Neutralization and containment of redox species produced by circumferential electrodes

Abstract

There is disclosed an electrode array architecture employing continuous and discontinuous circumferential electrodes. There is further disclosed a process for the neutralization of acid generated at anode(s) by base generated at cathode(s) circumferentially located to each other so as to confine a region of pH change. The cathodes can be displayed as concentric rings (continuous) or as counter electrodes in a cross pattern (discontinuous). In this way reagents, such as acid, generated in a center electrode are countered (neutralized) by reagents, such as base, generated at the corners or at the outer ring.

Claims

1. A method for electrochemical oligomer synthesis on a dense electrode array comprising: (a) providing a dense electrode array including a plurality of cells and a surface, where each cell of the plurality of cells includes an anode and a circumferential cathode, where each of the anodes are separately addressable electrodes, where a porous reaction layer is adsorbed to the surface, where the porous reaction layer is formed from one or more materials selected from the group consisting of a disaccharide, and a monosaccharide; (b) contacting the porous reaction layer with a bathing solution; and (c) bonding a first nucleotide having at least one protected chemical functional group to the porous reaction layer; (d) conducting a deblocking step by addressing each active anode with a first current or a first voltage to generate an acid, where each active anode is surrounded by a cathode that is biased with a second current or a second voltage, where the cathode produces a base to neutralize the acid, where the cathode is separated from each active anode by an electrode margin, where an insulating material is present in the electrode margin, where the at least one protected chemical functional group is removed by the action of the acid forming at least one deprotected chemical functional group; and (e) bonding a second nucleotide having at least one protected chemical functional group to the at least one deprotected chemical functional group.

2. The method of claim 1, where step (d) addresses each active anode with the first current.

3. The method of claim 1, where step (d) addresses the cathode with the second current.

4. The method of claim 1, where at least two neighboring electrodes are arranged in a pattern selected from the group consisting of nearest neighbor, neighbor electrodes, border of neighbor electrodes, border of nearest-neighbor, one closest available electrode, and electrode that is not approach.

5. The method of claim 1, where the insulating material is no more than 10 microns from the outer edge of each anode to the inner edge of the cathode.

6. A method for electrochemical oligomer synthesis on a dense electrode array comprising: (a) providing a dense electrode array including a plurality of cells and a surface, where each cell of the plurality of cells includes an anode and a cathode, where the cathode forms an outer ring around each anode, where each of the anodes are separately addressable, where a porous reaction layer comprising a disaccharide and/or a monosaccharide is adsorbed to the surface; (b) applying a bathing solution to the porous reaction layer; (c) bonding a first nucleotide having at least one protected chemical functional group to the porous reaction layer; (d) addressing one or more anodes with a first current or a first voltage to generate an acid; (e) applying a second current or a second voltage to the cathode to generate a base, where the base neutralizes the acid, where the cathode is separated from each active anode by an electrode margin, where an insulating material is present in the electrode margin, where the at least one protected chemical functional group is removed by the action of the acid forming at least one deprotected chemical functional group; and (f) bonding a second nucleotide having at least one protected chemical functional group to the at least one deprotected chemical functional group.

7. The method of claim 6, where the thickness of the insulating material is between: a lower limit of 10 microns; and an upper limit of 50 microns.

8. The method of claim 6, where an outer edge of the outer ring of a first cell is separated from an outer edge of the outer ring of a second cell by between: a lower limit of 33 microns; and an upper limit of 50 microns.

9. The method of claim 6, where the cell diameter is 44 microns.

10. The method of claim 6, where a center point of a first cell is separated from a center point of a second cell by 75 microns.

11. The method of claim 6, where step (d) addresses each active anode with the first current.

12. The method of claim 6, where step (e) applies the second current to the cathode.

13. A method for electrochemical oligomer synthesis on a dense electrode array comprising: (a) providing a dense electrode array including a plurality of cells and a surface, where each cell of the plurality of cells includes a cathode and an anode, where the anode forms an outer ring around each cathode, where each of the cathodes are separately addressable, where a porous reaction layer comprising a disaccharide and/or a monosaccharide is adsorbed to the surface; (b) applying a bathing solution to the porous reaction layer; (c) bonding a first nucleotide having at least one protected chemical functional group to the porous reaction layer; (d) addressing one or more cathodes with a first current or a first voltage to generate a base; (e) applying a second current or a second voltage to the anode to generate an acid, where the acid neutralizes the base, where the anode is separated from each active cathode by an electrode margin, where an insulating material is present in the electrode margin, where the at least one protected chemical functional group is removed by the action of the acid forming at least one deprotected chemical functional group; and (f) bonding a second nucleotide having at least one protected chemical functional group to the at least one deprotected chemical functional group.

14. The method of claim 13, where the thickness of the insulating material is between: a lower limit of 10 microns; and an upper limit of 50 microns.

