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Portable pH-inspired electrochemical detection of DNA amplification† Fang Zhang,ab Jian Wu,*ab Rui Wang,b Liu Wangb and Yibin Yingb
Received 23rd April 2014, Accepted 10th June 2014 DOI: 10.1039/c4cc03011g www.rsc.org/chemcomm
A portable and label-free pH-mediated electrochemical method for the detection of DNA amplification is described. With protons released as readouts, DNA amplifications were detected in realtime or at the end-point.
DNA assay has been an extremely important method in the fields ranging from agricultural product detection to the diagnosis of genetic diseases. Currently, most of the DNA assays are polymerase chain reaction (PCR)-based and employ a fluorescent probe or a dye to detect the amplicons optically. Thus, devices such as thermal blocks for amplification and precision optics for detection are required. Although numerous new amplification methods, such as loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA) and crosspriming isothermal amplification (CPA), have been developed to make DNA assays easier for implementation or miniaturization, some inherent drawbacks still cannot be avoided, including costly optical labels and bulky optical detection devices.1–6 A more portable detection method for DNA amplification is needed. Promising electrochemical approaches, which are simple, portable, low cost and highly sensitive, are well suited as alternatives to optical methods. Several groups have attempted to exploit this concept in the detection of DNA amplification, either by using DNA-intercalative electroactive molecules (e.g. methylene blue), labelled probes or by detecting the electrical conductivity changes of the reaction mixture during DNA amplification.7–20 However, these strategies are limited by the low signal resolution resulting from the weak binding affinity of electroactive molecules to dsDNA under PCR conditions, low efficiency of asymmetric amplification or disturbance from an excess amount of other ions in PCR buffer. Herein, we report the development of a novel
label-free pH-mediated electrochemical method as an alternative to fluorescent detection for DNA cross-priming isothermal amplification (CPA), which has been described in our previous work, making DNA assays more portable and much easier to miniaturize.2 Nucleotide incorporation occurs during DNA chain extension and replicates the template DNA. During this process there is nucleophilic attack on the a-phosphate group of a nucleotide from the terminal 3 0 -hydroxy group of the growing DNA strand and a proton on this terminal hydroxyl group is replaced by nucleotide base to be incorporated, that is, one proton is released from the sugar-phosphate backbone for every new phosphodiester bond formed (shown in Fig. 1).21,22 A semiconductor-based de novo nucleic acid sequencer developed by Ion Torrent Systems is also based on this concept using ISEFTs.23,24 Normally, CPA reaction would not exhibit a noticeable pH change during DNA amplification due to its buffered conditions, which was preferred for the DNA polymerase but would consume the protons released. To minimize the consumption of released protons of the reaction mixture and make the pH
a
The State Key Lab of Fluid Power Transmission and Control, Zhejiang University, Hangzhou 310027, China. E-mail: [email protected]; Tel: +86-571-88982180 b College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4cc03011g
8416 | Chem. Commun., 2014, 50, 8416--8419
Fig. 1
Mechanism for proton release during DNA amplification.
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Published on 11 June 2014. Downloaded by University of California - San Diego on 15/11/2014 12:58:40.
