Article pubs.acs.org/ac
Visual and Highly Sensitive Detection of Cancer Cells by a Colorimetric Aptasensor Based on Cell-Triggered Cyclic Enzymatic Signal Amplification Xianxia Zhang, Kunyi Xiao, Liwei Cheng, Hui Chen, Baohong Liu, Song Zhang,* and Jilie Kong* Department of Chemistry, Fudan University, Shanghai 200433, P. R. China S Supporting Information *
ABSTRACT: Rapid and efficient detection of cancer cells at their earliest stages is one of the central challenges in cancer diagnostics. We developed a simple, cost-effective, and highly sensitive colorimetric method for visually detecting rare cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and linker DNAs stably coexist in solution, and the linker DNA assembles DNA-AuNPs, producing a purple solution. In the presence of target cells, the specific binding of HAPs to the target cells triggers a conformational switch that results in linker DNA hybridization and cleavage by nicking endonuclease-strand scission cycles. Consequently, the cleaved fragments of linker DNA can no longer assemble into DNAAuNPs, resulting in a red color. UV−vis spectrometry and photograph analyses demonstrated that this CTCESA-based method exhibited selective and sensitive colorimetric responses to the presence of target CCRF-CEM cells, which could be detected by the naked eye. The linear response for CCRF-CEM cells in a concentration range from 102 to 104 cells was obtained with a detection limit of 40 cells, which is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. Given the high specificity and sensitivity of CTCESA, this colorimetric method provides a sensitive, labelfree, and cost-effective approach for early cancer diagnosis and point-to-care applications.
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tides originating from an in vitro strategy termed systematic evolution of ligands by exponential enrichment (SELEX),10 can bind a number of specific molecular targets, such as small chemicals, proteins, and even cells, with high affinity and specificity comparable to that of antibodies.11 Furthermore, aptamers offer distinct advantages over antibodies, such as small size, high stability, low cost, ease of modification, nontoxicity, and lack of immunogenicity.12,13 Many high-affinity aptamers that specifically bind to different target cancer cells have been created using cell-SELEX in recent years,1 and these efforts have created a great opportunity to develop aptamer-based biosensors for early cancer diagnosis. However, the direct detection of cancer cells using aptamers is severely hindered by the fact that most aptamer-cell interactions do not produce an easily measurable output. Many new aptasensors have been developed successfully for cancer cell analysis with several sensitive methods, such as colorimetry,14 fluorescence,15,16 electrochemistry,17 and magnetic field,18 and some signal amplification strategies, such as gold nanoparticles (AuNPs),19 magnetic core-gold shell nanoparticles,20 quantum dots (QDs),21 and PCR reaction,22 have been applied to improve the sensitivity of these aptamer-based approaches in
he development of a detection method with high selectivity and sensitivity for cancer at its earliest stages is one of the central challenges in cancer prevention, diagnostics, and treatment. By measuring specific cellular levels within the blood, a doctor can judge the patient’s responses to treatment or predict the probability of the onset of a specific disease.1 Most importantly, screening people with no symptoms to find early signs of cancer in a routine blood test is better, and the chances of cure are higher, than diagnosing suspected cancer.2,3 However, traditional analysis techniques, such as immunohistochemistry, flow cytometry, and polymerase chain reaction (PCR),4−6 do not meet the requirement of point-of-care (POC) diagnostics because they usually require costly instruments, long analytical time, and complicated operations.7 Meanwhile, considering the very low quantity of stray cancer cells,1−3 screening requires a new clinical platform with high specificity and ultrasensitivity in the detection of cancer cells, especially for preclinical diagnostics. Furthermore, the approach should reduce costs in order to screen more patients at the earliest stage and prevent cancer deaths, especially in developing countries. Thus, the development of a simple diagnostic tool for specific and ultrasensitive detection of rare cancer cells at low cost is necessary. Coupling nanomaterials and biomolecular recognition events represents a new direction toward the development of novel molecular diagnostic tools.8,9 Aptamers, artificial oligonucleo© 2014 American Chemical Society
Received: March 25, 2014 Accepted: May 12, 2014 Published: May 12, 2014 5567
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Figure 1. Schematic illustration of a highly sensitive colorimetric method for the detection of rare cancer cells based on CTCESA.
Table 1. Oligonucleotide Sequences Used in This Study sequencea
probe
hairpin aptamer probe-1 (HAP-1) 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAA ATC CTC AGC AGT3′ hairpin aptamer probe-2 (HAP-2) 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA3′ hairpin aptamer probe-3 (HAP-3) 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA TCC TCA GCA GTT AGA3′ linker DNA 5′-GCA AAC AAC TGC↓TGA GGA TAA ACG-3′ DNA1 5′-SH-(CH2)6-CGT TTA TCC TCA-3′ DNA2 5′-GCA GTT GTT TGC-(CH2)6-SH-3′ a
Bold letters are the CCRF-CEM cell aptamer sequence. Italic letters are the partially complementary sequence to the CCRF-CEM cell aptamer. Underlined letters are the NEase recognition sequence. The arrow indicates the nicking position.
and strong distance-dependent surface plasmon resonance (SPR) absorption. The color of the AuNP solution can change from red to purple in response to the SPR absorption of dispersed and aggregated nanoparticles,25 but the limited sensitivity and selectivity affects their use in early cancer diagnosis.33,34 To address these drawbacks, amplification of the detection signal is remarkably important. Until now, many signal amplification strategies have been reported,22,35 but a few amplified strategies are available for AuNP-based colorimetric methods. In addition, only a few highly sensitive colorimetric aptasensors have been developed by nicking endonucleaseassisted nanoparticle amplification (NEANA) for the detection of potassium,36 DNA (10 pM),30 and thrombin (20 pM).25 However, the colorimetric detection of rare cancer cells with such a NEANA technique has not been reported. Therefore, we tried to design a simple, sensitive, selective, and cost-effective colorimetric aptasensor for the detection of cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA).
