Supporting Information Real-Time Electrochemical PCR with a DNA ...
Supporting Information
Real-Time Electrochemical PCR with a DNA Intercalating Redox Probe Thibaut Deféver, Michel Druet, David Evrard, Damien Marchal* and Benoit Limoges*
S1
A. Electrochemical Cell Holder.
Carbon counter electrode Carbon working electrode Silver reference electrode
8-strip domed caps loaded with 50 µL PCR mix
Electrical contacts (2.54 mm pitch) for connection to the potentiostat
52.4 mm
Array of 8 3 band-electrodes screen-printed on a polyethylene terephthalate sheet
Bottom aluminum plate holder
Top plastic plate holder with the 8strip polypropylene domed caps whose opening is held up
8-electrochemical cell array To the potentiostat
The array of eight electrochemical cells once assembled
The array of eight electrochemical cells in place on the planar heating block of the thermocycler and connected to the multiplexed potentiostat through a specifically designed 24-pin connector
24-pin connector
Figure S1. Different views of the electrochemical cell array
B. Cyclic voltammetry of the DNA intercalating redox probes. Figure S2 show typical CVs of the different intercalating redox probes that have been investigated in this work. The voltammograms were recorded at carbon-based screen-printed electrodes and at room temperature. The shape and magnitude of the reversible waves of osmium complexes bearing a DPPZ ligand indicate significant adsorption of these compounds under both their oxidized and reduced states on the hydrophobic surface of the carbon electrodes. This is also the case with MB since it shows a much higher anodic peak current than the cathodic one. The hydrophobic adsorption
S2
of the redox probes on the electrode surface is advantageous since it allows for an increase in sensitivity of detection.
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Figure S2. CVs (v = 0.3 V s-1) of (A) 150 µM Os(bpy)2phen2+, (B) 5 µM Os(bpy)2DPPZ2+, (C) 5 µM Os(4,4’diamino-bpy)2DPPZ2+, and (D) 5 µM Os(4,4’-dimethyl-bpy)2DPPZ2+, all in a Tris buffer (pH 8.5), and (E) 20 µM MB (v = 0.1 V s-1) in a phosphate buffer (pH 7.4). Room temperature.
Fom the cyclic voltammograms, the following standard redox potential (vs. Ag/AgCl) were deduced: Os[(bpy)2phen]2+, E0 = 0.58 V; Os[(bpy)2DPPZ]2+, Os[(4,4’-diamino-bpy)2DPPZ]2+, E0 = 0.19 V; E0 = 0.66 V; Os[(4,4’-dimethyl-bpy)2DPPZ]2+ , E0 = 0.61 V; MB, E0’ = -0.26 V (at pH 8.5).
C. Influence of the DNA intercalating redox probes on the polymerase chain reaction. The inhibiting properties of the selected intercalating redox probes on the HotStartTaq DNA polymerase were evaluated by gel electrophoresis of the end-PCR product generated in standard PCR microtubes after PCR amplification of a target hCMV DNA sequence. At the beginning of our work, we have started with the PCR amplification of a 406-bp sequence of hCMV DNA, but we have rapidly changed for a shorter 283-bp sequence of hCMV DNA, simply because an optimized TaqMan-based real-time fluorescent PCR was commercially available for comparative studies.
S3
Figure S3. Gel electrophoresis image showing the influence of the Os(bpy) 2phen2+ on the endpoint PCR product yield of 406-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 100 µM each dNTPs, 0.5 µM each of forward and reverse primers, 1.25 units of HotStartTaq polymerase, 105 copies of 406-bp hCMV DNA template and different concentrations of Os(bpy) 2phen2+: (lane 1) 0, (lane 2) 1, and (lane 3) 5 µM. The overall microtubes were subjected to 40 PCR cycles of three temperature steps (denaturation at 95°C for 15 s, primer annealing at 58°C for 50 s and extension at 72°C for 30 s) and the end-PCR product was analyzed by gel electrophoresis.
Figure S6. Gel electrophoresis image showing the influence of Os(4,4’-diamino-bpy)2DPPZ2+ on the end-PCR product yield of 406-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 100 µM each dNTPs, 0.5 µM each of forward and reverse primers, 1.25 units of HotStartTaq polymerase, 105 copies of 406-bp hCMV DNA template and different concentrations of Os(4,4’diamino-bpy)2DPPZ2+: (lane 1) 1, (lane 2) 5 µM. The overall microtubes were subjected to 40 PCR cycles of three temperature steps (denaturation at 95°C for 15 s, primer annealing at 58°C for 50 s and extension at 72°C for 30 s) and the end-PCR product was analyzed by gel electrophoresis.
