Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal

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Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore Dongmei Xi, Zhi Li, Liping Liu, Shiyun Ai, and Shusheng Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04584 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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Analytical Chemistry

Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore Dongmei Xi,† Zhi Li,†,‡ Liping Liu,† Shiyun Ai,*,‡ Shusheng Zhang*,† * E-mail: [email protected]. Phone: +86-538-8249248 * E-mail: [email protected]. Phone: +86-539-8766867 † Shandong Provincial Key Laboratory of Detection Technology for Tumor Makers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, P. R. China. ‡ College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, P. R. China. ABSTRACT: Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer. Herein, we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells (human Burkitt’s lymphoma cells), by combining the enzymatic signal amplification with an aerolysin nanopore sensor. In this assay, an aptamer for Ramos cells was pre-hybridized with a short complementary DNA. The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA, which would subsequently trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce characteristic current events when translocated through aerolysin. The proposed method exhibits excellent sensitivity, and Ramos cells as low as 5 cells could be detectable. With good selectivity, the approach can allow for the determination of cancer cells in human serum, offering a powerful tool for biomedical research and clinical diagnosis.

Nanopore has proven to be an attractive, powerful, and sensitive tool in the single-molecule analysis.1-4 The principle of nanopore sensing is to monitor the ionic current fluctuation through nanopores when an analyte binds within the pore. 5 The characteristic current signature of the analyte binding reveals its identity, and the frequency of the binding events reveals the analyte concentration.6 Aerolysin is a heptameric pore-forming toxin from Aeromonas hydrophila. It allows spontaneous insertion into the lipid bilayer leading to a nanoscale pore whose diameter ranges from 1.0 to 1.7 nm.7,8 Since its emergence as a nanopore to analyze the translocation of α-helix peptides through single pore,9 aerolysin has been applied to study the dynamics of proteins 10-12 and oligosaccharides13 and kinetics of enzymatic degradation.14 Recently, aerolysin was first utilized to discriminate oligonucleotides of different lengths, exhibiting impressive advantage in sensing nucleic acids.15 Despite its good performance, aerolysin has been utilized rarely in quantitative determination of the analytes. To improve the sensitivity, a variety of signal amplification strategies have been developed for the fabrication of biosensors, such as polymerase chain reaction (PCR),16,17 targetinduced enzyme assistant cycling,18 hybridization chain reaction,19 and nanomaterials-based amplifying signal gain method.20 They have been introduced into various detection systems, including fluorescent, chemiluminescence, colorimetric, and electrochemical assays, and have become powerful techniques for diverse applications, such as bioassay and nanotechnology. However, up to now, amplification strategy has been rarely applied in the nanopore platform. Considering the advantages of the signal amplification strategies and nanopore

sensors, we envisioned their combination would greatly enhance the possibility of sensing analytes at the ultra-low level. In this report, we present an ultrasensitive nanopore-based strategy by combining the enzymatic cycling amplification with the aerolysin nanopore sensor, and employed Ramos cell (human Burkitt’s lymphoma cell), a kind of cancer cell, as a model. Cancer cell has specific intracellular or extracellular biomarkers, and is an important cancer biomarker to facilitate the monitoring of the relevant biological processes of cancers.21 Currently available analytical methods for quantitative detection of cancer cells include fuorescence,22 chemiluminescence,23 colorimetric analysis,24 and electrochemical assay.25,26 Herein, a probe was designed to contain DNA aptamer for Ramos cells which was conjugated to its partially complementary DNA (cDNA) (Scheme 1). Binding of the target cells to their aptamers induced the release of cDNA, which would then be used to trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce signature current events when subjected to aerolysin for the nanopore test. By introducing the signal amplification strategy into the nanopore sensor, the approach exhibits excellent sensitivity and high specificity, which can be further applied for the detection of the cancer cells in human serum.

EXPERIMENTAL SECTION Reagents and Chemicals. Decane (anhydrous, ≥ 99%) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). 1, 2-Diphytanoyl-sn-glycero-3-phosphocholine

1

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Schematic representation of the cancer cell detection based on aptamer recognition and enzymatic cycling amplification with an aerolysin nanopore.