15. The method of claim 13, where an outer edge of the outer ring of a first cell is separated from an outer edge of the outer ring of a second cell by between: a lower limit of 33 microns; and an upper limit of 50 microns.

16. The method of claim 13, where the cell diameter is 44 microns.

17. The method of claim 13, where a center point of a first cell is separated from a center point of a second cell by 75 microns.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a reaction scheme for electrochemical parallel DNA synthesis on a CMOS type of electrode array. The portion being shown is that of the deprotection step. O stands for the oxygen atom on the matrix covering the electrode. T stands for the nucleotide T and x is a protecting group, which in a preferred embodiment is DMT. A stands for the nucleotide A. As noted, the electrodes can be turned on and off on an individual basis.

(2) FIG. 2 shows a portion of a polypeptide or protein parallel electrochemical synthesis reaction scheme. In this case tBoc is a preferred amino moiety protecting group. The protecting moiety is removed by acid generated at an anode when the electrode is turned on.

(3) FIG. 3 shows a reaction scheme for the incorporation of an NHS-biotin moiety to a hydroxyl group located on a porous reaction layer on the electrode. In a preferred embodiment, this reaction is base catalyzed when the electrode acts as the cathode.

(4) FIG. 4 shows a diagram of a 3D-section of a continuous circumferential electrode. The inner and outer electrodes are separated by insulated gap material. The gap region is called the diffusion zone. In a preferred embodiment, a porous reaction layer covers both electrodes and the gap region.

(5) FIG. 5 shows a diagram showing a continuous circumferential electrode. The anode is one of the central electrodes, while the cathode ring is an extension of a neighboring electrode. The area called platinum is conducting.

(6) FIG. 6 shows a white light photograph of an electrode array containing the anode/cathode features diagrammed in FIG. 5.

(7) FIG. 7 shows synthesis results using an electrode-containing microarray device having continuous (circular) circumferential electrodes. A has oligonucleotide probe sequence (3 CCT CGA CCA CCG CAT 5) [SEQ ID NO. 1] was synthesized within a porous matrix located above an electrode functioning as an anode and having surrounding continuous circumferential electrodes acting as cathodes. The oligonucleotides were deblocked (by removing any remaining protecting groups after synthesis). The microarray was placed into a solution containing a complementary has sequence oligonucleotides having Texas Red (TR) bound to the 3 end (5 GGA GCT GGT GGC GTA-TR 3) [SEQ ID NO. 2] and hybridized at 40.degree. C. for 30 minutes. The electrode-containing microarray was washed to remove excess unbound material the bright circular areas over interior electrodes (used as anodes during synthesis) show that the presence of the circumferential electrode properly contained electrochemically generated acids at the anode.

(8) FIG. 8 shows a diagram and relative dimensions of the electrodes provided by a microarray or electrode array device having in excess of 12,000 electrodes in a region of semiconductor array of about 1 cm.sup.2. Circular electrode diameter was about 44 microns. The electrode distance-to-distance (or center point) was about 75 microns. The C in the circles represents that those electrodes set up to be cathodes and the A represent the single anode. It should be noted that an active anode can be surrounded by active cathodes in a cross configuration. This arrangement provides what is called discontinuous circumferential electrodes. During each step of synthesis and deblocking, a particular electrode can function as an anode if it is active for a step or later as a cathode if it is to be used as a counter electrode in a discontinuous circumferential pattern.

(9) FIG. 9 shows hybridization data derived from the synthesis of various oligonucleotide probes on the microarray device shown in FIG. 8. The lower section shows the data from an electrode array device when the discontinuous electrodes system (shown in FIG. 8) was not used. The upper rows are when the discontinuous electrodes (shown in FIG. 8) are used. Both synthesis modalities still used various buffers solutions to prevent cross talk or diffusion from the region of one selected electrode site to a non-selected neighboring site. FIG. 9 shows a significant improvement in the containment of electrochemically generated acids in the upper region (discontinuous circumferential electrodes) to the lower region (no discontinuous electrodes used). The pH bleeding stopped when the discontinuous circumferential electrodes were turned on. Therefore these comparative data show the benefits of using a discontinuous circumferential electrode pattern when there is a close or tight configuration between electrodes because the presence of a buffer (see, Montgomery patents) was not sufficient to contain electrochemically generated acids when there are tight densities between electrodes in an electrode array (see FIG. 8).

