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Electrochemical Detection of Circulating Tumor Cells Based on DNA Generated Electrochemical Current and Rolling Circle Amplification Congcong Shen, Shuping Liu, Xiaoqing Li, and Minghui Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01897 • Publication Date (Web): 27 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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Analytical Chemistry

Electrochemical Detection of Circulating Tumor Cells Based on DNA Generated Electrochemical Current and Rolling Circle Amplification

Congcong Shen,†‡ Shuping Liu,† Xiaoqing Li,† Minghui Yang*†

†Key

Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, College of Chemistry and Chemical Engineering, Central South University, Changsha, China, 410083 ‡School

of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang China, 453007 Email: [email protected] (M. Yang)

Tel: (+86) 731 88836954

1

ACS Paragon Plus Environment

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

ABSTRACT: Circulating tumor cells (CTCs) are important indicators for tumor diagnosis and tumor metastasis. However, the extremely low levels of CTCs in peripheral blood challenges the precise detection of CTCs. Herein, we report DNA generated electrochemical current combing with rolling circle amplification (RCA) as well as magnetic nanospheres for highly efficient magnetic capture and ultrasensitive detection of CTCs. The anti-epithelial-cell-adhesion-molecule (EpCAM) antibody modified magnetic nanospheres were used for enrich and capture CTCs. The following binding of aptamer onto the CTC surface and the subsequent RCA assembled significant amount of DNA molecules onto electrode. The reaction of the DNA molecules with molybdate can then form redox molybdophosphate and produce electrochemical current. Using breast cancer cell MCF-7 as model, the sensor displays good performances towards detection of MCF-7 that spiked into peripheral blood. The signal amplification strategy integrated with magnetic nanosphere platform exhibit good performance in efficient capture and detection of CTCs, which may find wide potential in cancer diagnostics and therapeutics.

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Circulating tumor cells (CTCs) are cells that shed into blood stream from a primary tumor and are carried around the body in the peripheral blood.[1-4] CTCs constitute seeds for the subsequent growth of additional tumors, called metastases, a highly frequent complication that is responsible for as much as 90% of cancer-related deaths.[5] So CTCs have been considered as prognostic biomarkers for cancer diagnosis and tumor metastasis.[6] However, as the number of CTCs in the peripheral blood is very small, ranging from 1 to 200 cells per milliliter of blood, sensitive and precise detection of CTCs from the whole blood still faces great challenges. Due to the extremely rare counts of CTCs in complex blood sample (only one CTC per 1 × 109 white blood cells, WBS),[7] efficient enrichment of CTCs become the first step for the precise detection of CTCs. Different studies reported the isolation of CTCs from peripheral blood utilizing physical (density, size, electric charges, deformability) or molecular (surface receptor expression) differences between blood cells and cancer cells. For example, the isolation of CTCs utilizing microfluidic technologies that based on the difference of size between cancer cells and normal cells.[8,

9]

However, the

microfluidic device fabrication process is rather complex and the throughput of the method is also limited due to the high flow resistance. On the contrary, the most widely used method for isolating CTCs is based on immunomagnetic beads which has the advantage of simplicity, low cost and high throughput.[10-13] CellSearch (Veridex) is the only technology approved by FDA of USA for CTCs enrichment, which utilizes functionalized magnetic nanospheres that bind with tumor specific antigens associated with CTCs, in most cases is the epithelial cell adhesion molecule, EpCAM.[14-16] 3



After enrichment of CTCs from blood, signal amplification is another critical step for the sensitive detection of CTCs. Formerly, our group reported electrochemical signal amplification strategy based on DNA generated electrochemical current, which utilize the reaction of the DNA phosphate backbone with molybdate to form redox molybdophosphate precipitate and then generate electrochemical current.[17-19] This signal amplification strategy is simple, can be combined with traditional nucleotide amplification techniques (e.g., polymerase chain reaction, hybridization chain reaction and rolling circle amplification) and find wide applications for the analysis of various targets. Herein, combing the above mentioned magnetic nanospheres (MNs) for enrichment of CTCs with DNA generated electrochemical current as well as rolling circle amplification (RCA) for signal amplification, we developed a sensitive electrochemical sensor for detection of CTCs in peripheral blood. Breast cancer tumor cell, MCF-7 was chosen to test the methodology. Enrichment and isolation of MCF-7 cells from peripheral blood was achieved using MNs modified with anti-EpCAM antibody (antiEpCAM-MNs) due to the specific binding of the MNs with EpCAM on the surfaces of MCF-7 cells. Then, aptamer-primer DNA sequence was linked onto MCF-7 cell through the binding of aptamer with MUC1 that overexpressed on MCF-7 cell surface. The following RCA triggered by the primer increased the loading of DNA molecules onto electrode, and the reaction of the DNA molecules with molybdate can generate electrochemical current that is proportional to the amount of CTCs detected. The application of the RCA greatly enhanced the sensitivity of the sensor, and this detection 4