(chloroform, ≥ 99%) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Aerolysin was kindly provided by Professor Yi-Tao Long (East China University of Science and Technology). Dynabeads MyOne™ Streptavidin T1 (10 mg/mL, 7 ~ 12×109 beads/mL; beads diameter: 1.0 μm, supplied in PBS; pH 7.4/0.1% BSA/0.02% sodium azide) was obtained from Invitrogen (California, U.S.A.). Phi29 DNA polymerase and Nt.BbvCI were provided by New England Biolabs (Ipswich, MA). The oligonucleotides (Table 1) were synthesized and HPLC-purified by Sangon Biotech Co. Ltd. (Shanghai, China). All of the other chemicals were of analytical grade unless otherwise indicated. All solutions for analytical studies were prepared with ultrapure water. Table 1 Sequences of the oligonucleotides Name

Sequence (5′ 3′)

Aptamer

TAC AGA ACA CCG GGA GGA TAG TTC GGT GGC TGT TCA GGG TCT CCT CCC G GT GTT TTT TTT TT - biotin

cDNA

CAC CGG GAG GAG

Template DNA

CTT CTT CTT GCT GAG GCT CCT CCC GGT GTT TTT TTT TT - biotin

Cell Culture and Collection. Ramos cells, human lung adenocarcinoma A549 cells, human T-acute lymphoblastic leukemia Jurkat cells, breast cancer MCF-7 cells, and human cervical carcinoma (HeLa) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37 °C in a humidified atmosphere with 5% CO2. A549, MCF-7, and Hela cells in the exponential phase of growth were collected and digested with trypsinization, washed twice with ice-cold PBS (137 mM NaCl, 2.68 mM KCl, 10 mM phosphate buffer, pH 7.4), and centrifuged at 900 rpm for 5 min at 4 °C. Ramos cells and Jurkat cells dispersed in RPMI 1640 cell media buffer were centrifuged at 900 rpm for 5 min. The supernatant was used for the cancer cell detection assay. For the cell suspension at high concentrations, cell density was determined using Luna-II™ automated cell counter prior to each experiment. Ramos cells at low concentrations are

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collected as follows. Cell suspension at high concentration is first harvested, and cell density is determined using Luna-II™ automated cell counter. Then this cell suspension is serially diluted with RPMI 1640 medium until the minimum cell number that can be observed in the following step. Finally, 1 μL of this cell suspension is dropped to the culture dish and counted with OLYMPUS IX73 Inverted Fluorescence Microscope. Either 5 Ramos cells or 10 Ramos cells are observed in a field. Preparation of Probe and Detection of Cancer Cells. Firstly, all oligonucleotides were diluted with the buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.9) to prepare the stocks. The double-stranded DNA (dsDNA) substrates were prepared by incubating cDNA and aptamer at desired concentrations at 90 °C for 5 min, followed by gradually cooling to room temperature. Meanwhile, Dynabeads MyOne™ Streptavidin T1 (50 μL, 10 mg/mL) was washed for three times with 1 mL 1× BW buffer (1 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5). Next, the DNA substrates (50 µL, 2 μM) were mixed with beads in 50 μL 2× BW buffer, and vortexed for 15 min. The suspension was decanted after the beads were concentrated with a centrifuge. The beads were washed for three times with 0.5 mL 1× BW buffer, marked as bead-aptamer/cDNA. Template DNA (50 µL, 2 μM) was immobilized onto the beads with the same method, marked as bead-Template DNA. Then, the bead-aptamer/cDNA complex was mixed with 100 μL various concentrations of cell suspension at 37 °C for 2 h in a constant temperature cell incubator. After incubation, the supernatant was collected with the aid of a magnet. Finally, the enzymatic cycling amplification reaction was carried out in 100 μL of reaction mixture containing the beadTemplate DNA, 1× Phi29 DNA polymerase reaction buffer (10mM (NH4)2SO4, 50 mM Tris-HCl, 10 mM MgCl2, 4 mM DTT, pH 7.5), 100 μg/mL BSA, 100 U/mL Phi29 DNA polymerase, 0.3 mM dNTP, 150 U/mL Nt.BbvCI nicking enzyme, 1× CutSmart buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9), and variable-concentration supernatant at 37 °C for 2 h with occasional vortexing. After incubation, the supernatant was collected with the aid of a magnet and heated to 80 °C for 15 min to deactivate the enzyme. The beads were washed with deionized water. The obtained sample was ready for nanopore analysis. Nanopore Electrical Recording and Data Analysis. The nanopore electrical recording was conducted according to our previous report with a minor change.27 The lipid bilayer membrane was formed spanning a 50 μm orifice in a Delrin bilayer cup (Warner Instruments, Hamden, CT) that was partitioned into two chambers, cis and trans. Both chambers were filled with 1 mL of buffer (cis: 0.5 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.8; trans: 3 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.8). DNA samples were then added into the cis chamber. Furthermore, the current trace was recorded with an Integrating Patch Clamp amplifier (Axon Instruments, Forest City, CA) equipped with a DigiData 1440A converter (Axon Instruments, Forest City, CA). The signals were collected using a 5 kHz lowpass Bessel filter at a sampling rate of 10 kHz by using a PC running PClamp 10.6 (Axon Instruments, Forest City, CA). Data analysis was performed using the software programmed by Long’s group28 and OriginLab 9.0 (OriginLab Corporation, Northampton, MA, USA). Nanopore measurements were carried out at 25 ± 2 °C. The frequency of the events was calculated from the data of at least 5 min. 2


