Electronic Supplementary Material

Surface molecularly imprinted polydopamine films for recognition of immunoglobulin G

Aleksei Tretjakov, Vitali Syritski, Jekaterina Reut, Roman Boroznjak, Olga Volobujeva, and Andres Öpik

Department of Materials Science, Tallinn University of Technology, Ehitajate tee 5, 19086 Tallinn ESTONIA,

1. Experimental part

1.1 Cyclic voltammetry

CV measurements were performed in the solution containing 4 mM of K3[Fe(CN)6]/K4[Fe(CN)6] redox probe in 1M KCl. The potential was cycled between 0 and 0.5 V at a scan rate of 50 mV•s-1. For each electrode modification stage 3 potential scans were applied.

1.2 EIS

EIS measurements were performed in the solution containing 4 mM of K3[Fe(CN)6]/K4[Fe(CN)6] redox couple in 0.1 M KCl as the supporting electrolyte at the formal potential of the system (E = 270 mV) and an alternating potential with amplitude of 10 mV at the frequency range from 0.1 Hz to 100 kHz. The experiments were repeated three times. The impedance spectra were fitted to an equivalent electrical circuit by using Gamry Echem Analyst software from Gamry Instruments, Inc.

2. Results and discussion

2.1 IgG immobilization on the gold electrode surface. Electrochemical impedance spectroscopy studies

The EIS spectra represented as experimental and simulated complex plane plots (Nyquist plots) for the bare and the modified Au electrodes are shown in Fig. S1. The plots are characterized by the depressed semicircle located at the high frequencies corresponding to the electron-transfer kinetics of the redox probe at the electrode interface, and by the linear region at the low frequencies where the impedance response is dominated by the mass transfer of the redox species to and from the electrode surface.

The equivalent circuits used for fitting the impedance data are presented in Fig. S2. The equivalent circuit in Fig. S2a consisting of a solution resistance (RU), a charge transfer resistance (RP), Warburg impedance resulting from the diffusion of ions from the bulk electrolyte to the electrode interface (Wd) and constant phase element (CPE) was used to describe the bare and 4-ATP modified Au electrodes. The use of CPE in the equivalent circuits instead of ideal capacitors was determined by the appearance of the depressed semicircles in the Nyquist plots. CPE reflects roughness and inhomogeneity of the surface, and was shown to be important for the modeling of primary protein layers on an electrode surface [1]. To fit the EIS data of the 4-ATP/DTSSP and 4-ATP/DTSSP/IgG modified electrodes the additional components were added to the circuit in order to account for the blocking effect due to the insulating multilayered structure formation on the electrode surface (Fig. S2b): Rp1 and corresponding CPE1 describe the resistance and capacitance associated with the 4-ATP monolayer on gold, while Rp2 and corresponding CPE2 describe the resistance and capacitance associated with the 4-ATP/DTSSP or DTSSP/IgG layers. The analogous circuit has been employed elsewhere for the similar interfaces [2]. The parameters obtained from EIS data fitting are presented in Table 1.

The modification of the Au surface by 4-ATP is clearly seen on the EIS spectra (Fig. S1). The semicircle measured at the bare Au electrode is poorly defined due to the fast electrode reaction. With the 4-ATP monolayer coated electrode Rp is clearly greater than Rp of the unmodified Au electrode due to inhibition of the electron transfer rate. After subsequent electrode modification by DTSSP Rp continues to increase indicating the more efficient electrode blocking. A significant increase of Rp after incubation of the 4-ATP/DTSSP modified electrode in IgG containing solution indicates that the electron transfer process is considerably hindered in this case, suggesting successful antibody immobilization.

Fig. S1. Impedance spectra of the bare Au (a), 4-ATP (b), 4-ATP/DTSSP (c), and 4ATP/DTSSP/IgG (d) modified electrodes. The data were recorded in 4mM Fe(CN)63−/Fe(CN)64− containing 1 M KCl.