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methodology can find wide applications clinically for cancer diagnosis and cancer therapeutics. EXPERIMENTAL SECTION Materials and Reagents: Iron trichloride hexahydrate (FeCl3·6H2O), ethylene glycol (EG), sodium acetate anhydrous (NaAc) and polyacrylic acid (PAA) were purchased from Mackin Biotech Co., Ltd. (Shanghai, China). anti-epithelial cell adhesion molecule antibody (antiEpCAM) was acquired from Abcam Co., Ltd. (Cambridge, MA). Sodium molybdate dihydrate

(Na2MoO4·2H2O),

propidium

iodide,

1-ethyl-3-[3-

dimethylaminopropyl]carbodiimide Hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Sigma Aldrich. DNA aptamer SYL3C-RCA primer and padlock were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Deoxynucleotides (dNTPs) and Cell Counting kit-8 (CCK-8) were obtained from Genview Scientific Inc. SYTOTM 9 green fluorescent nucleic acid stain (SYTO-9) was purchased from Thermo Fisher (USA). E.coli DNA polymerase and ligase were obtained from Takara (Dalian, China). 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (P/S) solution and trypsin were acquired from Gibco BRL, Invitrogen Corp., (Carlsbad, CA, USA). The living MCF-7, Hela cell and normal whole blood were obtained from Xiangya Hospital. Deionized water was used for all the experiments. Synthesis of MNs and anti-EpCAM-MNs Carboxylic group terminated Fe3O4 MNs were prepared according to previously 5



reported hydrothermal method.[20] In detail, 1.35 g FeCl3·6H2O, 3.2 g NaAc and 0.5 mL PAA were added into 38 mL of EG. The mixture was reacted in a sealed teflon reaction kettle at 200 °C for 6 h. After magnetic separation, the black products were washed with water and ethanol several times, and then dried in a vacuum drying oven at 100 °C. To conjugate anti-EpCAM onto the MNs, carboxylic groups on the surface of MNs(7.5 mg) were activated by 75 mM EDC and NHS (1:1) at room temperature for 30 min. The activated MNs were separated magnetically and washed with PBS (0.01 M, pH 7.2), and then reacted with 7.5 g of anti-EpCAM in 1 mL PBS (0.01 M, pH 7.2) for 4 h. The final products anti-EpCAM-MNs were separated and washed by deionized water thoroughly and stored at 4 °C. Cell Culture and Capture MCF-7 and Hela cell were cultured in 1640 medium supplemented with 10% fetal bovine serum, 100 U mL-1 penicillin, and 100 μg mL-1 streptomycin. The cells were incubated under a humidified atmosphere containing 5% CO2 at 37 °C to exponential phase. The cells were separated from the medium by centrifugation at 1000 rpm for 1 min and diluted with normal saline (0.9% NaCl) to different concentrations. For capture and enrichment of cells, 100 L of anti-EpCAM-MNs probe (the concentration of MNs was 7.5 mg mL-1) was added into 1 mL MCF-7 suspension solution with gentle shaking for 1 h at room temperature. The probe was separated by magnet, washed, and then stored at 4 °C. Construction of the electrochemical sensor 6


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The MNs probe was added onto the surface of clean magnetic gold electrode (diameter 2 mm). After thorough washing with deionized water and drying with nitrogen, 10 M aptamer SYL3C-RCA primer (the sequence of DNA used in this assay were listed in table S1) was added onto the electrode and incubated for 1 h. After washing, 10 M padlock probe was added and hybridized with two ends of primer for 1 h. Next, 4 U E.coli DNA Ligase, 1×E.coli DNA ligase buffer, 1×BSA (0.05%) were added and incubated for 30 min to make the padlock probe and primer form a circular template. Subsequently, 2 U E.coli DNA polymerase, 1 mM dNTPs and 1×E.coli DNA polymerase buffer were added onto the electrode for 1 h to complete the RCA reaction. The RCA processes were all achieved at 37 °C and thoroughly washing after every step of reaction. Finally, 5 mM Na2MoO4 was dropped onto the surface of electrode and incubated for 25 min before electrochemical testing. Detection of Spiked MCF-7 from Whole Blood Samples To mimic clinical samples, MCF-7 (10, 50, 100, 300, 600 cell mL-1) were spiked into 1 mL normal whole blood sample. Then, the spiked MCF-7 cells were captured, separated by the MN probe and then detected.