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change caused by DNA amplification noticeable, the potential buffering compounds (e.g. Tris-HCl) were wiped off and the concentration of weak electrolyte (e.g. NH4+) was also reduced as much as possible while retaining the amplification efficiency and specificity of the reaction system. Low buffered conditions were pH sensitive but not suitable for DNA polymerase exactly. DNA polymerase behaved well in an alkaline environment but the conditions without Tris-HCl and with low concentration of NH4+ were partially acidic. In this way, the addition of KOH is needed to keep the DNA polymerase active. We termed this pH sensitive amplification as pH-CPA and employed genetically modified rice (Huahui 1) as our model target and a set of primers to target Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) gene (see the ESI†). The pH-CPA was performed in PCR tubes in a simple heat block. The time-course changes of pH during DNA amplification were examined five times continuously in two minutes using a commercial pH sensor (8220 BNWP Thermo ROSS Ultra pH electrode, Thermo Fisher Scientific Inc., US) and averaged afterwards. As shown in Fig. 2, the negative control reaction did not display any change in pH (blue, up-triangle), but the target-containing reaction showed a clear reduction of pH during DNA amplification (black, diamond and red, circle). Similar to real-time fluorescent detection of DNA amplification, the time-course pH changes of pH-CPA occurred in an S-shaped curve, with a steady phase at the beginning, a sharp decrease when the CPA amplicons were generated efficiently and protons accumulated rapidly, and finally a stationary phase when the reaction reached its plateau. The reaction with 3 mM KOH changed at a pH of 0.31 after 55 min reaction at 63 1C, while the 6.5 mM KOH reaction at 0.14. Both these two cases were added with the same target DNA concentration (approximately 103 genome copies per 50 mL). And they reached almost the same point at which pH decreased most efficiently. The KOH added was helpful to maintain DNA amplification efficiency offering a favourable pH range for enzyme activity but it was also criticized to weaken pH changes induced by the released proton. In the pH-CPA reaction described in this communication, the sample with 3 mM KOH at the beginning showed the largest change in pH (data not shown). To ensure that the signal observed was not due to spurious amplification, the reaction mixture was then separated on agarose gel showing that DNA has been amplified specifically (Fig. 2). pH-CPA can be conducted as expected under modified conditions. To evaluate the potential quantitative application of the pH-mediated DNA amplification, we performed pH-CPA with different initial copy numbers of GM rice genomic DNA (ten-fold serial dilution from 104 to 102 copies) and compared with the conventional SYTO 9-based CPA method (previous work of our group, conducted on a Biorad MyiQ2 Real Time PCR Detection System with ‘‘buffered’’ CPA).2 By applying a similar methodology as in real-time PCR, we defined the signal threshold as the point at which amplification was most efficient and the corresponding pH decreased most rapidly (Fig. 3A). For each pH-CPA reaction, this threshold is reliably decided by identifying the local minimum in the corresponding pH-change derivative trace
This journal is © The Royal Society of Chemistry 2014
ChemComm
Fig. 2 Time-course pH-change traces during pH-CPA amplification at different concentrations of KOH and pH-CPA products separated on 3% agarose gel. Negative control (blue, up-triangle) represents the reaction performed without a DNA template. Both the 6.5 mM (black, diamond) and 3 mM KOH (red, circle) cases were added with the same target DNA concentration, approximately 103 genome copies per 50 mL.
Fig. 3 Potential quantitative application of pH-CPA with serial dilutions of target DNA. (A) Real-time pH traces for samples containing 100 (blue, up-triangle), 1000 (red, circle) and 10 000 copies (black, diamond) of target GM rice genomic DNA. (B) By taking the derivative of the pH-change trace, the signal threshold is revealed as the local minimum in the curve, facilitating determination of the time to threshold (tth, vertical dashed line, defined as the reaction time required to reach the signal threshold).
(Fig. 3B). Just like the ‘‘threshold cycle’’ (Ct) in real-time PCR, we subsequently defined the ‘‘time to threshold’’ (tth) as the time required for a particular sample reaction to reach the signal threshold (Fig. 3B). We observed distinct local minima separated by approximately 3 min for each of the pH traces, with a distinct
Chem. Commun., 2014, 50, 8416--8419 | 8417
View Article Online
Published on 11 June 2014. Downloaded by University of California - San Diego on 15/11/2014 12:58:40.
