Polynucleotide synthesis performed with a template independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.

The invention is directed to methods for massively parallel template-free enzymatic synthesis of a plurality of different polynucleotides of predetermined sequences. In one aspect, methods of the invention employ large scale arrays of reaction sites each associated with at least one working electrode for controlling deprotection and deblocking steps at predetermined user selected sites. In another aspect, the invention provides template-free enzymatic synthesis with proofreading, wherein completed polynucleotides at predetermined reaction sites are sequenced using a sequencing by synthesis technique, particularly employing electrochemically labile blocking groups.

According to an aspect of the present inventive concept there is provided a molecular synthesis array (100, 100) comprising: a substrate (208, 208); an insulating layer (202, 202) arranged on the substrate (208, 208); a plurality of lower electrode lines (104) extending in parallel along a column direction of the 5 molecular synthesis array (100, 100), and a plurality of upper electrode lines (102) extending in parallel along a row direction of the molecular synthesis array (100, 100), wherein the upper electrode lines (102) are vertically separated from the lower electrode lines (210, 210) and extend across the lower electrode lines (104), and wherein the lower and upper electrode lines (210, 210, 204, 0 204) are embedded in the insulating layer (202, 202); and a plurality of synthesis wells (106), wherein each well (200, 200) is formed at a crossing between a lower electrode line (210, 210) and an upper electrode line (204, 204) and extends from an upper surface (226) of the insulating layer (202, 202) to the lower electrode line (210, 210), through the insulating layer (202, 202) 5 and through the upper electrode line (204, 204), and exposes an electrode surface portion (222, 222) of the upper electrode line (204, 204) and an electrode surface portion (216, 216) of the lower electrode line (210, 210).

An apparatus for synthesizing nucleic acids and a method for synthesizing nucleic acids using the same. The apparatus includes a first substrate including a surface on which the nucleic acids are synthesized, a second substrate including a deprotection solution, a transporter configured to move the first substrate or the second substrate, and a transporter controller in operable communication with the transporter.

Polynucleotide synthesis performed with a template independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.

Disclosed herein include methods, devices, kits, and systems for nucleic acid sequencing, for example, to determine cell-cell interaction using a dielectrophoresis microfluidic device.

According to an aspect of the present inventive concept there is provided a molecular synthesis array comprising: a substrate; an insulating layer (202) arranged on the substrate; a plurality of column lines (102) extending in parallel along a column direction of the molecular synthesis array (100), and a plurality of row lines (104) extending in parallel along a row direction of the molecular synthesis array (100), wherein the column lines (102) are vertically separated from the row lines (104) and extend transverse to the row lines (104); a plurality of synthesis cells (105), wherein each cell (200) is coupled to a respective pair of a column line and a row line and comprises: a lower electrode (226) and an upper electrode (206) vertically separated from each other and embedded in the insulating layer (202), a synthesis well (223) extending from an upper surface (225) of the insulating layer (202) to the lower electrode (226), through the insulating layer (202) and through the upper electrode (206), wherein the well (223) exposes a surface portion (214) of the upper electrode (206) and a surface portion (220) of the lower electrode (226), and a select transistor (106) having a first terminal (114a), a second terminal (114b) and a gate terminal (114c), the first and second terminals (114a, 114b) forming respective source/drain terminals of the select transistor (106), wherein the gate terminal (114c) is coupled to the row line, the first terminal (114a) is coupled to the column line, the second terminal (114b) is coupled to the lower electrode (226), and the upper electrode (206) is coupled to a reference voltage, or wherein the gate terminal (114c) is coupled to the row line, the first terminal (114a) is coupled to the column line, the second terminal (114b) is coupled to the upper electrode (206), and the lower electrode (226) is coupled to a reference voltage.

Provided herein are compositions, devices, systems and methods for electrochemical synthesis. Further provided are devices comprising addressable electrodes controlling polynucleotide synthesis (deprotection, extension, or cleavage, etc.) The compositions, devices, systems and methods described herein provide improved synthesis, storage, density, and retrieval of biomolecule-based information.

The present invention relates to an electrode array comprising two-electrode assemblies and a corresponding device for use in performing electrochemical synthesis. The invention further relates to use of the device in the high-throughput electrochemical synthesis of chemical compounds, particularly for the purpose of chemical library synthesis and chemical reaction discovery.