Figure S3. Gel electrophoresis image showing the influence of MB on the end-PCR product yield of 283-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 200 µM each dNTPs, 0.25 µM each of forward and reverse primers, 1.25 units of HotStartTaq polymerase, (lane 1-5) 107 copies or (lane 6) 0 copies of hCMV DNA template (i.e., NTC), and different concentrations of MB (lane 1) 0, (lane 2) 0.2, (lane 3) 0.5, (lane 4) 1.0, (lane 5) 5.0, (lane 6) 0 µM. L: DNA ladder. The overall microtubes were subjected to 45 PCR cycles of two temperature steps (denaturation at 95°C for 30 s, primer annealing/extension at 60°C for 60 s) and the end-PCR product was analyzed by gel electrophoresis.
S4
Figure S4. Gel electrophoresis image showing the influence of Os(4,4’-dimethyl-bpy)2DPPZ2+ on the end-PCR product yield of 283-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 200 µM each dNTPs, 0.25 µM each of forward and reverse primers, 2 units of HotStartTaq polymerase, 107 copies of hCMV DNA template and different concentrations of Os(4,4’-dimethylbpy)2DPPZ2+: (lane 1) 0, (lane 2) 0.1, (lane 3) 0.2, (lane 4) 0.4, (lane 5) 0.8, (lane 6) 1.6, and (lane 7) 3.2 µM. L: DNA ladder. The overall microtubes were subjected to 40 PCR cycles of three temperature steps (denaturation at 95°C for 15 s, primer annealing at 58°C for 50 s and extension at 72°C for 30 s) and the end-PCR product was analyzed by gel electrophoresis.
Figure S5. Gel electrophoresis image showing the influence of Os(bpy)2DPPZ2+ on the end-PCR product yield of 283-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 100 µM each dNTPs, 0.5 µM each of forward and reverse primers, 2.5 units of HotStartTaq polymerase, 5× 104 copies of hCMV DNA template and different concentrations of Os(bpy)2DPPZ2+ (total volume of 25 µL): (lane 1) 0, (lane 2) 5, (lane 3) 1, (lane 4) 0.5, (lane 5) 0.2 µM. L: DNA ladder. The overall microtubes were subjected to 45 PCR cycles with each consisting of two temperature steps (denaturation at 95°C for 10 s followed by primers annealing/extension at 60°C for 40 s) and the end-PCR product analyzed by gel electrophoresis.
S5
D. Stability of the SWV peak current response of the DNA intercalating redox probes during thermal PCR cycling. Figures S7 and S8 give an overall view of the stability and reproducibility of the SWV responses of Os(bpy)2phen2+, Os(bpy)2DPPZ2+ and MB under thermal PCR-like cycling conditions and for different concentrations (the SWV peaks were systematically corrected from the background current by simply subtracting a tangent line under the peak). It is worth to note that during the first cycles a slight increase of the peak current as a function of the cycle number was reproducibly observed with Os(bpy)2DPPZ2+. This behavior was attributed to an adsorption of the osmium complex on the hydrophobic surface of the carbon-based electrode.
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Figure S7. Series of SWV curves (f = 300 Hz, Ep = 25 mV, and Esw =1 mV) recorded during the thermal cycling of 50 µL containing (A, D) 5 µM or (B, E) 1 µM of (A, B) Os(bpy) 2phen2+ or (D, E) Os(bpy)2DPPZ2+ in a Qiagen PCR buffer. The thermal cycling parameters were 15 min at 95°C, followed by 30 cycles of 90 s at 95°C, 120 s at 58°C and 120 s at 72°C. The SWV curves were acquired at the end of the 58°C heating phase once every three cycles for the Os(bpy)2phen2+ or once every cycles for the Os(bpy)2DPPZ2+ (the overall scans were overlaid in A-B and D-E). (C, F) Plots of SWV peak current density, jp, as a function of cycle number.
