Ferrocene

Electrochemical measurements were performed on a CHI660C workstation (CH Instruments) with a three electrode cell comprised of the modified Au working electrode, a platinum wire counter electrode, and an Ag/AgCl/3 mol L⁻¹ NaCl reference electrode (Bioanalytical Systems; 0.209 V vs NHE at 25 °C). All potentials are reported relative to this reference. A glass sleeve salt bridge was used to guard against leakage of NaCl from the reference electrode's reservoir into the electrolyte. The electrolyte, which also served as the hybridization buffer, was 0.2 mol L⁻¹ pH 7.0 sodium phosphate buffer. A fixed target concentration of 25 nmol L⁻¹ and probe coverages of about 5 × 10¹² probes cm⁻² were used. When data were not being collected the electrochemical cell was kept off.
Cyclic voltammetry (CV) measurements to determine the instantaneous coverage of ferrocene-labeled strands used a scan rate of 20 V s⁻¹ from 0 V to 0.6 V or to 0.65 V, requiring approximately 0.07 s per cycle. Probe and target surface coverages, S_P and S_T, were calculated from the charge Q associated with oxidation of their ferrocene tags: $$S_{\mathrm{P}}=Q_{\text{FC}1}/(e{A}_{\mathrm{g}}r) S_{\mathrm{T}}=Q_{\mathrm{F}2}(e{A}_{\mathrm{g}}r)$$ where e = 1.60 × 10⁻¹⁹ C is the elementary charge, A_g is the geometric area occupied by the probe layer, and r is the measured roughness factor. Q_FC1 and Q_F2 are total charges from the oxidation FC1 → FC1⁺ + e⁻ and F2 → F2⁺ + e⁻, respectively, corresponding to integration of the blue and green areas in Figure 1 after converting the potential axis to time. Each probe and target possesses one ferrocene tag. The "T" peak near 0.25 V represents increased current due to oxidation of F2, and confirms presence of surface-bound target molecules. The probe FC1 signal, labeled "P", is observed near 0.45 V. On the reverse scan the tags are reset back to the neutral ferrocene state. The figure also shows fits to the data from which Q_FC1 and Q_F2 were determined. Fits were calculated by an automated computer routine described in the Supporting Information.
In AC impedance (ACI) measurements, (1) a steady bias, V_DC, is imposed to set up the surface environment (e.g. distribution of mobile ions) and, (2) the charge-flow (current) response of this environment to perturbations in potential is sampled using a weak sinusoidal read-out function added to V_DC. Under the experimental conditions used, the response consisted only of charging currents, with the electrochemical cell behaving as a series combination of a resistance, R, representing the electrolyte, and a differential capacitance per area, C_d, representing the probe-modified working electrode. C_d characterizes the surface organization of the probe layer and, for a series RC arrangement, is calculated from the measured out-of-phase impedance Z" using |Z"| = 1/(2πfA_grC_d). f is the read-out frequency. Z" is related to experimental quantities via Z" = − V_acI_op/(I_ip² + I_op²) with V_ac the magnitude of the imposed read-out function, and I_ip and I_op the magnitudes of the measured in-phase and out-of-phase current components, respectively. A useful interpretation of C_d is as a metric of the near-surface screening of electric fields: more effective screening correlates with higher capacitance because greater charge dσ₀ must be placed on the electrode to achieve a potential increment dV (see equation 2 below). Screening can be provided by polarization of the surface environment, as governed by the local dielectric properties, and/or by redistribution of mobile ionic charge.
An ACI measurement consisted of stepping the surface bias V_DC from 0.25 V to - 0.2 V in 0.025 V steps, and back, with C_d determined at each step. A full C_d-loop took 1 min, and was performed once every 5 min during the course of hybridization. A read-out frequency f = 5435 Hz and ac potential magnitude of 5 mV rms were used. This frequency corresponded to a phase angle of 45 ° to 50 °, and was sufficiently low to avoid secondary capacitive charging observed in the presence of the salt bridge at high frequencies, yet high enough to minimize contributions from spurious interfacial charge transfer that become more prominent at low frequencies.

All compounds were obtained from SigmaAldrich and used as received unless otherwise indicated. TEMPO was purified by sublimation under vacuum prior to use. Solvents were purchased from Fisher and deuterated solvents were purchased from Cambridge Isotope Labs. Methylene chloride was dried by running through a column of activated alumina, degassed, and stored over 4 Å molecular sieves before use. TEMPOH was synthesized following the literature reported anhydrous synthesis.^{45 (link)} Methanol-D (CH₃OD) was dried over 4 Å molecular sieves and stored under inert atmosphere. Anhydrous TEMPOD was synthesized by stirring TEMPOH with CH₃OD for 1h then removing volatiles under vacuum. Pyridine bases were distilled and dried over 4 Å molecular sieves. 1,2,3,4,5-pentamethylferrocene⁴⁶ was synthesized from iron(II)bis(acetylacetonate)⁴⁷ according to literature procedures. Ferrocene and 1,2,3,4,5-pentamethylferrocne were oxidized with AgPF₆ according to literature procedures.^{48 (link) 1}H NMR spectra were recorded at ambient temperature on either an A400 or A500 DD2 Agilent spectrometer operating at 400 MHz or 500 MHz, respectively. Spectra were referenced to the residual solvent signal (5.32 ppm) for CHDCl₂. UV-vis spectra were recorded on an Agilent Cary 60 spectrophotometer. IR spectra were recorded inside a Nitrogen filled glovebox with a Bruker Alpha FT-IR spectrometer using a CsF liquid cell with a 1 mm pathlength. Kinetic measurements were recorded on an OLIS-RSM 1000 single mixing stopped flow spectrophotometer in anaerobic methylene chloride solvent. Full details of these and other measurements are given in the Supplementary Information.