DNA-Modified Electrodes Fabricated Using Copper-Free Click Chemistry for Enhanced Protein Detection

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development

Electrochemical Patterning and Detection of DNA Arrays on a Two-Electrode Platform

We report a novel method of DNA array formation that is electrochemically formed and addressed with a two-electrode platform. Electrochemical activation of a copper catalyst, patterned with one electrode, enables precise placement of multiple sequences of DNA onto a second electrode surface. The two-electrode patterning and detection platform allows for both spatial resolution of the patterned DNA array and optimization of detection through DNA-mediated charge transport with electrocatalysis. This two-electrode platform has been used to form arrays that enable differentiation between well-matched and mismatched sequences, the detection of TATA-binding protein, and sequence-selective DNA hybridization

Electron Flow through Nitrotyrosinate in Pseudomonas aeruginosa Azurin

We have designed ruthenium-modified Pseudomonas aeruginosa azurins that incorporate 3-nitrotyrosine (NO₂YOH) between Ru­(2,2′-bipyridine)₂(imidazole)­(histidine) and Cu redox centers in electron transfer (ET) pathways. We investigated the structures and reactivities of three different systems: RuH107NO₂YOH109, RuH124NO₂YOH122, and RuH126NO₂YOH122. RuH107NO₂YOH109, unlabeled H124NO₂YOH122, and unlabeled H126NO₂YOH122 were structurally characterized. The p<i>K</i>_a’s of NO₂YOH at positions 122 and 109 are 7.2 and 6.0, respectively. Reduction potentials of 3-nitrotyrosinate (NO₂YO^–)-modified azurins were estimated from cyclic and differential pulse voltammetry data: oxidation of NO₂YO^–122 occurs near 1.1 versus NHE; oxidation of NO₂YO^–109 is near 1.2 V. Our analysis of transient optical spectroscopic experiments indicates that hopping via NO₂YO^– enhances Cu^I oxidation rates over single-step ET by factors of 32 (RuH107NO₂YO^–109), 46 (RuH126NO₂YO^–122), and 13 (RuH124NO₂YO^–122)

Reduced and Superreduced Diplatinum Complexes

A d⁸–d⁸ complex [Pt₂(μ-P₂O₅(BF₂)₄]^4– (abbreviated Pt­(pop-BF₂)^4–) undergoes two 1e^– reductions at <i>E</i>_1/2 = −1.68 and <i>E</i>_p = −2.46 V (vs Fc⁺/Fc) producing reduced Pt­(pop-BF₂)^5– and superreduced Pt­(pop-BF₂)^6– species, respectively. The EPR spectrum of Pt­(pop-BF₂)^5– and UV–vis spectra of both the reduced and the superreduced complexes, together with TD-DFT calculations, reveal successive filling of the 6pσ orbital accompanied by gradual strengthening of Pt–Pt bonding interactions and, because of 6pσ delocalization, of Pt–P bonds in the course of the two reductions. Mayer–Millikan Pt–Pt bond orders of 0.173, 0.268, and 0.340 were calculated for the parent, reduced, and superreduced complexes, respectively. The second (5–/6−) reduction is accompanied by a structural distortion that is experimentally manifested by electrochemical irreversibility. Both reduction steps proceed without changing either d⁸ Pt electronic configuration, making the superreduced Pt­(pop-BF₂)^6– a very rare 6p² σ-bonded binuclear complex. However, the Pt–Pt σ bonding interaction is limited by the relatively long bridging-ligand-imposed Pt–Pt distance accompanied by repulsive electronic congestion. Pt­(pop-BF₂)^4– is predicted to be a very strong photooxidant (potentials of +1.57 and +0.86 V are estimated for the singlet and triplet dσ*pσ excited states, respectively)