A critical view on electrochemical impedance spectroscopy (EIS) using
A critical view on electrochemical impedance spectroscopy (EIS) using the ferri/ferrocyanide redox couple at gold electrodes Stephan Vogta†, Qiang Sua†, Cristina Gutiérrez-Sáncheza, and Gilbert Nöll*a a) Nöll Junior Research Group, Organic Chemistry, Chem. Biol. Dept., Faculty IV, Siegen University, Adolf-Reichwein-Str. 2, 57068 Siegen, Germany †
= contributed equally
*E-mail: [email protected]
Supporting Information Materials and Methods Chemicals Flow cells used for EIS combined with SPR and QCM-D measurements Preparation of sensor chips for QCM-D measurements Preparation of the gold substrates for SPR Deprotection of the thiol modified capture DNA Surface modification for SPR and QCM-D measurements DNA hybridization and dehybridization Impedimetric measurements Calculation of the center-to-center distance of hybridized DNA on the sensor chips Preparation of sensors with low DNA surface coverages for SPR measurements Combined EIS and SPR measurements Combined EIS and QCM measurements Parameters obtained by fitting the measurements depicted in Figure 6B References
S-2 S-2 S-3 S-3 S-4 S-4 S-4 S-5 S-5 S-6 S-8 S-11 S-13 S-13
S-1
Chemicals Hellmanex II was bought from Hellma (Müllheim, Germany). Bio-Spin P-6 gel Chromatography columns were supplied by Bio-Rad Laboratories (München, Germany). All buffers and solutions were prepared from water which was purified in two steps using an Elix advantage 3 and a Simplicity system from Merck Millipore (Schwalbach, Germany), resulting in a resistivity of 18.2 M cm-1 at 25 °C. All chemicals were used as received. Experiments were carried out at pH 7.0 in phosphate buffers with different ionic strength. While the concentrations of phosphate varied from 20 mM to 100 mM, the KCl content ranged from 50 mM to 1 M. All buffers comprised a constant MgCl2 concentration (5 mM). Flow cells used for EIS combined with SPR and QCM-D measurements For SPR measurements combined with EIS we used a homemade flow cell. A drawing of this cell is depicted in Figure S-1. A platinum wire coated with Ag and subsequently oxidized to AgCl was used as reference electrode and a platinum sheet served as reference electrode. The diameter of the inner cylindrical compartment is 14 mm, and the height is 2.5 mm. As neither the flow cell nor our laboratories are temperature controlled, there might be a small temperature dependent drift of the SPR-signal with time.
Figure S-1. Homemade flow cell used for combined EIS and SPR experiments.
S-2
For combined EIS and QCM-D experiments a commercially available QEM 401 flow cell (Biolin Scientific, Stockholm, Sweden) was used. It is equipped with a World-Precision Dri-Ref 2SH reference electrode (World Precision Instruments, Sarasota, Florida, USA) and a platinum sheet as counter electrode, respectively. Detailed information about the QEM 401 can be found here: http://www.biolinscientific.com/product/electrochemistry-module/ Preparation of sensor chips for QCM-D measurements Commercially available gold coated quartz crystals with a resonance frequency at 4.95 MHz comprising either a titanium, (QSX 338) or a chromium adhesion layer (QSX 301) underneath the gold surface from Q-Sense (Q-Sense AB, Göteborg, Sweden) were used. Before each experiment, the QCM crystals were exposed to UV-ozonation for 15 min, followed by immersion in a mixture of water, hydrogen peroxide (30%) and ammonia solution (25%) (v/v/v = 5 : 1 : 1) at 70 °C for 5 min (CAUTION: This solution is highly corrosive and should be handled with special care). Subsequently, the sensors were thoroughly washed with water and then dried with argon before they were placed in the ozone chamber for additional 15 minutes. After mounting the cleaned QCM-D slides in the corresponding sensor module, they were allowed to equilibrate in phosphate buffer until a stable signal was reached. After each experiment, the QCM-D chambers were cleaned with 2% Hellmanex® II solution followed by extensive rinsing with pure water. Preparation of the gold substrates for SPR Gold substrates were prepared by vacuum evaporation of gold (about 48 nm layer thickness) onto cleaned glass slides (nBK7 = 1.515 at 633 nm) which were precoated with a titanium layer (about 1.5 nm) to improve adhesion. The gold substrates were freshly cleaned prior to use by immersion in hot piranha solution (a 3:1 mixture of concentrated H2SO4 (95%) and H2O2 (30%), CAUTION: piranha solution reacts violently with most organic S-3