(10) FIG. 10 shows an expanded portion of the electrodes show in FIG. 9. The electrode pattern on the left shows the portion of the electrode array device undergoing electrochemical synthesis of oligonucleotides using the inventive discontinuous circumferential method. The photo on the right shows the synthesis of oligonucleotides via the traditional electrochemical synthetic route (e.g., Montgomery U.S. Pat. No. 6,093,302) and relying on buffering methods. The picture on the left is much crisper and clearer, indicating in this direct, intrachip comparison (i.e., different regions of the same electrode array device done at the same time) that the inventive discontinuous electrode system does indeed perform electrochemical synthesis in a superior manner (that is, crisper regions of oligomer synthesis and lack of cross talk when sing very dense electrode arrays. The higher density electrode array in the right shows bleed over in the various steps.

(11) FIG. 11 shows another segment of the electrode array device shown in FIG. 9. The figure on the left is using the inventive discontinuous circumferential electrode synthesis method, while the figure on the right does not.

(12) FIG. 12 shows another inventive embodiment wherein an array of electrodes in row and column format (marked anodes in the figure) has a surrounding line-based grid of counter electrodes marked cathodes in the figure. This embodiment is often referred to as the tic-tac-toe embodiment.

DETAILED DESCRIPTION OF THE INVENTION

(13) The basis of this invention is that a reactive chemical (protons in the case of DNA synthesis) and a reagent, which neutralizes that chemical (in the case of DNA synthesis this is some form of base methoxide or deprotonated hydroquinone) is produced at opposite electrodes to form a reagent wall (in the case of nucleic acid as the oligomer to be synthesized) to prevent crosstalk. By surrounding the functional or active electrode (the one producing the desired reagent) with the counter electrode (the one producing the neutralizing reagent) the inventive microarray device architecture, when used with the inventive process, creates a wall of concentrated highly reactive neutralizing agent. Moreover, unlike Southern WO/020415, the oligomer synthesized by the inventive process on the inventive electrode array device is synthesized within a porous matrix that overlies an active electrode and not in rows on a glass slide overlaying rows of electrodes. The inventive architecture and inventive method for electrochemical synthesis of oligomers enhances containment of the desired electrochemically-generated reactants to the specific area adjacent to the active electrode and the overlaying porous reaction layer but not to neighbor (inactive) electrodes and their overlaying porous reaction layers.

(14) In the case of nucleic acid as the oligomer to be synthesized, the pH of the plume that diffuses away from the active or selected electrode can be measured in terms of the acid concentration and the conjugate base. Thus, the decrease in hydrogen ion concentration is exponential and related to the pK.sub.i of the weak acid. In addition the buffer capacity (related to weak acid concentration) of the media also plays a role in the containment of the protons. However, buffering alone, as described in the Montgomery patents, appear to be insufficient to contain electrochemically-generated reactants when such electrode borders are less than 50 microns apart.

(15) Oligonucleotide Synthesis

(16) One of the steps in the oligonucleotide (DNA) synthesis on any solid surface is the deprotection of the C-5 hydroxyl group of the sugar ring. The protecting group is usually a bulky trityl moiety (dimethyltrityl or DMT) that can be removed in the presence of a trace of acid (Wang et al., Proceedings of the 219.sup.th ACS meeting August 1998, poster #3, page 184; Dill et al., Anal. Chim. Acta 444:69, 2001). Generation of acid at the anode removes the protecting group so that the next activated nucleotide may be attached. A scheme for the removal of the trityl group (DMT) is shown in FIG. 1.

(17) The electrochemical reaction can only be confined and effective locally if enough H.sup.+ is generated at an electrode functioning as an anode and it is localized to the electrode in question. Any bleeding of acid to the neighboring electrode will produce incorrect oligonucleotide sequences at that electrode. The net result would be not only sequence infidelity, but also oligomers that would be composed of various lengths. The reliability of the whole synthesis chemistry would come into question. Therefore, the need to confine the pH to the region surrounding the selected electrode and not neighboring electrodes is so critical.

(18) Peptide Synthesis

(19) Peptide synthesis is carried out in a similar manner as the oligonucleotide synthesis. Often, the blocking group is tBoc, which can be removed in acid media. The schematic for the synthesis procedure is shown in FIG. 2. Again, acid generated at the electrode removes the protecting group. As in the case of oligonucleotide synthesis, it is crucial that the acid produced be contained within the area of the electrode.

(20) Organic ReactionsElectro-Generation of Base

(21) NHS esters may be attached to the porous matrix above the chip surface by the electrogeneration of base. FIG. 3 shows a suggested reactive route, although the exact mechanism is in question. In this case, the NHS ester is introduced (or reacted) with the hydroxyl on the surface of the membrane. However, a CC bond formation has also been reported using slightly modified reaction conditions.

(22) Inventive Method Advantages

(23) The present inventive method confines synthesis conditions (such as pH) to selected areas surrounding a selected electrode on a microarray semiconductor device comprising a plurality of separately addressable electrodes. This method can also be used when producing and neutralizing redox species.