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tth for each initial target copy number (Fig. 3B). In particular, the tth of pH-CPA reaction with 100, 1000 and 10 000 copies of initial genomic DNA templates was 26, 29 and 32 min respectively. Decreased quantities of the template DNA resulted in slower accumulation of released protons, which were in turn reflected by larger tth. Compared with the optical real-time CPA (reported previously), the testing time of DNA amplification was saved by about 20%.2 To further develop a portable pH-mediated electrochemical platform for the detection of DNA amplification at the point-ofcare, a disposable and low-cost pH sensor is needed. We fabricated pH-sensitive disposable screen printed electrodes (pH SPEs) and detected CPA at the end-point. With the SPEs’ feature of single-use, opening of the tube-cap can be avoided. The SPE has a size of 23.5 mm 5 mm 0.7 mm (Fig. 4), and can be directly inserted into the 200 mL microtubes. It was modified according to the research published by Kampouris and Musa.25,26 As both pH active compounds (phenanthraquinone, PAQ) and the other pH inactive compounds (dimethylferrocene, Fc) are combined within a single working electrode, pH can be revealed as the potential difference between the two compounds (see the ESI,† Fig. S1). Therefore, potential drift of the reference half-cell was not as detrimental as for common electrochemical platforms. As shown in Fig. 5, with pH sensitive screen printed electrodes described, the potential peak
Communication
spacing in the square-wave voltammetry (SWV) curve of samples that has been amplified at 63 1C about 55 min displayed a larger dispersion degree, compared with those samples without any heat process. We speculate that such discrepancy may be due to the detection temperature variance between different treatments. And the potential peak spacing changed very little between the negative control samples before and after amplification, which meant DNA had not been amplified. While for the positive samples with initial templates about 1000 copies of genomic DNA, the potential peak spacing decreased by about 28.3 mV after pH-CPA amplification. From the standard curve of our pH sensitive disposable electrode (DEp = 0.075 pH – 0.2674, R2 = 0.9918, Fig. S2, ESI†), pH-changes at 28.3 mV can be calculated to be 0.38, which is comparable with the result obtained from a commercial pH sensor (approximately 0.35 pH). Thus, this disposable SPE can be used for DNA amplification using a pH-mediated electrochemical strategy. In conclusion, the present study has successfully developed a portable and label-free pH-mediated method for the detection of DNA amplification in CPA. Protons were detected as readouts for DNA amplification. Additionally, we devised reliable signal threshold and time-to-threshold metrics for pH-CPA analysing, demonstrating that this method possesses quantitative application ability. And with a disposable pH-sensitive screen printed electrode, DNA amplification of pH-CPA can be detected ‘‘in-the-field’’. This work is supported by the National Natural Science Foundation of China (31271617) and the National Key Technology Support Program (2012BAK08B05).
Notes and references
Fig. 4 Photograph and sketch map of the screen-printed electrode.
Fig. 5 Endpoint detection of pH-CPA using screen printed electrodes. Square-wave voltammetry (SWV) was used. Potential peak spacing between PAQ and Fc was calculated for each sample. Samples of negative control (without the DNA template) and the ones with 1000 copies of target DNA were detected before and after pH-CPA reaction at room temperature separately.
8418 | Chem. Commun., 2014, 50, 8416--8419
1 T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino and T. Hase, Nucleic Acids Res., 2000, 28, e63. 2 F. Zhang, L. Wang, K. Fan, J. Wu and Y. Ying, Anal. Bioanal. Chem., 2014, 406, 3069–3078. 3 G. T. Walker, M. S. Fraiser, J. L. Schram, M. C. Little, J. G. Nadeau and D. P. Malinowski, Nucleic Acids Res., 1992, 20, 1691–1696. 4 G. Xu, L. Hu, H. Zhong, H. Wang, S.-i. Yusa, T. C. Weiss, P. J. Romaniuk, S. Pickerill and Q. You, Sci. Rep., 2012, 2, 246. 5 N. Tomita, Y. Mori, H. Kanda and T. Notomi, Nat. Protoc., 2008, 3, 877–882. 6 M. Goto, E. Honda, A. Ogura, A. Nomoto and K.-I. Hanaki, Biotechniques, 2009, 46, 167–172. 7 M. U. Ahmed, M. Saito, M. M. Hossain, S. R. Rao, S. Furui, A. Hino, Y. Takamura, M. Takagi and E. Tamiya, Analyst, 2009, 134, 966–972. 8 B. Arneth, Anal. Biochem., 2009, 385, 26–33. 9 M. U. Ahmed, Q. Hasan, M. Mosharraf Hossain, M. Saito and E. Tamiya, Food Control, 2010, 21, 599–605. 10 N. Nagatani, K. Yamanaka, M. Saito, R. Koketsu, T. Sasaki, K. Ikuta, T. Miyahara and E. Tamiya, Analyst, 2011, 136, 5143–5150. 11 B. Y. Won, S. Shin, S. Baek, Y. L. Jung, T. Li, S. C. Shin, D. Y. Cho, S. B. Lee and H. G. Park, Analyst, 2011, 136, 1573–1579. 12 K. Yamanaka, M. Saito, K. Kondoh, M. M. Hossain, R. Koketsu, T. Sasaki, N. Nagatani, K. Ikuta and E. Tamiya, Analyst, 2011, 136, 2064–2068. 13 M. U. Ahmed, S. Nahar, M. Safavieh and M. Zourob, Analyst, 2013, 138, 907–915. 14 X. Fang, Q. Jin, F. Jing, H. Zhang, F. Zhang, H. Mao, B. Xu and J. Zhao, Biosens. Bioelectron., 2013, 44, 241–247. 15 M. Safavieh, M. U. Ahmed, A. Ng and M. Zourob, Biosens. Bioelectron., 2014, 58, 101–106. 16 M. Safavieh, M. U. Ahmed, E. Sokullu, A. Ng, L. Braescu and M. Zourob, Analyst, 2014, 139, 482–487.