Fig. S2. The equivalent circuit used to fit the impedance spectra for the bare and 4-ATP modified Au electrodes (a), 4-ATP/DTSSP and 4-ATP/DTSSP/IgG modified Au electrodes (b).

Table S1. Model parameters extracted from EIS data (Fig. S1) by fitting to the equivalent circuits in Fig. S2. α is a CPE exponent reflecting the extent of system inhomogeneity.

Au / Au/4-ATP / Au/4-ATP/DTSSP / Au/4-ATP/DTSSP/IgG
Applied model / CPE with diffusion (Fig. S2a) / CPE with diffusion (Fig. S2a) / two CPE elements with diffusion (Fig. S2b) / two CPE elements with diffusion (Fig. S2b)
Ru (Ohm) / 16.25 / 21.81 / 23.42 / 21.25
CPE1 (S·sα) / 2.58 ×10-5 / 2.25 ×10-6 / 8.14 ×10-7 / 8.44 ×10-7
α1 / 94.41 ×10-2 / 82.06 ×10-2 / 85.37 ×10-2 / 84.81 ×10-2
Rp1 (Ohm) / 3.44 / 94.32 / 15.04 ×102 / 50.79 ×10 2
CPE2 (S·sα) / - / - / 77.73 ×10-7 / 55.34 ×10-7
α2 / - / - / 68.84 ×10-2 / 66.71 ×10-2
Rp2 (Ohm) / - / - / 13.12 ×103 / 64.13 ×10 3
Wd (S·s1/2) / 80.90 ×10-4 / 59.85 ×10-5 / 51.82 ×10-5 / 89.92 ×10-5
Goodness of Fit / 51.96 ×10-6 / 57.29 ×10-5 / 16.03 ×10-5 / 30.35 ×10-6

2.2 Effect of mercaptoethanolic treatment on PDA thin films stability

In the presented strategy the procedure of template protein removal from the PDA matrix includes mercaptoethanolic treatment at approx. 100 ºC to disrupt the disulfide bonds of the DTSSP linker. Thus, there is a potential risk of the polymer degradation under such harsh washing conditions leading to its detachment from the electrode surface. In order to evaluate the influence of the mercaptoethanolic treatment on PDA stability the cyclic voltammograms of the PDA coated Au electrode before and after the treatment were recorded and compared with the cyclic volatmmogram of the bare Au electrode. As it clearly seen redox peaks of Fe(CN)63−/Fe(CN)64− pair completely disappear when the bare Au electrode is coated by the PDA film (Fig. S3a and b) yielding a featureless cyclic voltammogram, which remains very similar to that after the mercaptoethanolic treatment (Fig. S3b and c). This supports that the PDA film is stable enough to withstand this treatment and keep its original structure, what is additionally proven by the SEM micrographs (Fig. 6b and c)

Fig.S3. Cyclic voltammograms of the bare Au electrode (a), PDA coated Au electrode before (b) and after (c) the mercaptoethanolic treatment. The data were recorded in 1M KCl containing 4mM Fe(CN)63−/Fe(CN)64− at scan rate 50 mV•s-1.

2.3 SEM microscopy

The SEM micrographs in Fig. S4 provide additional evidence for the PDA film deposition on the 4-ATP/DTSSP/IgG modified electrode, where this polymer with morphology of uniformly sized agglomerates of fine particles can be seen.

Fig. S4. SEM images of the bare Au electrode surface of the QCM sensor (a), the PDA film electrodeposited on the Au/4-ATP/DTSSP/IgG modified Au electrode (b) and the PDA film after mercaptoethanolic treatment (c).

2.4 Evaluation of IgG-SIPs films rebinding capability

Fig. S5. The log plot of the IgG adsorption isotherms for the IgG-SIP (black circle) and NIP (hollow circle) of the PDA various thicknesses. Curves represent fits of the data to Freundlich-Langmuir (FL) isotherm model.

Fig. S6. The IgA and IgG adsorption isotherms on the IgA-SIP, IgG-SIP and corresponding NIP films. The curves represent fits of the data to Freundlich-Langmuir (FL) isotherm model.

References

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