RESULTS AND DISCUSSION Scheme 1 shows the schematic representation for the preparation of the MN probe, the isolation and separation of CTCs, and the sensor preparation as well as detection process. The design of the electrochemical sensor was straight forward. We utilized MNs to enrich and isolate MCF-7 cells, and using DNA generated electrochemical 7



current as detection signal. For signal generation, the phosphate in the DNA backbone reacted with Na2MoO4 to form redox molybdophosphate precipitate and the current generated was measured.[17, 21] To amplify the detection signal, RCA was performed to enhance the loading of DNA molecules onto electrode and then increase the current intensity.[22-24]

A

B

S N

C dNTP

Current (μA)

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

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a

Na2MO4 H+

SWV b

a: without RCA b: with RCA

E vs Ag/AgCl (V)

MNs RBC

Anti-EpCAM Padlock probe

CTCs ligase

WBS Phi29

Aptamer-primer PMo12O43-

Scheme 1. The illustration of CTCs measurement in whole blood based on MNs isolation and RCA signal amplification:(A) Formation of Anti-EpCAM-MNs; (B) Recognition and isolation of CTCs by Anti-EpCAM-MNs; (C) Electrochemical detection of CTCs. Characterization of MNs and anti-EpCAM-MN Probe Hydrothermal method was used to synthesize uniform, carboxylic group terminated MNs. As shown in Figure 1A, the MNs display uniform sphere shape with size around 70 nm. The MNs can be easily separated, and it took only 8s to separate 8


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MNs from solution completely using a magnet, which demonstrated the MNs has good magnetic properties (Figure 1B). Due to expression of EpCAM on many CTC surface, anti-EpCAM antibody was conjugated onto the MNs via the carboxylic groups of the MNs and amino groups of the antibody. After conjugation of anti-EpCAM antibody onto the MNs, as characterized by dynamic light scattering (DLS) and shown in Figure 1C, the size of anti-EpCAM-MNs was larger than bare MNs (Supporting Information, Figure S1). Due to carboxyl group functional MNs will form hydrated nanospheres in aqueous solution, the size measured by DLS was larger than that characterized by SEM. Meanwhile, in Figure 1D, the zeta potential of MNs was -30.8 mV. When modified with anti-EpCAM, the zeta potential became more negative (-43 mV). After the enrichment and separation of MCF-7, the potential increased to -16.1 mV. UV-vis spectroscopy was used to characterize the conjugation of antibody onto MNs, and an absorption peak at 260 nm arise after modification of anti-EpCAM (Supporting Information, Figure S1). In addition, microscopic images demonstrated the successful binding of anti-EpCAM-MNs with MCF-7 cells (Supporting Information, Figure S2). SYTO-9 and PI staining of MCF-7 before and after incubating with anti-EpCAM-MNs in normal saline proved the MCF-7 keep alive after incubated with anti-EpCAM-MNs

for 8 h (Supporting Information, Figure S2). The toxicity of the probe was also studied using CCK-8 kit. The results demonstrated the probe has low toxicity to MCF-7 ( Supporting Information, Figure S3).

9



A

B

200 nm

C

0s

15

a

10 5 0 10

100

1000

2s 0

a: MNs 160.8 nm b b: MNs-EpCAM 248.9 nm

Zeta Potential (mV)

20

Intensity (%)

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

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10000

4s

6s

8s

D

-10 -20 -30 -40 -50

MNs

Size (nm)

MNs

AM CF7 -EpC M-M pCA E s MN

Figure 1. Characterization of MNs and MNs-EpCAM probe, (A) SEM image of MNs; (B) digital photos of MNs before and after interaction with a magnet for 2,4,6, and 8 s; (C) size of MNs before and after anti-EpCAM conjugation; (D) the change of zeta potential during the modification of MNs-EpCAM probe.