(24) In prior Montgomery method, each individual electrode is turned on, so as to produce acid or base at some specific time. The electrochemically-generated reagent is confined to selected electrode areas based upon the buffer present in solution. The acid (or base) diffuses, until it reaches base (or acid), which neutralizes the electrochemically-generated species. Within a certain region, the acid (or base) produced eliminates all base (or acid) present and the pH of the local hot spot is very low (or very high) but not in the surrounding area. This allows deprotection to take place under appropriate pH conditions. If the electrode is turned on for a longer period of time (or set very high), the acid generated could exceed to base in the surrounding area and this would allow the acid to bleed from one electrode to another (crosstalk). Thus, the control of the acid generation is finely tuned to avoid a situation where the acid generated would override the buffering capacity of the solution in the vicinity of the electrode. This method has limitations because higher density electrode arrays have smaller distances between electrodes and a greater chance for crosstalk or pH bleed.

(25) Therefore, the present invention uses a circumferential electrode as a counter electrode to contain the acid and neutralize the electrochemically generated reagents. A buffer may or may not need to be used.

(26) In a first embodiment of the inventive method, a reactive chemical (protons in the case of DNA synthesis) and a reagent which neutralizes that chemical (in the case of DNA synthesis this is some form of base such as methoxide or deprotonated hydroquinone) is produced at circumferential counter electrodes. By biasing counter electrodes, when a selected electrode is an anode and generates acid (protons) electrochemically, the circumferential electrode is also biased to form a cathode (that is, a counter electrode to the anode) and generate base electrochemically. The base generated will act to neutralize acids diffusing away from the region of the selected (anode) electrode. By surrounding the selected electrode (the one producing the desired reagent) with the counter electrode (the one producing the neutralizing reagent) one creates a wall of concentrated highly reactive neutralizing agent to contain the desired reactant to the specific area of the active electrode and its overlaying porous reaction layer.

(27) For example in FIG. 4 protons are produced at a central anode. These protons are used to produce the desired reaction and their spatial containment is important to specific sequence formation. In the surrounding (ring) cathode, alkoxide or phenoxide bases are formed. Both of these materials diffuse some distance from their point of origin, however if a proton diffuses away from the anode area it crosses in to an area permeated with alkoxide and is rapidly eliminated ensuring containment of the acid to the anode area.

(28) With regard to the make up of the solution, Montgomery discloses that the solution be composed of a buffer or scavenging agent in order to effect pH confinement surrounding an active electrode. However, when the distance between the outer edges of electrodes is 50 microns or less, even strong buffer solutions may not be sufficient to prevent bleeding over of pH changes. In the present inventive method and electrode array design for denser electrode arrays, the solution used during a deblocking step may or may not contain a buffer of varying strength.

(29) Fabrication of Continuous Circumferential Electrodes

(30) Continuous circular circumferential electrode array devices were fabricated using standard CMOS technology. This device utilized alternating array of circular active electrodes and continuous circumferential counter electrodes. In a CMOS process, the semiconductor silicon wafer is fabricated using aluminum wiring and electrodes and then post-processed by sputtering another metal, preferably Pt or Au. The post process masks (Platinum metal and passivation opening) for this device were modified to define a circumferential cathode around each anode (see diagram below).

(31) As H.sup.+ diffuses away from the anode, the proton encounters base produced at the circumferential cathode and is neutralized. The circumferential electrode design is simple to produce under the current fabrication procedures.

(32) FIG. 6 shows a bright field image of a semiconductor electrode array device manufactured with circumferential electrodes (called cathode ring in the image). The functional active electrode is somewhat larger than the pad used for the counter electrode. The downside to this is that an active anode (functional electrode) is produced and the counter electrode pad cannot be used for synthesis.

(33) Another format that is not depicted is to have a standard electrode array device made with circular electrodes arranged in rows and columns (see, for example, FIG. 10). In FIG. 10, there are lines separating each cell of the electrode array. A cell comprises an electrode and the associated circuitry needed to independently electronically access each electrode individually. In a preferred embodiment, the wires separating each cell can be raised to the surface of the electrode array (where the electrodes have surface exposure) and function as an array-wide grid of counter electrode for which ever electrodes are turned on in each electrochemical synthesis step. In prior embodiments of electrode arrays, one must view the electrode array device in three dimensions and not two dimensions (although the Figures are shows with a top-down view of two dimensions). The a grid wire counter electrode embodiment, the wires that provide for electronic communication between cells (that allow for independent electrodes to be separately addressed) are raised to the surface of the device (from a location within the device having at least one layer of passivation coating or insulating material separating the wire from the surface. Only when the wires are exposed to the surface of the electrode array device can the wires act as counter electrodes.

