Direct Plasmon-Enhanced Electrochemistry Enables Ultrasensitive

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Direct Plasmon-Enhanced Electrochemistry Enables Ultrasensitive and Label-Free Detection of Circulating Tumor Cells in Blood Shan-Shan Wang, Xiao-Ping Zhao, Fei-Fei Liu, Muhammad Rizwan Younis, Xing-Hua Xia, and Chen Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04908 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Direct

Plasmon-Enhanced

Electrochemistry

Enables

Ultrasensitive and Label-Free Detection of Circulating Tumor Cells in Blood Shan-Shan Wang,† Xiao-Ping Zhao,† Fei-Fei Liu,† Muhammad Rizwan Younis,‡ Xing-Hua Xia,*‡ Chen Wang*†

†Key

Laboratory of Drug Quality Control and Pharmacovigilance (China

Pharmaceutical University), Ministry of Education; Key Laboratory of Biomedical Functional Materials, School of Science, China Pharmaceutical University, Nanjing, 211198, China ‡State

Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210093, China *To whom correspondence should be addressed. E-mail: [email protected]; [email protected]

ABSTRACT: In this work, we developed a simple electrochemical method for ultrasensitive and label-free detection of CTCs based on direct plasmon-enhanced electrochemistry (DPEE). After plasmonic gold nanostars (AuNSs) were modified on the glassy carbon (GC) electrode, the aptamer probe was immobilized on AuNSs surface, which can selectively capture the CTCs in samples. Upon LSPR excitation, the electrochemical current response can be enhanced remarkably due to efficient hot electrons transport from AuNSs to the external circuit. The captured cells on AuNSs surface will influence the hot electrons transport efficiency, leading to a decreased current response. Using ascorbic acid (AA) as the electroactive probe, it was found that the current responses of AuNSs/GC electrode upon light irradiation decrease with the cell concentration. Due to the special molecular recognition of aptamer and enhanced electrochemical performance of plasmon, the proposed method enables an ultrasensitive and label-free detection of CTCs with excellent selectivity. The experimental results show that CCRF-CEM cells concentration as low as 5 cells mL-1

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can be successfully detected, which is superior to most reported work up to now. Using the present method, the MCF-7 cells as low as 10 cells mL-1 can be also successfully detected, indicating the universality of the proposed method for CTCs detection. Furthermore, the cytosensor can successfully distinguish CTCs from normal cells in blood samples. The as-proposed strategy provides a promising application of DPEE in the development of novel biosensors for nondestructive analysis of biological samples. Keywords: Direct plasmon-enhanced electrochemistry, circulating tumor cells, gold nanostars, cancer, biosensor

Circulating tumor cells (CTCs) are viable cancer cells that transfer from the primary tumor site into peripheral blood. As CTCs pass through the circulatory system, they can form metastasis in the resident organs, eventually leading to cancer-related deaths.1,2 Therefore, CTCs have been considered as prognostic biomarkers in tumor metastasis and cancer diagnostics.3 The detection of CTCs is of great significance toward effective monitoring and early diagnosis of cancers.4-6 However, the major challenge lies in the extremely rare counts in an complex blood sample (just a few CTCs mixed with the approximately 10 million leukocytes and 5 billion erythrocytes in 1 mL of blood).7,8 It is of urgent demand to develop simple and ultrasensitive approaches to efficiently detect the trace amounts of CTCs in blood samples. Up to now, various techniques have been developed for CTCs detection including chromatometry,9-11 near-infrared fluorescence,12,13 photoluminescence,14 fluorescent imaging,15 inductively coupled plasma-mass spectrometry,16,17 Raman imaging,18 and Dielectrophoresis.19 For example, Kelley and co-workers integrated circuit that combines the capture of CTCs with the profiling of their gene expression signatures to rapid monitoring of CTCs in prostate cancer.6 The conventional CTCs detection techniques help us to understand the tumor metastasis mechanism in depth.20-22 However, most of these methods suffer from either time-consuming experimental procedures or sophisticated instruments, which ultimately limit their


