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Cite this: DOI: 10.1039/c4cc08954e Received 10th November 2014, Accepted 2nd December 2014 DOI: 10.1039/c4cc08954e
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Ultrasensitive electrochemical detection of breast cancer cells based on DNA-rolling-circleamplification-directed enzyme-catalyzed polymerization† Qinglin Sheng, Ni Cheng, Wushuang Bai and Jianbin Zheng*
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An ultrasensitive cytosensor based on DNA-rolling-circle-amplificationdirected enzyme-catalyzed polymerization is demonstrated. As a proof of concept, the cytosensor shows excellent sensitivity for MCF-7 cell detection with a lower detection limit of 12 cells per mL.
Identification and sensitive detection of cancer cells is of great importance due to the urgent demand for rapid and highlyeffective methods for the early diagnosis of cancer diseases.1 However, because only very low concentrations of circulating tumor cells exist in peripheral blood,2 the detection of cancer cells with high specificity and sensitivity remains challenging. Currently, there are some technologies that have been applied for cancer diagnosis, including the polymerase chain reaction, immunohistochemistry, and flow cytometry.3 However, some of those methods require relatively complicated, time-consuming and expensive processes, while others still have unsatisfactory sensitivity and specificity. To overcome these problems, amplified signal schemes based on chemical and/or physical amplification have recently been investigated and developed to improve and enhance the efficiency of signal amplification.4 In most existing methods, aptamers are one of the most important and ideal molecular probes for signal amplification units for the highly specific recognition of cancer cells due to their high affinity and specificity, ease of chemical modification and lack of immunogenicity.5 Aptamers can also be easily assembled on nanomaterials and electrode surfaces to realize multi-function signal conversion between biological events and chemical and/or physical signals.6 Especially by combining specific aptamers with enzymatic reactions, electrochemical signal amplification for cancer cell detection could be realized successfully. For example, the coimmobilization of both recombinant human tumor necrosis factor-related apoptosis-inducing ligand and horseradish peroxidase
Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, China. E-mail: [email protected]; Fax: +86-29-88303448; Tel: +86-29-88303448 † Electronic supplementary information (ESI) available: Experimental section, table and figures. See DOI: 10.1039/c4cc08954e
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(HRP) on Au nanoparticle-decorated magnetic Fe3O4 beads achieved ultrasensitive detection of leukemia cells with a detection limit of B40 cells.7 Wu et al. proposed an ultrasensitive electrochemical telomerase activity sensing strategy based on nanoparticlemediated signal amplification through a solid-state Ag/AgCl reaction with DNA exonuclease III-assisted background current suppression.4b Moreover, the combination of cell–aptamer binding events with enzymatically catalyzed reactions resulted in a further improvement in cancer cell detection.8 However, certain limitations of those methods still exist, such as complex fabrication with multiple modification steps and unsatisfactory detection limits. More recently, the rolling circle amplification (RCA) reaction was found to be a very promising strategy for signal amplification and was applied for cancer cell diagnosis.9 It has been shown that sensitive and highly multiplexed assays of protein analytes with zeptomole sensitivity and broad dynamic range can be successfully realized.10 Thus, it is obvious that the biosensing of biological events, especially the detection of extremely low abundance cancer cells, could be further extended by integrating a series of amplification methods. Herein, we re-engineered the RCA reaction to trigger enzymatically catalyzed polymerization, through which cancer cells are released from the electrode surface and multiple streptavidin labeled HRPs can be attached for efficient signal amplification as illustrated in Scheme 1. A hairpin-structured aptamer sequence, SYL3C,10d with a strong binding affinity, excellent selectivity and
Scheme 1
Schematic illustration of the cytosensor assembly process.