cancer analysis. Using the primary cell-detecting strategy, “always-on” aptamer probes were designed and labeled with a sensitive reporter that can specifically bind to the target cells and produce obviously enhanced signals. An intrinsic disadvantage of “always-on” probes is that they emit constant signals into the surrounding solution, resulting in considerable background signal. To improve the signal-to-background ratio, the unbound aptamers need to be washed carefully before each measurement. Recently, a more efficient “signal-on” strategy was developed by an activated aptamer probe; its binding to target cancer cells triggers a conformational change and activates fluorescence based on Förster resonance energy transfer (FRET).15 In a previous study, we developed a graphene oxide-based FRET biosensor with a dye-labeled aptamer probe and implemented it in a miniature multiplex chip for visual detection of cancer cells with high sensitivity and specificity.16 However, the application of this strategy has some limitations, including the need for expensive instruments and the compulsory modification of a fluorophore and quencher pair at its two terminals (or a terminal). Compared to costly fluorescence instruments, AuNP-based colorimetric methods23 have attracted significant interest in clinic diagnostics due to their visualization, simplicity, and low cost. T-cell acute lymphoblastic leukemia (ALL) is a malignant thymocyte disease, accounting for 10%−15% of pediatric ALL cases and 25% of adult ALL cases.24 In the present study, CCRF-CEM cells were chosen as a model of rare cancer and a label-free hairpin aptamer probe (HAP) designed to specifically bind the target cancer cells and produce a spontaneous conformational change (Figure 1). AuNP-based colorimetric sensors have been widely applied for the detection of a broad range of analytes, including proteins,25−27 DNA,28−30 and metal ions.31,32 With these methods, AuNPs have emerged as colorimetric reporters due to their high extinction coefficients
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EXPERIMENTAL SECTION Materials. The oligonucleotides used in this study were synthesized by Sangon Biotechnology Co. (Shanghai, China) (Table 1). NEase (Nb.BbvCI) and 10× NEB buffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, and 10 mM dithiothreitol, pH 7.9) were purchased from New England Biolabs (USA). Chloroauric acid (HAuCl 4 ), tris(2carboxyethyl)phosphine (TCEP), and trisodium citrate were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS), normal bovine calf serum (NBCS), Dulbecco’s modified eagle medium (DMEM), and penicillin/streptomycin were purchased from Gibco (USA). SYBR Green II (10,000× stock solution in dimethyl sulfoxide, 50 μL) was purchased from Bio5568
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CCRF-CEM cells triggers its conformational change, resulting in a nicking endonuclease-strand scission cycle and detection by the three-component sandwich assay.33 Our detection system consisted of a HAP, linker DNA, two sets of DNA-modified AuNPs (Table 1), and NEase. The HAP contained three domains: I, II, and III. Region I was the sequence of the aptamer for CCRF-CEM cells,15,39 which was partially caged in the duplex structure of the stem by hybridization with region III. Region II was a single-stranded loop, and region III was complementary to the linker DNA, where the associated DNA duplex contained the recognition sequence for NEase. NEase is a nicking endonuclease that cleaves only one strand of DNA on a double-stranded DNA substrate.40,41 The NEase used in this study was Nb.BbvCI, which recognizes a simple asymmetric sequence: 5′-GCTGAGG-3′. The asymmetric sequence was designed in the middle of the linker DNA (Table 1). According to the design and calculation by Oligo Analyzer 3.1 software from IDT, the stability of the HAP should be greater than that of HAP-Linker DNA and less than that of the HAP-cell complex, which plays important roles in the selectivity and sensitivity of the cell aptasensor. In the absence of target cells, the HAP was in a stable hairpin structure and coexisted with linker DNA in solution. In a threecomponent sandwich assay (Figure 1), the linker DNA assembled two sets of DNA-AuNPs due to their complementary sequences, inducing aggregation and a clear color change from red to purple. In contrast, when the HAP was incubated with target cancer cells, the binding of the HAP to protein receptors on the cell membrane triggered spontaneous conformational reorganization15 and region III was exposed to form a switched HAP. Consequently, the exposed region III could hybridize to the linker DNA and form the new HAP/ linker DNA duplex, which is recognized and cleaved by NEase. The cleaved linker DNA fragments dissociated from the unstable duplex and released free switched HAP. Then, the switched HAP could hybridize with another intact linker DNA to form a new substrate for NEase and initiate new nicking endonuclease-strand scission cycles. In the subsequent threecomponent sandwich assay, the cleaved fragments of linker DNA could no longer assemble two DNA-AuNPs and the red color of separated AuNPs was observed. Because the increased number of target cells allowed more linker DNA to be cleaved by CTCESA, more separated (rather than aggregated) AuNPs were present and the quantitative and visual information about target cells was detected by the AuNP-based colorimetric aptasensor. Feasibility of the Method for the Detection of CCRFCEM Cells. The cell-triggered nicking endonuclease-strand scission cycle was verified by PAGE. The HAP and linker DNA stably coexisted in solution when target CCRF-CEM cells were absent, and no cleaved fragments of linker DNA were found with NEase treatment (Figure 2A, lanes 1−3). After adding CCRF-CEM cells and treating the mixture with NEase, cleaved fragments of linker DNA appeared, which migrated faster than the linker DNA (Figure 2A, lane 4). Furthermore, during the cell-triggered nicking endonuclease-strand scission cycle, the HAP was stable. To further confirm that the cleavage of linker DNA by CTCESA can alter the optical properties of DNA-modified AuNPs, UV−visible spectroscopy and TEM were carried out to test the feasibility of the colorimetric design. Figure 2B shows the absorption spectra and photographs of DNA-modified AuNP solutions for cancer cell analysis under different
Vision Biotechnology (Xiamen, China). All other reagents and solvents were analytical grade and used as received without further purification. Double distilled water was used for all experiments. Cells. T-cell acute lymphoblastic leukemia cells (CCRFCEM cells), human cervix carcinoma cells (HeLa cells), and mouse fibroblast cells (NIH-3T3 cells) were purchased from the cell bank of the Chinese Academy of Sciences. CCRF-CEM cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 100 units/mL penicillin/streptomycin and incubated at 37 °C in a humidified incubator containing 5 wt %/vol CO2. HeLa cells were cultured in DMEM medium supplemented with 10% FBS. NIH-3T3 cells were cultured in DMEM medium supplemented with 10% NBCS. Cell density was determined using a hemocytometer prior to any experiments. Apparatus. The size and morphology of AuNPs were characterized by transmission electron microscopy (TEM) using a JEOL model JEM-2011(HR) at 200 kV. The UV−vis absorption spectra were recorded in a Shimadzu UV-3150 spectrophotometer using 1 cm path length quartz microcuvettes. Polyacrylamide gel electrophoresis (PAGE) of the HAP, linker DNA, HAP and linker DNA mixture, and cleavage products was carried out with a 16% denaturing polyacrylamide gel in Tris/borate/EDTA (TBE) buffer (1× ) at 400 V for 30 min. The gel was stained with SYBR Green II and imaged under an ultraviolet transilluminator. Preparation of DNA-Modified AuNPs. AuNPs with an average diameter of 13 nm were prepared via citrate reduction of HAuCl4 as described previously,23 and the size of the AuNPs was verified by TEM and their concentration estimated by UV−vis spectroscopy. The AuNPs were modified with two different thiol-modified oligonucleotides (DNA1 and DNA2) as described previously, with minor modifications.37,38 Briefly, thiol-modified DNA1 and DNA2 were activated by 10 mM of freshly prepared TCEP for 1 h. These TCEP-activated DNA1 and DNA2 were incubated separately with AuNPs and mixed at room temperature for 16 h. The mixture was then aged in salt and brought to a final concentration of 0.1 M NaCl through a stepwise process over 2 days. To remove excess reagents, the solution was centrifuged for 15 min at 16,000 rpm. After removal of the supernatant, the red oily precipitate was washed three times with 1 mL of 10 mM phosphate buffer (PB, pH 7.0) containing 0.1 M NaCl, which was finally dispersed in the PB solution and stored at 4 °C until use. Cancer Cell Detection by CTCESA-Based Colorimetric Assay. The detailed procedure for the homogeneous CTCESA-based colorimetric detection of cancer cells was as follows. First, HAP-2 was incubated with different concentrations of CCRF-CEM cells at 37 °C for 30 min to form the switched HAP. Second, linker DNA, NEase, and NEB buffer 2 (10× ) were added to the solution at 37 °C for 90 min for nicking endonuclease-strand scission cycles. Third, the mixtures were added to the solution containing DNA1-AuNPs and DNA2-AuNPs to allow assembly of the AuNPs and detect a signal for 30 min. The resulting samples were photographed and tested with an UV−vis spectrometer.