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application in bioanalysis and clinical diagnose. In comparison, the electrochemical technique such as Differential pulse voltammetry (DPV)23, Electrochemical impedance spectroscopy (EIS)24 and Chronoamperometry (CA)25 has been a particularly attractive tool for bioanalysis and detection due to fast response, low background, high sensitivity, ease of operation and low manufacturing cost.25-28 For example, Zhu et al. reported a ultrasensitive electrochemical cytosensing based on Fe3O4@nanocage core-satellite nanohybrids by DPV with a detection limits for MCF-7 to be ∼34 cells mL−1 at 3σ.23 Isik et al. designed a low-cost, highly sensitive and selective cytosensor using EIS, which exhibits a detection limit of 10 cells mL−1.24 Kelley and his co-workers developed a highly specific electrochemical method for detection of CTCs using nanoparticle labels. The analysis method requires less time and the instrumentation required is cost-effective. Due to the special binding of labels to the target cells, the electrochemical assay enables selectively discrimination between cancer cells and normal blood cells.29 Recently, Dai’s group developed a simple and sensitive photoelectrochemical biosensor for the nondestructive analysis of living cells. A wide linear range from 1×102 to 1×107 cells/mL with a detection limit of 100 cells/mL was achieved.25 Localized surface plasmon resonance (LSPR) arises from the collective oscillation of conduction electrons of metal nanostructures driven by the applied electromagnetic field of incident light.30,31 As a result of LSPR excitation, a strong electromagnetic field and a high concentration of energetic charge carriers (electron-hole pairs) are generated at the nanostructured surface. Due to the higher energy of the generated charge carriers than those of thermal excitations at ambient temperatures, they are also called as “hot electrons” and “hot holes”.32 LSPR enables the noble metal nanoparticles several unique benefits including the enhanced local electromagnetic fields, efficient charge carrier separation and the heat effect during the photon dissipating. These features make noble metallic nanoparticles be widely applied in chemical, physical and biological research field.32-44 It has been reported that LSPR can be used for surface-enhanced spectroscopies including surface-enhanced Raman scattering,38-40 infrared spectroscop41 and ﬂuorescence.42,43 Recently, it is found that



the LSPR effect can not only enhance the spectroscopies but the electrochemistry.44 In this work, the hot electrons are driven into the external circuit by a positive potential. The remaining hot holes are driven to AuNPs surface for accelerating the glucose oxidation. By investigating the effect of the LSPR wavelength, light intensity and temperature, a direct plasmon enhanced electrochemistry (DPEE) mechanism was proposed. Based on the DPEE mechanism, a plasmon-improved glucose electrochemical detection was constructed, demonstrating the remarkably enhanced performance of the sensor by LSPR effect. This work provides a novel strategy for designing powerful electrochemical devices and assays using LSPR effect. In this work, the principle of DPEE is first used for ultrasensitive and label-free detection of CTCs in blood. The detection principle is illustrated in Scheme 1. First, the gold nanostars (AuNSs) were dropped on GC electrode surface, forming AuNSs/GC electrode. The thiol aptamer probe sgc8c was then immobilized on the AuNSs/GC electrode by the Au-S bond, which can specially bind with the transmembrane receptor protein tyrosine kinase 7 highly expressed on CCRF-CEM cells (one type of CTCs) membrane, thus enabling the selective trapping of CCRF-CEM cells.26,45 The trapped CTCs efficiently cover the AuNSs/GC electrode surface, resulting in a decreased AuNSs surface. The ascorbic acid (AA) is used as the electroactive probe, which can be oxidized by AuNSs/GC electrode. Upon light irradiation, the current response increases remarkably due to DPEE. It was found that the enhanced current responses by light change with the CTCs concentration, based on which, an ultrasensitive and label-free electrochemical detection method for CTCs can be achieved. Due to the special aptamer recognition capacity and the DPEE mechanism, the present method enables ultrasensitive and label-free detection of CTCs in blood with excellent selectivity. The results showed that CCRF-CEM cells concentration as low as 5 cells/mL can be successfully detected. In addition, we also detect the MCF-7 cells with the concentration as low as 10 cells, indicating this is a universal method for different CTCs detection. This work provided a promising application of DPEE in the nondestructive analysis of biological samples, which would hold great potential in the early clinical diagnosis and treatment of cancers, as


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well as the constructing of sensitive biosensors.

