Construction of a Robust Entropy-Driven DNA Nanomachine for

Letter Cite This: Anal. Chem. 2019, 91, 7505−7509

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Construction of a Robust Entropy-Driven DNA Nanomachine for Single-Molecule Detection of Rare Cancer Cells Fei Ma,‡ Shu-hua Wei,‡ and Chun-yang Zhang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, Shandong 250014, China

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S Supporting Information *

ABSTRACT: Accurate and sensitive detection of rare cancer cells such as circulating tumor cells (CTCs) has attracted great interest in the fields of clinical diagnosis and cancer research. However, the reported methods for cancer cell detection often involve a complicated platform and laborious procedures with a limited sensitivity. Herein, we construct a new entropy-driven DNA nanomachine for single-molecule detection of rare cancer cells. This assay employs the entropy-driven DNA nanomachine for efficient cancer cell recognition and signal amplification without the involvement of any expensive and unstable antibodies and enzymes, and it enables one-step detection of living cancer cells with high sensitivity and good specificity. This DNA nanomachine can be further applied for rare CTC detection in whole blood samples.

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sensitivity due to the lack of efficient signal amplification approaches, unsuitable for the detection of extremely rare cells such as circulating tumor cells (CTCs). The DNA nanomachines are switchable DNA assemblies that perform machine-like functions (e.g., tweezer, walker, pendulum, and amplifier) in response to the molecular/ environmental signals.19 Due to their excellent specificity, simplicity, structural robustness, programmability, and biocompatibility, a variety of DNA nanomachines have been established to sense pH,20 nucleic acids,21−23 and proteins.24 However, the use of enzyme-free DNA nanomachines for living cancer cell detection has never been explored. In this research, we construct a new entropy-driven DNA nanomachine with the integration of total internal reflection fluorescence (TIRF) imaging-based single-molecule detection25,26 for rare cancer cell detection. The DNA nanomachine can transform and amplify the target cell signal to the detectable fluorescence signal, enabling one-step, enzyme-free, sensitive, and specific detection of living cancer cells at the single-molecule level (Scheme 1A). The DNA nanomachine consists of three components including a detection probe, a

ancer is one of the most deadly human diseases, causing about 10 million deaths each year around the world.1,2 Accurate and sensitive detection of cancer cells is of great importance to cancer diagnosis and research.3,4 Polymerase chain reaction (PCR) enables sensitive detection of cancer cells indirectly, but it involves time-consuming procedures for the lysis of cancer cells and the extraction of DNAs/RNAs, expensive and unstable reverse transcriptase and polymerase for signal amplification, and precise temperature control for thermal cycling.5−7 Alternatively, great efforts have been put into the direct detection of intact cancer cells. Antibodies are the most commonly used probes for living cancer cell detection,8 but they are relatively expensive, unstable, large in size, and difficult for modification. To overcome these issues, the aptamers are introduced with significant advantages of high stability, convenient synthesis, low cost, small size, high binding affinity, and easy functionalization,9 which makes them promising alternative to antibodies for cancer cell detection.10−12 Until now, a series of methods have been developed for cancer cell detection by using antibodies and aptamers in combination with fluorescence detection,13,14 flow cytometry,15 surface-enhanced Raman scattering,16 electrochemical measurement,17 and aerolysin nanopore.18 Despite their impressive assay performance, the existing methods often involve a complicated platform, laborious procedures, and poor stability. In addition, most of these methods exhibit a limited © 2019 American Chemical Society

Received: April 1, 2019 Accepted: June 4, 2019 Published: June 4, 2019 7505

DOI: 10.1021/acs.analchem.9b01617 Anal. Chem. 2019, 91, 7505−7509

Letter

Analytical Chemistry

Scheme 1. Schematic Illustration of the Entropy-Driven DNA Nanomachine for Single-Molecule Detection of Cancer Cellsa

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(A) Overview of a DNA nanomachine with the integration of single-molecule counting for cancer cell detection. The simple mixing of target cells with three DNA components can activate the DNA nanomachine to release abundant dye-labeled oligonucleotides, which can be simply quantified by TIRF imaging-based single-molecule detection. (B) Schematic illustration of DNA nanomachine for cancer cell detection. The lowercase letters denote the functional domains, and domain x is complementary to the domain x*.