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optimized length to recognize human breast cancer MCF-7 cells, was selected as a proof-of-concept for the proposed ultrasensitive sensing method. In this protocol, the hairpin structure P1 was composed of five fragments: a short T linker (A), a cancer-targeted aptamer sequence (B and C), a short T linker (D) and a short sequence complementary to the C fragment (E), with a thiol-C6 linked at the end of the 50 terminus (see Fig. S1 in the ESI†). In the absence of the target, the conformation of P1 is stable. When it encounters the target cancer cell, the hairpin P1 is capable of binding to protein receptors on the cell surface, leading a spontaneous conformational reorganization of the hairpin structure. The aptamer SYL3C was first used to demonstrate the feasibility of the RCA-directed enzymatically-catalyzed amplification-based strategy for electrochemical cancer detection. The SYL3C aptamer can specifically recognize human cancer cells that overexpress the epithelial cell adhesion molecule.10d Confocal fluorescence microscopy was first utilized to image the intracellular fluorescence resulting from the uptake of the SYL3C aptamer in MCF-7 cells. Typical confocal fluorescence microscopy images of MCF-7 cells without (A and B) or with treatment with P1 (C and D) are shown in Fig. S2 (ESI†). The MCF-7 cells were distributed separately before the addition of P1. After the incubation of the MCF-7 cells with P1, the cells were obviously aggregated. This result indicates that the added P1 is capable of binding to protein receptors on the cell surface, and the conformational changes lead to the exposure of the E-strand of the capturing probe P1. The hybrid interactions of those E-strands exposed to the outside of the MCF-7 cell surfaces result in the aggregation of cells. In order to have further evidence of the recognition by P1 of MCF-7 cells, comparison experiments were also conducted by incubating MCF-7 cells with P1, P3 (a DNA sequence containing another aptamer sequence that can also recognize MCF-7 cells) and P4 (a random DNA sequence with equal length to P1). As shown in Fig. 1, the confocal fluorescence microscopy images of MCF-7 cells treated with P4 (Fig. 1D) are almost the same as those of native state MCF-7 cells (Fig. 1A), whereas MCF-7 cells treated with P1 (Fig. 1B) or P3 (Fig. 1C) in both cases show obvious cell aggregation. Fig. 1E shows the native
Fig. 1 Confocal fluorescence microscopy images of MCF-7 cells treated without (A) or with the hairpin-aptamer P1 (B), P3 (C) and the random DNA sequence P5 (D). Native PAGE (12%) analysis (E) of the recognition of hairpin-aptamers with MCF-7 cells: (a) P1, (b) P3, (c) P5, (d) MCF-7 cells, (e) P1-MCF-7 cells, (f) P3-MCF-7 cells and (g) P5-MCF-7 cells.
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polyacrylamide gel electrophoresis image of different DNA sequences and MCF-7 cells before and after incubation. Through comparing the bands in these lanes, we can see that the mobilities of the P1 and P3 bands were almost blocked after incubation with MCF-7 cells, while for the P4 strand, the band after incubation of MCF-7 cells was almost the same as that before incubation. Therefore, the polyacrylamide gel electrophoresis results and confocal fluorescence data match well with each other, suggesting a reliable DNA sequence that functions as we designed. To achieve the electrochemical sensing of MCF-7 cells with the RCA-directed enzymatically catalyzed amplification-based strategy, the response of the biosensor towards MCF-7 target cells was first evaluated. Fig. 2A shows the cyclic voltammograms of the biosensor before (curve a) and after (curve b) incubation with 1 106 MCF-7 target cells per mL. After treatment, the electrodes were washed and transferred into a blank 0.1 M HAc–NaAc solution (pH 4.3) to record the electrochemical signals. As shown in curve a, no redox peaks were observed in the potential range of 0.2 to 0.7 V. However, one pair of well-defined redox peaks was observed in curve b. This result indicated that polyaniline (PANI) was formed after the RCA-directed enzymatically catalyzed deposition. The reduction and oxidation peak potentials at 0.217 V and 0.273 V (vs. SCE) were similar to those observed in some previous studies and can be ascribed to the leucoemeraldine/emeraldine transition of PANI.11 Additional experiments showed that the redox peak currents increased linearly with increasing scan rates, suggesting a surface-confined process of the electrode surface (Fig. S3, ESI†). The electrochemical responses of the formed PANI after sensing of MCF-7 cells have been further proven by the results of square wave voltammetry (SWV). As is shown in Fig. 2B, almost symmetric SWV curves were obtained, suggesting
Fig. 2 (A) Cyclic voltammograms of the modified electrodes in 0.1 M HAc– NaAc solution (pH 4.3) before (a) and after (b) the RCA-directed enzymatically catalytic reaction; (B) SWV of the modified electrodes in 0.1 M HAc–NaAc solution (pH 4.3) before (a) and after (b) the RCA-directed enzymatically catalytic reaction; (C) comparison of the recognition abilities of the aptamerhairpin probes P1 and P3 recorded by SWV signals after incubation with 1 105 MCF-17 cells per mL; AFM images of (D) MCF-7 cells and (E) PANItrapped DNA on the electrode surface; (F) Nyquist diagrams corresponding to each step of the modification of the Au electrode: (a) the bare Au electrode, (b) the electrode after hairpin-aptamer P1 modification, (c) after treatment with 1 106 MCF-7 cells per mL, (d) after the RCA reaction, and (e) after the deposition of PANI.