Signal amplification by RCA For MCF-7 cell detection, the MNs with enriched MCF-7 cells were dropped onto magnetic electrode surface, and the electrode was then incubated with aptamer-primer DNA sequence. The aptamer was specific to MUC1 that overexpressed on MCF-7 cell surface. After binding of the aptamer with MCF-7 cell, the primer moiety on the DNA sequence can trigger RCA reaction. In the presence of ligase and the circular template, the primer can hybridize with the circular template and initiate the RCA to produce long single-strand DNA with the assistance of DNA polymerase. 10


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To demonstrate the capability of the sensor for MCF-7 detection and the efficiency of signal amplification with RCA, the sensors were characterized by cyclic voltammetry (CV) and square wave voltammetry (SWV). As displayed in Figure 2A, the bare gold electrode reacted with sodium molybdate results in no redox peaks (curve a). Of the sensor for 1.5×103 cell mL-1 MCF-7 without RCA, two pairs of redox peaks appeared, which was attributed to the reaction of aptamer with sodium molybdate that formed the redox-active molybdophosphate precipitate (curve c). However, of the sensor for 0 cell mL-1 MCF-7 with RCA, there are also two pairs of rather weak current peaks, which may be caused by a small amount of DNA nonspecifically adsorbed onto electrode during RCA process (curve b). As the concentration of MCF-7 increased from 6×10 to 6×103 cell mL-1, the current intensity enhanced significantly (curve d and e). Especially, even the concentration of MCF-7 cell is much lower, the current intensity of the sensor for 6×10 cell mL-1 of MCF-7 with RCA is higher than that of the sensor for 1.5×103 cell mL-1 of MCF-7 without RCA. In addition, the SWV responses of the sensor for 1.5*103 cell mL-1 MCF-7 after RCA is about 3 time higher than that without RCA (Supporting Information, Figure S4). These data demonstrated the signal amplification by RCA can significantly enhance the sensitivity of the sensor. The corresponding SWV responses of the sensors are shown in Figure 2B, which are in accordance with CV results. The principle of the signal generation was study in our previous reports.[25, 26] In brief, the two pairs of peaks (about 0.22 V and 0.37 V) were caused by the electrons transfer within molybdophosphates.

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-2

A

0

-1

1

a b c d

2

e

0

3 0.1

B a b

-2

0.2

Current (A)

Current (A)

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0.3

0.4

-4 -6

a: 0 Cell/mL with RCA

-8

b: 1.5*103 Cell/mL without RCA c: 6*101 Cell/mL with RCA

-10 0.10

0.5

c

d 0.15

0.20

E vs AgCl (V)

d: 6*103 Cell/mL with RCA

0.25 0.30 0.35 E vs AgCl (V)

0.40

0.45

Figure 2. (A) CV responses of the electrochemical sensor after reaction with molybdate: (a) bare gold electrode; (b) sensor for 0 cell mL-1 with RCA; (c) sensor for 1.5×103 cell mL-1 without RCA; (d) sensor for 6×10 cell mL-1 with RCA; (e) 6×103 cell mL-1 with RCA. (B): The corresponding SWV responses of the sensors. Supporting electrolyte, 0.5 M H2SO4.

Analytical performance of the sensor towards MCF-7 detection To achieve quantitative detection of MCF-7, different concentrations of MCF-7 were detected by the sensor with SWV measurement. As depicted in Figure 3, the current response of the sensor at about 0.2 V is increased along with the increasing of MCF-7 concentrations. This result is because higher concentration of MCF-7 leads to more aptamer-RCA primer captured onto sensor surface. The relationship between the change of current intensity and the logarithm of MCF-7 concentration is linear in the range from 5 cell mL-1 to 3×104 cell mL-1(as exhibited in inset of Figure 3) with a linear correlation coefficient of 0.992. On the base of S/N = 3, the calculated limit of detection was 1 cell mL-1. When the concentration of MCF-7 is further increased, the current intensity decreased, on the contrary (Supporting Information, Figure S5). This result 12


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may be ascribed to excess MCF-7 on the electrode increased the impedance of the sensor and slowed down the electron transfer rate of the sensor. 12

0

a

-4

Current (A)

10

-2

Current (A)

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


8 6 4 2 0

1

-6

10 100 1000 10000 LogC（ Cell mL-1（

-8 -10

g

-12 0.10

0.15

0.20

0.25 0.30 0.35 E vs AgCl (V)

0.40

0.45

Figure 3. SWV responses of the electrochemical sensor to different concentrations of MCF-7, from (a) to (g):0, 1, 5, 6×10, 6×102, 6×103, 3×104 cell mL-1. The inset is the calibration curve. Supporting electrolyte, 0.5 M H2SO4.

The analytical performance of this electrochemical sensor was compared to literature reported biosensors for CTCs detection. As exhibited in table 1, this sensor displays higher sensitivity and lower detection limit.