Scheme 1. Schematic illustration of the strategy for ultrasensitive and label-free detection of CTCs by the DPEE mechanism.

EXPERIMENTAL SECTION Materials and Reagents. Aptamer sgc8c, sequence 5ʹ-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTT TTT-3ʹ-(CH2)6-SH, 6-FAM-5ʹ- ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTT TTT-3ʹ (FAM-sgc8c) and red cell lysis buffer were ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China). Aptamer SYL3C, sequence HS-(CH2)6-5ʹ-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3ʹ was ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China). The CCRF-CEM (CCL-119, T cell line, human, acute lymphoblastic leukemia, ALL) ， the MCF-7 (human breast cancer cells), Ramos (B-cell lymphoma cell line) and the Raji (CCL-86, B lymphocyte, homo sapiens, human) were from the Shanghai Institutes for Biological Sciences (China). The K562 (CCL-243, human CML) was obtained from Beijing Xiehe Hospital. Roswell Park Memorial Institute-1640 (RPMI-1640) culture medium, Dulbecco's Modified Eagle Medium (DMEM), Acridine orange (AO), propidium iodide (PI) were purchased



from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Penicillin-streptomycin solution and Fetal bovine serum (FBS) were from Corning Co. (Manassas). Ascorbic acid (AA), poly (vinylpyrrolidone) (PVP, average MW=10,000), Nafion perfluorinated resin solution and 6-mercapto-1-hexanol (MCH) were from Sigma-Aldrich. Gold chloride (HAuCl4·4H2O) was from the First Reagent Factory (Shanghai, China). The 10 mM phosphate buffer (PBS, pH 7.4) was used as the rinsing solution. All solutions were prepared by ultrapure water (18.2 MΩ/cm) and all reagents were of analytical grade. Instrumentation. The morphology of the prepared gold nanostars was characterized on a transmission electron microscopy (TEM, JEM-2100, Japan) and scanning electron microscope (SEM, S-4800, Japan). UV−vis absorbance was performed on a UV-1800 spectrophotometer. Cells were imaged using a inversion microscope system (Olympus IX53). Synthesis of AuNSs. AuNSs were synthesized according to the method as described before.46 First the gold seeds (15 nm, PVP-coated) were synthesized based on the reported method.47 The PVP was dissolved in water by ultrasonication for 10 min. Subsequently, the PVP and the as-prepared gold colloidal solutions were mixed. To ensure the efficient adsorption, the mixed system was stirred at least for 24 h at room temperature. The as-prepared particles were then added into ethanol. The synthesized gold seeds were centrifuged to remove the unbound PVP. After that, the precipitate was re-dispersed in ethanol for growth. The chloroauric acid (110 μL, 25 mM) was mixed with PVP (10 mL, 10 mM), followed by rapid addition of 29 μL 15 nm PVP-coated gold seeds under continuous stirring. When the solution color changed from pink to blue, the AuNSs formed successfully. Fabrication of AuNSs and Aptamer Modified Electrodes. The fabrication of AuNSs modified electrode is illustrated in Scheme 1. After the GC electrode (diameter of 3 mm) was polished by 0.05 μm alumina slurries and washed by water, 5 μL AuNSs were dropped on the pretreated GC electrode, forming AuNSs/GC electrode. Then Nafion (5 μL, 0.05% in alcohol) was dropped and covered on the GC surface, preventing the leaking of the catalysts. For aptamer modification, 10 μL