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the RCA-directed enzyme-catalyzed polymerization of aniline to PANI was accomplished with predominantly head-to-tail coupling, and deterred parasitic branching.11,12 The FT-IR absorption spectra of the PANI formed during the RCA-directed enzymatically catalyzed deposition also suggested a head-to-tail coupling pattern of PANI (Fig. S4, ESI†).11a To demonstrate the designed RCA-directed enzymatically catalyzed amplification strategy for cell assay, an RCA-free process was designed as a control. Results showed that the peak current obtained with the RCA-directed enzymatically catalyzed amplification strategy was 10 times higher than obtained with the RCA-free control. This suggests that the rolling cyclic process accompanied by the biotin–streptavidin interaction induced a dramatic increase in the HRP-tagged DNA duplex on the electrode surface and resulted in a significant signal amplification. Confocal fluorescence microscopy results have shown that the aptamer-hairpin probe P3 also has recognition ability towards MCF-17 cells. Thus, the recognition ability of the aptamer-hairpin probe P3 was also tested by evaluating the SWV signal in the presence of 1 105 MCF-17 cells per mL (Fig. 2C). The results showed that the current response of the aptamer-hairpin probe P1 was two times higher than that of the aptamer-hairpin probe P3, suggesting that the aptamer–cell interaction equilibrium dissociation constant (Kd) of P3 is higher than that of P1, which is in agreement with the previous results.10d Thus, the aptamer-hairpin probe P1 was selected for recognition of MCF-7 cells in the following experiments. The specific recognition of MCF-7 cells as well as the formation of PANI on the Au electrode surfaces were also confirmed using atomic force microscopy (AFM) images. As shown in Fig. 2D, the MCF-7 cells were assembled at the Au electrode surface. After incubating the MCF-7-cell-assembled Au electrode in the RCA reaction solution and enzymatic reaction solution, no cells were observed at the electrode surface, but many short bunched nanostructures were observed (Fig. 2E). Those short bunched nanostructures can be ascribed to the formation of PANI, templated by the DNA strands. The AFM images further proved the feasibility of the proposed scheme. Electrochemical impedance spectroscopy (EIS) is also an effective method for probing the features of surface changes at the electrode surfaces.13 Fig. 2F shows the EIS results during the sensing of MCF-7 cells. Curve a in Fig. 2F shows no semicircle but instead an almost straight line of the bare gold electrode, suggesting a diffusional limiting step of the electrochemical process. Curve b shows an enhancement of electron-transfer resistance (Ret) after the assembly of aptamer-hairpin P1 on the Au electrode, indicating a repulsion of the redox label was anticipated to inhibit the interfacial electron transfer. The further increase of Ret suggested a specific binding of MCF-17 cells to the aptamer assembled electrode (curve c). The binding of the aptamer to MCF-17 cells leads to a conformation change of the P1 probe. The opened site of the P1 probe will then hybridize with the biotinylated-primer, and then trigger the primer extension reaction and the biotin–streptavidin interaction. After the formation of the HRP-tagged DNA duplex (curve d), a significant increase in the Ret value was observed (curve d). Nevertheless, after the enzyme-catalyzed deposition of PANI around the DNA duplex, the Ret value decreased
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Fig. 3 (A) SWVs obtained at the cytosensor toward MCF-7 cells. From a to h: 0, 20, 50, 100, 103, 104, 105, and 5 106 cells per mL. (B) The calibration plot of the peak current versus the logarithm of the concentration of MCF7 cells. The error bars represent the standard deviations (n = 5).