As a proof of concept, we used a human lung cancer cell line (A549 cells) as the model target. Lung cancer is the most frequently diagnosed cancer worldwide with 11.6% of the total cases.1 We employed a specific aptamer of A549 cells with a high affinity constant (Kd) of 28.2 nM to prepare a detection probe (see Figure S1).29 After incubation of the DNA nanomachine with the cell samples for 30 min, the reaction products are subjected to single-molecule detection (Figure 1). A high Cy5 fluorescence signal is observed in response to A549 cells (Figure 1B), while no significant signal is detected in the control group without target cells (Figure 1A), indicating that the A549 cells can activate the DNA nanomachine to produce a significant fluorescence signal. To further confirm that this DNA nanomachine is specific to the target cells, A549 cells were pretreated with 0.25% trypsin prior to measurement. The treatment with trypsin will digest the membrane proteins to prevent the binding of aptamers to A549 cells.29 As expected, the Cy5 fluorescence signal disappears upon the treatment of A459 cells with trypsin (Figure 1C), because the DNA nanomachine cannot be activated. The Cy5 counts increase with the reaction time upon the addition of A549 cells. In contrast, a near-zero background signal is observed in either the control group without A549 cells or the A459 cells treated with trypsin (Figure 1C). These results are further confirmed

signal probe, and a fuel strand (Scheme 1B). The initiator sequence is hidden in the hairpin structure of the detection probe and unable to activate the DNA nanomachine. As a result, the signal probe remains a three-stranded structure with the Cy5 fluorescence being efficiently quenched by black hole quencher (BHQ), and no signal is detected. In contrast, when the detection probe binds to the target cancer cell, the initiator is exposed and its subsequent binding to the toehold domain d* of the quencher strand displaces the assistant strand through the toehold-mediated strand displacement,27 exposing the toehold domain b* of the quencher strand. The subsequent binding of the quencher strand to the fuel strand enables the release of the reporter strand from the signal probe. Meanwhile, the detection probe is regenerated, and it can bind to a new signal probe to initiate the next round of the strand displacement reaction, leading to the release of abundant Cy5labeled reporters. While the DNA nanomachine proceeds, large amounts of Cy5-labeled reporter are released, which can be simply quantified by single-molecule counting. Notably, this DNA nanomachine-induced signal amplification reaction is driven by the entropy, without the involvement of any enzymes that are usually required in a signal amplification approach,28 greatly reducing the experimental cost and improving the assay robustness. 7506

DOI: 10.1021/acs.analchem.9b01617 Anal. Chem. 2019, 91, 7505−7509

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Analytical Chemistry

Figure 1. Single-molecule detection of Cy5-labeled reporters in the absence of target cells (A) and in the presence of A549 cells (B) and A549 cells pretreated with trypsin (C). The scale bar is 5 μm. (D) Variance of Cy5 counts with reaction time in the absence of A549 cells (control, black color) and in the presence of A549 cells (red color) and A549 cells pretreated with trypsin (blue color). Error bars represent the standard deviation of three experiments.

Figure 2. (A) Measurement of Cy5 counts in response to different cell lines. (B) Measurement of Cy5 counts in response to an increasing number of A549 cells from 0 to 2000 cells. (C) Measurement of Cy5 counts in response to reaction temperature ranging from 25 to 45 °C in the absence of target cells (control, black color) and in the presence of A549 cells (red color). (C) Measurement of Cy5 counts in response to NaCl concentration ranging from 100 to 1000 mM in the absence of target cells (control, black color) and in the presence of A549 cells (red color). Error bars represent the standard deviation of three experiments.

by the ensemble fluorescence measurement, which shows a high Cy5 signal in response to A549 cells (see Figure S2). In addition, the release of Cy5-labeled reporters induced by target A549 cells is verified by gel analysis (see Figure S3). To demonstrate the selectivity of the DNA nanomachine, we measured the Cy5 counts in response to A549 cells, a human embryonic kidney cell line (HEK cells), human fetal lung fibroblasts (MRC-5 cells), a human cervical cancer cell line (HeLa cells), and a human T-lymphocyte cell line (Jurkat cells). In theory, none of HEK cells, MRC-5 cells, HeLa cells, and Jurkat cells can activate the DNA nanomachine because the detection probe is only specific to A549 cells. As expected, a high Cy5 signal is observed only in response to A549 cells, while no distinct signal is detected in response to HEK cells, MRC-5 cells, HeLa cells, and Jurkat cells (Figure 2A), demonstrating the high specificity of the proposed DNA nanomachine. We further investigated the detection sensitivity by measuring the Cy5 counts in response to different numbers of A549 cells. As shown in Figure 2B, the Cy5 counts are enhanced with the increasing number of A549 cells from 0 to 2000 cells, and the signal from even one A549 cell can be well distinguished from the background signal, indicating a detection limit of 1 single cell, which is superior to the reported methods for cancer cell detection based on fluorescence measurement (250 cells),13 flow cytometry (118 cells),15 surface-enhanced Raman scattering (10 cells),16 and electrochemical measurement (2 cells)17 (see Table S2). The high sensitivity of this DNA nanomachine may be attributed to the low background signal of single-molecule detection and the efficient signal amplification of the DNA nanomachine. The accurate quantification of cancer cells is often challenged by complex environmental conditions such as varying temperature and salt concentrations. We further investigated the stability of the DNA nanomachine. As shown in Figure 2C, the presence of A549 cells generates a