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sgc8c (10 μM) was dropped to the AuNSs/GC and incubated in the refrigerator overnight. After that, the electrode was washed by PBS buffer (pH 7.4) and immersed in MCH (100 nM, 100 μL) for 30 min to block the nonspecific binding sites and control the orientation and spatial distribution of the aptamer.48 Finally the fabricated Aptamer/AuNSs/GC electrode was washed by water and dried for use. Cell Culture. CCRF-CEM cells, Ramos cells, Raji cells and K562 cells were cultured in RPMI-1640 medium at 37 °C, supplemented with penicillin (100 μg mL−1), FBS (10%) and streptomycin (100 μg mL−1) in the cell incubator. MCF-7 cells were cultured with DMEM under the same experiment condition. Cell Capture and Detection. For cell capture, the fabricated Aptamer/AuNSs/GC electrode was immersed in 1 mL of the cell suspension, incubating at 37 °C for 45 min. It was considered that 37 ℃ is the adaptable environment for cells activity, which will be beneficial for the binding of cells with aptamer. The experimental method was used in some previous references.23,49 After cells capture, the electrode was washed by PBS buffer, removing the cells nonspecifically adsorbed. Finally, the as-prepared electrode was inserted in 1mM AA to record the current response with 808 nm light (200 mW/cm2) on and off. The electrochemical experiments of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometric curve (I-t) were performed on a CHI 660E instrument (Chenhua, China) at 25℃. The prepared AuNSs/GC electrode was working electrode, and the reference electrode and auxiliary electrode is respectively Ag/AgCl and Pt wire. The CVs were conducted from -0.2 to 0.6 V with the scan rate of 50 mV/s. The I-t curves were performed at 0.4 V (vs Ag/AgCl) in 1mM AA. The impedance

spectra

were

tested

in

10

mM

PBS

buffer

containing

K3Fe(CN)6/K4Fe(CN)6 (10 mM, 1:1) and 0.1 M KCl. The frequency range is 0.1-106 Hz, and the signal amplitude is 5 mV. Fluorescence Microscope Analysis. The modified ITO glass was prepared as described for modified GC electrode, and then incubated with CCRF-CEM cells suspension at 37 °C for 45 min. After being rinsed with PBS, the cells were stained with AO fluorescent dye in the dark, then thoroughly washed via PBS and detected



using the fluorescence inversion microscope (Olympus IX53). Cell Viability Analysis. Cell viability was detected using well-established AO/PI staining assay.26 The cells suspension was dropped into an AO/PI solution (1 μg/mL AO and 1 μg/mL PI in 10 mM PBS) for 5 min. After staining, viable and dead cells were observed by the fluorescence inversion microscope. Blood Sample Preparation. The blood sample was collected from healthy volunteers. Different amount of CCRF-CEM cells were spiked into the sample. After being centrifuged at 1500 rpm for 5 min, the supernatant of the blood sample was discarded. The red blood cell lysis buffer (1 mL) were added into the system, incubating for 5 min. Then the sediment was washed and suspended using PBS, and the cell suspension was obtained.27 Confocal microscopy. Fluorescent images were taken by a confocal laser scanning microscope (LSM800, CLSM, Zeiss, Germany) and processed using the ZEN imaging software. Cells were treated with FAM-sgc8c in cell culture medium and incubated for 2 h, followed by washing with PBS. The resultant cells were then observed by confocal microscopy. Flow cytometry. Flow cytometry analysis was performed on a FACScan cytometer (MACSQuant Analyzer 10). Different cells including the CCRF-CEM cells, K562 cells and Raji cells with a concentration of 1 × 106 cells/mL were incubated with FAM-sgc8c aptamer (500 nM) at 4C for 30 min. Then the cells were centrifuged, washed by PBS, and then re-dispersed in the binding buffer for flow cytometry analysis.