(34) Similarly with regard to continuous circumferential electrodes, the continuous circumferential electrodes must be exposed to the surface of the device (and any solution bathing the device) in order to properly function as a counter electrode during electrochemical synthesis. Similar architectures (at least when looked at in two dimensions instead of three dimensions) have been made with getter electrodes added to an electrode array (see, for example, Montgomery patents). In a getter structure embodiment, by contrast, the circumferential getter electrode is located completely within the structure of the electrode array without surface exposure. The getter electrode can only function as a getter structure to scavenge ions within the device if it cannot act as a counter electrode during electrochemical synthesis. Therefore, although appearing similar from a two dimensional perspective, a getter electrode is substantially different both in location (located within instead of on the surface) and function from a continuous circumferential counter electrode of the present invention.

(35) In FIGS. 9 and 10, an electrode array having over 12,000 electrode cells was subject to electrochemical oligonucleotide synthesis using a discontinuous electrode method according to the present invention (top panels in FIG. 9 and left panel in FIG. 10). Synthesis of the same oligonucleotide probes were made with an anhydrous solution containing base (e.g., acetonitrile/methanol) solution (buffered deblocking solution) in the bottom panels of FIG. 9 and the right panel of FIG. 10. In the electrode array, the distance between the outer edges of neighboring electrodes is 33 microns. The oligonucleotides were synthesized with oligonucleotides of an average length of 35 bases complementary to regions of various human genes. A collection of random 9-mer oligonucleotides (that is oligonucleotides with an average length of 9 bases) were labeled with a fluorescent probe and hybridized to the synthesized electrode array. The hybridization signal was measured with an appropriate fluorescent filter. The results (FIGS. 9 and 10) show confinement of the electrochemical reaction at the anode region to allow the oligo-synthesis to occur.

(36) Discontinuous Circumferential Electrodes as Counter Electrodes

(37) The following is a list of types of anode-cathode arrangements when having a grid of separately addressable electrodes and a solution bathing the electrode array. In a preferred embodiment, there is current flow set up between anodes and cathodes such that there is an acidic environment in the solution created around the anodes and a basic environment in the solution created around the cathodes. Because of this, there are different physical arrangements of anodes and cathodes that can help confine the acidic environment to particular anodes or sets of anodes.

(38) In one embodiment, there is a common cathode or common counter-electrode. Specifically, this arrangement has a large plate of conductive material overlaying the electrode array and already disclosed in WO 02/090963 A1. In a second embodiment, there is a continuous circumferential electrode. In the continuous circumferential electrode, a preferred form is a ring counter electrode wherein a circular active electrode is surrounded by a concentric ring counter electrode. However, other geometries, such as a square active electrode and concentric counter electrode, or a hexagon to hexagon or an octagon to octagon configuration are also within the scope of the present invention embodiment for continuous or discontinuous concentric counter electrodes having insulating material located between the outer edge of an active electrode and the inner edge of the counter electrode.

(39) As a generalization, one might have any arrangement or pattern of anodes and cathodes where the electrodes in the array are used as anodes, cathodes, or are floating (and thus not used as either anodes or cathodes). In the examples provided for oligonucleotide synthesis, an acidic environment is generally needed for deblocking to occur and an acidic environment is made surrounding an anode. Thus, in the example, the anode is the active electrode and a cathode is the counter electrode. However, in other embodiments where a basic environment is needed at an active site for electrochemical synthesis, the currents would be reversed and a cathode would be considered the active electrode and an anode considered the counter electrode. Because these involve using electrodes either as anodes or cathodes, some electrochemical steps that could be one step for the common cathode approach might be multiple electrochemical steps.

(40) One example is that every anode has its nearest-neighbor electrodes turned off (cathodes). In the following diagram (a grid of an electrode array having rows and columns of electrodes), an a denotes an anode, a c denotes a cathode, and a . denotes an electrode that is floating (i.e., used as neither an anode nor a cathode).

(41) ac........

(42) c...c.....

(43) ...cac....

(44) ....c.....

(45) ..........

(46) ....c.....

(47) ...cac....

(48) ....cac...

(49) .....c....

(50) With respect to multiple electrochemical steps, as an example, if one wanted to deprotect material over the following electrodes (denoted by a and which would be anodes in the common cathode approach).

(51) aa........

(52) ..........

(53) ....aa....

(54) ..........

(55) ..........

(56) ..........

(57) ....aa....

(58) ....aa....

(59) ..........

(60) This could be accomplished by two successive electrochemical steps, shown as follows with the first step on the left and the second step on the right.

(61) ac........ cac.......

(62) c...c..... .c...c....

(63) ...cac.... ....cac...

(64) ....cac..... .....cac....

(65) .......... ..........

(66) ....c..... .....c....

(67) ...cac.... ....cac...

(68) ....cac... ...cac....

(69) .....c.... ....c.....

(70) Another example is having every anode surrounded by neighbor electrodes as cathodes, as in the following. By neighboring it is meant the horizontal, vertical and diagonal electrodes.