high signal in the tested temperature ranging from 25 to 45 °C, indicating that the DNA nanomachine performs well over a large temperature range. We further investigated the influences of salt concentrations upon the assay performance. As shown in Figure 2D, the presence of A549 cells produces a high Cy5 signal in the tested NaCl concentrations ranging from 100 to 1000 mM, indicating that the DNA nanomachine performs well in changing salt conditions, which may somewhat influence the cell viability (see Figure S4B). Notably, the background signal of the control group without target cells remains extremely low in the tested temperatures and salt concentrations. These results clearly demonstrate the high robustness of the DNA nanomachine in complex conditions. CTCs are cancer cells in peripheral blood, which shed from original tumors into the bloodstream to cause cancer metastasis30 and are considered as the major cause of cancerassociated death.31,32 Therefore, CTCs are emerging as the promising biomarkers in a liquid biopsy for early cancer diagnosis, prognosis, treatment, and cancer research.33,34 The detection of CTCs is very challenging because they are extremely rare in blood (a few to hundreds per milliliter).35 To verify the feasibility of the DNA nanomachine for CTC detection, we constructed a CTC capture platform using aptamer-labeled magnetic beads (Figure 3A). The capture probe is a 5′ biotinylated aptamer of A549 cells (Kd = 28.2 nM),29 and the superparamagnetic beads are coated with streptavidin. The capture platform is obtained by the selfassembly of capture probes and magnetic beads through the biotin / streptavidin interaction. Under the optimized conditions, the capture platform can achieve a capture efficiency of 72% (see Figure S5) with high capture purity.14,36,37 The A549 cells are spiked into whole blood to prepare the artificial CTC samples.16,17 The CTCs are captured by the magnetic capture platform and quantified by the DNA nanomachine (Figure 3A). As shown in Figure 3B, no distinct Cy5 signal is detected in the absence of CTCs. In 7507

DOI: 10.1021/acs.analchem.9b01617 Anal. Chem. 2019, 91, 7505−7509

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Figure 3. (A) Principle of the DNA nanomachine with the integration of capture probe-labeled magnetic beads and single-molecule counting for CTC detection. (B) Single-molecule detection of A549 CTCs in whole blood. The scale bar is 5 μm. (C) Linear relationship between the Cy5 counts and the number of CTCs ranging from 5 to 500 cells. Error bars represent the standard deviation of three experiments.

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contrast, distinct Cy5 signals are observed in the presence of CTCs, with the Cy5 signals increasing with the number of CTCs. In addition, no distinct Cy5 signal is detected in the absence of capture probe. Notably, the Cy5 counts exhibit a linear correlation with the number of CTCs in the range from 5 to 1000 cells (Figure 3C) with a regression equation of F = 4.26 + 0.639N (R2 = 0.997), where F is the Cy5 counts and N is the number of CTCs. The detection limit is calculated to be 3 cells according to the 3σ/K rule (σ is the standard deviation of the control sample, and K is the slope of the linear regression curve), suggesting the high accuracy and good stability of the DNA nanomachine. Moreover, the DNA nanomachine can be performed without the involvement of CTC washing steps (see Figure S6), greatly simplifying the experimental procedure and reducing the assay time. In conclusion, we have constructed a new entropy-driven DNA nanomachine with the integration of single-molecule detection for rare cancer cell detection. This DNA nanomachine is composed of a detection probe, a signal probe, and a fuel, and it can efficiently recognize and amplify the target cell signal without the involvement of any expensive and unstable enzymes/antibodies. The combination of efficient DNA nanomachine-assisted signal amplification with a high signal-to-noise ratio of single-molecule detection enables onestep sensitive detection of target A549 cells with a detection limit of 1 cancer cell, superior to the reported cancer cell assays (with a detection limit ranging from a few to hundreds of cells; see Table S2).13,15−18,38 This DNA nanomachine can be applied for rare CTC detection in human whole blood samples, and it can be extended to the detection of other types of cancer cells including EpCAM-positive HT-29 and MCF-7 cells by using the corresponding detection probes (see Figure S7), holding great potential in early clinical diagnosis and cancer research.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01617. Experimental details, structure switching of the detection probe, ensemble fluorescence detection of A549 cells, PAGE analysis, measurement of cell viability, optimization of CTC capture system, influence of washing steps on assay performance, demonstration of the assay generality, and comparison of different cancer cell assays (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chun-yang Zhang: 0000-0002-8010-1981 Author Contributions ‡

F.M. and S.-h.W. contributed equally.

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 (Grant Nos. 21527811, 21735003, and 21705096) and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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DOI: 10.1021/acs.analchem.9b01617 Anal. Chem. 2019, 91, 7505−7509