materials and must be handled with care) for 5 min and then rinsed thoroughly with pure water. Deprotection of thiol modified capture DNA To cleave the disulfide bond of the protected capture DNA, a DNA aliquot was dissolved in 90 µL phosphate buffer and incubated overnight at 10 °C and 400 rpm with an appropriate volume of a tris (2-carboxyethyl)-phosphine hydrochloride solution (100 mM in water).1 Thereafter, the mixture was purified using a Bio-Spin® P-6 gel chromatography column (which was pre-equilibrated with phosphate buffer) and the elution was subsequently diluted with phosphate buffer to a final volume of 400 µL. Surface modification for SPR and QCM-D measurements In the first step, 400 µL of the deprotected capture probe DNA (concentrations between 5 µM and 8µM were used in individual experiments) were injected to the flow cell and allowed to adsorb at the gold surface for a period of about 90 minutes. After rinsing with buffer and water, a mixture of mercaptobutanol (MCB) and mercaptopropionic acid (MPA) in water, 1 mM each, was incubated for one hour to remove non-specifically adsorbed DNA strands. After rinsing with buffer solution, the system was allowed to equilibrate overnight. DNA hybridization and dehybridization Hybridization of the complementary DNA sequence was performed in phosphate buffer (pH 7.0) by adding 400 µL of a complementary DNA solution (5 µM). For dehybridization the electrode surface was rinsed with approximately 50 mL of deionized water at the maximum flow rate (3 mL ∙ min-1). For the first dehybridization step on a freshly prepared sensor chip even a larger volume of pure water (100-200 mL) was required for rinsing in order to achieve complete dehybridization.2-4
S-4
Impedimetric Measurements Impedimetric measurements were performed in (oxygen-free) degassed phosphate buffer pH 7.0, containing potassium phosphate and potassium chloride at different concentrations as well as the ferri/ferrocyanide redox couple. The concentration of ferri/ferrocyanide was varied from 1 mM to 10 mM, each. EIS measurements spanned a frequency range between 10 Hz and 100 kHz at a bias potential of +408 mV vs. NHE which was modulated with an amplitude of 5 mV. For stand-alone EIS measurements, a three electrode setup using a disc shaped gold working electrode, an Ag/AgCl/KClsat reference electrode and a platinum wire serving as counter electrode were used. For experiments illustrating the etching effect of ferri/ferrocyanide on bare gold, impedance measurements were performed in the QEM electrochemistry module of the QCM-D on non-modified surfaces with an applied minimum flow rate (approx. 0.1 mL∙min-1) to ensure the constant availability of fresh ferri/ferrocyanide. Calculation of the center-to-center distance of hybridized DNA on the sensor chips For the calculation of the distance between two adjacent DNA strands standing on a surface homogeneous distribution is assumed. The values in brackets correspond to the numbers determined in this study. The surface coverage value given in molecules · cm-2 (11.2·1012 molecules · cm-2) is multiplied by 10,000 to obtain the value for molecules · m-2 (11.2·1016 molecules · m-2). To obtain the statistical space occupied by one DNA strand in m² · molecule-1, this value is inverted (8.93·10-18 m2 · molecule-1). Taking the square root of this number leads to the center-to-center distance of DNA (about 3 nm). To calculate the statistical center-to-center distance of dsDNA, the surface coverage was multiplied by the normalized hybridization efficiency (hybridization efficiency in % divided by 100) and the calculation again was S-5
