Electrode patterning in accordance with a predetermined design facilitates the quasi-permanent modification of the electrical impedance of the EIS (Electrolyte-Insulator-Semiconductor) structure of interest here. Electrode-patterning modifies the EIS impedance spatially to determine the ionic current near the electrode. Beads can either seek out or avoid high ionic current regions depending on the frequency of the electric field. Spatial patterning allows for explicit external control over the shape and placement of beads arrays.
There are many methods for patterning, but two techniques offer the most advantages. The first is UV-mediated regrowth of a thin layer of oxide on a properly prepared silicon substrate. This method avoids photolithographic resist patterning or etching. The UV illumination mediates the transformation of exposed silicon to oxide in the presence of oxygen. The thickness of the oxide layer is dependent on its exposure time. This can be spatially modulated through the use of patterned masks in the UV illumination path. This thickness modulation, which is typically around 10 Angstroms in size, results in spatial modulations in the impedance at the Si/SiOx interface. However, the top surface remains flat and chemically homogeneous to the electrolyte solution. A second method that can produce spatial modulations in electrode surface charge distribution is UV-mediated photochemical oxygenation of a suitable chemical substance. This first occurs on the SiOx surface as a monolayer film. This allows fine control of local features and electrokinetic flow.
“A variation on this photochemical modulation involves the creation of lateral variations in the EIS impedance, and thus in the current generated by the applied electric field. This can be achieved by controlling UV exposure to cause a slow lateral variation of the oxide thickness and/or the surface charge density. Controlling lateral gradients is key to inducing lateral bead transportation. It also facilitates fundamental operations such as the capture and channeling beads to a predetermined destination using conduits made of impedance elements embedded in Si/SiOx interface. Photochemical patterning of functionalized chemicals overlayers can also be applied to other types electrode surfaces, including ITO.
“III?Light-controlled Modulation of the Interfacial Impedance”
The basis for controlling the electrokinetic forces that mediate the aggregation of beads is the spatial and temporal modulation in the EIS-impedance according to a pattern or external illumination. Remote control of the formation, placement, and rearrangement bead arrays is possible via light-modulated electrokinetic assembly. This allows for a wide variety of interactive manipulations of colloidal beads as well as biomolecules.
It will help to review the photoelectric properties of semiconductors in order to understand the basic principle of this method. This includes the EIS structure, the Insulating SiOx and Semiconductor structures (I), E (Electrolyte solution) and S (Semiconductor). The photoelectric characteristics of this structure are closely related to those of a standard Metal-Insulator-Semiconductor (MIS) or Metal-Oxide-Semiconductor (MOS) devices which are described in S. M. Sze, ?The Physics of Semiconductors?, 2nd Edition, Chapt. 7 (Wiley Interscience 1981), whose contents are incorporated by reference.
“The interface between semiconductor and insulating oxide layers deserves special attention. Understanding the MOS structure’s electrical response to light is crucial. This concept refers to a small, but finite space charge region that forms at the Si/SiOx Interface in the presence of an applied bias potential. An effective bias in the form a junction potential is present for the EIS structure under all conditions. Space charge regions form due to distortions of the semiconductor’s conduction and valence bands (?bandbending?). The interface is in its vicinity. This is due to the fact that the interface has a bias potential. However, the insulating oxide prevents charge transfer. In electrochemical terms, this means that the EIS structure eliminates Faradaic effect. Instead, opposite sign charges accumulate on either side the insulating oxide layer to generate a finite degree of polarization.
“In the presence a reverse bias, the conduction and valence band edges of an N-doped semiconductor bend upwards near the Si/SiOx Interface and electrons flow out from the interfacial area in response to the corresponding voltage gradient. A majority carrier depletion layer forms near the Si/SiOx Interface. This region is able to produce electron-hole pairs through light absorption. If they don’t recombine instantaneously, electron-hole pairs can be split by the local acting electric field and a corresponding current flows. This latter effect allows for control over the electrokinetic assembly beads in an electrolyte solution.