S6
10 µM MB 10 µM MB 2 µM MB 2 µM MB
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Figure S8. (A) Series of SWV curves (f = 100 Hz, Ep = 50 mV, and Esw = 1 mV) recorded during the thermal PCR cycling of 50 µL of 2 µM MB in a Qiagen PCR buffer. The thermal cycling parameters were 15 min at 95°C followed by 40 thermal PCR cycles of 30 s at 95°C, 30 s at 58°C and 30 s at 72°C. The SWV curves were acquired once at the end of the 72°C heating phase of each cycle (the overall scans were overlaid). (B) Plot of the SWV peak current density, jp, as a function of cycle number for two duplicated MB concentrations (i.e., 2 and 20 µM). (C) Same as (B) after normalization to the highest jp value.
E. Evolution of the SWV peak current response of Os(bpy)2phen2+ and MB during PCR amplification of hCMV DNA target.
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Figure S9. (A) Kinetic PCR plots of the SWV peak current density of Os(bpy) 2phen2+ (5 µM) as a function of cycle number obtained during the PCR amplification of two positive (5 × 10 4 copies) and two negative controls (NTC) of 406-bp hCMV DNA (see the legend for the color code). The experiments were performed in 50 µL PCR mix (0.1 mM dNTPs, 0.5 µM of forward and reverse primers, 2.5 units of HotStartTaq polymerase) and the thermal cycling parameters were 15 min at 95°C followed by 40 thermal PCR cycles of 15 s at 95°C, 50 s at 58°C and 30 s at 72°C. The SWV curves (f = 300 Hz, Ep = 25 mV, and Esw =1 mV) were acquired once at the end of the 58°C heating phase of each cycle. (B) Gel electrophoresis image of the end-PCR solutions: (lanes +) positive and (lanes -) negative controls. Lane L: DNA ladder.
S7
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10 µM MB, positive control 10 µM MB, positive control 10 µM MB, NTC 10 µM MB, no dNTPs 2 µM MB, positive control 2 µM MB, positive control 2 µM MB, NTC 2 µM MB, no dNTPs
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Figure S10. (A) Kinetic PCR plots of the SWV peak current density of MB (at two duplicated MB concentrations of 2 and 10 µM) as a function of cycle number obtained during the PCR amplification of positive (108 copies) and negative controls (NTC) of 283-bp hCMV DNA (see the legend for the color code). The experiments were performed in 50 µL PCR mix (0.2 mM dNTPs, 0.25 µM of forward and reverse primers, 2.5 units of HotStartTaq polymerase) and the thermal cycling parameters were 15 min at 95°C followed by 40 thermal PCR cycles of 30 s at 95°C, 30 s at 58°C and 30 s at 72°C. The SWV curves (f = 100 Hz, Ep = 50 mV, and Esw = 1 mV) were acquired once at the end of the 72°C heating phase of each cycle. (B) Same as (A) after normalization to the highest jp value. (C) Gel electrophoresis image of the overall end-PCR solutions containing 2 µM MB: (lanes 1, 2) positive and (lanes 3, 4) negative controls. Lanes 5 and 6: positive and negative controls carried out in standard PCR microtubes. Lane L: DNA ladder.
F. Determination of the affinity binding constant (Kb) of Os(bpy)2DPPZ2+ toward dsDNA.
The determination of the DNA-binding constant of Os(bpy)2DPPZ2+
was achieved
electrochemically by measuring at 25°C the SWV peak current of a 0.5 µM osmium complex solution (in a PCR buffer) as a function of increasing concentrations of a calibrated solution of DNA amplicon. For obtaining reproducible results, the current were measured at a screen-printed carbon-based electrode that was pre-incubated for 30 min in a Tris buffer containing 0.1% bovine serum albumin. Under the assumption of reversible, diffusion-controlled electron transfer, and the hypothesis of a slow interconversion of free and bound intercalator on the time scale of SWV experiments, the peak current is thus given by:
S8
i p A D1f 2C f Db1 2Cb A D1f 2 CI Cb Db1 2Cb (S1) where A is a proportionality factor, Df and Db are the diffusion coefficients of the free and DNA-bound intercalating probe, and Cf and Cb the concentrations of free and DNA-bound complex in the bulk PCR solution (Carter M. T., Rodriguez M., Bard A. J., J. Am. Chem. Soc., 1989, 111, 8901–8911). The concentration of DNA-bound complex, Cb, can be calculated from eq S2 which takes into account a classical equilibrium binding with a predetermined number of noncooperative binding sites per unit of ds-DNA (i.e., only one type of noninteracting discrete binding site). 12 Cb b b2 2Kb2CI CDNA / s / 2Kb