significantly (curve e), suggesting that the formed PANI accelerated the electron transfer of [Fe(CN)6]3/4. To elucidate the analytical performance of the assay, a calibration experiment was conducted. As illustrated in Scheme 1, the peak current is related to the concentration of MCF-7 cells added to the incubation solution. As shown in Fig. 3, the SWV peak currents increased with increasing cell concentration and displayed a linear relationship between DIp and the logarithm of the MCF-7 cell concentration over the range from 20 to 5 106 cells per mL. A lower detection limit of 12 cells per mL at 3s for MCF-7 cells was obtained. This value is significantly lower or similar to those of leaky surface acoustic wave aptasensor arrays14 and other electrochemical cytosensing approaches,15 suggesting an enhanced signal amplification in the RCA-directed enzymatically catalyzed amplification. The precision and reproducibility of the proposed method was evaluated by measuring the responses of the cytosensor towards 1.0 105 MCF-7 cells per mL. The results suggested that relative standard deviations of 8.0% are acceptable. When not in use, the cytosensor was stored at 4 1C and the analytical performance did not decline obviously. Thus, the designed strategy shows good performance as a cancer cell assay. In summary, we have demonstrated an ultrasensitive electrochemical cytosensor based on RCA-directed enzymatically catalyzed amplification, and successfully applied it for the electrochemical cytosensing of MCF-7 cells. The RCA-directed enzymatically catalyzed amplification strategy demonstrates remarkable amplification performance by transferring an aptamer–cell recognition event to a lot of enzyme-tagged DNA duplexes, utilizing the biotin–streptavidin interaction unit and the enzymatically catalyzed deposition of PANI. By designing more target-specific aptamer-hairpin probes and combining more efficient signal amplification strategies, this electrochemical approach may be readily extended to the cytosensing of other types of cancer cells and clinical diagnostic applications. This work was supported by the NSFC (No. 21105080, 21275116), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20126101120023), the Natural Science Foundation of Shaanxi Province of China (No. 2012JM2013 and 2013KJXX-25), the Scientific Research Foundation of Shaanxi Provincial Key Laboratory (12JS087, 13JS097, 13JS098) and the Foundation of Shaanxi Province Educational Committee of China (No. 12JK0576).
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Cite this: DOI: 10.1039/c4cc08954e Received 10th November 2014, Accepted 2nd December 2014 DOI: 10.1039/c4cc08954e
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Ultrasensitive electrochemical detection of breast cancer cells based on DNA-rolling-circleamplification-directed enzyme-catalyzed polymerization† Qinglin Sheng, Ni Cheng, Wushuang Bai and Jianbin Zheng*
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An ultrasensitive cytosensor based on DNA-rolling-circle-amplificationdirected enzyme-catalyzed polymerization is demonstrated. As a proof of concept, the cytosensor shows excellent sensitivity for MCF-7 cell detection with a lower detection limit of 12 cells per mL.
Identification and sensitive detection of cancer cells is of great importance due to the urgent demand for rapid and highlyeffective methods for the early diagnosis of cancer diseases.1 However, because only very low concentrations of circulating tumor cells exist in peripheral blood,2 the detection of cancer cells with high specificity and sensitivity remains challenging. Currently, there are some technologies that have been applied for cancer diagnosis, including the polymerase chain reaction, immunohistochemistry, and flow cytometry.3 However, some of those methods require relatively complicated, time-consuming and expensive processes, while others still have unsatisfactory sensitivity and specificity. To overcome these problems, amplified signal schemes based on chemical and/or physical amplification have recently been investigated and developed to improve and enhance the efficiency of signal amplification.4 In most existing methods, aptamers are one of the most important and ideal molecular probes for signal amplification units for the highly specific recognition of cancer cells due to their high affinity and specificity, ease of chemical modification and lack of immunogenicity.5 Aptamers can also be easily assembled on nanomaterials and electrode surfaces to realize multi-function signal conversion between biological events and chemical and/or physical signals.6 Especially by combining specific aptamers with enzymatic reactions, electrochemical signal amplification for cancer cell detection could be realized successfully. For example, the coimmobilization of both recombinant human tumor necrosis factor-related apoptosis-inducing ligand and horseradish peroxidase
Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, China. E-mail: [email protected]; Fax: +86-29-88303448; Tel: +86-29-88303448 † Electronic supplementary information (ESI) available: Experimental section, table and figures. See DOI: 10.1039/c4cc08954e
This journal is © The Royal Society of Chemistry 2014
(HRP) on Au nanoparticle-decorated magnetic Fe3O4 beads achieved ultrasensitive detection of leukemia cells with a detection limit of B40 cells.7 Wu et al. proposed an ultrasensitive electrochemical telomerase activity sensing strategy based on nanoparticlemediated signal amplification through a solid-state Ag/AgCl reaction with DNA exonuclease III-assisted background current suppression.4b Moreover, the combination of cell–aptamer binding events with enzymatically catalyzed reactions resulted in a further improvement in cancer cell detection.8 However, certain limitations of those methods still exist, such as complex fabrication with multiple modification steps and unsatisfactory detection limits. More recently, the rolling circle amplification (RCA) reaction was found to be a very promising strategy for signal amplification and was applied for cancer cell diagnosis.9 It has been shown that sensitive and highly multiplexed assays of protein analytes with zeptomole sensitivity and broad dynamic range can be successfully realized.10 Thus, it is obvious that the biosensing of biological events, especially the detection of extremely low abundance cancer cells, could be further extended by integrating a series of amplification methods. Herein, we re-engineered the RCA reaction to trigger enzymatically catalyzed polymerization, through which cancer cells are released from the electrode surface and multiple streptavidin labeled HRPs can be attached for efficient signal amplification as illustrated in Scheme 1. A hairpin-structured aptamer sequence, SYL3C,10d with a strong binding affinity, excellent selectivity and
Scheme 1
Schematic illustration of the cytosensor assembly process.
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optimized length to recognize human breast cancer MCF-7 cells, was selected as a proof-of-concept for the proposed ultrasensitive sensing method. In this protocol, the hairpin structure P1 was composed of five fragments: a short T linker (A), a cancer-targeted aptamer sequence (B and C), a short T linker (D) and a short sequence complementary to the C fragment (E), with a thiol-C6 linked at the end of the 50 terminus (see Fig. S1 in the ESI†). In the absence of the target, the conformation of P1 is stable. When it encounters the target cancer cell, the hairpin P1 is capable of binding to protein receptors on the cell surface, leading a spontaneous conformational reorganization of the hairpin structure. The aptamer SYL3C was first used to demonstrate the feasibility of the RCA-directed enzymatically-catalyzed amplification-based strategy for electrochemical cancer detection. The SYL3C aptamer can specifically recognize human cancer cells that overexpress the epithelial cell adhesion molecule.10d Confocal fluorescence microscopy was first utilized to image the intracellular fluorescence resulting from the uptake of the SYL3C aptamer in MCF-7 cells. Typical confocal fluorescence microscopy images of MCF-7 cells without (A and B) or with treatment with P1 (C and D) are shown in Fig. S2 (ESI†). The MCF-7 cells were distributed separately before the addition of P1. After the incubation of the MCF-7 cells with P1, the cells were obviously aggregated. This result indicates that the added P1 is capable of binding to protein receptors on the cell surface, and the conformational changes lead to the exposure of the E-strand of the capturing probe P1. The hybrid interactions of those E-strands exposed to the outside of the MCF-7 cell surfaces result in the aggregation of cells. In order to have further evidence of the recognition by P1 of MCF-7 cells, comparison experiments were also conducted by incubating MCF-7 cells with P1, P3 (a DNA sequence containing another aptamer sequence that can also recognize MCF-7 cells) and P4 (a random DNA sequence with equal length to P1). As shown in Fig. 1, the confocal fluorescence microscopy images of MCF-7 cells treated with P4 (Fig. 1D) are almost the same as those of native state MCF-7 cells (Fig. 1A), whereas MCF-7 cells treated with P1 (Fig. 1B) or P3 (Fig. 1C) in both cases show obvious cell aggregation. Fig. 1E shows the native
Fig. 1 Confocal fluorescence microscopy images of MCF-7 cells treated without (A) or with the hairpin-aptamer P1 (B), P3 (C) and the random DNA sequence P5 (D). Native PAGE (12%) analysis (E) of the recognition of hairpin-aptamers with MCF-7 cells: (a) P1, (b) P3, (c) P5, (d) MCF-7 cells, (e) P1-MCF-7 cells, (f) P3-MCF-7 cells and (g) P5-MCF-7 cells.