Two aspects of an equivalent circuit that represents the EIS structure can be used to better understand the frequency dependence of light-induced modulation. The first is a close analogy between the detailed electrical characteristics at the electrolyteoxide interface and the depletion layer at interface between semiconductor and insulator. The depletion layer has similar electrical characteristics to the double layer. It also exhibits a voltage-dependent capacitance. The depletion layer’s impedance can be reduced by illumination, as we have already discussed. The second reason is that the oxide layer’s capacitive electrical response means that it will only pass current above a threshold (?threshold?) frequency. If the applied voltage frequency exceeds the threshold, illumination may lower the effective impedance for the entire EIS structure.
“Effective reduction of EIS impedance is also dependent on light intensity, which affects the rate at which electron-hole pairs are generated. The majority of photogenerated electrons are free to flow out of the depletion area and contribute to the photocurrent, even if there is no significant recombination. The rest of the hole charge builds up near the Si/SiOx Interface and blocks the electric field from the depletion area. The rate of recombination decreases and electron-hole separation efficiency (and hence the photocurrent) decreases. Given the values of frequency, amplitude, and voltage applied, it is expected that the current will increase in intensity as the illumination intensifies. Then, it will decrease. The impedance decreases initially to a minimum (at maximum current), and then it decreases.”
This intensity dependence can be used to inducing the lateral displacement beads between partially covered and fully masked regions. The fully exposed areas will correspond to regions of interface with the lowest impedance and therefore the highest current. Beads will be drawn into these areas as the illumination intensities increase. The effective EIS impedance of fully exposed regions will decrease as the photocurrent increases. This can lead to an inversion of current’s lateral gradient. The fully exposed areas will be used to draw beads. You can also use time-varying variations in the illumination pattern to influence bead movement.
“IV?Integration in Biochemical Analysis in a Miniaturized and Planar Format”
“Planar array assays are a good option for biomolecular screening and medical diagnostics. They have the advantage of high parallelity and automation, which allows them to achieve high throughput in multi-step, complex analytical protocols. Miniaturization will reduce pertinent mixing times due to the small spatial scale. It will also result in reduced sample and reagent volumes, as well a reduction in power requirements. Integration of biochemical analytical methods into a miniaturized system surfaced on a planar substrate (chip?) would be significant. This would result in significant improvements in performance and cost reductions in diagnostic and analytical procedures.
“In the context of DNA manipulation, and analysis, initial steps were taken (i.e. miniaturization) by combining on glass substrate, the restriction enzyme treat of DNA, and the subsequent separation enzyme digests using capillary electrophoresis. See, for instance, Ramsey, PCT Publication Number. WO 96/04547 is incorporated by reference. Alternatively, the amplifying of DNA sequences using the polymerase chain react (PCR) and subsequent electrophoretic separation are described in U.S. Pat. Nos. Nos.
These standard laboratory processes were demonstrated in a miniature format but have not been applied to create a complete system. Additional manipulations will be required to create a complete system, such as binding and functional assays, front-end sample processing and small signal detection followed by information processing. Complete functional integration is the real challenge. This is where system architecture and design limitations on individual components will become apparent. A fluidic process is needed to concatenate analytic steps that require spatial separation and transport to new places of sets of analytes. There are many options, including electroosmotic pumps and droplet transport by temperature-induced local surface tension gradients. These techniques are possible in demonstration experiments but require a lot of system design to manage the large DC voltages needed for electroosmotic mixing.
The present invention combines three functional elements to provide an apparatus and method that allow for the interactive spatial manipulation of colloidal particle (?beads?) in real-time. The interface between an electrolyte solution and a light sensitive electrode is used to allow for the interaction of molecules and colloidal particles. These three functional elements include the electric field-induced assembly planar particle arrays at the interface between an insulating electrode or a conductor electrode and an electrolyte. The spatial modulation and control of interfacial resistance by UV-mediated oxide growth or surface-chemical patterning. Finally, the interactive, real-time control of interfacial Impedance by light. External intervention is possible to adjust the spatial distribution of ionic electrons and the fluid flow mediating array assembly. This invention has many advantages. It is important to introduce spatial non-uniformities within the EIS structure. These inhomogeneities can be either permanent or temporary as a result of taking advantage of the EIS structures’ physical and chemical properties.
“The invention concerns the realization of an integrated, functionally complete system for performing biochemical analysis on a silicon wafer surface or other substrate. The present invention also allows for the fabrication of surface-mounted optical elements, such as lens arrays, that can be fabricated using the method and apparatus.