(71) ..........

(72) ...ccc....

(73) ...cac....

(74) ...ccc..cc

(75) ........ca

(76) Another example is having each anode or group of anodes surrounded by a border of neighbor cathodes, as in the following arrangement.

(77) aac.......

(78) ccccccc...

(79) ...caac...

(80) ...cccc...

(81) .....ccc..

(82) ...cccac..

(83) ...caaaccc

(84) ...caaaaac

(85) ...ccccaaa

(86) Another example is having each anode or group of anodes surrounded by a border of nearest-neighbor cathodes, as in the following.

(87) aac.......

(88) cc..cc....

(89) ...caac...

(90) ....cc....

(91) ......c...

(92) ....ccac..

(93) ...caaacc.

(94) ...caaaaac

(95) ....cccaaa

(96) Another example is having each anode with one closest-available electrode (i.e., a closest electrode that is not already being used as an anode or cathode) used as a cathode, as in the following.

(97) aa........

(98) cc........

(99) ....aa....

(100) ....cc....

(101) ......c...

(102) ....ccacc.

(103) ....aaaccc

(104) ....aaaaac

(105) ....cccaaa

(106) Another example is having any electrode that-is-not an anode being set as a cathode, as in the following.

(107) aacccccccc

(108) cccccccccc

(109) ccccaacccc

(110) cccccccccc

(111) cccccccccc

(112) ccccccaccc

(113) ccccaaaccc

(114) ccccaaaaac

(115) cccccccaaa

(116) In the foregoing diagrams, a software program can configure each step of an electrochemical oligomer synthesis reaction in any of the nearest neighbor, neighbor electrodes, border of neighbor electrodes, border of nearest-neighbor, one closest available electrode, electrode that is not approach, or a combination of various exemplified and unexemplified approaches.

EXAMPLE 1

(117) This example provides a chemical description of an electrochemical buffer formulation using an electrode array having a plurality of discontinuous circumferential electrodes. The central electrode producing protons (i.e., anode) is surrounded by a discontinuous set of electrodes (i.e., acting as cathodes) that help neutralize the protons, which are produced and diffuse away from the anode (active electrode) as shown in FIG. 8. In FIG. 8, the active electrode marked A represents the anode. The surrounding electrodes marked C are the cathodes. The active electrode can be turned on (i.e., generate current flow or voltage) to generate acid, while the surrounding electrodes can also be turned on (opposite voltage or current flow) to produce base. The discontinuous circumferential ring of electrodes (cathodes or counter electrodes in this example) also represents a form of circumferential electrodes, but they are not attached to each other (i.e., are discontinuous). However, each of the ring of discontinuous circumferential electrodes is controlled one at a time or in concert. Thus, the resulting electrochemical synthesis step to form an oligomer synthesis is still run in a solution (preferably a buffered solution), but using this form of circumferential electrodes. This configuration is less costly than a continuous circumferential counter electrode system because it can employ a dense grid of separately controlled electrodes.

(118) It is interesting that not all four surrounding electrodes surrounding an active electrode need to be turned on to confine the acid production. However, we have found this to be optimal in terms of the resulting quality of the electrochemical synthesis.

EXAMPLE 2

(119) This example illustrates different confinement techniques for electrochemical synthesis of oligonucleotides. We synthesized approximately 1,000 sequences of different 35-mer oligonucleotides on two separate CUSTOMARRAY oligonucleotide microarray devices each having over 12,000 electrode sites with a distance between the outer edges of circular electrodes of approximately 33 microns. For the control oligonucleotide microarray, we synthesized oligonucleotide probes electrochemically using the method disclosed in the Montgomery patents having a buffered deblocking solution to try to confine the acid generated at the active (anode) electrodes. This technique further utilized an off chip Pt electrode as a common counter electrode. In addition, four-fold extra systemic base was added to the deblocking solution for the control oligonucleotide array as compared with the inventive oligonucleotide array using a discontinuous circumferential electrode pattern. The pattern of oligonucleotide probe sequences synthesized was the same for both the control and inventive electrode arrays. Selected areas were utilized for comparisons.

(120) For the discontinuous circumferential electrode synthesis procedure, we developed a checkered pattern where a central electrode is the anode and the surrounding electrodes play the role of the cathode. In order to always have 4 surrounding electrodes available for use as cathodes, a checker filter was applied to the electrode array before the anode locations were mapped. This ensured that even if two neighboring electrodes need to be activated during the same cycle of deblocking, it can only do so when neighboring electrodes in use are cathodes. This cyclical procedure for each electrode proceeded until complete deblocking was achieved for one electrode and it then switched functions. This way all the necessary or designated electrodes for a particular cycle had seen deblocking conditions.