(S2) with, b 1 KbCI KbCDNA / 2s and where CI is the total concentration of intercalator added to the solution (i.e., CI Cb C f ), CDNA is the total concentration of DNA in nucleotide phosphate, and s the binding size site in base pairs. From a combination of eqs S1 and S2, we obtained the general expression S3:
12 12 i p A D1f 2 CI b b 2 2Kb2CI CDNA / s / 2Kb Db1 2 b b 2 2Kb2CI CDNA / s / 2Kb
(S3)
The latter equation allows fitting the experimental binding titration curve shown in Figure S11 (green curve). From the best fitting using Df = 5 × 10-6 cm2 s-1 and Db = 2.5 × 10-8 cm2 s-1, values of s = 3 and Kb = 5 × 106 M-1 were finally obtained at T = 25°C. 50
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Figure S11. Plot of the SWV peak current (f = 100 Hz, Ep = 50 mV, and Esw =1 mV) of a 0.5 µM Os(bpy)2DPPZ2+ solution as a function of DNA concentration (CDNA), which is given here as the ratio R = CDNA/CI. T = 25°C. Red circle symbol: experimental data. Green curve: nonlinear regression fitting to a combination of eq S1 and S2 using CI = 0.5 µM, A = 4.2 × 104, Kb = 5 × 106 M-1, s = 3, Df = 5 × 10-6 cm2 s-1, Db = 2.5 × 10-8 cm2 s-1. S9
When eq S3 is combined to the basic equation of PCR kinetics, i.e. NC = N0 × C (where NC is the amount of amplicon after C cycles, N0 the initial amount of target, and the amplification efficiency), we obtained the general expression S4 which allow to calculate the current response as a function of the PCR cycles. 12 12 2 2 2 LN0 C 2 LN 0 C 12 2 12 2 i p A D f C I b b 2 K b C I / 2 Kb Db b b 2Kb CI / 2 Kb sV sV
where L is the DNA length and V the volume of PCR solution.
S10
(S4)
Real-Time Electrochemical PCR with a DNA Intercalating Redox Probe Thibaut Deféver, Michel Druet, David Evrard, Damien Marchal* and Benoit Limoges*
S1
A. Electrochemical Cell Holder.
Carbon counter electrode Carbon working electrode Silver reference electrode
8-strip domed caps loaded with 50 µL PCR mix
Electrical contacts (2.54 mm pitch) for connection to the potentiostat
52.4 mm
Array of 8 3 band-electrodes screen-printed on a polyethylene terephthalate sheet
Bottom aluminum plate holder
Top plastic plate holder with the 8strip polypropylene domed caps whose opening is held up
8-electrochemical cell array To the potentiostat
The array of eight electrochemical cells once assembled
The array of eight electrochemical cells in place on the planar heating block of the thermocycler and connected to the multiplexed potentiostat through a specifically designed 24-pin connector
24-pin connector
Figure S1. Different views of the electrochemical cell array
B. Cyclic voltammetry of the DNA intercalating redox probes. Figure S2 show typical CVs of the different intercalating redox probes that have been investigated in this work. The voltammograms were recorded at carbon-based screen-printed electrodes and at room temperature. The shape and magnitude of the reversible waves of osmium complexes bearing a DPPZ ligand indicate significant adsorption of these compounds under both their oxidized and reduced states on the hydrophobic surface of the carbon electrodes. This is also the case with MB since it shows a much higher anodic peak current than the cathodic one. The hydrophobic adsorption
S2
of the redox probes on the electrode surface is advantageous since it allows for an increase in sensitivity of detection.
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Figure S2. CVs (v = 0.3 V s-1) of (A) 150 µM Os(bpy)2phen2+, (B) 5 µM Os(bpy)2DPPZ2+, (C) 5 µM Os(4,4’diamino-bpy)2DPPZ2+, and (D) 5 µM Os(4,4’-dimethyl-bpy)2DPPZ2+, all in a Tris buffer (pH 8.5), and (E) 20 µM MB (v = 0.1 V s-1) in a phosphate buffer (pH 7.4). Room temperature.
Fom the cyclic voltammograms, the following standard redox potential (vs. Ag/AgCl) were deduced: Os[(bpy)2phen]2+, E0 = 0.58 V; Os[(bpy)2DPPZ]2+, Os[(4,4’-diamino-bpy)2DPPZ]2+, E0 = 0.19 V; E0 = 0.66 V; Os[(4,4’-dimethyl-bpy)2DPPZ]2+ , E0 = 0.61 V; MB, E0’ = -0.26 V (at pH 8.5).