The combination of these three functional elements gives the invention the ability to manipulate beads and bead arrays with planar geometry. This allows for the application of biochemical analytical methods. These fundamental operations are applicable to aggregates or arrays of colloidal particles, including beaded polymer resins (also referred to by latices), whole chromosomes cells, biomolecules such as proteins and DNA as well as metal and semiconductor colloids and clusters.
“Sets of colloidal particles can be captured and arrays formed on designated areas of the electrode surface (FIGS. 1 a, 1 band FIGS. 2 a?d). The particles and their arrays formed in response to the applied fields can be channeled along conduits in any configuration. These conduits may either be embedded in Si/SiOx by UV-oxide patterning, or delineated using an external pattern. This channeling (FIGS. 1 c, 1 d, 1 e, FIGS. 3 c, 3 D), in a direction that is normal to the applied electric field. This relies on lateral gradients of the EIS structure’s impedance and, therefore, in the field-induced field. FIGS. These gradients can be introduced using appropriate patterns of illumination. This allows for the implementation of a gated version (FIG. 1 e). For the alignment of long-lived particles such as DNA near the electrode’s surface, the electrokinetic flow that mediates the array assembly process can also be used. The present invention also allows for methods of sorting and separating particles.
“Colloidal particles can be placed in designated areas, and kept there until they are released or disassembled. You can define the overall shape of an array by UV-oxide patterning, or by changing the pattern of illumination. This allows the creation of functionally distinct compartments on the electrode surface, whether permanent or temporary. The arrays can be modified in real-time and may be combined with other arrays (FIG. 1 f) Or split into multiple subarrays, or clusters (FIG. 1 g, FIGS. 4 a, 4 b). You can also adjust the local order of the array and the lateral particle densities by using the external electric field, or by adding a second, chemically inert component to the array.
“The invention allows the combination of fundamental operations to create increasingly complex products and process. The following examples illustrate how analytical procedures can be used to solve a variety of problems in materials science and pharmaceutical drug discovery. These and other functionalities can be integrated in a planar geometries. The present invention provides the ability to create temporary or permanent compartmentalization to spatially isolate concurrent processes and subsequentail steps in a protocol. It also allows for the manipulation of sets of particles in such a way that they can be concatenated in different areas of the substrate surfaces.
“This invention is for a system for programming illumination patterns. The present invention discloses a novel method and apparatus to generate patterns of illumination and project them onto planar surfaces or onto planar interfaces such as the interface formed by an electrolyte-insulator-semiconductor (EIS), e.g., as described herein. The present invention allows the creation of patterns or sequences thereof using graphical design software or drawing software on a personal computers and the projection of those patterns or sequences (?time-varying pattern?). The LCD panel is projected onto the interface by an optical design that images the LCD panel onto the surface. LCD technology allows flexibility and control over spatial layout, time sequences and intensities. You can also create illumination patterns. This allows you to create patterns with rapidly changing light intensities, or patterns with slowly changing intensity profiles.
“The present invention uses patterns of illumination to control assembly and lateral motion of colloidal particle within an enclosed fluid environment. Particles can be inducible to move between the two electrode surfaces that are planar and bound by the liquid with the help of an electric field. This field is time-varying and can be applied between the electrode’s planar electrode surfaces. It can also induce movement into and out of the illuminated areas of the electrode depending upon the layout of the patterns, the frequency and intensity of the transmitted light, the electric field strength, the junction gap separation, and the semiconductor doping levels.
“In conjunction the present invention disclosing an programmable illumination generator, advanced operations in array reconfiguration, segmentation, and (spatial encoding) are possible which, in turn, lead to a variety advanced operations and applications.”
“Applications according to the invention are described where patterns are created by projecting fixed masks that define bright and dark areas of illumination on the substrate. The present invention describes a programmable generator that allows for flexibility and control over the placement and direction of colloidal particles. Particles can be?dragged? to form dense planar layers. You can also?drop them? Interactively by?dragging? You can also?drop? The graphical design displayed on a computer monitor using a mouse. Alternately, you can program a sequence of patterns or a transformation pattern to manipulate arrays in a planned manner. Multiple ?sub-assemblies? Under illumination, multiple particles can be controlled simultaneously in different areas of the substrate.
“BRIEF DESCRIPTION DES DRAWINGS”
“Other objects features and advantages of this invention will be easier to understand when combined with the detailed description of an embodiment. This will be understood only as an illustration and the accompanying drawings will reflect aspects of that embodiment.