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RESULTS AND DISCUSSION Principle of the CTCESA-Based Colorimetric Method for Cancer Cell Analysis. The principle of the homogeneous colorimetric assay for rare cancer cells based on CTCESA is presented in Figure 1; the specific binding of the HAP to target 5569
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Figure 2. Feasibility of the colorimetric method for detecting CCRFCEM cells. (A) Polyacrylamide gel electrophoresis (PAGE) image. Lane 1, HAP; lane 2, linker DNA; lane 3, HAP+linker DNA+NEase; and lane 4, HAP+linker DNA+NEase+cells. (B) Absorption spectra and photograph of DNA-AuNPs under different conditions. (a) The system contained HAP (10 nM), linker DNA (30 nM), CCRF-CEM cells (1.0 × 105 cells), and NEase (20 U). The control systems were under the same conditions as (a) but without (b) NEase, (c) linker DNA, or (d) CCRF-CEM cells.
Figure 3. Signaling profile of the system using different HAP (HAP-1, HAP-2, and HAP-3) in the absence (black bars) or presence of target cells (1.0 × 105 cells; red bars). The system contained HAP (10 nM), linker DNA (30 nM), CCRF-CEM cells (1.0 × 105 cells), and NEase (20 U). The data represent the means and standard deviations of three independent experiments.
ment. HAP-1 had a relatively less stable structure that may open and directly hybridize with linker DNA to form duplex DNA, which could be recognized and cleaved by the NEase, giving high background signal and low sensitivity. Thus, the HAP-2 probe was chosen as the optimum probe in the experiment. Both the concentration of NEase and nicking time played important roles in the detection of rare cancer cells and signal amplification. Different concentrations of NEase (ranging from 0 to 30 U) were incubated with HAP-2, linker DNA, and with or without target cells (blank control). As seen in Figure 4A, the A525/A610 ratio began to increase with the concentration of NEase and attained a relatively stable plateau at 20 U NEase (red line). The noise ratio did not change significantly for the concentration range (Figure 4A, black line), which indicates that the increased NEase concentration is beneficial for the
conditions. As expected, in the presence of CCRF-CEM cells and treatment with NEase, a narrow absorption peak appeared at 525 nm, representing dispersed DNA-AuNPs, and a red solution was observed (Figure 2B, curve a), which implies that most of the linker DNAs were cleaved by CTCESA and could not assemble DNA-AuNPs. A similar phenomenon was observed in the absence of linker DNA (Figure 2B, curve c) under the same experimental conditions, and no aggregation of AuNPs occurred. In other control experiments, purple solutions were observed and an absorption peak representing the linker DNA-induced aggregation of AuNPs increased clearly at 610 nm in the absence of NEase (Figure 2B, curve b) and CCRECEM cells (Figure 2B, curve d), suggesting that linker DNA cannot be cleaved into fragments and produce a visual color change without CTCESA. TEM verified that the aggregation of DNA-modified AuNPs (Supporting Information, Figure S1A) was observed for the purple solution in the presence of linker DNA, whereas dispersed DNA-modified AuNPs (Supporting Information, Figure S1B) were found for the red solution without the addition of linker DNA. Therefore, the ratio of absorbance at 525 and 610 nm (A525/A610) was used for the quantitative analysis of cancer cells; a high ratio indicates redcolored dispersed DNA-modified AuNPs, and a low ratio indicates purple-colored aggregates. Optimization of Experimental Conditions for CCRFCEM Cell Detection. In order to achieve the best performance for identifying cancer cells, the stability of the HAP, NEase concentrations, and nicking time were investigated. The key point of our proposed strategy was based on the conformational change in the HAP triggered by CCRF-CEM cells (Figure 1); therefore, the stability of the HAP was optimized and tested. Three HAPs (HAP-1, HAP-2, and HAP-3) with increased stability were synthesized (Table 1) that included 7, 9, and 11 base pairs in their stem region, respectively. As shown in Figure 3, the highest signal-to-noise ratio was observed for HAP-2 (2.69), followed by HAP-3 (2.58) and HAP-1 (2.47), according to the change in A525/A610 under conditions with or without target CCRF-CEM cells (1.0 × 105 cells). The HAP-2 probe had suitable stability and exhibited the best signal amplification for CCRF-CEM cells among the three HAP probes. HAP-3 had the most stable structure but a rather low ability to bind target cells, causing decreased signal enhance-
Figure 4. Absorption ratio was plotted as a function of (A) the NEase concentration and (B) the nicking time in the absence (control, black line) or presence (red line) of target cells. The system contained HAP2 (10 nM), linker DNA (30 nM), CCRF-CEM cells (1.0 × 105 cells), and NEase (20 U). 5570
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increasing number of CCRF-CEM cells until a plateau was reached at approximately 5.0 × 104 cells (Figure 5C). By plotting the absorption ratio versus the number of cells, a linear range of 100 to 10,000 cells was obtained with a regression coefficient of 0.998 (Figure 5C, inset). The detection limit was 40 cells, which is roughly 20 times lower than the normal AuNP-based method without amplification42 and 2.25 times lower than the limit with enhanced SPR signal using 20 nm AuNP-labeled aptamer.14 The selectivity of the CTCESA-based colorimetric aptasensor toward CCRF-CEM cells was evaluated by measuring the optical responses of systems to other cell lines, including HeLa cells and NIH-3T3 cells (1.0 × 105 cells). As shown as Figure 6,
cleavage of linker DNA by CTCESA, and 20 U NEase can attain a better signal-to-noise ratio for the analysis of rare cancer cells. The effect of nicking time was tested by measuring A525/A610 under the same conditions (Figure 4B). The signal ratio elevated quickly during the initial stage and increased slowly after 90 min (Figure 4B, red line), but the noise ratio did not obviously change (black line). Therefore, 20 U NEase and 90 min of nicking were selected to attain a good signal-to-noise ratio for the analysis of rare cancer cells. Application of the Colorimetric Aptasensor for Detection of CCRF-CEM Cells. The sensitivity of the CTCESA-based colorimetric aptasensor was investigated with different numbers of CCRF-CEM cells (0 to 1.0 × 105 cells; Figure 5). An obvious color change from purple to red was
Figure 6. Absorption spectra and (inset) photograph of CCRF-CEM cells (1.0 × 105 cells) and different nontarget cells (1.0 × 105 cells). (a) CCRF-CEM cells, (b) HeLa cells, and (c) NIH-3T3 cells.