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RESULTS AND DISCUSSION Principle of the Nanopore-Based Assay for Cancer Cells. The nanopore-based sensing strategy was proposed by the specific recognition of the aptamer by Ramos cells and subsequent enzymatic cycling amplification, of which the yielded output DNA were analyzed with an aerolysin nanopore. As illustrated in Scheme 1, the biotinlylated aptamer for Ramos cell was first hybridized partially with the complementary DNA (cDNA), and the double-stranded DNA (dsDNA) obtained were immobilized onto the magnetic beads. Since the bead-aptamer-cDNA complex is too large to enter aerolysin, no translocation events could be observed when the complex is subjected to a nanopore test. However, the addition of Ramos cells would cause the aptamer-cDNA duplex to unwind and lead to the release of the cDNA, which could subsequently trigger the enzymatic cycling amplification in the presence of Phi29 DNA polymerase and nickase Nt.BbvCI. This process eventually produced a large number of output DNA which would generate characteristic current events when translocated through aerolysin under the transmembrane potential. It was previously reported that a gradient of salt concentration across α-hemolysin nanopore greatly increases the capture rate of DNA29 and miRNA.27 We speculate that this strategy might also work on the aerolysin nanopore system to improve the sensitivity. As expected, 100 nM of the output DNA in asymmetrical KCl solutions (0.5 M/3 M, cis/trans) afforded ~70 events per min, while 100 nM of the DNA in 1 M KCl (cis/trans) only produced ~ 4 events per min (Figure 1). Prompted by this result, we carried out the following experiments with a salt gradient to focus the DNA into the nanopore, aiming to enhance the possibility of analyzing cancer cells at extremely low level.

Figure 1. Comparison of the frequency of output DNA events under symmetrical and asymmetric salt conditions. 100 pmol of output DNA were added to the cis chamber, and its final concentration was 100 nM. Current traces were recorded at +120 mV.