RESULTS AND DISCUSSION Characterization of AuNSs. The morphology of AuNSs was characterized using TEM. The image in Figure 1A indicates the synthesized AuNSs resemble a star-like assembly with some sharp tips. The average size of the AuNSs is 60 nm in diameter with good monodispersity and uniform morphology. Figure 1B is the UV/vis extinction spectra of the prepared AuNSs. A dominant LSPR band with the absorption peak at ~767 nm occurs in the near-infrared region.46 To confirm that the AuNSs have


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been successfully modified on GC electrode, the electrocatalysis of AuNSs toward AA was investigated. AuNSs/GC electrode shows a well-defined anodic peak and higher current value for the elelctrochemical oxidation of AA in dark (black curve). In comparison, the bare GC electrode shows no electochemical response towards AA oxidation in the dark (Figure 1C, blue curve). The result demonstrates AuNSs have been successfully modified on GC electrode. More importantly, upon illumination of AuNSs/GC electrode by 808 nm light source, the current response increases significantly (Figure 1C, red curve), while no obvious change occurs on the bare GC electrode (Figure 1C, pink and blue curves). The phenomenon is caused by the previous reported DPEE mechanism.44 To confirm the well adhering of AuNSs to GC surface, the same amount of AuNSs was dropped on a GC slide and then 5 μL of Nafion (0.05% in alcohol) was dropped on the catalysts surface to prevent being leaked during the experiment process. The fabricated AuNSs/GC slide was used as the electrode for the same electrochemical experiment. The SEM images of the AuNSs/GC slide before and after experiment were taken, which were added in the inset of Figure 1A and Figure S1. The similar density suggests the stability of AuNSs. In addition, the prepared AuNSs/GC electrode was also tested by scanning the electrode in 1 mM AA continuously (Figure S2). The current response does not changed obviously even after 100 consecutive cycles, indicating that the quite good stability of the prepared electrode during the whole experimental process.



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Figure 1. (A) TEM image of the prepared AuNSs. Inset: SEM image of AuNSs deposited on a GC slide before experiment. (B) UV−vis extinction spectrum of the prepared AuNSs. (C) CVs for 1 mM AA oxidation on the AuNSs/GC and GC electrode with light irradiation on and off. (D) EIS of different electrodes: (a) GC, (b) AuNSs/GC, (c) Aptamer/AuNSs/GC, (d) AuNSs/aptamer/MCH/GC and (e) after incubation with CCRF-CEM (1 × 105 cells/mL) for 45 min. The inset shows the partial enlarged circuit in Figure 1D.

EIS characterizes the properties of electrode surface, and can be used to detect the stepwise construction process of the prepared electrode. It is known that the impedance spectrum consists of a semicircular portion and a linear portion. The semicircle portion is relevant to the process at high frequencies (electron transfer limited),

and

the

linear

portion

relates

to

process

at

low

frequencies

(diffusion-limited). The semicircle diameter at high frequency region is the value of the charge-transfer resistance (Rct).28 In this work, the redox probe [Fe(CN)6]4−/3− was used for EIS measurement. As shown in Figure 1D, the Rct value of the AuNSs/GC


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electrode is relatively smaller (curve b, Rct=402.190 Ω) than GC electrode (curve a, Rct=444.523Ω), indicating a lower electron transfer resistance and a faster charge exchange of [Fe(CN)6]4−/3− on AuNSs/GC electrode compared to naked GC electrode. Nevertheless, the Rct value increases after being modified by the aptamer (curve c, Rct=520.683Ω) and MCH (curve d, Rct=587.951Ω) on the electrode surface due to the weakly conductive properties of aptamer and MCH. In our experiment, the pore size of Nafion is around 5 nm, with minor changes under different ionic strength conditions.50,51 The sgc8c aptamer is single-stranded DNA, and the diameter of the aptamer is no more than 2 nm. Thus the pores within Nafion layer are large enough for aptamer (with the width less than 2 nm) to pass through Nafion layer to reach the AuNSs. After the CCRF-CEM cells were incubated on the electrode, a remarkable increase in Rct appears (curve e, Rct=7117.912Ω) owing to the dramatic blocking of electron transfer process by cells. The values of Rct for the cytosensor could be seen in Table S1. The results of the stepwise EIS characterizations suggest successful fabrication of Aptamer/AuNSs/GC electrode for selectively capture of cells. DPEE Based Electrochemical Detection of CTCs. After incubation of the Aptamer/AuNSs/GC electrode with the CTCs in PBS, the CVs of the electrode before and after cells capture were investigated. As shown in Figure 2A, after cell capture, the current response decreases obviously due to the blocking effect of captured cells on the electrode surface (∆I1 from curve a to b). However, upon light irradiation, the current increases remarkably due to DPEE mechanism (∆I2 from curve b to c). The increased current (∆I2 = 0.4 μA) is more than 2 folds higher than the reduced current (∆I1= 0.16 μA), thus enables a more sensitive analysis. Then the photocurrent response was measured at 0.4 V (vs. Ag/AgCl) upon illumination of the electrode. Curves (a-d) in Figure 2B are the varied current responses of different electrodes towards AA oxidation with light on and off. The AuNSs/GC electrode has the highest current enhancement (curve a, ~1.44 µA) upon light on. When the aptamer and MCH was attached on the electrode surface, the enhanced current by DPEE decreased slightly to 1.37 µA and 1.32 µA (curve b, c) due to the steric blocking effect. After the electrode was incubated with CCRF-CEM cells (1×105 cell/mL) for 45 min, the