performed as described above (leading to a center-to-center distance of about 4.7 nm for dsDNA). Preparation of sensor chips with low surface coverage of capture probe for SPR measurements The hybridization efficiency reported in the main file is about 40% for a sensor surface modified with 11.2 ∙ 1012 molecules cm-2 (values determined by SPR). Since it was reported that hybridization efficiencies of 100% may be reached if the surface coverage of the capture probe is significantly lower,5 we also prepared sensor chips with low surface coverage. Therefore a buffer solution containing a rather low concentration of probe DNA (0.5 µM) and MCB/MPA (25 µM each) was incubated for 90 minutes. After rinsing the cell with buffer solution and water, a mixture of MCB and MPA (0.5 mM each) in water was injected and incubated for one hour. In the next step, the system was rinsed with water and buffer. In Figure S-2A a kinetic SPR scan curve collected during three hybridization and two dehybridization steps is shown (dehybridization was achieved by intense rinsing with pure water). In between the individual steps entire angular scan curves were collected, the minima of the corresponding angular scan curves are shown in Figure S-2B. As the concentration of capture probe was rather low, the signal contribution from MCB/MPA could not be neglected anymore when the surface coverage was calculated. Therefore in a control experiment SPR sensor chips were modified with MCB/MPA only, following the same procedure (not shown). To determine the surface coverage of the capture probe DNA, the surface bound mass calculated for the control experiment was subtracted from the surface bound mass calculated for the sensor chips modified with DNA and MCB/MPA. Using this method, for ssDNA a surface bound mass of 27 ng · cm-2 or a surface coverage of 4.19 · 10-12 mol · cm-2 and 2.5 · 1012 molecules · cm-2, respectively, was calculated. After hybridization the surface
S-6
bound mass increased on average by 26 ng · cm-2, which corresponds to 4.2 · 10-12 mol · cm-2 or 2.5 · 1012 molecules · cm-2. This yields a hybridization efficiency close to 100%. Even though at low surface coverage the hybridization efficiency is nearly 100%, the overall increase in reflectivity during hybridization with target is rather low, and more sensitive results will be obtained if the sensor chips are prepared by a procedure resulting in higher surface coverage of the capture probe strands.
A 34 33
complementary DNA (5 µM in buffer) angular stop scan after rinsing rinsing
32 reflectivity (%)
31 30
rinsing with buffer
29 28 27 26 25 24
angular scan on ssDNA angular scan on dsDNA
rinsing with pure water 300 350 400 time (min)
450
S-7
B
bare gold surface ssDNA1 ssDNA2 ssDNA3 dsDNA1 before rinsing dsDNA2 before rinsing dsDNA3 before rinsing dsDNA1 after rinsing dsDNA2 after rinsing dsDNA3 after rinsing
12
reflectivity (%)
10 8 6 4 2 0 67.2
67.5
67.8 68.1 68.4 incident angle ()
68.7
69.0
Figure S-2. (A) SPR kinetic scan and B) SPR angular scan curves of consecutive hybridization/dehybridization cycles on a sensor chip modified with a low surface coverage of capture probe DNA strands. In comparison to Figure 1 in the main manuscript, the angular shift between the bare gold, dehybridization, and hybridization state is very low due to the low coverage of capture probe. Angular scan curves were measured before and after hybridization as well as after a short period of rinsing with buffer to remove unspecifically bound DNA. Measurements were performed in 20 mM phosphate buffer, pH 7.0, also comprising 50 mM KCl and 5 mM MgCl2.
Combined EIS and SPR measurements at a bare gold surface To prove the assumption that free CN- released from the ferri/ferrocyanide redox couple etches the gold surface, we performed combined EIS and SPR measurements over a period of 24 hours using a deoxygenated buffer solution containing 1 mM ferri/ferrocyanide, each, independently at two gold coated SPR slides. For one of these slides, the measured and fitted angular scan curves (including the fit parameters which were calculated by using the software WinSpall) recorded before and after the EIS measurements are shown in Figure S-3.