Nanopore-Based Analysis of Cancer Cells. The assay was dependent on the successful recognition of aptamers by Ramos cells and subsequent amplification of the output DNA. To identify the target signals in the nanopore test, we first used the output DNA to examine the signature current blocks. As illustrated in Figure 2A, large amounts of unambiguous

Figure 2. Detection of the output DNA with aerolysin nanopore. (A) Current trace, (B) Scattering plot, (C) I/I0 histogram, (D) P1 duration histogram, and (E) P2 duration histogram of the output DNA monitored at +100 mV in 0.5 M/3 M (cis/trans) KCl. (F) Duration time versus applied voltage for P1. A single-exponential function was used to fit the duration times from +80 to +120 mV.

current signals were observed after addition of the output DNA in the cis chamber. Scatter plot showing current blockage and duration time yielded two populations (Figure 2B), a major one labeled as P1 and a minor one labeled as P2. Compared with the P2 population, the P1 events featured a more concentrated blockage and a substantially longer duration. For current amplitude analysis, I0 was defined as the open pore current and I as the blockage current when the analyte stayed within the pore. Statistical analysis showed that I/I0 of P1 was fitted to the Gauss distribution with a value of 0.92 ± 0.01, while P2 had a wide range of I/I0 from 0.25 to 0.8 (Figure 2C). The duration time of P1 mostly lied within the range of 0.25 – 10 ms, and was statistically fitted to the exponential function with a value of 0.96 ± 0.08 ms (Figure 2D). Furthermore, as the applied voltage increasing from 80 to 120 mV, the duration of P1 displays strong voltage dependence (Figure 2F and Figure S1), whereas that of P2 remains consistent (Figure S2). These results, integrated with the previous reports of aerolysin30 and α-hemolysin,31 suggesting that P1 is produced by the translocation of the output DNA through aerolysin, whereas P2 resulted from the collision of the DNA with the cis opening of the nanopore. Accordingly, the translocation events with current blockage higher than 80% and the duration within the range of 0.2 – 10 ms could serve as the output DNA signatures for the identification of Ramos cells. Next, on the basis of the signal features, we set out to fully investigate and evaluate this nanopore-based strategy for the detection of Ramos cells. As shown in Figure 3A, in the control experiment without addition of target cells, only a few noise-like blocks were observed in the current trace, which is due to the collision of the reaction mixture with the opening of the aerolysin. Once the Ramos cells were introduced, a large 3



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Figure 3. Detection of Ramos cells with aerolysin nanopore. (A) Representative current traces for the nanopore in the absence (Blank) and presence of Ramos cells recorded at +100 mV. The number of Ramos cells is 1000. (B) I/I0 histogram of the events produced by reaction products of Ramos cells. (C) Duration histogram of the events generated by reaction products of Ramos cells.

Figure 4. Quantification of Ramos cells with aerolysin. (A) Images of the 5 cells and 10 cells obtained with the inverted fluorescence microscope. (B) Representative current traces of the events in the presence of different amounts of Ramos cells. Blue triangles represent the signature events. (C) Correlation of the frequency of signature events with the number of Ramos cells. Data were recorded at +100 mV.

number of distinct blocks with large current blockage appeared that were easily discriminated from the noise-like events. The properties of the translocation events, including the current amplitude (Figure 3B) and duration (Figure 3C), were similar to that observed in the presence of only output DNA. These data unambiguously indicates the presence of the released output DNA, demonstrating the realization of a successful recognition of aptamer by Ramos cells and subsequent enzymatic cycling amplification. Detection Sensitivity of the Nanopore-Based Assay. One of the most important factors affecting the sensitivity of the assay is the reaction time of the enzymatic cycling amplification, whose products are used to quantify the number of the target cells. Thus, the change in the frequencies of the signature events in the nanopore test with the reaction time was investigated. As shown in Figure S3, the frequency was ob-

served to increase gradually with the increase in reaction time and reached a plateau at 2 h. Accordingly, 2 h was selected as the optimum incubation time for the enzymatic cycling amplification reaction in the following experiments. Under the optimal conditions, the sensitivity of the proposed assay was evaluated by monitoring the variance of signal frequency with variable amounts of Ramos cells. To eliminate the effect on time-dependent data, continuous recordings for 5 minutes using a single nanopore for each analyte were analyzed. As shown in Figure 4B, the blank sample reveals only noise-like blocks in the absence of Ramos cells. Upon increasing the target number ranging from 5 (Figure 4A, B) to 10000, we observe a consistent increase of characteristic current events. The corresponding calibration plots display good linear correlation between the frequency of signature events and the logarithm of cell number in this wide range (Figure 4C). The 4