the colorimetric signal of the aptasensor clearly changed for CCRE-CEM cells (red against purple) and negligibly changed with the addition of control cells (purple). The good selectivity was ascribed to a high affinity of the HAP for CCRF-CEM cells (∼0.80 nM dissociation constant, Kd)15 and the specificity of CTCESA (Figure 2). The relative standard deviation (RSD) of the colorimetric aptasensor toward the same CCRF-CEM cells (1.0 × 105 cells/mL) in eight parallel tests was found to be 0.52%. Therefore, the CTCESA-based colorimetric aptasensor can be applied in the detection of CCRF-CEM cells with visualization, high specificity, and acceptable reproducibility.
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Figure 5. (A) Photograph and (B) absorption spectra after incubation with different numbers of CCRF-CEM cells (a−i, 0, 50, 1 × 102, 5.0 × 102, 1 × 103, 5.0 × 103, 1 × 104, 5.0 × 104, and 1 × 105 cells, respectively). (C) The A520/A610 absorption ratio was plotted as a function of the number of CCRF-CEM cells. Inset: The A520/A610 absorption ratio had a linear correlation with the number of CCRFCEM cells in the range of 100 to 10,000 cells. Error bars indicate the standard deviation of three experiments.
CONCLUSION In conclusion, we have demonstrated a CTCESA-based colorimetric method for highly efficient detection of rare CCRF-CEM cells. First, this method can detect CCRF-CEM cells with high sensitivity and high selectivity. Its detection limit of 40 cells is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. The improvement in analytical properties is likely due to the good amplification efficiency and specificity of CTCESA, which were verified by UV−visible spectroscopy and PAGE. Second, this method has significant advantages, namely, visualization and simple operation. In contrast to expensive fluorescencebased assays,15,16 this colorimetric method uses normal UV−vis spectroscopy, even just the naked eye. Also, it is a separationfree method for cell analysis and avoids complex operations, such as cell-immobilization and washing. Third, the method is generic and can be applied to other cell analyses and clinical diagnosis. With this method, the nicking site is in the DNA linker, not the HAP. Also, the label-free HAP design benefits
observed with an increasing number of target cells (Figure 5A), which was even seen with the naked eye. The high sensitivity of this colorimetric aptasensor was ascribed to the good signal amplification of CTCESA, as more linker DNAs are cleaved and cannot assemble DNA-modified AuNPs, resulting in more separated AuNPs to produce a feasible colorimetric reading. The cell number-dependent change in the colorimetric signal was verified by UV−vis spectrometry (Figure 5B,C). The absorbance at 525 nm increased, and the absorbance at 610 nm decreased with an increasing number of target cells (Figure 5B). Also, the A520/A610 signal continued to increase with an 5571
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conservation of its biorecognition to target cancer cells. Given its easy operation, visualization, high sensitivity, and selectivity, this colorimetric assay is potentially useful for preclinical and clinical cancer diagnosis and treatment.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. Tel.: +86 21 65642138. Fax: +86 21 65641740. *E-mail: [email protected]. Tel.: +86 21 65642138. Fax: +86 21 65641740. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21075021, 21175029, and 21335002) and the Shanghai Leading Academic Discipline Project (B109).