C. Influence of the DNA intercalating redox probes on the polymerase chain reaction. The inhibiting properties of the selected intercalating redox probes on the HotStartTaq DNA polymerase were evaluated by gel electrophoresis of the end-PCR product generated in standard PCR microtubes after PCR amplification of a target hCMV DNA sequence. At the beginning of our work, we have started with the PCR amplification of a 406-bp sequence of hCMV DNA, but we have rapidly changed for a shorter 283-bp sequence of hCMV DNA, simply because an optimized TaqMan-based real-time fluorescent PCR was commercially available for comparative studies.
S3
Figure S3. Gel electrophoresis image showing the influence of the Os(bpy) 2phen2+ on the endpoint PCR product yield of 406-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 100 µM each dNTPs, 0.5 µM each of forward and reverse primers, 1.25 units of HotStartTaq polymerase, 105 copies of 406-bp hCMV DNA template and different concentrations of Os(bpy) 2phen2+: (lane 1) 0, (lane 2) 1, and (lane 3) 5 µM. The overall microtubes were subjected to 40 PCR cycles of three temperature steps (denaturation at 95°C for 15 s, primer annealing at 58°C for 50 s and extension at 72°C for 30 s) and the end-PCR product was analyzed by gel electrophoresis.
Figure S6. Gel electrophoresis image showing the influence of Os(4,4’-diamino-bpy)2DPPZ2+ on the end-PCR product yield of 406-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 100 µM each dNTPs, 0.5 µM each of forward and reverse primers, 1.25 units of HotStartTaq polymerase, 105 copies of 406-bp hCMV DNA template and different concentrations of Os(4,4’diamino-bpy)2DPPZ2+: (lane 1) 1, (lane 2) 5 µM. The overall microtubes were subjected to 40 PCR cycles of three temperature steps (denaturation at 95°C for 15 s, primer annealing at 58°C for 50 s and extension at 72°C for 30 s) and the end-PCR product was analyzed by gel electrophoresis.
Figure S3. Gel electrophoresis image showing the influence of MB on the end-PCR product yield of 283-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 200 µM each dNTPs, 0.25 µM each of forward and reverse primers, 1.25 units of HotStartTaq polymerase, (lane 1-5) 107 copies or (lane 6) 0 copies of hCMV DNA template (i.e., NTC), and different concentrations of MB (lane 1) 0, (lane 2) 0.2, (lane 3) 0.5, (lane 4) 1.0, (lane 5) 5.0, (lane 6) 0 µM. L: DNA ladder. The overall microtubes were subjected to 45 PCR cycles of two temperature steps (denaturation at 95°C for 30 s, primer annealing/extension at 60°C for 60 s) and the end-PCR product was analyzed by gel electrophoresis.
S4
Figure S4. Gel electrophoresis image showing the influence of Os(4,4’-dimethyl-bpy)2DPPZ2+ on the end-PCR product yield of 283-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 200 µM each dNTPs, 0.25 µM each of forward and reverse primers, 2 units of HotStartTaq polymerase, 107 copies of hCMV DNA template and different concentrations of Os(4,4’-dimethylbpy)2DPPZ2+: (lane 1) 0, (lane 2) 0.1, (lane 3) 0.2, (lane 4) 0.4, (lane 5) 0.8, (lane 6) 1.6, and (lane 7) 3.2 µM. L: DNA ladder. The overall microtubes were subjected to 40 PCR cycles of three temperature steps (denaturation at 95°C for 15 s, primer annealing at 58°C for 50 s and extension at 72°C for 30 s) and the end-PCR product was analyzed by gel electrophoresis.
Figure S5. Gel electrophoresis image showing the influence of Os(bpy)2DPPZ2+ on the end-PCR product yield of 283-bp hCMV DNA template. The PCR amplification was carried out in standard PCR microtubes in the presence of 100 µM each dNTPs, 0.5 µM each of forward and reverse primers, 2.5 units of HotStartTaq polymerase, 5× 104 copies of hCMV DNA template and different concentrations of Os(bpy)2DPPZ2+ (total volume of 25 µL): (lane 1) 0, (lane 2) 5, (lane 3) 1, (lane 4) 0.5, (lane 5) 0.2 µM. L: DNA ladder. The overall microtubes were subjected to 45 PCR cycles with each consisting of two temperature steps (denaturation at 95°C for 10 s followed by primers annealing/extension at 60°C for 40 s) and the end-PCR product analyzed by gel electrophoresis.