S-8
A before EIS after EIS
100
reflectivity (%)
80 60 40 20 0 56
58
60 62 64 66 68 incident angle ()
70
72
S-9
Figure S-3. (A) Angular scan curve measurements recorded for an unmodified SPR chip before and after 24 h of EIS measurements using 1 mM ferri-/ferrocyanide, each. Panels (B) and (C) show the overlay of a measured and simulated angular scan curve before (B) and after 24 h of EIS (C), respectively. By fitting the simulation parameters to the measured curves, it was found that the average thickness of the gold layer on the SPR chip decreased by 2.3 nm during the impedance measurements. For fitting, the software WinSpall was used. S-10
Combined QCM-D and EIS measurements In order to study the etching of gold surfaces by ferri/ferrocyanide, we performed EIS measurements on bare gold-coated QCM chips either with chromium or titanium adhesion layer. In Figure 5 in the main manuscript a QCM-D measurement performed on a gold coated sensor chip at continuous flow (0.1 mL·min-1) in oxygen-free buffer containing ferri/ferrocyanide, 5 mM each, is shown at the top. At the bottom a microscopic image of a fresh QCM-D chip and the chip used above after 26 h of combined EIS/QCM measurements using the ferri/ferrocyanide redox couple is shown. In Figure S-4 EIS spectra collected during the first 20 h of the combined EIS/QCM measurements corresponding to Figure 5 in the main manuscript are shown. The EIS spectra were recorded periodically every 30 minutes in a frequency range from 10 mHz to 100 kHz. In Figure S-4 only every second spectrum is depicted. After 20 h of EIS, the RCT did not further decrease but started to increase again (not shown), possibly because at some pinholes the gold layer had been removed completely.
10
-ZIm ()
8 6 4 2 20 h EIS
0 0
5
10
15
20
ZRe ()
Figure S-4. Series of EIS spectra measured on a bare QCM-D chip in 20 mM phosphate buffer, pH 7.0 with 50 mM KCl as well as additional 5 mM MgCl2, and 5 mM ferri/ferrocyanide, each. Spectra were recorded every 30 minutes in a frequency range between 10 mHz and 100 kHz. For clarity only every second spectrum is shown.
S-11
As described in the main manuscript, we optimized the conditions for EIS measurements on modified QCM-D sensor chips in terms of minimized acquisition times, freshly prepared redox probe solutions with low ferri-/ferrocyanide concentrations and shielding these solutions from daylight. We applied these conditions to different QCM-D sensor chips modified with ssDNA and short chain thiols to test whether hybridization and dehybridization can be tracked reliably with EIS and QCM-D. Only one out of four different QCM-D sensor chips showed three repeatable hybridization/dehybridization cycles in EIS measurements. The EIS data recorded on this sensor was fitted to the Randles circuit (depicted Figure S-5) equipped with a constant phase element (CPE) to account for the surface roughness of the sensor. The corresponding fitting results, i.e. the values for RCT as well as RS, CDL, α and W can be found in Table S-1.
Figure S-5. The Randles circuit was used to fit the EIS data. A constant phase element (CPE) was used instead of a capacitor.
S-12
Table S-1. Parameters obtained by fitting the measurements depicted in Figure 6B in the main manuscript to a Randles circuit equipped with a constant phase element (CPE).
W (DW)
ssDNA (1) ssDNA (2) dsDNA (1) dsDNA (2)
1.09E-03 1.45E-03 6.11E-04 6.41E-04
ssDNA (1) ssDNA (2) dsDNA (1) dsDNA (2)
6.44E-03 8.19E-03 3.02E-03 3.64E-03
ssDNA (1) ssDNA (2) dsDNA (1) dsDNA (2)
3.32E-02 4.74E-02 1.80E-02 1.95E-02
RCT ()
CDL (F) First cycle 884.7 1.08E-05 888.9 1.08E-05 1040 1.07E-05 1041 1.07E-05 Second cycle 851.9 1.09E-05 862.1 1.09E-05 1066 1.08E-05 1057 1.08E-05 Third cycle 866.2 1.09E-05 882.1 1.09E-05 1026 1.09E-05 1025 1.09E-05
α
RS ()
0.9273 0.9294 0.938 0.9383
8.950 8.965 9.006 9.010
0.9289 0.9314 0.9412 0.9407
8.974 8.980 9.002 9.014
0.9299 0.932 0.9404 0.9403
9.008 9.009 9.016 9.017
References (1) Nogues, C.; Cohen, S. R.; Daube, S. S.; Naaman, R. Phys. Chem. Chem. Phys. 2004, 6, 4459-4466. (2) Nöll, G.; Su, Q.; Heidel, B.; Yu, Y. Adv. Healthcare Mater. 2014, 3, 42-46. (3) Oh, B.-K.; Nam, J.-M.; Lee, S. W.; Mirkin, C. A. Small 2006, 2, 103-108. (4) Ricci, F.; Zari, N.; Caprio, F.; Recine, S.; Amine, A.; Moscone, D.; Palleschi, G.; Plaxco, K. W. Bioelectrochemistry 2009, 76, 208-213. (5) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168.