(121) The oligonucleotides were synthesized with oligonucleotides of an average length of 35 bases complementary to regions of various human genes. A collection of random 9-mer oligonucleotides (that is oligonucleotides with an average length of 9 bases) were labeled with a fluorescent probe and hybridized to the synthesized electrode array. The hybridization signal was measured with an appropriate fluorescent filter. The results (FIGS. 9 and 10) show confinement of the electrochemical reaction at the anode region to allow the oligo-synthesis to occur. The results and comparisons between the two synthesis procedures are show in FIG. 9. The upper figure shows the fluorescence data for the synthesis of the oligonucleotide probes (average length of 35 nucleotides or 35 mers) using the inventive discontinuous circumferential electrodes. The lower panels show the control microarray device with the same oligonucleotide probes synthesized at the same locations by using the electrochemical synthesis approach disclosed in the Montgomery patents using buffered solutions to enclose a region of pH change and a Pt common counter electrode. The improvements in synthesis quality for electrode arrays with the 33 micron distance between outer edges of electrodes is a striking. In the inventive electrode array the confinement of synthesis is to each electrode area. The blurry mess for the buffered electrochemical synthesis approach described in the Montgomery patents method indicates that some bleeding (crosstalk) has occurred.

(122) FIG. 10 shows an expanded area of identical regions of a given the comparison oligonucleotide arrays. Here the difference in the results from the two methods is stark and better visualized. The use of discontinuous circumferential electrodes does provide much better containment of the acid produced. The dark areas of the array are regions where no synthesis has taken place. This region is not so clean on the synthesis performed on the control (right side). Protons have diffused to these neighboring electrodes and some synthesis has taken place.

(123) FIG. 11, shows the identical expanded areas for regions of the array shown in FIG. 9. Note that the quality of spots (and background) is much better when the discontinuous circumferential electrode method was used.

(124) The foregoing objects have been accomplished in accordance with this invention by providing a method for electrochemical placement of a material at a specific location on a substrate, which comprises the steps of:

(125) providing a substrate having at its surface at least one electrode that is proximate to at least one molecule that is reactive with an electrochemically generated reagent,

(126) applying a potential to the electrode sufficient to generate electrochemical reagents capable of reacting to the at least one molecule proximate to the electrode, and

(127) producing a chemical reaction thereby.

(128) The present invention also includes a method for the electrochemical placement of a material at a specific location on a substrate comprising the steps of:

(129) providing a substrate having at its surface at least one electrode that is proximate to at least one molecule bearing at least one protected chemical functional group,

(130) applying a potential to the electrode sufficient to generate electrochemical reagents capable of deprotecting at least one of the protected chemical functional groups of the molecule, and

(131) bonding the deprotected chemical functional group with a monomer or a pre-formed molecule.

(132) The present invention also includes a method for electrochemical synthesis of an array of separately formed polymers on a substrate, which comprises the steps of:

(133) placing a buffering or scavenging solution in contact with an array of electrodes that is proximate to a substrate surface, said surface being proximate to one or more molecules bearing at least one protected chemical functional group attached thereto,

(134) selectively deprotecting at least one protected chemical functional group on at least one of the molecules;

(135) bonding a first monomer having at least one protected chemical functional group to one or more deprotected chemical functional groups of the molecule;

(136) selectively deprotecting a chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group;

(137) bonding a second monomer having at least one protected chemical functional group to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule; and

(138) repeating the selective deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule and the subsequent bonding of an additional monomer to the deprotected chemical functional group until at least two separate polymers of desired length are formed on the substrate surface.

(139) Another embodiment of the present invention also includes a method for electrochemical synthesis of an array of separately formed oligonucleotides on a substrate, which comprises the steps of:

(140) placing a buffering or scavenging solution in contact with an array of electrodes that is proximate to a substrate surface, said surface being proximate to one or more molecules bearing at least one protected chemical functional group attached thereto,

(141) selectively deprotecting at least one protected chemical functional group on at least one of the molecules;

(142) bonding a first nucleotide having at least one protected chemical functional group to one or more deprotected chemical functional groups of the molecule;

(143) selectively deprotecting a chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group;

(144) bonding a second nucleotide having at least one protected chemical functional group to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule; and

(145) repeating the selective deprotection of a chemical functional group on a bonded protected nucleotide or a bonded protected molecule and the subsequent bonding of an additional nucleotide to the deprotected chemical functional group until at least two separate oligonucleotides of desired length are formed on the substrate surface.

(146) The present invention provides methods for the preparation and use of a substrate having one or a plurality of chemical species in selected regions. The present invention is described herein primarily with regard to the preparation of molecules containing sequences of amino acids, but could be readily applied to the preparation of other polymers, as well as to the preparation of sequences of nucleic acids. Such polymers include, for example, both linear and cyclic polymers of nucleic acids, polysaccharides, phospholipids, and peptides having either alpha-, beta-, or omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or other polymers which will be apparent upon review of this disclosure. In a preferred embodiment, the invention herein is used in the synthesis of peptides. In another preferred embodiment, the present invention is used for the synthesis of oligonucleotides and/or DNA.