S5
D. Stability of the SWV peak current response of the DNA intercalating redox probes during thermal PCR cycling. Figures S7 and S8 give an overall view of the stability and reproducibility of the SWV responses of Os(bpy)2phen2+, Os(bpy)2DPPZ2+ and MB under thermal PCR-like cycling conditions and for different concentrations (the SWV peaks were systematically corrected from the background current by simply subtracting a tangent line under the peak). It is worth to note that during the first cycles a slight increase of the peak current as a function of the cycle number was reproducibly observed with Os(bpy)2DPPZ2+. This behavior was attributed to an adsorption of the osmium complex on the hydrophobic surface of the carbon-based electrode.
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Figure S7. Series of SWV curves (f = 300 Hz, Ep = 25 mV, and Esw =1 mV) recorded during the thermal cycling of 50 µL containing (A, D) 5 µM or (B, E) 1 µM of (A, B) Os(bpy) 2phen2+ or (D, E) Os(bpy)2DPPZ2+ in a Qiagen PCR buffer. The thermal cycling parameters were 15 min at 95°C, followed by 30 cycles of 90 s at 95°C, 120 s at 58°C and 120 s at 72°C. The SWV curves were acquired at the end of the 58°C heating phase once every three cycles for the Os(bpy)2phen2+ or once every cycles for the Os(bpy)2DPPZ2+ (the overall scans were overlaid in A-B and D-E). (C, F) Plots of SWV peak current density, jp, as a function of cycle number.
S6
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Cycle number
Cycle number
Figure S8. (A) Series of SWV curves (f = 100 Hz, Ep = 50 mV, and Esw = 1 mV) recorded during the thermal PCR cycling of 50 µL of 2 µM MB in a Qiagen PCR buffer. The thermal cycling parameters were 15 min at 95°C followed by 40 thermal PCR cycles of 30 s at 95°C, 30 s at 58°C and 30 s at 72°C. The SWV curves were acquired once at the end of the 72°C heating phase of each cycle (the overall scans were overlaid). (B) Plot of the SWV peak current density, jp, as a function of cycle number for two duplicated MB concentrations (i.e., 2 and 20 µM). (C) Same as (B) after normalization to the highest jp value.
E. Evolution of the SWV peak current response of Os(bpy)2phen2+ and MB during PCR amplification of hCMV DNA target.
A
1.2
B
Normalized response
1.0
2000 bp 1200 bp 800 bp
0.8 0.6
400 bp 200 bp 100 bp
NTC positive control positive control NTC
0.4 0.2 0.0 0
5
10 15 20 25 30 35 40
PCR cycle number
Figure S9. (A) Kinetic PCR plots of the SWV peak current density of Os(bpy) 2phen2+ (5 µM) as a function of cycle number obtained during the PCR amplification of two positive (5 × 10 4 copies) and two negative controls (NTC) of 406-bp hCMV DNA (see the legend for the color code). The experiments were performed in 50 µL PCR mix (0.1 mM dNTPs, 0.5 µM of forward and reverse primers, 2.5 units of HotStartTaq polymerase) and the thermal cycling parameters were 15 min at 95°C followed by 40 thermal PCR cycles of 15 s at 95°C, 50 s at 58°C and 30 s at 72°C. The SWV curves (f = 300 Hz, Ep = 25 mV, and Esw =1 mV) were acquired once at the end of the 58°C heating phase of each cycle. (B) Gel electrophoresis image of the end-PCR solutions: (lanes +) positive and (lanes -) negative controls. Lane L: DNA ladder.