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REFERENCES
(1) Tan, W. H.; Donovan, M. J.; Jiang, J. H. Chem. Rev. 2013, 113, 2842−2862. (2) Savage, N. Nature 2011, 471, S14−S15. (3) Yu, L.; Ng, S. R.; Xu, Y.; Dong, H.; Wang, Y. J.; Li, C. M. Lab Chip 2013, 13, 3163−3182. (4) Singh, S. K.; Hawkins, C.; Clarke, I. D.; Squire, J. A.; Bayani, J.; Hide, T.; Henkelman, R. M.; Cusimano, M. D.; Dirks, P. B. Nature 2004, 432, 396−401. (5) Schamhart, D.; Swinnen, J.; Kurth, K. H.; Westerhof, A.; Kusters, R.; Borchers, H.; Sternberg, C. Clin. Chem. 2003, 49, 1458−1466. (6) Phillips, J. A.; Xu, Y.; Xia, Z.; Fan, Z. H.; Tan, W. H. Anal. Chem. 2009, 81, 1033−1039. (7) Gubala, V.; Harris, L. F.; Ricco, A. J.; Tan, M. X.; Williams, D. E. Anal. Chem. 2012, 84, 487−515. (8) He, S. J.; Song, B.; Li, D.; Zhu, C. F.; Qi, W. P.; Wen, Y. Q.; Wang, L. H.; Song, S. P.; Fang, H. P.; Fan, C. H. Adv. Funct. Mater. 2010, 20, 453−459. (9) Wang, Y.; Li, Z.; Hu, D.; Lin, C.-T.; Li, J.; Lin, Y. J. Am. Chem. Soc. 2010, 132, 9274−9276. (10) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818−822. (11) Baker, B. R.; Lai, R. Y.; Wood, M. S.; Doctor, E. H.; Heeger, A. J.; Plaxco, K. W. J. Am. Chem. Soc. 2006, 128, 3138−3139. (12) Tombelli, S.; Minunni, A.; Mascini, A. Biosens. Bioelectron. 2005, 20, 2424−2434. (13) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 4540−4545. (14) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. H. Anal. Chem. 2008, 80, 1067−1072. (15) Shi, H.; He, X. X.; Wang, K. M.; Wu, X.; Ye, X. S.; Guo, Q. P.; Tan, W. H.; Qing, Z. H.; Yang, X. H.; Zhou, B. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 3900−3905. (16) Cao, L.; Cheng, L.; Zhang, Z.; Wang, Y.; Zhang, X.; Chen, H.; Liu, B.; Zhang, S.; Kong, J. Lab Chip 2012, 12, 4864−4869. (17) Zhang, M.; Liu, H.; Chen, L.; Yan, M.; Ge, L.; Ge, S.; Yu, J. Biosens. Bioelectron. 2013, 49, 79−85. (18) Medley, C. D.; Bamrungsap, S.; Tan, W.; Smith, J. E. Anal. Chem. 2011, 83, 727−734. (19) Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 9, 3258−3261. (20) Fan, Z.; Shelton, M.; Singh, A. K.; Senapati, D.; Khan, S. A.; Ray, P. C. ACS Nano 2012, 6, 1065−1073. 5572
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Visual and Highly Sensitive Detection of Cancer Cells by a Colorimetric Aptasensor Based on Cell-Triggered Cyclic Enzymatic Signal Amplification Xianxia Zhang, Kunyi Xiao, Liwei Cheng, Hui Chen, Baohong Liu, Song Zhang,* and Jilie Kong* Department of Chemistry, Fudan University, Shanghai 200433, P. R. China S Supporting Information *
ABSTRACT: Rapid and efficient detection of cancer cells at their earliest stages is one of the central challenges in cancer diagnostics. We developed a simple, cost-effective, and highly sensitive colorimetric method for visually detecting rare cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and linker DNAs stably coexist in solution, and the linker DNA assembles DNA-AuNPs, producing a purple solution. In the presence of target cells, the specific binding of HAPs to the target cells triggers a conformational switch that results in linker DNA hybridization and cleavage by nicking endonuclease-strand scission cycles. Consequently, the cleaved fragments of linker DNA can no longer assemble into DNAAuNPs, resulting in a red color. UV−vis spectrometry and photograph analyses demonstrated that this CTCESA-based method exhibited selective and sensitive colorimetric responses to the presence of target CCRF-CEM cells, which could be detected by the naked eye. The linear response for CCRF-CEM cells in a concentration range from 102 to 104 cells was obtained with a detection limit of 40 cells, which is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. Given the high specificity and sensitivity of CTCESA, this colorimetric method provides a sensitive, labelfree, and cost-effective approach for early cancer diagnosis and point-to-care applications.
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tides originating from an in vitro strategy termed systematic evolution of ligands by exponential enrichment (SELEX),10 can bind a number of specific molecular targets, such as small chemicals, proteins, and even cells, with high affinity and specificity comparable to that of antibodies.11 Furthermore, aptamers offer distinct advantages over antibodies, such as small size, high stability, low cost, ease of modification, nontoxicity, and lack of immunogenicity.12,13 Many high-affinity aptamers that specifically bind to different target cancer cells have been created using cell-SELEX in recent years,1 and these efforts have created a great opportunity to develop aptamer-based biosensors for early cancer diagnosis. However, the direct detection of cancer cells using aptamers is severely hindered by the fact that most aptamer-cell interactions do not produce an easily measurable output. Many new aptasensors have been developed successfully for cancer cell analysis with several sensitive methods, such as colorimetry,14 fluorescence,15,16 electrochemistry,17 and magnetic field,18 and some signal amplification strategies, such as gold nanoparticles (AuNPs),19 magnetic core-gold shell nanoparticles,20 quantum dots (QDs),21 and PCR reaction,22 have been applied to improve the sensitivity of these aptamer-based approaches in
he development of a detection method with high selectivity and sensitivity for cancer at its earliest stages is one of the central challenges in cancer prevention, diagnostics, and treatment. By measuring specific cellular levels within the blood, a doctor can judge the patient’s responses to treatment or predict the probability of the onset of a specific disease.1 Most importantly, screening people with no symptoms to find early signs of cancer in a routine blood test is better, and the chances of cure are higher, than diagnosing suspected cancer.2,3 However, traditional analysis techniques, such as immunohistochemistry, flow cytometry, and polymerase chain reaction (PCR),4−6 do not meet the requirement of point-of-care (POC) diagnostics because they usually require costly instruments, long analytical time, and complicated operations.7 Meanwhile, considering the very low quantity of stray cancer cells,1−3 screening requires a new clinical platform with high specificity and ultrasensitivity in the detection of cancer cells, especially for preclinical diagnostics. Furthermore, the approach should reduce costs in order to screen more patients at the earliest stage and prevent cancer deaths, especially in developing countries. Thus, the development of a simple diagnostic tool for specific and ultrasensitive detection of rare cancer cells at low cost is necessary. Coupling nanomaterials and biomolecular recognition events represents a new direction toward the development of novel molecular diagnostic tools.8,9 Aptamers, artificial oligonucleo© 2014 American Chemical Society
Received: March 25, 2014 Accepted: May 12, 2014 Published: May 12, 2014 5567
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Figure 1. Schematic illustration of a highly sensitive colorimetric method for the detection of rare cancer cells based on CTCESA.