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Communication 17 B. Y. Won, S. Shin, S. Baek, Y. L. Jung, T. Li, S. C. Shin, D.-Y. Cho, S. B. Lee and H. G. Park, Analyst, 2011, 136, 1573–1579. ´ver, M. Druet, D. Evrard, D. Marchal and B. Limoges, Anal. 18 T. Defe Chem., 2011, 83, 1815–1821. ´ver, M. Druet, M. Rochelet-Dequaire, M. Joannes, 19 T. Defe C. Grossiord, B. Limoges and D. Marchal, J. Am. Chem. Soc., 2009, 131, 11433–11441. 20 S. S. W. Yeung, T. M. H. Lee and I. M. Hsing, J. Am. Chem. Soc., 2006, 128, 13374–13375. 21 C. K. Mathews, K. G. Ahern and K. E. V. Holde, Biochemistry, 3rd edn, Benjamin Cummings, San Francisco, 2000. 22 S. Purushothaman, C. Toumazou and C.-P. Ou, Sens. Actuators, B, 2006, 114, 964–968. 23 J. M. Rothberg, W. Hinz, T. M. Rearick, J. Schultz, W. Mileski, M. Davey, J. H. Leamon, K. Johnson, M. J. Milgrew, M. Edwards, J. Hoon, J. F. Simons, D. Marran, J. W. Myers, J. F. Davidson, A. Branting, J. R. Nobile, B. P. Puc, D. Light, T. A. Clark,
This journal is © The Royal Society of Chemistry 2014
ChemComm M. Huber, J. T. Branciforte, I. B. Stoner, S. E. Cawley, M. Lyons, Y. Fu, N. Homer, M. Sedova, X. Miao, B. Reed, J. Sabina, E. Feierstein, M. Schorn, M. Alanjary, E. Dimalanta, D. Dressman, R. Kasinskas, T. Sokolsky, J. A. Fidanza, E. Namsaraev, K. J. McKernan, A. Williams, G. T. Roth and J. Bustillo, Nature, 2011, 475, 348–352. 24 C. Toumazou, L. M. Shepherd, S. C. Reed, G. I. Chen, A. Patel, D. M. Garner, C.-J. A. Wang, C.-P. Ou, K. Amin-Desai, P. Athanasiou, H. Bai, I. M. Q. Brizido, B. Caldwell, D. Coomber-Alford, P. Georgiou, K. S. Jordan, J. C. Joyce, M. La Mura, D. Morley, S. Sathyavruthan, S. Temelso, R. E. Thomas and L. Zhang, Nat. Methods, 2013, 10, 641–646. 25 D. K. Kampouris, R. O. Kadara, N. Jenkinson and C. E. Banks, Anal. Methods, 2009, 1, 25–28. 26 A. E. Musa, M. A. Alonso-Lomillo, F. J. del Campo, N. Abramova, O. Domı´nguez-Renedo, M. J. Arcos-Martı´nez and J. P. Kutter, Talanta, 2012, 99, 737–743.
Chem. Commun., 2014, 50, 8416--8419 | 8419
Published on 11 June 2014. Downloaded by University of California - San Diego on 15/11/2014 12:58:40.
COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 8416
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Portable pH-inspired electrochemical detection of DNA amplification† Fang Zhang,ab Jian Wu,*ab Rui Wang,b Liu Wangb and Yibin Yingb
Received 23rd April 2014, Accepted 10th June 2014 DOI: 10.1039/c4cc03011g www.rsc.org/chemcomm
A portable and label-free pH-mediated electrochemical method for the detection of DNA amplification is described. With protons released as readouts, DNA amplifications were detected in realtime or at the end-point.