S7
B
A 150
10 µM MB, positive control 10 µM MB, positive control 10 µM MB, NTC 10 µM MB, no dNTPs 2 µM MB, positive control 2 µM MB, positive control 2 µM MB, NTC 2 µM MB, no dNTPs
2
jp (µA/cm )
100
50
0
Normalized response
1.0 0.8 0.6 0.4 0.2 0.0 0
5
10 15 20 25 30 35 40
0
5
10 15 20 25 30 35 40
PCR cycle number
PCR cycle number
C
Figure S10. (A) Kinetic PCR plots of the SWV peak current density of MB (at two duplicated MB concentrations of 2 and 10 µM) as a function of cycle number obtained during the PCR amplification of positive (108 copies) and negative controls (NTC) of 283-bp hCMV DNA (see the legend for the color code). The experiments were performed in 50 µL PCR mix (0.2 mM dNTPs, 0.25 µM of forward and reverse primers, 2.5 units of HotStartTaq polymerase) and the thermal cycling parameters were 15 min at 95°C followed by 40 thermal PCR cycles of 30 s at 95°C, 30 s at 58°C and 30 s at 72°C. The SWV curves (f = 100 Hz, Ep = 50 mV, and Esw = 1 mV) were acquired once at the end of the 72°C heating phase of each cycle. (B) Same as (A) after normalization to the highest jp value. (C) Gel electrophoresis image of the overall end-PCR solutions containing 2 µM MB: (lanes 1, 2) positive and (lanes 3, 4) negative controls. Lanes 5 and 6: positive and negative controls carried out in standard PCR microtubes. Lane L: DNA ladder.
F. Determination of the affinity binding constant (Kb) of Os(bpy)2DPPZ2+ toward dsDNA.
The determination of the DNA-binding constant of Os(bpy)2DPPZ2+
was achieved
electrochemically by measuring at 25°C the SWV peak current of a 0.5 µM osmium complex solution (in a PCR buffer) as a function of increasing concentrations of a calibrated solution of DNA amplicon. For obtaining reproducible results, the current were measured at a screen-printed carbon-based electrode that was pre-incubated for 30 min in a Tris buffer containing 0.1% bovine serum albumin. Under the assumption of reversible, diffusion-controlled electron transfer, and the hypothesis of a slow interconversion of free and bound intercalator on the time scale of SWV experiments, the peak current is thus given by:
S8
i p A D1f 2C f Db1 2Cb A D1f 2 CI Cb Db1 2Cb (S1) where A is a proportionality factor, Df and Db are the diffusion coefficients of the free and DNA-bound intercalating probe, and Cf and Cb the concentrations of free and DNA-bound complex in the bulk PCR solution (Carter M. T., Rodriguez M., Bard A. J., J. Am. Chem. Soc., 1989, 111, 8901–8911). The concentration of DNA-bound complex, Cb, can be calculated from eq S2 which takes into account a classical equilibrium binding with a predetermined number of noncooperative binding sites per unit of ds-DNA (i.e., only one type of noninteracting discrete binding site). 12 Cb b b2 2Kb2CI CDNA / s / 2Kb
(S2) with, b 1 KbCI KbCDNA / 2s and where CI is the total concentration of intercalator added to the solution (i.e., CI Cb C f ), CDNA is the total concentration of DNA in nucleotide phosphate, and s the binding size site in base pairs. From a combination of eqs S1 and S2, we obtained the general expression S3:
12 12 i p A D1f 2 CI b b 2 2Kb2CI CDNA / s / 2Kb Db1 2 b b 2 2Kb2CI CDNA / s / 2Kb
(S3)
The latter equation allows fitting the experimental binding titration curve shown in Figure S11 (green curve). From the best fitting using Df = 5 × 10-6 cm2 s-1 and Db = 2.5 × 10-8 cm2 s-1, values of s = 3 and Kb = 5 × 106 M-1 were finally obtained at T = 25°C. 50
ip (nA)
40 30 20 10 0 0
10
20
30
40
50
R
Figure S11. Plot of the SWV peak current (f = 100 Hz, Ep = 50 mV, and Esw =1 mV) of a 0.5 µM Os(bpy)2DPPZ2+ solution as a function of DNA concentration (CDNA), which is given here as the ratio R = CDNA/CI. T = 25°C. Red circle symbol: experimental data. Green curve: nonlinear regression fitting to a combination of eq S1 and S2 using CI = 0.5 µM, A = 4.2 × 104, Kb = 5 × 106 M-1, s = 3, Df = 5 × 10-6 cm2 s-1, Db = 2.5 × 10-8 cm2 s-1. S9
When eq S3 is combined to the basic equation of PCR kinetics, i.e. NC = N0 × C (where NC is the amount of amplicon after C cycles, N0 the initial amount of target, and the amplification efficiency), we obtained the general expression S4 which allow to calculate the current response as a function of the PCR cycles. 12 12 2 2 2 LN0 C 2 LN 0 C 12 2 12 2 i p A D f C I b b 2 K b C I / 2 Kb Db b b 2Kb CI / 2 Kb sV sV
where L is the DNA length and V the volume of PCR solution.
S10
(S4)