“FIGS. “FIGS.
“FIGS. “FIGS.
“FIGS. “FIGS.
“FIGS. “FIGS.
“FIGS. “FIGS.
“FIGS. “FIGS.
“FIG. “FIG.
“FIGS. 6 a.c are side views of a layout-preserving process to transfer a microtiter plate from a planar cells to a microtiter plates;
“FIG. 7 shows the inclusion of spacer particles in bead clusters.
“FIG. “FIG.8 is an example of binding assay variations;
“FIGS. “FIGS.
“FIG. “FIG.
“FIG. “FIG.
“FIG. “FIG.
“FIG. “FIG.
“FIGS. 14 a.d are photos of different forms of light-induced arrays;
“FIG. “FIG.
“FIG. “FIG.
“FIG. 16 is a photo showing a “drag and drop?” operation applied to particles
“FIG. 17 shows how an illumination profile can be used to create a subarray border;
“FIGS. 18 a and 18b are photos that illustrate the creation and maintenance of particle confinement patterns.
“FIG. 19 is a photo that illustrates the preferential collection only of one type of particle in the mixture into an illuminated space under conditions that ensure the exclusion of all other particles.
“FIGS. “FIGS.
“FIGS. 21 a and 21b are photos taken in succession while sweeping an illumination pattern across an sample containing small colloidal particles (2.8?m in diameter), which were deposited at random locations on a planar substrate.
“FIGS. 22 a and 22b show examples of the methods and procedures for chemical and spatial encoding arrays and decoding arrays using selective anchoring individual beads to substrates, segmentation and fractionation.
“FIG. 23 is an example of random sequential injection.
“FIG. “FIG.
“FIG. “FIG.
“FIG. 26 a?b shows a method for producing a composite particle arrangement exhibiting a concentric collection of discrete compositional bands;
“FIG. “FIG. 27 illustrates the principle that imposing conditions favoring expulsion particles from sub-regions illuminated with high intensities;
“FIG. 29 shows the light-induced fluid flow at the boundary of illuminated and unilluminated areas of a substrate.
“The functional elements of the invention can be combined to create a set fundamental operations for interactive spatial manipulation of colloidal molecules and particles, as well as planar aggregates near an electrode surface. The following describes the fundamental operations of this “toolset”. These operations are listed in ascending order of complexity. It is helpful to use a classification scheme that takes into account the number of inputs or outputs involved in any given operation. A?three-terminal’ would be the combination of two different arrays or sets of particles into one. operation that requires two inputs as well as one output. Three-terminal reverse operation involves one input and two outputs. It is the division of an array into two subarrays.
“Capture-and-hold is the fundamental one-terminal operation. operation (FIG. 1a), which creates an array of particles in a defined area of arbitrary outline on a surface, delineated either by UV-mediated Oxid patterning or a corresponding pattern projected onto a Si/SiOx substrate. FIGS. FIGS. FIG. FIG. FIG. FIG. 2 b shows an electric field (10Vp-p source at 1 kHz), and bead capture takes place within the thin oxide zone 22. The array will grow in less than one second. It will continue to grow for approximately 10 seconds after that, as beads arrive at greater distances to increase the region 22’s outward growth perimeter. The array stops growing when it reaches the outer limit of its target area. This is the area that is defined by the thin oxide with a low impedance. The applied voltage determines the internal state of the beads. Higher values encourage denser packing and eventually form ordered arrays with hexagonal crystalline configurations in the form of bubble rafts. The array will remain in place as long the voltage is applied. The array will be disassembled if the voltage is removed.
“The ?capture-and-hold? “Capture-and-hold” operation can also be performed under visible or infrared illumination. For example, a mask with the desired layout may be projected onto the Si/SiOx electro. On a Zeiss UEM microscope, a 100 W quartz microscope light source was used. Affixing masks or apertures in the intermediate image plane provided the desired shape in the electrode’s plane (when the microscope is properly focused under Koehler illumination) has been possible. An IR laser diode of 3 mW output at 650-680 nm has also been used. External illumination is preferred to oxide patterning in order to modify the spatial confinement pattern of particles.