Table 1. Oligonucleotide Sequences Used in This Study sequencea
probe
hairpin aptamer probe-1 (HAP-1) 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAA ATC CTC AGC AGT3′ hairpin aptamer probe-2 (HAP-2) 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA3′ hairpin aptamer probe-3 (HAP-3) 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA TCC TCA GCA GTT AGA3′ linker DNA 5′-GCA AAC AAC TGC↓TGA GGA TAA ACG-3′ DNA1 5′-SH-(CH2)6-CGT TTA TCC TCA-3′ DNA2 5′-GCA GTT GTT TGC-(CH2)6-SH-3′ a
Bold letters are the CCRF-CEM cell aptamer sequence. Italic letters are the partially complementary sequence to the CCRF-CEM cell aptamer. Underlined letters are the NEase recognition sequence. The arrow indicates the nicking position.
and strong distance-dependent surface plasmon resonance (SPR) absorption. The color of the AuNP solution can change from red to purple in response to the SPR absorption of dispersed and aggregated nanoparticles,25 but the limited sensitivity and selectivity affects their use in early cancer diagnosis.33,34 To address these drawbacks, amplification of the detection signal is remarkably important. Until now, many signal amplification strategies have been reported,22,35 but a few amplified strategies are available for AuNP-based colorimetric methods. In addition, only a few highly sensitive colorimetric aptasensors have been developed by nicking endonucleaseassisted nanoparticle amplification (NEANA) for the detection of potassium,36 DNA (10 pM),30 and thrombin (20 pM).25 However, the colorimetric detection of rare cancer cells with such a NEANA technique has not been reported. Therefore, we tried to design a simple, sensitive, selective, and cost-effective colorimetric aptasensor for the detection of cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA).
cancer analysis. Using the primary cell-detecting strategy, “always-on” aptamer probes were designed and labeled with a sensitive reporter that can specifically bind to the target cells and produce obviously enhanced signals. An intrinsic disadvantage of “always-on” probes is that they emit constant signals into the surrounding solution, resulting in considerable background signal. To improve the signal-to-background ratio, the unbound aptamers need to be washed carefully before each measurement. Recently, a more efficient “signal-on” strategy was developed by an activated aptamer probe; its binding to target cancer cells triggers a conformational change and activates fluorescence based on Förster resonance energy transfer (FRET).15 In a previous study, we developed a graphene oxide-based FRET biosensor with a dye-labeled aptamer probe and implemented it in a miniature multiplex chip for visual detection of cancer cells with high sensitivity and specificity.16 However, the application of this strategy has some limitations, including the need for expensive instruments and the compulsory modification of a fluorophore and quencher pair at its two terminals (or a terminal). Compared to costly fluorescence instruments, AuNP-based colorimetric methods23 have attracted significant interest in clinic diagnostics due to their visualization, simplicity, and low cost. T-cell acute lymphoblastic leukemia (ALL) is a malignant thymocyte disease, accounting for 10%−15% of pediatric ALL cases and 25% of adult ALL cases.24 In the present study, CCRF-CEM cells were chosen as a model of rare cancer and a label-free hairpin aptamer probe (HAP) designed to specifically bind the target cancer cells and produce a spontaneous conformational change (Figure 1). AuNP-based colorimetric sensors have been widely applied for the detection of a broad range of analytes, including proteins,25−27 DNA,28−30 and metal ions.31,32 With these methods, AuNPs have emerged as colorimetric reporters due to their high extinction coefficients
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EXPERIMENTAL SECTION Materials. The oligonucleotides used in this study were synthesized by Sangon Biotechnology Co. (Shanghai, China) (Table 1). NEase (Nb.BbvCI) and 10× NEB buffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, and 10 mM dithiothreitol, pH 7.9) were purchased from New England Biolabs (USA). Chloroauric acid (HAuCl 4 ), tris(2carboxyethyl)phosphine (TCEP), and trisodium citrate were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS), normal bovine calf serum (NBCS), Dulbecco’s modified eagle medium (DMEM), and penicillin/streptomycin were purchased from Gibco (USA). SYBR Green II (10,000× stock solution in dimethyl sulfoxide, 50 μL) was purchased from Bio5568
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CCRF-CEM cells triggers its conformational change, resulting in a nicking endonuclease-strand scission cycle and detection by the three-component sandwich assay.33 Our detection system consisted of a HAP, linker DNA, two sets of DNA-modified AuNPs (Table 1), and NEase. The HAP contained three domains: I, II, and III. Region I was the sequence of the aptamer for CCRF-CEM cells,15,39 which was partially caged in the duplex structure of the stem by hybridization with region III. Region II was a single-stranded loop, and region III was complementary to the linker DNA, where the associated DNA duplex contained the recognition sequence for NEase. NEase is a nicking endonuclease that cleaves only one strand of DNA on a double-stranded DNA substrate.40,41 The NEase used in this study was Nb.BbvCI, which recognizes a simple asymmetric sequence: 5′-GCTGAGG-3′. The asymmetric sequence was designed in the middle of the linker DNA (Table 1). According to the design and calculation by Oligo Analyzer 3.1 software from IDT, the stability of the HAP should be greater than that of HAP-Linker DNA and less than that of the HAP-cell complex, which plays important roles in the selectivity and sensitivity of the cell aptasensor. In the absence of target cells, the HAP was in a stable hairpin structure and coexisted with linker DNA in solution. In a threecomponent sandwich assay (Figure 1), the linker DNA assembled two sets of DNA-AuNPs due to their complementary sequences, inducing aggregation and a clear color change from red to purple. In contrast, when the HAP was incubated with target cancer cells, the binding of the HAP to protein receptors on the cell membrane triggered spontaneous conformational reorganization15 and region III was exposed to form a switched HAP. Consequently, the exposed region III could hybridize to the linker DNA and form the new HAP/ linker DNA duplex, which is recognized and cleaved by NEase. The cleaved linker DNA fragments dissociated from the unstable duplex and released free switched HAP. Then, the switched HAP could hybridize with another intact linker DNA to form a new substrate for NEase and initiate new nicking endonuclease-strand scission cycles. In the subsequent threecomponent sandwich assay, the cleaved fragments of linker DNA could no longer assemble two DNA-AuNPs and the red color of separated AuNPs was observed. Because the increased number of target cells allowed more linker DNA to be cleaved by CTCESA, more separated (rather than aggregated) AuNPs were present and the quantitative and visual information about target cells was detected by the AuNP-based colorimetric aptasensor. Feasibility of the Method for the Detection of CCRFCEM Cells. The cell-triggered nicking endonuclease-strand scission cycle was verified by PAGE. The HAP and linker DNA stably coexisted in solution when target CCRF-CEM cells were absent, and no cleaved fragments of linker DNA were found with NEase treatment (Figure 2A, lanes 1−3). After adding CCRF-CEM cells and treating the mixture with NEase, cleaved fragments of linker DNA appeared, which migrated faster than the linker DNA (Figure 2A, lane 4). Furthermore, during the cell-triggered nicking endonuclease-strand scission cycle, the HAP was stable. To further confirm that the cleavage of linker DNA by CTCESA can alter the optical properties of DNA-modified AuNPs, UV−visible spectroscopy and TEM were carried out to test the feasibility of the colorimetric design. Figure 2B shows the absorption spectra and photographs of DNA-modified AuNP solutions for cancer cell analysis under different