S-13
= contributed equally
*E-mail: [email protected]
Supporting Information Materials and Methods Chemicals Flow cells used for EIS combined with SPR and QCM-D measurements Preparation of sensor chips for QCM-D measurements Preparation of the gold substrates for SPR Deprotection of the thiol modified capture DNA Surface modification for SPR and QCM-D measurements DNA hybridization and dehybridization Impedimetric measurements Calculation of the center-to-center distance of hybridized DNA on the sensor chips Preparation of sensors with low DNA surface coverages for SPR measurements Combined EIS and SPR measurements Combined EIS and QCM measurements Parameters obtained by fitting the measurements depicted in Figure 6B References
S-2 S-2 S-3 S-3 S-4 S-4 S-4 S-5 S-5 S-6 S-8 S-11 S-13 S-13
S-1
Chemicals Hellmanex II was bought from Hellma (Müllheim, Germany). Bio-Spin P-6 gel Chromatography columns were supplied by Bio-Rad Laboratories (München, Germany). All buffers and solutions were prepared from water which was purified in two steps using an Elix advantage 3 and a Simplicity system from Merck Millipore (Schwalbach, Germany), resulting in a resistivity of 18.2 M cm-1 at 25 °C. All chemicals were used as received. Experiments were carried out at pH 7.0 in phosphate buffers with different ionic strength. While the concentrations of phosphate varied from 20 mM to 100 mM, the KCl content ranged from 50 mM to 1 M. All buffers comprised a constant MgCl2 concentration (5 mM). Flow cells used for EIS combined with SPR and QCM-D measurements For SPR measurements combined with EIS we used a homemade flow cell. A drawing of this cell is depicted in Figure S-1. A platinum wire coated with Ag and subsequently oxidized to AgCl was used as reference electrode and a platinum sheet served as reference electrode. The diameter of the inner cylindrical compartment is 14 mm, and the height is 2.5 mm. As neither the flow cell nor our laboratories are temperature controlled, there might be a small temperature dependent drift of the SPR-signal with time.
Figure S-1. Homemade flow cell used for combined EIS and SPR experiments.
S-2
For combined EIS and QCM-D experiments a commercially available QEM 401 flow cell (Biolin Scientific, Stockholm, Sweden) was used. It is equipped with a World-Precision Dri-Ref 2SH reference electrode (World Precision Instruments, Sarasota, Florida, USA) and a platinum sheet as counter electrode, respectively. Detailed information about the QEM 401 can be found here: http://www.biolinscientific.com/product/electrochemistry-module/ Preparation of sensor chips for QCM-D measurements Commercially available gold coated quartz crystals with a resonance frequency at 4.95 MHz comprising either a titanium, (QSX 338) or a chromium adhesion layer (QSX 301) underneath the gold surface from Q-Sense (Q-Sense AB, Göteborg, Sweden) were used. Before each experiment, the QCM crystals were exposed to UV-ozonation for 15 min, followed by immersion in a mixture of water, hydrogen peroxide (30%) and ammonia solution (25%) (v/v/v = 5 : 1 : 1) at 70 °C for 5 min (CAUTION: This solution is highly corrosive and should be handled with special care). Subsequently, the sensors were thoroughly washed with water and then dried with argon before they were placed in the ozone chamber for additional 15 minutes. After mounting the cleaned QCM-D slides in the corresponding sensor module, they were allowed to equilibrate in phosphate buffer until a stable signal was reached. After each experiment, the QCM-D chambers were cleaned with 2% Hellmanex® II solution followed by extensive rinsing with pure water. Preparation of the gold substrates for SPR Gold substrates were prepared by vacuum evaporation of gold (about 48 nm layer thickness) onto cleaned glass slides (nBK7 = 1.515 at 633 nm) which were precoated with a titanium layer (about 1.5 nm) to improve adhesion. The gold substrates were freshly cleaned prior to use by immersion in hot piranha solution (a 3:1 mixture of concentrated H2SO4 (95%) and H2O2 (30%), CAUTION: piranha solution reacts violently with most organic S-3