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Analytical Chemistry linear equation can be expressed as the function f = 2.372 + 7.756 log N (R = 0.995), where f is the signal frequency and N represents the cell number. Notably, Ramos cells with the number as low as 5 (Figure 4A) were detected in this assay. To the best of our knowledge, this is the lowest number of cancer cells that can be detectable in the actual measurement, in contrast to those previously reported (Table S1). The excellent sensitivity is likely ascribed to not only the high amplification efficiency of the enzymatic cycling, but also the greatly improved capture rate of DNA by using the salt gradient in the aerolysin nanopore assays. Their effective combination enables the realization of the trace detection of cancer cells. Furthermore, to determine the amount of DNA that is produced from the amplification using 5 cell-binding released DNA as primers, different concentrations of free output DNA were subjected to nanopore test. We constructed the standard working curve of event frequency versus DNA concentration ranging from 5 pM to 500 pM (Figure S4). Based on the frequency of current events produced by the 5 cell reaction products, the amplified DNA was estimated to be 6.9 fmol. This amount is found to be close to the minimum DNA amount (5 fmol) requirement for detection under the condition in this work. Detection Selectivity of the Nanopore-Based Assay. To assess the selectivity of this sensing platform toward Ramos cells, different cancer cells were detected comparably including A549, Jurkat, MCF-7, and Hela with the final amount of 100 cells. As shown in Figure 5A, the appreciable

translocation blocks of output DNA are only produced in response to Ramos cells. In contrast, the addition of the other cells generated few signature events at a frequency close to that of the blank control (Figure 5B). Thus, the strategy exhibited excellent performances for discriminating Ramos cells from the interfering analytes. This could be attributed to the highly specific recognition of aptamer to its target, combined with the precise replication of DNA polymerase and accurate digestion of the nicking enzyme. Real Sample Assay. To further evaluate the real applicability of the strategy, a series of different amounts of Ramos cells were spiked into human serum and detected using the standard method. From the results presented in Table 2, the recovery was found to vary from 92.0% to 102.6%, which are in the acceptable ranges of real sample assay. Therefore, the nanopore sensing platform has a great potential for the applications in complex biological samples. Table 2 Recovery tests of Ramos cells in healthy human serum samples by the nanpore-based strategy a Sample

Added (cell number)

Founded (cell number)

Recovery (%)

1

100

92

92.0

2

1000

1026

102.6

3

10000

9527

95.3

a

The human serum samples are diluted 10-fold before use.

CONCLUSION In summary, we developed for the first time an ultrasensitive nanopore sensor for label-free detection of cancer cells with aerolysin based on aptamer recognition and signal amplification. By introducing a robust, one-step enzymatic cycling amplification into the aerolysin nanopore system, Ramos cells with the number low to 5 cells could be determined in this assay. Compared with the conventional methods, this simple, label-free nanopore-based strategy opens a new horizon for the ultrasensitive detection of cancer cells, holding great promise for potential applications in early diagnosis of cancers. Meanwhile, this assay would open up a wide application of aerolysin to the quantitative determination of a variety of analytes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

P1 duration histograms of output DNA at different voltages (Figure S1), P2 duration histograms of output DNA at different voltages (Figure S2), Dependence of the event frequency on the time of enzymatic cycling reaction (Figure S3), The correlation of event frequency versus the concentration of free output DNA (Figure S4), Comparison between the current assay and other reported methods for cancer cell detection (Table S1) (PDF). Figure 5. Investigation of the selectivity of the assay. (A) Representative current traces in the presence of Ramos cells, A549, Jurkat, MCF-7, Hela, and 0 cell (Blank), recorded at +100 mV. Blue triangles represent the signature events. (B) Comparison of the frequency of translocation events generated by different cancer cells. The final number of the different cells is 100.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected] 5



Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful for the financial supported from the National Natural Science Foundation of China (21405073, 21535002, 21775063), the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201502), and the Shandong Provincial Natural Science Foundation (ZR2016QZ001). We thank Prof. Yi-Tao Long’s help for kindly providing aerolysin and the data analysis software.

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Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal

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