DNA assay has been an extremely important method in the fields ranging from agricultural product detection to the diagnosis of genetic diseases. Currently, most of the DNA assays are polymerase chain reaction (PCR)-based and employ a fluorescent probe or a dye to detect the amplicons optically. Thus, devices such as thermal blocks for amplification and precision optics for detection are required. Although numerous new amplification methods, such as loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA) and crosspriming isothermal amplification (CPA), have been developed to make DNA assays easier for implementation or miniaturization, some inherent drawbacks still cannot be avoided, including costly optical labels and bulky optical detection devices.1–6 A more portable detection method for DNA amplification is needed. Promising electrochemical approaches, which are simple, portable, low cost and highly sensitive, are well suited as alternatives to optical methods. Several groups have attempted to exploit this concept in the detection of DNA amplification, either by using DNA-intercalative electroactive molecules (e.g. methylene blue), labelled probes or by detecting the electrical conductivity changes of the reaction mixture during DNA amplification.7–20 However, these strategies are limited by the low signal resolution resulting from the weak binding affinity of electroactive molecules to dsDNA under PCR conditions, low efficiency of asymmetric amplification or disturbance from an excess amount of other ions in PCR buffer. Herein, we report the development of a novel
label-free pH-mediated electrochemical method as an alternative to fluorescent detection for DNA cross-priming isothermal amplification (CPA), which has been described in our previous work, making DNA assays more portable and much easier to miniaturize.2 Nucleotide incorporation occurs during DNA chain extension and replicates the template DNA. During this process there is nucleophilic attack on the a-phosphate group of a nucleotide from the terminal 3 0 -hydroxy group of the growing DNA strand and a proton on this terminal hydroxyl group is replaced by nucleotide base to be incorporated, that is, one proton is released from the sugar-phosphate backbone for every new phosphodiester bond formed (shown in Fig. 1).21,22 A semiconductor-based de novo nucleic acid sequencer developed by Ion Torrent Systems is also based on this concept using ISEFTs.23,24 Normally, CPA reaction would not exhibit a noticeable pH change during DNA amplification due to its buffered conditions, which was preferred for the DNA polymerase but would consume the protons released. To minimize the consumption of released protons of the reaction mixture and make the pH
a
The State Key Lab of Fluid Power Transmission and Control, Zhejiang University, Hangzhou 310027, China. E-mail: [email protected]; Tel: +86-571-88982180 b College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4cc03011g
8416 | Chem. Commun., 2014, 50, 8416--8419
Fig. 1
Mechanism for proton release during DNA amplification.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 11 June 2014. Downloaded by University of California - San Diego on 15/11/2014 12:58:40.
Communication
change caused by DNA amplification noticeable, the potential buffering compounds (e.g. Tris-HCl) were wiped off and the concentration of weak electrolyte (e.g. NH4+) was also reduced as much as possible while retaining the amplification efficiency and specificity of the reaction system. Low buffered conditions were pH sensitive but not suitable for DNA polymerase exactly. DNA polymerase behaved well in an alkaline environment but the conditions without Tris-HCl and with low concentration of NH4+ were partially acidic. In this way, the addition of KOH is needed to keep the DNA polymerase active. We termed this pH sensitive amplification as pH-CPA and employed genetically modified rice (Huahui 1) as our model target and a set of primers to target Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) gene (see the ESI†). The pH-CPA was performed in PCR tubes in a simple heat block. The time-course changes of pH during DNA amplification were examined five times continuously in two minutes using a commercial pH sensor (8220 BNWP Thermo ROSS Ultra pH electrode, Thermo Fisher Scientific Inc., US) and averaged afterwards. As shown in Fig. 2, the negative control reaction did not display any change in pH (blue, up-triangle), but the target-containing reaction showed a clear reduction of pH during DNA amplification (black, diamond and red, circle). Similar to real-time fluorescent detection of DNA amplification, the time-course pH changes of pH-CPA occurred in an S-shaped curve, with a steady phase at the beginning, a sharp decrease when the CPA amplicons were generated efficiently and protons accumulated rapidly, and finally a stationary phase when the reaction reached its plateau. The reaction with 3 mM KOH changed at a pH of 0.31 after 55 min reaction at 63 1C, while the 6.5 mM KOH reaction at 0.14. Both these two cases were added with the same target DNA concentration (approximately 103 genome copies per 50 mL). And they reached almost the same point at which pH decreased most efficiently. The KOH added was helpful to maintain DNA amplification efficiency offering a favourable pH range for enzyme activity but it was also criticized to weaken pH changes induced by the released proton. In the pH-CPA reaction described in this communication, the sample with 3 mM KOH at the beginning showed the largest change in pH (data not shown). To ensure that the