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polyacrylamide gel electrophoresis image of different DNA sequences and MCF-7 cells before and after incubation. Through comparing the bands in these lanes, we can see that the mobilities of the P1 and P3 bands were almost blocked after incubation with MCF-7 cells, while for the P4 strand, the band after incubation of MCF-7 cells was almost the same as that before incubation. Therefore, the polyacrylamide gel electrophoresis results and confocal fluorescence data match well with each other, suggesting a reliable DNA sequence that functions as we designed. To achieve the electrochemical sensing of MCF-7 cells with the RCA-directed enzymatically catalyzed amplification-based strategy, the response of the biosensor towards MCF-7 target cells was first evaluated. Fig. 2A shows the cyclic voltammograms of the biosensor before (curve a) and after (curve b) incubation with 1 106 MCF-7 target cells per mL. After treatment, the electrodes were washed and transferred into a blank 0.1 M HAc–NaAc solution (pH 4.3) to record the electrochemical signals. As shown in curve a, no redox peaks were observed in the potential range of 0.2 to 0.7 V. However, one pair of well-defined redox peaks was observed in curve b. This result indicated that polyaniline (PANI) was formed after the RCA-directed enzymatically catalyzed deposition. The reduction and oxidation peak potentials at 0.217 V and 0.273 V (vs. SCE) were similar to those observed in some previous studies and can be ascribed to the leucoemeraldine/emeraldine transition of PANI.11 Additional experiments showed that the redox peak currents increased linearly with increasing scan rates, suggesting a surface-confined process of the electrode surface (Fig. S3, ESI†). The electrochemical responses of the formed PANI after sensing of MCF-7 cells have been further proven by the results of square wave voltammetry (SWV). As is shown in Fig. 2B, almost symmetric SWV curves were obtained, suggesting
Fig. 2 (A) Cyclic voltammograms of the modified electrodes in 0.1 M HAc– NaAc solution (pH 4.3) before (a) and after (b) the RCA-directed enzymatically catalytic reaction; (B) SWV of the modified electrodes in 0.1 M HAc–NaAc solution (pH 4.3) before (a) and after (b) the RCA-directed enzymatically catalytic reaction; (C) comparison of the recognition abilities of the aptamerhairpin probes P1 and P3 recorded by SWV signals after incubation with 1 105 MCF-17 cells per mL; AFM images of (D) MCF-7 cells and (E) PANItrapped DNA on the electrode surface; (F) Nyquist diagrams corresponding to each step of the modification of the Au electrode: (a) the bare Au electrode, (b) the electrode after hairpin-aptamer P1 modification, (c) after treatment with 1 106 MCF-7 cells per mL, (d) after the RCA reaction, and (e) after the deposition of PANI.
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the RCA-directed enzyme-catalyzed polymerization of aniline to PANI was accomplished with predominantly head-to-tail coupling, and deterred parasitic branching.11,12 The FT-IR absorption spectra of the PANI formed during the RCA-directed enzymatically catalyzed deposition also suggested a head-to-tail coupling pattern of PANI (Fig. S4, ESI†).11a To demonstrate the designed RCA-directed enzymatically catalyzed amplification strategy for cell assay, an RCA-free process was designed as a control. Results showed that the peak current obtained with the RCA-directed enzymatically catalyzed amplification strategy was 10 times higher than obtained with the RCA-free control. This suggests that the rolling cyclic process accompanied by the biotin–streptavidin interaction induced a dramatic increase in the HRP-tagged DNA duplex on the electrode surface and resulted in a significant signal amplification. Confocal fluorescence microscopy results have shown that the aptamer-hairpin probe P3 also has recognition ability towards MCF-17 cells. Thus, the recognition ability of the aptamer-hairpin probe P3 was also tested by evaluating the SWV signal in the presence of 1 105 MCF-17 cells per mL (Fig. 2C). The results showed that the current response of the aptamer-hairpin probe P1 was two times higher than that of the aptamer-hairpin probe P3, suggesting that the aptamer–cell interaction equilibrium dissociation constant (Kd) of P3 is higher than that of P1, which is in agreement with the previous results.10d Thus, the aptamer-hairpin probe P1 was selected for recognition of MCF-7 cells in the following experiments. The specific recognition of MCF-7 cells as well as the formation of PANI on the Au electrode surfaces were also confirmed using