Table 1.Comparison of analytical performance for CTCs detection Method

Target cell

Detection limit （cell mL-1）

Detection range （cell mL-1）

References

ICP-MS

MCF-7

50

2×102-4×103

[27]

I-t

MCF-7

4

0-1×105

[28]

LSV

CCRF-CEM

100

1×102-1×106

[29]

13



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DPV

MCF-7

3

2×101-1×106

[30]

DPV

MCF-7

34

5×101-1×107

[20]

Fluorescence

CCRF-CEM

400

4×102-5×106

[31]

SWV

MCF-7

1

5×100-3×104

Our work

Selectivity of the sensor After proved the high sensitivity and low detection limit of the sensor, the selectivity of the sensor was studied. Since the MUC1 protein are not overexpressed on the surface of Hela cells, Hela cells are selected as control to study the selectivity of the senor. As shown in Figure 4, the SWV response of the sensor to 1000 cell mL-1 of Hela cells is very weak, similar to the response of the sensor to blank sample. In addition, after 1000 cell mL-1 of Hela cell was mixed with different concentrations of MCF-7, the peak current was increased gradually, and the current intensity was in consistent with individual MCF-7 sample without Hela cell. This phenomenon proved that the sensor has high specificity, which can be ascribed to MCF-7 identifying with both antiEpCAM antibody and SYL3C aptamer. What’s more, different concentrations of Hela (0, 10, 100, 1000 cell mL-1) was mixed with 100 cell mL-1 MCF-7 and detected. The results indicated Hela cells had negligible effects on the MCF-7 enrichment and detection (Supporting Information, Figure S6).

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10

Hela+MCF-7 MCF-7

8

Current (A)

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


6 4 2 0

0

10

100

CMCF-7 (cell mL-1)

1000

Figure 4. Comparison the specific recognition of electrochemical cystosensor for Hela cells and MCF-7 cells: Various concentrations of MCF-7 cells (0, 10, 100, 1000 cell mL-1) mixed with 1000 cell mL-1 Hela, and the corresponding MCF-7 as control.

Detection of MCF-7 in Whole Blood As an important biomarker of breast cancer, the monitoring of MCF-7 in peripheral blood has significant clinical impact. Then, the capability of the sensor for detection of MCF-7 in whole blood sample was investigated. The samples were prepared by spiking various concentrations of MCF-7 cell (10, 50, 100, 300, 600 cell mL-1) into 1 mL whole blood sample that without any pre-treatment. The recovery test results of MCF-7 in whole blood and 0.9% NaCl are shown in Figure 5 The regression analysis of detected cell numbers versus spiked cell number were expressed by y= 2.817+0.6295x (R2=0.966) in blood, and y= 2.013+0.968x (R2=0.999) in 0.9% NaCl, respectively. The recovery rate of MCF-7 from whole blood is between the range from 50.4% to 77.4%. The relatively low recovery may mainly be ascribed to the high 15



viscosity of whole blood, and a larger number of red cell, white cell as well as proteins as potential interferences.

600

Captured cell numbers

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

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0.9% NaCl blood

500 400 300 200 100 0 0

100

200 300 400 Spiked cell numbers

500

600

Figure 5. Comparison of detection efficiency for MCF-7 cell in 0.9% NaCl and whole blood using the electrochemical sensor.

CONCLUSION In summary, we reported electrochemical sensor for rapid, efficient and sensitive detection of CTCs in whole blood based on magnetic nanosphere separation and DNA generated electrochemical current. MNs (with size about 70 nm ) were modified with anti-EpCAM antibodies, and then can enrich and isolate CTCs from samples. For proofof-concept demonstration, the breast cancer cell MCF-7 was tested. The utilization of RCA signal amplification strategy significantly enhanced the detection sensitivity, leading to low detection limit of the sensor. The sensor was successfully employed for detecting MCF-7 in peripheral blood samples, indicating potential clinical applications. In addition, the sensor can be easily adapted to the sensitive detection of other 16


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biomarkers.

ASSOCIATED CONTENT

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors thank the support of this work by the National Natural Science Foundation of China (No. 21575165). Supporting Information The supporting Information is available free of charge on the ACS Publications website at DOI:A table with the DNA sequence used in this study. Characterization of Fe3O4 magnetic nanospheres (MNs) and MNs-EpCAM probe. Bright, fluorescence field and overlay micrographs of MCF-7 incubated before and after with anti-EpCAM-MNs, which stained with SYTO-9 and DAPI. The toxicity of different concentrations of antiEpCAM-MNs for MCF-7 cell. SWV responses of the cystosensor to MCF-7 before and after with RCA. SWV responses of the electrochemical cystosensor to different concentrations of MCF-7. Responses of the sensor to sample containing different concentrations of Hela mixed with MCF-7.

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SWV

Current (μA)

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a

b

a: without RCA b: with RCA

E vs Ag/AgCl (V)

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Electrochemical Detection of Circulating Tumor ... - ACS Publications

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