signal observed was not due to spurious amplification, the reaction mixture was then separated on agarose gel showing that DNA has been amplified specifically (Fig. 2). pH-CPA can be conducted as expected under modified conditions. To evaluate the potential quantitative application of the pH-mediated DNA amplification, we performed pH-CPA with different initial copy numbers of GM rice genomic DNA (ten-fold serial dilution from 104 to 102 copies) and compared with the conventional SYTO 9-based CPA method (previous work of our group, conducted on a Biorad MyiQ2 Real Time PCR Detection System with ‘‘buffered’’ CPA).2 By applying a similar methodology as in real-time PCR, we defined the signal threshold as the point at which amplification was most efficient and the corresponding pH decreased most rapidly (Fig. 3A). For each pH-CPA reaction, this threshold is reliably decided by identifying the local minimum in the corresponding pH-change derivative trace
This journal is © The Royal Society of Chemistry 2014
ChemComm
Fig. 2 Time-course pH-change traces during pH-CPA amplification at different concentrations of KOH and pH-CPA products separated on 3% agarose gel. Negative control (blue, up-triangle) represents the reaction performed without a DNA template. Both the 6.5 mM (black, diamond) and 3 mM KOH (red, circle) cases were added with the same target DNA concentration, approximately 103 genome copies per 50 mL.
Fig. 3 Potential quantitative application of pH-CPA with serial dilutions of target DNA. (A) Real-time pH traces for samples containing 100 (blue, up-triangle), 1000 (red, circle) and 10 000 copies (black, diamond) of target GM rice genomic DNA. (B) By taking the derivative of the pH-change trace, the signal threshold is revealed as the local minimum in the curve, facilitating determination of the time to threshold (tth, vertical dashed line, defined as the reaction time required to reach the signal threshold).
(Fig. 3B). Just like the ‘‘threshold cycle’’ (Ct) in real-time PCR, we subsequently defined the ‘‘time to threshold’’ (tth) as the time required for a particular sample reaction to reach the signal threshold (Fig. 3B). We observed distinct local minima separated by approximately 3 min for each of the pH traces, with a distinct
Chem. Commun., 2014, 50, 8416--8419 | 8417
View Article Online
Published on 11 June 2014. Downloaded by University of California - San Diego on 15/11/2014 12:58:40.
ChemComm
tth for each initial target copy number (Fig. 3B). In particular, the tth of pH-CPA reaction with 100, 1000 and 10 000 copies of initial genomic DNA templates was 26, 29 and 32 min respectively. Decreased quantities of the template DNA resulted in slower accumulation of released protons, which were in turn reflected by larger tth. Compared with the optical real-time CPA (reported previously), the testing time of DNA amplification was saved by about 20%.2 To further develop a portable pH-mediated electrochemical platform for the detection of DNA amplification at the point-ofcare, a disposable and low-cost pH sensor is needed. We fabricated pH-sensitive disposable screen printed electrodes (pH SPEs) and detected CPA at the end-point. With the SPEs’ feature of single-use, opening of the tube-cap can be avoided. The SPE has a size of 23.5 mm 5 mm 0.7 mm (Fig. 4), and can be directly inserted into the 200 mL microtubes. It was modified according to the research published by Kampouris and Musa.25,26 As both pH active compounds (phenanthraquinone, PAQ) and the other pH inactive compounds (dimethylferrocene, Fc) are combined within a single working electrode, pH can be revealed as the potential difference between the two compounds (see the ESI,† Fig. S1). Therefore, potential drift of the reference half-cell was not as detrimental as for common electrochemical platforms. As shown in Fig. 5, with pH sensitive screen printed electrodes described, the potential peak
Communication
spacing in the square-wave voltammetry (SWV) curve of samples that has been amplified at 63 1C about 55 min displayed a larger dispersion degree, compared with those samples without any heat process. We speculate that such discrepancy may be due to the detection temperature variance between different treatments. And the potential peak spacing changed very little between the negative control samples before and after amplification, which meant DNA had not been amplified. While for the positive samples with initial templates about 1000 copies of genomic DNA, the potential peak spacing decreased by about 28.3 mV after pH-CPA amplification. From the standard curve of our pH sensitive disposable electrode (DEp = 0.075 pH – 0.2674, R2 = 0.9918, Fig. S2, ESI†), pH-changes at 28.3 mV can be calculated to be 0.38, which is comparable with the result obtained from a commercial pH sensor (approximately 0.35 pH). Thus, this disposable SPE can be used for DNA amplification using a pH-mediated electrochemical strategy. In conclusion, the present study has successfully developed a portable and label-free pH-mediated method for the detection of DNA amplification in CPA. Protons were detected as readouts for DNA amplification. Additionally, we devised reliable signal threshold and time-to-threshold metrics for pH-CPA analysing, demonstrating that this method possesses quantitative application ability. And with a disposable pH-sensitive screen printed electrode, DNA amplification of pH-CPA can be detected ‘‘in-the-field’’. This work is supported by the National Natural Science Foundation of China (31271617) and the National Key Technology Support Program (2012BAK08B05).