“Related To?capture and-hold?” “Related to?capture-and-hold?” (FIG. 1 b), which removes particles from a specified area of the surface. An inversion occurs when the frequency of the applied voltage is increased to around 100 kHz. This results in particles that assemble in the thin oxide portion of the surface (e.g. region 22 FIG. 2 b) instead of forming structures that surround the perimeter of the target area. This effect is not sufficient. The exclusion of particles from desired areas can also be achieved, analogously to the original “capture-and-hold” method. Operations by simply embedding a corresponding structure into the Si/SiOx interface via UV-mediated oxide regrowth. FIG. 2 c, 2 d are achieved under conditions that are identical to those in FIGS. 2 a, 2 b are achieved by applying 20V (pp), at 10 kHz. The oxide thickness in non-designated areas 24 is about 30 Angstroms. However, the value for the designated square areas 26 are approximately 40 Angstroms. This indicates a higher impedance at the applied frequency.
“The ?capture-and-hold? “Capture-and-hold” allows the spatial compartmentalization and separation of functionally distinct areas on the substrate surface. This operation allows for the spatial isolation of particles of different chemical types, which can be introduced to the electrochemical cells at different times and injected at different locations.
Translocation (FIG.) is the fundamental two-terminal operation. 1 c, or the controlled transporting of a set particles from location O on the surface to location F; O and F are the target areas to which one-terminal operations can be applied. Translocation uses a one-dimensional, lateral beam transport. This is done by applying a current along a conduit connecting O and F. FIGS. 3. a and 3. b, or projecting a linear pattern of illumination. This channeling operation causes beads to move in the direction that has lower impedance than the arrow in FIGS. 3. a and 3. b are in line with the electrokinetic flow.
To create a lateral current at the Si/SiOx interface, Oxide patterning can be used in two ways. FIG. 3 c shows the simplest way to do this. FIG. 3 c shows the simplest method. It depicts a large holding area 32 that is fed by three narrow conduits 34. These conduits are formed by thermal oxide etching. To form a bead array, beads move along narrow conduits 34 to reach the holding area 32. FIG. FIG. 3 d shows a larger view of FIG. 3 c. This is a large-scale view of FIG. 3 a. FIGS. FIGS. 3 c, 3 d show that the voltage applied was 10V (pp), at 10 kHz. Alternate methods for creating bead transportation, such as UV-mediated oxide growth, include controlling the thickness of the oxide along the conduit. You can achieve this by UV exposure using a graduated filter. To cause lateral transport, there are only 5-10 Angstroms difference in oxide thickness between O & F. This situation does not require that the aspect ratios of the conduit or holding areas be changed. FIG. 3 b.”
External illumination is used to define conduits. The illumination intensity along the conduit can be varied to create the required impedance gradient. This has the advantage of making the conduit a temporary structure. Also, the direction of motion can be reversed or modified if necessary. The present invention provides mechanisms for light-mediated active linear transport (light-mediated) of planar aggregates made of beads. This can be controlled interactively. This can be achieved by moving an external pattern of illumination across a substrate surface to entrain the array of beads or electronically changing the shape of that pattern to incite particles to move.
“Two modes light-mediated, active transportation arc:
“i) Direct Translocation? Tractor beam? This is a method for translocating arrays and delineating their overall shapes by setting parameters to favor particle assembly in illuminated areas of the surface. The pattern is simply followed by arrays. The fluid’s mobility limits the rate of motion and so depends on the particle diameter and fluid viscosity.
“ii] Transverse Array Contraction is a bead transport system that allows fluids to flow through flexible tubing. This very general idea can be implemented using the light-control component. Multi-component planar beads are confined to a rectangular channel by UV-patterning, if desired, or by simply using light. The channel is free of beads by diffusion. A transverse illumination pattern is created that matches the channel’s dimension. The time is then varied to create a transverse constriction, which travels in one direction. This constriction wave can be created in many ways. One method to create a constriction wave is to project a mask onto the sample. The mask pattern can then be moved in the desired manner. This technique can also be applied electronically by controlling the illumination patterns of suitable light sources.
“The control of lateral beam transport through changing or moving patterns in illumination has the advantage of being able to apply it whenever and wherever (on any given substrate surface) is required. It does not require the imposition of gradients in impedance using predefined UV patterning. A pre-defined impedance pattern, on the other hand can offer additional capabilities when used in conjunction with light control. It may be possible to transport beads using a substrate-embedded, impedance gradient in order to separate the beads according to mobility.