atomic force microscopy (AFM) images. As shown in Fig. 2D, the MCF-7 cells were assembled at the Au electrode surface. After incubating the MCF-7-cell-assembled Au electrode in the RCA reaction solution and enzymatic reaction solution, no cells were observed at the electrode surface, but many short bunched nanostructures were observed (Fig. 2E). Those short bunched nanostructures can be ascribed to the formation of PANI, templated by the DNA strands. The AFM images further proved the feasibility of the proposed scheme. Electrochemical impedance spectroscopy (EIS) is also an effective method for probing the features of surface changes at the electrode surfaces.13 Fig. 2F shows the EIS results during the sensing of MCF-7 cells. Curve a in Fig. 2F shows no semicircle but instead an almost straight line of the bare gold electrode, suggesting a diffusional limiting step of the electrochemical process. Curve b shows an enhancement of electron-transfer resistance (Ret) after the assembly of aptamer-hairpin P1 on the Au electrode, indicating a repulsion of the redox label was anticipated to inhibit the interfacial electron transfer. The further increase of Ret suggested a specific binding of MCF-17 cells to the aptamer assembled electrode (curve c). The binding of the aptamer to MCF-17 cells leads to a conformation change of the P1 probe. The opened site of the P1 probe will then hybridize with the biotinylated-primer, and then trigger the primer extension reaction and the biotin–streptavidin interaction. After the formation of the HRP-tagged DNA duplex (curve d), a significant increase in the Ret value was observed (curve d). Nevertheless, after the enzyme-catalyzed deposition of PANI around the DNA duplex, the Ret value decreased
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Fig. 3 (A) SWVs obtained at the cytosensor toward MCF-7 cells. From a to h: 0, 20, 50, 100, 103, 104, 105, and 5 106 cells per mL. (B) The calibration plot of the peak current versus the logarithm of the concentration of MCF7 cells. The error bars represent the standard deviations (n = 5).
significantly (curve e), suggesting that the formed PANI accelerated the electron transfer of [Fe(CN)6]3/4. To elucidate the analytical performance of the assay, a calibration experiment was conducted. As illustrated in Scheme 1, the peak current is related to the concentration of MCF-7 cells added to the incubation solution. As shown in Fig. 3, the SWV peak currents increased with increasing cell concentration and displayed a linear relationship between DIp and the logarithm of the MCF-7 cell concentration over the range from 20 to 5 106 cells per mL. A lower detection limit of 12 cells per mL at 3s for MCF-7 cells was obtained. This value is significantly lower or similar to those of leaky surface acoustic wave aptasensor arrays14 and other electrochemical cytosensing approaches,15 suggesting an enhanced signal amplification in the RCA-directed enzymatically catalyzed amplification. The precision and reproducibility of the proposed method was evaluated by measuring the responses of the cytosensor towards 1.0 105 MCF-7 cells per mL. The results suggested that relative standard deviations of 8.0% are acceptable. When not in use, the cytosensor was stored at 4 1C and the analytical performance did not decline obviously. Thus, the designed strategy shows good performance as a cancer cell assay. In summary, we have demonstrated an ultrasensitive electrochemical cytosensor based on RCA-directed enzymatically catalyzed amplification, and successfully applied it for the electrochemical cytosensing of MCF-7 cells. The RCA-directed enzymatically catalyzed amplification strategy demonstrates remarkable amplification performance by transferring an aptamer–cell recognition event to a lot of enzyme-tagged DNA duplexes, utilizing the biotin–streptavidin interaction unit and the enzymatically catalyzed deposition of PANI. By designing more target-specific aptamer-hairpin probes and combining more efficient signal amplification strategies, this electrochemical approach may be readily extended to the cytosensing of other types of cancer cells and clinical diagnostic applications. This work was supported by the NSFC (No. 21105080, 21275116), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20126101120023), the Natural Science Foundation of Shaanxi Province of China (No. 2012JM2013 and 2013KJXX-25), the Scientific Research Foundation of Shaanxi Provincial Key Laboratory (12JS087, 13JS097, 13JS098) and the Foundation of Shaanxi Province Educational Committee of China (No. 12JK0576).
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