(147) The present invention is directed to placing molecules, selected generally from monomers, linker molecules and pre-formed molecules, including, in particular, nucleic acids, at a specific location on a substrate. The present invention is more particularly directed to the synthesis of polymers at a specific location on a substrate, and in particular polypeptides, by means of a solid phase polymerization technique, which generally involves the electrochemical removal of a protecting group from a molecule provided on a substrate that is proximate at least one electrode. The present invention is also particularly directed to the synthesis of oligonucleotides and/or DNA at selected locations on a substrate, by means of the disclosed solid phase polymerization technique.

(148) Electrochemical reagents capable of electrochemically removing protecting groups from chemical functional groups on the molecule are generated at selected electrodes by applying a sufficient electrical potential to the selected electrodes. Removal of a protecting group, or deprotection, in accordance with the invention, occurs at selected molecules when a chemical reagent generated by the electrode acts to deprotect or remove, for example, an acid or base labile protecting group from the selected molecules.

(149) In one embodiment of the present invention, a terminal end of a monomer nucleotide, or linker molecule (i.e., a molecule which links, for example, a monomer or nucleotide to a substrate) is provided with at least one reactive functional group, which is protected with a protecting group removable by an electrochemically generated reagent. The protecting group(s) is exposed to reagents electrochemically generated at the electrode and removed from the monomer, nucleotide or linker molecule in a first selected region to expose a reactive functional group. The substrate is then contacted with a first monomer or pre-formed molecule, which bonds with the exposed functional group(s). This first monomer or pre-formed molecule may also bear at least one protected chemical functional group removable by an electrochemically generated reagent.

(150) The monomers or pre-formed molecules can then be deprotected in the same manner to yield a second set of reactive chemical functional groups. A second monomer or pre-formed molecule, which may also bear at least one protecting group removable by an electrochemically generated reagent, is subsequently brought into contact with the substrate to bond with the second set of exposed functional groups. Any unreacted functional groups can optionally be capped at any point during the synthesis process. The deprotection and bonding steps can be repeated sequentially at this site on the substrate until polymers or oligonucleotides of a desired sequence and length are obtained.

(151) In another embodiment of the present invention, the substrate having one or more molecules bearing at least one protected chemical functional group bonded thereto is proximate an array of electrodes, which array is in contact with a buffering or scavenging solution. Following application of an electric potential to selected electrodes in the array sufficient to generate electrochemical reagents capable of deprotecting the protected chemical functional groups, molecules proximate the selected electrodes are deprotected to expose reactive functional groups, thereby preparing them for bonding. A monomer solution or a solution of pre-formed molecules, such as proteins, nucleic acids, polysaccharides, and porphyrins, is then contacted with the substrate surface and the monomers or pre-formed molecules bond with the deprotected chemical functional groups.

(152) Another sufficient potential is subsequently applied to select electrodes in the array to deprotect at least one chemical functional group on the bonded molecule or another of the molecules bearing at least one protected chemical functional group. A second monomer or pre-formed molecule having at least one protected chemical functional group is subsequently bonded to a deprotected chemical functional group of the bonded molecule or the other deprotected molecule. The selective deprotection and bonding steps can be repeated sequentially until polymers or oligonucleotides of a desired sequence and length are obtained. The selective deprotection step is repeated by applying another potential sufficient to effect deprotection of a chemical functional group on a bonded protected monomer or a bonded protected molecule. The subsequent bonding of an additional monomer or pre-formed molecule to the deprotected chemical functional group(s) until at least two separate polymers or oligonucleotides of desired length are formed on the substrate.

(153) Preferred embodiments of the present invention use a buffering or scavenging solution in contact with each electrode, which is buffered towards the electrochemically generated reagents, in particular, towards protons and/or hydroxyl ions, and that actively prevents chemical cross-talk caused by diffusion of the electrochemically generated ions from one electrode to another electrode in an array. For example, when an electrode exposed to an aqueous or partially aqueous media is biased to a sufficiently positive (or negative) potential, protons (or hydroxyl ions) are produced as products of water hydrolysis. Protons, for example, are useful for removing electrochemical protecting groups from several molecules useful in combinatorial synthesis, for example, peptides, nucleic acids, and polysaccharides.

(154) In order to produce separate and pure polymers, it is desirable to keep these protons (or hydroxyl ions) confined to the area immediately proximate the selected electrode(s) in order to minimize, and, if possible to eliminate, chemical cross-talk between nearby electrodes in an array.