“Conduits connecting O to F do not have to be straight. As tracks direct the movement of trains, conduits can be bent in any way you like (FIG. 1 d). 1 d. 1 e permits transport of particles between O and F after the conduit has been opened (or made in real-time) by a gated version of translocation (FIG. This operation uses UV oxide patterning to create two holding areas O and F. Light control is used to temporarily establish O and F. The conduit is lit with sufficient intensity to block the passage. The conduit can be opened by reducing the intensity or removing the illumination. The former allows beads to be transported by light, while the latter prevents them from being transported.
“The three fundamental operations that are essential to be successful are the merging or splitting of sets of beads (FIGS). 1 f, 1 g FIG. 1 f involves the two previous fundamental operations of?capture and-hold? applied to two spatially separated sets of beads at locations O1 and O2, their respective channeling along merging channels into a common target zone, and their eventual channeling into a final destination, a third area, F.
“The division of an array into subarrays (FIG. 1 g is an exception to a more complicated sorting operation. Sorting is the process of dividing beads from a set or array into two subsets. This can be done by analyzing their fluorescence intensity. The simpler case is that an array held in O must be divided into two subarrays following a demarcation line. Subarrays must be moved to F1 or F2 as the target areas. This is done according to the above conditions. operation to the array in O. Conduits link O to F1 or F2. The array is divided by high intensity illumination along a narrowly defined line. This again relies on gated translocation to control transport away from the holding space O. Another version, called indiscriminate split, randomly assigns particles to F1 or F2 through gated translocation of O’s array into F1 or F2 after conduits have been opened as previously described.
“FIGS. 4 a and 4b depict a variant where beads are located in the region O (FIG. 4 a) can be divided into multiple regions F1, F2, and. . . Fn (FIG. 4 b). For carboxylated polystyrenespheres with 2 micron diameter, this is achieved by increasing the voltage, typically from 5V (pp), to 20V (20 pp). The field-induced particle Polarization causes the array to fragment into smaller clusters. Splitting is used to distribute particles over a larger substrate area for presentation to potential analytes in solutions and subsequent scanning of individual clusters using analytical instruments to make individual readings.
The three functional elements of this invention may be combined to produce additional fundamental operations that control the orientation anisotropic objects embedded within the electroosmotic flow. This is created by the applied electrical field at the electrode’s surface. Gradients in the impedance are used to control the direction of flow in the substrate’s plane. These gradients can be shaped in accordance with the channeling operation. This is used to controllably align anisotropic items as shown in FIG. 1 h and can be used to align biomolecules such as DNA.
“Permanently anchoring an array on a substrate is another fundamental operation. Anchoring chemistries that are analogous to those using heterobifunctional crosslinking agents to anchor proteins via the formation of amide bonds can be used to accomplish this best. Another class of coupling chemicals for permanent anchoring is molecular recognition. This can be done, for instance, between biotinylated and surface-anchored streptavidin.
“General Experimental Conditions”
Experimental studies have demonstrated that the functional elements of the invention, which include the electric-field induced assembly planar particle arrays, spatial modulation of interfacial resistance by UV-mediated oxide and surface-chemical patterning, and finally the control over the interfacial impingance by light, were successfully demonstrated. These experiments used n-doped silicon wafers with resistivities in the range 0.01 Ohm cm. They were capped with thermally grown oxide layers varying in thickness from several thousand Angstrom to thin oxide layers that were regrown after the original “native” was removed. Under UV illumination from a source of deuterium in the presence oxygen, oxide was grown in HF to thicknesses ranging between 10 and 50 Angstroms. To produce features within the range of several microns, thermally grown oxide was lithographed using standard techniques.
“Surfaces were cleaned according to industry standards RCA and Piranha cleaning protocol. Before use, substrates were kept in Millipore water purification water. The contact angle of a 20 microliter droplet placed on the surface was measured and then viewed through a telescope. The contact angle refers to the angle that is subtended by the surface and the angle that is tangent (in side view), at the point of contact. A contact angle of 90 degrees would be for a droplet that is perfectly hemispherical. Surface chemical derivatization with mercapto-propyl-trimethoxysilanc (2% in dry toluene) produced surfaces giving typical contact angles of 70 degrees. The contact angle was reduced to zero by oxidation of terminal thiol functionality in the presence UV radiation. This occurred in less than 10 minutes of exposure to the UV source. Similar silane reagents could also be used to create hydrophobic surfaces. These surfaces had contact angles exceeding 110 degrees.