enhanced current response remarkably fell to 0.56 µA (curve d). Owing to the efficient hot electron transfer by light irradiation, AuNSs can dramatically enhance the current response of AA electrocatalysis. The captured cells on AuNSs surface will influence the hot electrons transport efficiency from AuNSs to the external circuit, leading to a decreased current response. The experimental results demonstrate the feasibility of the DPEE based label-free and sensitive detection of CTCs.

Figure 2. (A) CVs of 1 mM AA (in 10 mM PBS, pH 7.4) on aptamer/AuNSs/GC electrode. Blue curve: CV in 1 mM AA; Black curve: CV after being incubated with CCRF-CEM cells (1×105 cell/mL) for 45 min; Red curve: CV after the electrode was incubated with CCRF-CEM cells (1×105 cell/mL) for 45 min with light on. (B) I-t curves on different electrodes in 1 mM AA with light on and off: (a) AuNSs/GC, (b) Aptamer/AuNSs/GC, (c) MCH/aptamer/AuNSs/GC, (d) after incubation with 1 × 105 cells/mL CCRF-CEM for 45 min. The potential was set at 0.4 V (vs. Ag/AgCl).

Effect of the Aptamer Concentration. Aptamers are well known for their high affinity and specificity toward the target molecules. To achieve efficient cell capture, the effect of the aptamer probe concentration was investigated. As shown in Figure 3A, the current response firstly decreases with the increase of aptamer concentration from 1 to 10 μM, then becomes steady at concentration higher than 10 μM. Figure 3B shows the corresponding current values with light on at varied aptamer concentration, from which we can see that 10 μM of aptamer is optimal caoncentration for cell capture. The bright-field microscope images were used to observe the cell capture


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results under different aptamer concentrations. As can be seen from Figure 3C, the as-prepared aptamer/AuNSs/GC electrodes could efficiently capture the CCRF-CEM cells. The amount of the captured cells increases obviously with aptamer concentration until the aptamer reaches 10 μM. Under this circumstance, the trapped cells nearly cover the whole electrode surface (Figure 3C). Therefore, the similar current is expected when further increasing aptamer concentration from 10 to 50 μM. Therefore 10 μM is chosen as the aptamer concentration in the following work.

Figure 3. (A) I−t curves of different concentrations of aptamer sgc8c with light on and off. (B) The enhanced current values (from Figure 3A) versus the aptamer concentrations. (C) Bright-field microscope images of the cells captured on the as-prepared aptamer/AuNSs/GC electrodes. The sgc8c concentrations vary from 1-50 μM. The cell concentration is 1× 105 cell/mL.