Notes and references
Fig. 4 Photograph and sketch map of the screen-printed electrode.
Fig. 5 Endpoint detection of pH-CPA using screen printed electrodes. Square-wave voltammetry (SWV) was used. Potential peak spacing between PAQ and Fc was calculated for each sample. Samples of negative control (without the DNA template) and the ones with 1000 copies of target DNA were detected before and after pH-CPA reaction at room temperature separately.
8418 | Chem. Commun., 2014, 50, 8416--8419
1 T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino and T. Hase, Nucleic Acids Res., 2000, 28, e63. 2 F. Zhang, L. Wang, K. Fan, J. Wu and Y. Ying, Anal. Bioanal. Chem., 2014, 406, 3069–3078. 3 G. T. Walker, M. S. Fraiser, J. L. Schram, M. C. Little, J. G. Nadeau and D. P. Malinowski, Nucleic Acids Res., 1992, 20, 1691–1696. 4 G. Xu, L. Hu, H. Zhong, H. Wang, S.-i. Yusa, T. C. Weiss, P. J. Romaniuk, S. Pickerill and Q. You, Sci. Rep., 2012, 2, 246. 5 N. Tomita, Y. Mori, H. Kanda and T. Notomi, Nat. Protoc., 2008, 3, 877–882. 6 M. Goto, E. Honda, A. Ogura, A. Nomoto and K.-I. Hanaki, Biotechniques, 2009, 46, 167–172. 7 M. U. Ahmed, M. Saito, M. M. Hossain, S. R. Rao, S. Furui, A. Hino, Y. Takamura, M. Takagi and E. Tamiya, Analyst, 2009, 134, 966–972. 8 B. Arneth, Anal. Biochem., 2009, 385, 26–33. 9 M. U. Ahmed, Q. Hasan, M. Mosharraf Hossain, M. Saito and E. Tamiya, Food Control, 2010, 21, 599–605. 10 N. Nagatani, K. Yamanaka, M. Saito, R. Koketsu, T. Sasaki, K. Ikuta, T. Miyahara and E. Tamiya, Analyst, 2011, 136, 5143–5150. 11 B. Y. Won, S. Shin, S. Baek, Y. L. Jung, T. Li, S. C. Shin, D. Y. Cho, S. B. Lee and H. G. Park, Analyst, 2011, 136, 1573–1579. 12 K. Yamanaka, M. Saito, K. Kondoh, M. M. Hossain, R. Koketsu, T. Sasaki, N. Nagatani, K. Ikuta and E. Tamiya, Analyst, 2011, 136, 2064–2068. 13 M. U. Ahmed, S. Nahar, M. Safavieh and M. Zourob, Analyst, 2013, 138, 907–915. 14 X. Fang, Q. Jin, F. Jing, H. Zhang, F. Zhang, H. Mao, B. Xu and J. Zhao, Biosens. Bioelectron., 2013, 44, 241–247. 15 M. Safavieh, M. U. Ahmed, A. Ng and M. Zourob, Biosens. Bioelectron., 2014, 58, 101–106. 16 M. Safavieh, M. U. Ahmed, E. Sokullu, A. Ng, L. Braescu and M. Zourob, Analyst, 2014, 139, 482–487.
This journal is © The Royal Society of Chemistry 2014
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Chem. Commun., 2014, 50, 8416--8419 | 8419