materials and must be handled with care) for 5 min and then rinsed thoroughly with pure water. Deprotection of thiol modified capture DNA To cleave the disulfide bond of the protected capture DNA, a DNA aliquot was dissolved in 90 µL phosphate buffer and incubated overnight at 10 °C and 400 rpm with an appropriate volume of a tris (2-carboxyethyl)-phosphine hydrochloride solution (100 mM in water).1 Thereafter, the mixture was purified using a Bio-Spin® P-6 gel chromatography column (which was pre-equilibrated with phosphate buffer) and the elution was subsequently diluted with phosphate buffer to a final volume of 400 µL. Surface modification for SPR and QCM-D measurements In the first step, 400 µL of the deprotected capture probe DNA (concentrations between 5 µM and 8µM were used in individual experiments) were injected to the flow cell and allowed to adsorb at the gold surface for a period of about 90 minutes. After rinsing with buffer and water, a mixture of mercaptobutanol (MCB) and mercaptopropionic acid (MPA) in water, 1 mM each, was incubated for one hour to remove non-specifically adsorbed DNA strands. After rinsing with buffer solution, the system was allowed to equilibrate overnight. DNA hybridization and dehybridization Hybridization of the complementary DNA sequence was performed in phosphate buffer (pH 7.0) by adding 400 µL of a complementary DNA solution (5 µM). For dehybridization the electrode surface was rinsed with approximately 50 mL of deionized water at the maximum flow rate (3 mL ∙ min-1). For the first dehybridization step on a freshly prepared sensor chip even a larger volume of pure water (100-200 mL) was required for rinsing in order to achieve complete dehybridization.2-4
S-4
Impedimetric Measurements Impedimetric measurements were performed in (oxygen-free) degassed phosphate buffer pH 7.0, containing potassium phosphate and potassium chloride at different concentrations as well as the ferri/ferrocyanide redox couple. The concentration of ferri/ferrocyanide was varied from 1 mM to 10 mM, each. EIS measurements spanned a frequency range between 10 Hz and 100 kHz at a bias potential of +408 mV vs. NHE which was modulated with an amplitude of 5 mV. For stand-alone EIS measurements, a three electrode setup using a disc shaped gold working electrode, an Ag/AgCl/KClsat reference electrode and a platinum wire serving as counter electrode were used. For experiments illustrating the etching effect of ferri/ferrocyanide on bare gold, impedance measurements were performed in the QEM electrochemistry module of the QCM-D on non-modified surfaces with an applied minimum flow rate (approx. 0.1 mL∙min-1) to ensure the constant availability of fresh ferri/ferrocyanide. Calculation of the center-to-center distance of hybridized DNA on the sensor chips For the calculation of the distance between two adjacent DNA strands standing on a surface homogeneous distribution is assumed. The values in brackets correspond to the numbers determined in this study. The surface coverage value given in molecules · cm-2 (11.2·1012 molecules · cm-2) is multiplied by 10,000 to obtain the value for molecules · m-2 (11.2·1016 molecules · m-2). To obtain the statistical space occupied by one DNA strand in m² · molecule-1, this value is inverted (8.93·10-18 m2 · molecule-1). Taking the square root of this number leads to the center-to-center distance of DNA (about 3 nm). To calculate the statistical center-to-center distance of dsDNA, the surface coverage was multiplied by the normalized hybridization efficiency (hybridization efficiency in % divided by 100) and the calculation again was S-5
performed as described above (leading to a center-to-center distance of about 4.7 nm for dsDNA). Preparation of sensor chips with low surface coverage of capture probe for SPR measurements The hybridization efficiency reported in the main file is about 40% for a sensor surface modified with 11.2 ∙ 1012 molecules cm-2 (values determined by SPR). Since it was reported that hybridization efficiencies of 100% may be reached if the surface coverage of the capture probe is significantly lower,5 we also prepared sensor chips with low surface coverage. Therefore a buffer solution containing a rather low concentration of probe DNA (0.5 µM) and MCB/MPA (25 µM each) was incubated for 90 minutes. After rinsing the cell with buffer solution and water, a mixture of MCB and MPA (0.5 mM each) in water was injected and incubated for one hour. In the next step, the system was rinsed with water and buffer. In Figure S-2A a kinetic SPR scan curve collected during three hybridization and two dehybridization steps is shown (dehybridization was achieved by intense rinsing with pure water). In between the individual steps entire angular scan curves were collected, the minima of the corresponding angular scan curves are shown in Figure S-2B. As the concentration of capture probe was rather low, the signal contribution from MCB/MPA could not be neglected anymore when the surface coverage was calculated. Therefore in a control experiment SPR sensor chips were modified with MCB/MPA only, following the same procedure (not shown). To determine the surface coverage of the capture probe DNA, the surface bound mass calculated for the control experiment was subtracted from the surface bound mass calculated for the sensor chips modified with DNA and MCB/MPA. Using this method, for ssDNA a surface bound mass of 27 ng · cm-2 or a surface coverage of 4.19 · 10-12 mol · cm-2 and 2.5 · 1012 molecules · cm-2, respectively, was calculated. After hybridization the surface
