Electrochemical substrate patterning and analyte detection on a two-electrode platform

1. A two-electrode detection system comprising more than one type of target substrate selected from the group consisting of a nucleic acid, a protein, and/or a small molecule, the detection system further comprising:

(1) a substrate surface comprising a single working substrate electrode, wherein the single working substrate electrode comprises gold or carbon;

(2) a substrate surface linker comprising mixed monolayers self-assembled onto the working substrate electrodes, such that the substrate surface linker comprises;

(a) a first moiety attached to a plurality of positions on the substrate surface, wherein the first moiety comprises a thiol or diazonium salt that attaches to the substrate surface; and

(b) more than one second attachment moiety and at least one second passivating moiety, the more than one second attachment moiety being reactive and capable of attaching to a single type of target substrate of the more than one type of target substrate and the at least one passivating moiety being unreactive;

(i) wherein the more than one second attachment moiety of the substrate surface linker comprises an azide or an alkyne,

(ii) wherein each of the more than one type of target substrate comprises a target linker moiety, the target linker moiety of each of the more than one type of target substrate comprises an azide or an alkyne that covalently binds to a corresponding azide or alkyne of the more than one second attachment moiety, and

(iii) wherein the at least one second passivating moiety comprises a passivating compound;

(3) an inactive copper catalyst precursor;

(4) a patterning electrode pad in an interdigitated pattern of lines comprising more than one working probe electrodes;

(i) wherein the more than one working probe electrodes are individually addressable and interspersed with at least one reference electrodes,

(ii) wherein each of the one or more working probe electrodes corresponds to a different one of the more than one type of target substrate, and

(iii) wherein each of the more than one working probe electrodes is positioned facing a different selected position of the plurality of positions on the substrate surface of the working substrate electrode, such that each of the at least more than one working probe electrodes is capable of applying a negative potential at its corresponding selected position on the substrate surface to activate the inactive copper catalyst precursor by electrochemical reduction of the inactive copper catalyst precursor at specific working probe electrodes on the patterning pad; and

(5) a plurality of more than one type of target substrate spatially patterned and isolated by covalent bond to selected positions of the second attachment moiety of the substrate surface via a catalyst azide/alkyne coupling.

2. The detection system of claim 1 , wherein:

the inactive catalyst precursor comprises copper (II).

3. The detection system of claim 1 , wherein the more than one second attachment moiety of the substrate surface linker and/or the linker moiety of at least one of the more than one type of target substrate is selected the group consisting of a 6-carbon azide, a 9-carbon azide, and a 12-carbon azide.

4. The detection system of claim 2 , wherein the inactive catalyst precursor comprises copper(II)(bathophen) 2 and/or copper(II)(phendione) 2 .

5. The detection system of claim 1 , wherein the at least one second passivating moiety is selected from the group consisting of alkanes, alcohols, carboxylic acids, phosphates, and combinations thereof.

6. The detection system of claim 1 , wherein the passivating compound is selected from the group consisting of mercaptoethanol, mercaptohexanol, mercaptoundecanol, mercaptohexane, mercaptohexanoic acid, mercaptoundecanoic acid, mercaptoundecylphosphoric acid, and combinations thereof.

7. The detection system of claim 1 , wherein the amount of the more than one second attachment moiety of the substrate surface linker relative to the amount of the at least one second passivating moiety is 1% to 20%.

8. The detection system of claim 1 , wherein at least one of the more than one type of target substrate comprises DNA and reactants that are selected to undergo a redox reaction, wherein the reactants comprise methylene blue and ferricyanide, such that the redox reaction comprises reduction of the ferricyanide.

9. The detection system of claim 1 , The detection system of claim 1 , wherein at least one of the more than one type of target substrate is hemoglobin, and wherein hemoglobin is selected to undergo a redox reaction, the redox reaction being a dioxygen reaction.

10. The detection system of claim 1 , further comprising an analyte sample.

11. The detection system of claim 10 , wherein the analyte sample is selected from the group consisting of nucleic acids, proteins, small molecules, cell suspensions, and cell lysates.

12. The detection system of claim 1 , wherein the substrate surface comprises a flat surface or a hollow cylinder.

13. The detection system of claim 1 , wherein the substrate surface is a hollow cylinder and each of the more than one working probe electrodes of the patterning electrode pad has a rod shape.