Vision Biotechnology (Xiamen, China). All other reagents and solvents were analytical grade and used as received without further purification. Double distilled water was used for all experiments. Cells. T-cell acute lymphoblastic leukemia cells (CCRFCEM cells), human cervix carcinoma cells (HeLa cells), and mouse fibroblast cells (NIH-3T3 cells) were purchased from the cell bank of the Chinese Academy of Sciences. CCRF-CEM cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 100 units/mL penicillin/streptomycin and incubated at 37 °C in a humidified incubator containing 5 wt %/vol CO2. HeLa cells were cultured in DMEM medium supplemented with 10% FBS. NIH-3T3 cells were cultured in DMEM medium supplemented with 10% NBCS. Cell density was determined using a hemocytometer prior to any experiments. Apparatus. The size and morphology of AuNPs were characterized by transmission electron microscopy (TEM) using a JEOL model JEM-2011(HR) at 200 kV. The UV−vis absorption spectra were recorded in a Shimadzu UV-3150 spectrophotometer using 1 cm path length quartz microcuvettes. Polyacrylamide gel electrophoresis (PAGE) of the HAP, linker DNA, HAP and linker DNA mixture, and cleavage products was carried out with a 16% denaturing polyacrylamide gel in Tris/borate/EDTA (TBE) buffer (1× ) at 400 V for 30 min. The gel was stained with SYBR Green II and imaged under an ultraviolet transilluminator. Preparation of DNA-Modified AuNPs. AuNPs with an average diameter of 13 nm were prepared via citrate reduction of HAuCl4 as described previously,23 and the size of the AuNPs was verified by TEM and their concentration estimated by UV−vis spectroscopy. The AuNPs were modified with two different thiol-modified oligonucleotides (DNA1 and DNA2) as described previously, with minor modifications.37,38 Briefly, thiol-modified DNA1 and DNA2 were activated by 10 mM of freshly prepared TCEP for 1 h. These TCEP-activated DNA1 and DNA2 were incubated separately with AuNPs and mixed at room temperature for 16 h. The mixture was then aged in salt and brought to a final concentration of 0.1 M NaCl through a stepwise process over 2 days. To remove excess reagents, the solution was centrifuged for 15 min at 16,000 rpm. After removal of the supernatant, the red oily precipitate was washed three times with 1 mL of 10 mM phosphate buffer (PB, pH 7.0) containing 0.1 M NaCl, which was finally dispersed in the PB solution and stored at 4 °C until use. Cancer Cell Detection by CTCESA-Based Colorimetric Assay. The detailed procedure for the homogeneous CTCESA-based colorimetric detection of cancer cells was as follows. First, HAP-2 was incubated with different concentrations of CCRF-CEM cells at 37 °C for 30 min to form the switched HAP. Second, linker DNA, NEase, and NEB buffer 2 (10× ) were added to the solution at 37 °C for 90 min for nicking endonuclease-strand scission cycles. Third, the mixtures were added to the solution containing DNA1-AuNPs and DNA2-AuNPs to allow assembly of the AuNPs and detect a signal for 30 min. The resulting samples were photographed and tested with an UV−vis spectrometer.
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RESULTS AND DISCUSSION Principle of the CTCESA-Based Colorimetric Method for Cancer Cell Analysis. The principle of the homogeneous colorimetric assay for rare cancer cells based on CTCESA is presented in Figure 1; the specific binding of the HAP to target 5569
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Figure 2. Feasibility of the colorimetric method for detecting CCRFCEM cells. (A) Polyacrylamide gel electrophoresis (PAGE) image. Lane 1, HAP; lane 2, linker DNA; lane 3, HAP+linker DNA+NEase; and lane 4, HAP+linker DNA+NEase+cells. (B) Absorption spectra and photograph of DNA-AuNPs under different conditions. (a) The system contained HAP (10 nM), linker DNA (30 nM), CCRF-CEM cells (1.0 × 105 cells), and NEase (20 U). The control systems were under the same conditions as (a) but without (b) NEase, (c) linker DNA, or (d) CCRF-CEM cells.
Figure 3. Signaling profile of the system using different HAP (HAP-1, HAP-2, and HAP-3) in the absence (black bars) or presence of target cells (1.0 × 105 cells; red bars). The system contained HAP (10 nM), linker DNA (30 nM), CCRF-CEM cells (1.0 × 105 cells), and NEase (20 U). The data represent the means and standard deviations of three independent experiments.
ment. HAP-1 had a relatively less stable structure that may open and directly hybridize with linker DNA to form duplex DNA, which could be recognized and cleaved by the NEase, giving high background signal and low sensitivity. Thus, the HAP-2 probe was chosen as the optimum probe in the experiment. Both the concentration of NEase and nicking time played important roles in the detection of rare cancer cells and signal amplification. Different concentrations of NEase (ranging from 0 to 30 U) were incubated with HAP-2, linker DNA, and with or without target cells (blank control). As seen in Figure 4A, the A525/A610 ratio began to increase with the concentration of NEase and attained a relatively stable plateau at 20 U NEase (red line). The noise ratio did not change significantly for the concentration range (Figure 4A, black line), which indicates that the increased NEase concentration is beneficial for the