S-6
bound mass increased on average by 26 ng · cm-2, which corresponds to 4.2 · 10-12 mol · cm-2 or 2.5 · 1012 molecules · cm-2. This yields a hybridization efficiency close to 100%. Even though at low surface coverage the hybridization efficiency is nearly 100%, the overall increase in reflectivity during hybridization with target is rather low, and more sensitive results will be obtained if the sensor chips are prepared by a procedure resulting in higher surface coverage of the capture probe strands.
A 34 33
complementary DNA (5 µM in buffer) angular stop scan after rinsing rinsing
32 reflectivity (%)
31 30
rinsing with buffer
29 28 27 26 25 24
angular scan on ssDNA angular scan on dsDNA
rinsing with pure water 300 350 400 time (min)
450
S-7
B
bare gold surface ssDNA1 ssDNA2 ssDNA3 dsDNA1 before rinsing dsDNA2 before rinsing dsDNA3 before rinsing dsDNA1 after rinsing dsDNA2 after rinsing dsDNA3 after rinsing
12
reflectivity (%)
10 8 6 4 2 0 67.2
67.5
67.8 68.1 68.4 incident angle ()
68.7
69.0
Figure S-2. (A) SPR kinetic scan and B) SPR angular scan curves of consecutive hybridization/dehybridization cycles on a sensor chip modified with a low surface coverage of capture probe DNA strands. In comparison to Figure 1 in the main manuscript, the angular shift between the bare gold, dehybridization, and hybridization state is very low due to the low coverage of capture probe. Angular scan curves were measured before and after hybridization as well as after a short period of rinsing with buffer to remove unspecifically bound DNA. Measurements were performed in 20 mM phosphate buffer, pH 7.0, also comprising 50 mM KCl and 5 mM MgCl2.
Combined EIS and SPR measurements at a bare gold surface To prove the assumption that free CN- released from the ferri/ferrocyanide redox couple etches the gold surface, we performed combined EIS and SPR measurements over a period of 24 hours using a deoxygenated buffer solution containing 1 mM ferri/ferrocyanide, each, independently at two gold coated SPR slides. For one of these slides, the measured and fitted angular scan curves (including the fit parameters which were calculated by using the software WinSpall) recorded before and after the EIS measurements are shown in Figure S-3.
S-8
A before EIS after EIS
100
reflectivity (%)
80 60 40 20 0 56
58
60 62 64 66 68 incident angle ()
70
72
S-9
Figure S-3. (A) Angular scan curve measurements recorded for an unmodified SPR chip before and after 24 h of EIS measurements using 1 mM ferri-/ferrocyanide, each. Panels (B) and (C) show the overlay of a measured and simulated angular scan curve before (B) and after 24 h of EIS (C), respectively. By fitting the simulation parameters to the measured curves, it was found that the average thickness of the gold layer on the SPR chip decreased by 2.3 nm during the impedance measurements. For fitting, the software WinSpall was used. S-10
Combined QCM-D and EIS measurements In order to study the etching of gold surfaces by ferri/ferrocyanide, we performed EIS measurements on bare gold-coated QCM chips either with chromium or titanium adhesion layer. In Figure 5 in the main manuscript a QCM-D measurement performed on a gold coated sensor chip at continuous flow (0.1 mL·min-1) in oxygen-free buffer containing ferri/ferrocyanide, 5 mM each, is shown at the top. At the bottom a microscopic image of a fresh QCM-D chip and the chip used above after 26 h of combined EIS/QCM measurements using the ferri/ferrocyanide redox couple is shown. In Figure S-4 EIS spectra collected during the first 20 h of the combined EIS/QCM measurements corresponding to Figure 5 in the main manuscript are shown. The EIS spectra were recorded periodically every 30 minutes in a frequency range from 10 mHz to 100 kHz. In Figure S-4 only every second spectrum is depicted. After 20 h of EIS, the RCT did not further decrease but started to increase again (not shown), possibly because at some pinholes the gold layer had been removed completely.