“Simple ?sandwich? Electrochemical cells were made by using kapton film to act as a spacer between Si/SiOx (ITO) and conductive indium Tin oxide (SiOx), which was deposited on a thin layer of glass. Silver epoxy was used to make contacts to platinum leads. It was applied directly to the ITO electrode’s top and (oxide-stripped to) backside of Si electrode. AC fields were generated by a function generator in this two-electrode arrangement. The applied voltages ranged from 20V to 1 MHZ and the frequencies varied from DC to 1 Mhz. High frequencies favour the formation of particle chains linking the electrodes. The currents were displayed on an oscilloscope and monitored by a potentiostat. Laser illumination was used for epi-fluorescence and reflection differential interference contrast microscopy. A simple microscope illuminator of 100 W was used to produce light-induced modulations in EIS impenetration. The laser diode emitting light at 680?680 nm also worked well.
“Colloidal beads were used, anionic and cationic, as well as nominally neutre, with a diameter ranging from several hundred Angstroms up to 20 microns. They were stored in NaN2 solutions.”
To avoid any non-specific interactions between particles or between particles and electrode surfaces, colloidal stability was carefully considered. Colloid suspensions were carefully avoided from bacteria contamination.
“Typical operating conditions that produced, except where otherwise noted, most of these results were: 0.2mM NaN2 solution (sodium azide), containing particles at a concentration such as to produce not more then a monolayer of particles when deposited onto the electrode; DC potentials in excess of 1?4V and AC potentials between 500 Hz.10 kHz and 500 Hz.10 kHz; electrode gap of 50 microns; anionic beads (carboxylated polystyrene beads) of 2 micron in diameter as well as nominally neutral polystyrene beads with a 2?20 micron in diameter;
“The present invention’s method and apparatus can be used in many different areas. We will discuss some of these in detail. Each example contains background information, followed by an application of the invention to the particular application.
“EXAMPLE I”
“Fabrication Surfaces and Coatings with Designed Properties.”
The present invention can be used to create planar surfaces or coatings with desired properties. The functional elements of this invention allow the formation of arrays of particles with a wide variety of sizes (approximately 100 Angstrom up to 10 microns), chemical composition, or surface functionality. These arrays can be placed on designated substrate areas and delineated. The interparticle spacing or internal order of the array can be controlled by changing the applied field before anchoring it to the substrate. These newly formed surfaces have pre-designed optical, mechanical and chemical characteristics that can be modified by subsequent treatments such as chemical cross-linking.
“The mechanical or chemical modification of surfaces or coatings principally determines how materials interact in a wide variety of applications that depend upon low adhesion (e.g. the familiar?nonstick?). surfaces that are important for housewares, low friction (e.g. to reduce wear in computer hard drives), hydrophobicity (the ability to repel water), catalytic activation or chemical functionality to either suppress or promote molecular interactions with surfaces. This last area is crucial for the development of biosensors and other bioelectronic devices. A large number of applications rely on surfaces with defined topography and/or chemical function to act as templates for controlling the growth morphology or command surfaces. Controlling the alignment of optically active molecules in thin organic films deposited, such as liquid crystal display applications.
“It has been extensively researched how thin organic films can be absorbed from liquids or gases using a variety of methods. Despite their simplicity and widespread use, these methods can prove difficult to manage in order to produce reliable and reproducible results. Molecular films cannot be used to create surfaces with a consistent topography.
“An alternative solution to the problem is modification of conductive surface by electrophoretic deposit of suspended particulates. This technique is widely used in industry to coat metal parts with paint and deposit phosphor on display screens. In practical applications, active deposition significantly increases the kinetics for formation. This is in contrast to passive adsorption organic films from solution. Electrophoretic deposition is a process that uses high DC electric fields to create layers where particles are permanently attracted to the surface. Although particles found in monolayers so-deposited are often randomly distributed, it is possible to form monolayers of polycrystalline gold colloids (150 Angstrom) on carbon-coated copper grids. In most cases, however, carbon-coated copper grids are not recommended as substrates.
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