conditions. As expected, in the presence of CCRF-CEM cells and treatment with NEase, a narrow absorption peak appeared at 525 nm, representing dispersed DNA-AuNPs, and a red solution was observed (Figure 2B, curve a), which implies that most of the linker DNAs were cleaved by CTCESA and could not assemble DNA-AuNPs. A similar phenomenon was observed in the absence of linker DNA (Figure 2B, curve c) under the same experimental conditions, and no aggregation of AuNPs occurred. In other control experiments, purple solutions were observed and an absorption peak representing the linker DNA-induced aggregation of AuNPs increased clearly at 610 nm in the absence of NEase (Figure 2B, curve b) and CCRECEM cells (Figure 2B, curve d), suggesting that linker DNA cannot be cleaved into fragments and produce a visual color change without CTCESA. TEM verified that the aggregation of DNA-modified AuNPs (Supporting Information, Figure S1A) was observed for the purple solution in the presence of linker DNA, whereas dispersed DNA-modified AuNPs (Supporting Information, Figure S1B) were found for the red solution without the addition of linker DNA. Therefore, the ratio of absorbance at 525 and 610 nm (A525/A610) was used for the quantitative analysis of cancer cells; a high ratio indicates redcolored dispersed DNA-modified AuNPs, and a low ratio indicates purple-colored aggregates. Optimization of Experimental Conditions for CCRFCEM Cell Detection. In order to achieve the best performance for identifying cancer cells, the stability of the HAP, NEase concentrations, and nicking time were investigated. The key point of our proposed strategy was based on the conformational change in the HAP triggered by CCRF-CEM cells (Figure 1); therefore, the stability of the HAP was optimized and tested. Three HAPs (HAP-1, HAP-2, and HAP-3) with increased stability were synthesized (Table 1) that included 7, 9, and 11 base pairs in their stem region, respectively. As shown in Figure 3, the highest signal-to-noise ratio was observed for HAP-2 (2.69), followed by HAP-3 (2.58) and HAP-1 (2.47), according to the change in A525/A610 under conditions with or without target CCRF-CEM cells (1.0 × 105 cells). The HAP-2 probe had suitable stability and exhibited the best signal amplification for CCRF-CEM cells among the three HAP probes. HAP-3 had the most stable structure but a rather low ability to bind target cells, causing decreased signal enhance-
Figure 4. Absorption ratio was plotted as a function of (A) the NEase concentration and (B) the nicking time in the absence (control, black line) or presence (red line) of target cells. The system contained HAP2 (10 nM), linker DNA (30 nM), CCRF-CEM cells (1.0 × 105 cells), and NEase (20 U). 5570
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increasing number of CCRF-CEM cells until a plateau was reached at approximately 5.0 × 104 cells (Figure 5C). By plotting the absorption ratio versus the number of cells, a linear range of 100 to 10,000 cells was obtained with a regression coefficient of 0.998 (Figure 5C, inset). The detection limit was 40 cells, which is roughly 20 times lower than the normal AuNP-based method without amplification42 and 2.25 times lower than the limit with enhanced SPR signal using 20 nm AuNP-labeled aptamer.14 The selectivity of the CTCESA-based colorimetric aptasensor toward CCRF-CEM cells was evaluated by measuring the optical responses of systems to other cell lines, including HeLa cells and NIH-3T3 cells (1.0 × 105 cells). As shown as Figure 6,
cleavage of linker DNA by CTCESA, and 20 U NEase can attain a better signal-to-noise ratio for the analysis of rare cancer cells. The effect of nicking time was tested by measuring A525/A610 under the same conditions (Figure 4B). The signal ratio elevated quickly during the initial stage and increased slowly after 90 min (Figure 4B, red line), but the noise ratio did not obviously change (black line). Therefore, 20 U NEase and 90 min of nicking were selected to attain a good signal-to-noise ratio for the analysis of rare cancer cells. Application of the Colorimetric Aptasensor for Detection of CCRF-CEM Cells. The sensitivity of the CTCESA-based colorimetric aptasensor was investigated with different numbers of CCRF-CEM cells (0 to 1.0 × 105 cells; Figure 5). An obvious color change from purple to red was
Figure 6. Absorption spectra and (inset) photograph of CCRF-CEM cells (1.0 × 105 cells) and different nontarget cells (1.0 × 105 cells). (a) CCRF-CEM cells, (b) HeLa cells, and (c) NIH-3T3 cells.
the colorimetric signal of the aptasensor clearly changed for CCRE-CEM cells (red against purple) and negligibly changed with the addition of control cells (purple). The good selectivity was ascribed to a high affinity of the HAP for CCRF-CEM cells (∼0.80 nM dissociation constant, Kd)15 and the specificity of CTCESA (Figure 2). The relative standard deviation (RSD) of the colorimetric aptasensor toward the same CCRF-CEM cells (1.0 × 105 cells/mL) in eight parallel tests was found to be 0.52%. Therefore, the CTCESA-based colorimetric aptasensor can be applied in the detection of CCRF-CEM cells with visualization, high specificity, and acceptable reproducibility.
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Figure 5. (A) Photograph and (B) absorption spectra after incubation with different numbers of CCRF-CEM cells (a−i, 0, 50, 1 × 102, 5.0 × 102, 1 × 103, 5.0 × 103, 1 × 104, 5.0 × 104, and 1 × 105 cells, respectively). (C) The A520/A610 absorption ratio was plotted as a function of the number of CCRF-CEM cells. Inset: The A520/A610 absorption ratio had a linear correlation with the number of CCRFCEM cells in the range of 100 to 10,000 cells. Error bars indicate the standard deviation of three experiments.
CONCLUSION In conclusion, we have demonstrated a CTCESA-based colorimetric method for highly efficient detection of rare CCRF-CEM cells. First, this method can detect CCRF-CEM cells with high sensitivity and high selectivity. Its detection limit of 40 cells is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. The improvement in analytical properties is likely due to the good amplification efficiency and specificity of CTCESA, which were verified by UV−visible spectroscopy and PAGE. Second, this method has significant advantages, namely, visualization and simple operation. In contrast to expensive fluorescencebased assays,15,16 this colorimetric method uses normal UV−vis spectroscopy, even just the naked eye. Also, it is a separationfree method for cell analysis and avoids complex operations, such as cell-immobilization and washing. Third, the method is generic and can be applied to other cell analyses and clinical diagnosis. With this method, the nicking site is in the DNA linker, not the HAP. Also, the label-free HAP design benefits
observed with an increasing number of target cells (Figure 5A), which was even seen with the naked eye. The high sensitivity of this colorimetric aptasensor was ascribed to the good signal amplification of CTCESA, as more linker DNAs are cleaved and cannot assemble DNA-modified AuNPs, resulting in more separated AuNPs to produce a feasible colorimetric reading. The cell number-dependent change in the colorimetric signal was verified by UV−vis spectrometry (Figure 5B,C). The absorbance at 525 nm increased, and the absorbance at 610 nm decreased with an increasing number of target cells (Figure 5B). Also, the A520/A610 signal continued to increase with an 5571
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Analytical Chemistry
Article
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conservation of its biorecognition to target cancer cells. Given its easy operation, visualization, high sensitivity, and selectivity, this colorimetric assay is potentially useful for preclinical and clinical cancer diagnosis and treatment.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. Tel.: +86 21 65642138. Fax: +86 21 65641740. *E-mail: [email protected]. Tel.: +86 21 65642138. Fax: +86 21 65641740. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21075021, 21175029, and 21335002) and the Shanghai Leading Academic Discipline Project (B109).
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REFERENCES
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dx.doi.org/10.1021/ac501068k | Anal. Chem. 2014, 86, 5567−5572