10
-ZIm ()
8 6 4 2 20 h EIS
0 0
5
10
15
20
ZRe ()
Figure S-4. Series of EIS spectra measured on a bare QCM-D chip in 20 mM phosphate buffer, pH 7.0 with 50 mM KCl as well as additional 5 mM MgCl2, and 5 mM ferri/ferrocyanide, each. Spectra were recorded every 30 minutes in a frequency range between 10 mHz and 100 kHz. For clarity only every second spectrum is shown.
S-11
As described in the main manuscript, we optimized the conditions for EIS measurements on modified QCM-D sensor chips in terms of minimized acquisition times, freshly prepared redox probe solutions with low ferri-/ferrocyanide concentrations and shielding these solutions from daylight. We applied these conditions to different QCM-D sensor chips modified with ssDNA and short chain thiols to test whether hybridization and dehybridization can be tracked reliably with EIS and QCM-D. Only one out of four different QCM-D sensor chips showed three repeatable hybridization/dehybridization cycles in EIS measurements. The EIS data recorded on this sensor was fitted to the Randles circuit (depicted Figure S-5) equipped with a constant phase element (CPE) to account for the surface roughness of the sensor. The corresponding fitting results, i.e. the values for RCT as well as RS, CDL, α and W can be found in Table S-1.
Figure S-5. The Randles circuit was used to fit the EIS data. A constant phase element (CPE) was used instead of a capacitor.
S-12
Table S-1. Parameters obtained by fitting the measurements depicted in Figure 6B in the main manuscript to a Randles circuit equipped with a constant phase element (CPE).
W (DW)
ssDNA (1) ssDNA (2) dsDNA (1) dsDNA (2)
1.09E-03 1.45E-03 6.11E-04 6.41E-04
ssDNA (1) ssDNA (2) dsDNA (1) dsDNA (2)
6.44E-03 8.19E-03 3.02E-03 3.64E-03
ssDNA (1) ssDNA (2) dsDNA (1) dsDNA (2)
3.32E-02 4.74E-02 1.80E-02 1.95E-02
RCT ()
CDL (F) First cycle 884.7 1.08E-05 888.9 1.08E-05 1040 1.07E-05 1041 1.07E-05 Second cycle 851.9 1.09E-05 862.1 1.09E-05 1066 1.08E-05 1057 1.08E-05 Third cycle 866.2 1.09E-05 882.1 1.09E-05 1026 1.09E-05 1025 1.09E-05
α
RS ()
0.9273 0.9294 0.938 0.9383
8.950 8.965 9.006 9.010
0.9289 0.9314 0.9412 0.9407
8.974 8.980 9.002 9.014
0.9299 0.932 0.9404 0.9403
9.008 9.009 9.016 9.017
References (1) Nogues, C.; Cohen, S. R.; Daube, S. S.; Naaman, R. Phys. Chem. Chem. Phys. 2004, 6, 4459-4466. (2) Nöll, G.; Su, Q.; Heidel, B.; Yu, Y. Adv. Healthcare Mater. 2014, 3, 42-46. (3) Oh, B.-K.; Nam, J.-M.; Lee, S. W.; Mirkin, C. A. Small 2006, 2, 103-108. (4) Ricci, F.; Zari, N.; Caprio, F.; Recine, S.; Amine, A.; Moscone, D.; Palleschi, G.; Plaxco, K. W. Bioelectrochemistry 2009, 76, 208-213. (5) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168.
S-13