Cells Capture Kinetics. The incubation time of CCRF-CEM cells is another critical factor for efficient cells capture. In order to achieve the optimum capture time, the cells capture kinetics was investigated. The electrode was incubated with CCRF-CEM cells solution for varied times (0, 10, 30, 45, 60, 90) at 37 °C, and the electrochemistry of AA was recorded accordingly (Figure 4A). The oxidation current



of AA decreases with the incubation time significantly at first (from 10 min to 45 min), then levels off after 45 min. The peak current values were displayed as the black curve in Figure 4C, indicating efficient cells capture within 45 min. In addition to CVs, the I-t curves were also investigated with varied cell capture time (Figure 4B). The current values were displayed as red curve in Figure 4C. It is clear that both CVs and I-t curves offer the similar change trend, demonstrating both are available for capture kinetics study. However, the change in I-t curves is more obvious compared to CVs, suggesting a more sensitive technique by light irradiation based on DPEE mechanism. In our work, the cell incubation time was 45 min. Figure 4D shows the fluorescence microscope image of captured cells on ITO glass which were stained by AO/PI method. The inset shows the electrode surface before cell capture. The image confirms the successful capture of CTCs by the aptamer modified electrodes.

Figure 4. (A) CVs of 1 mM AA on Aptamer/AuNSs/GC electrode after incubation with CCRF-CEM cells for different time. (B) I-t curves of the Aptamer/AuNSs/GC electrodes in 1 mM AA after incubation with CCRF-CEM cells for different time with light on and off. (C) The peak current values in Figure 4 A (black dots) and the


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current response with light on in Figure 4B (red dots) versus the cells incubation time. (D) The fluorescence image of the ITO electrode after cells capture and followed by AO/PI staining. The inset shows the fluorescence image of the electrode before cells capture.

It is worthy to note that the baseline in the current versus time in Figure 4B does not return to the same baseline after the peak. This phenomenon is caused by the heat effect under light irradiation. It is well known that the non-radiative decay the excited free electrons upon LSPR excitation may lead to the effective conversion of photon energy to thermal effect.44 In our work, when light is turned on, the surface temperature of working electrode is slightly higher than that before light irradiation, which resulting in the slight change of the baseline after light off. Detection of CTCs. Under the optimum conditions, the DPEE based electrochemical method was used for detection of CCRF-CEM cells. The I-t curves upon light irradiation after capture of different concentrations of CTCs were recorded, as shown in Figure 5A. The relationship between the changed current value with light on-off and the cell concentrations is displayed in Figure S4A. It is clear that the changed current values increase with the cell concentration due to the growing steric hindrance by cells. The changed current values upon light irradiation are proportional to the logarithm of cell concentrations. The calibration equation is ΔI (μA) = 0.1312 lgC + 0.09745 with a correlation coefficient of 0.999 (ΔI = I0 - I, where I is the photocurrent value at different cell concentrations, and I0 is the photocurrent response without cells. C is the cell concentration). The linear range is 5~1×105 cells/mL (Figure 5B). The detection limit is calculated as 5 cells/mL based on 3 S/m method, where S is the standard deviation of blank experiments, and m is the slope of calibration curve.52,53 The achieved sensitivity is higher than mostly reported works (Table S2). To demonstrate the excellent performance of our present DPEE based detection method, another electrochemical quantification technique using the varied CVs of AA electrocatalysis upon incubation with different concentrations of cells (Figure 5C). It was found that the peak current values in CVs decrease with the increase of cell



concentration due to the same blocking effect by captured cells. The relationship between the peak current values and cell concentration is shown in Figure S4B. It is found that the linear range is from 1×102 to 1×105 cells/mL (Figure 5D) with the detection limit of 100 cells/mL. It is clear that the present CTCs detection strategy based on DPEE mechanism shows a 20 folds higher sensitivity compared to the conventional method without light irradiation, showing promising potential of DPEE mechanism in construction of robust biosensors and clinic diagnostic techniques with excellent performance. In order to demonstrate the universality of the proposed strategy, we also detected the MCF-7 cells (breast cancer cell lines) using the same method (Figure S5). The used aptamer was SYL3C, which can specifically recognize the epithelial cell adhesion molecule (EpCAM) overexpressed on MCF-7 cells.23 Results show that MCF-7 as low as 10 cells/mL could be successfully detected. The calibration equation is ΔI (μA) = 0.1457 lgC - 0.0200 with a correlation coefficient of 0.997 and linear range of 10~1×105 cells/mL.

Figure 5. (A) I−t curves of the electrode after capturing different concentrations of CCRF-CEM cells with light on and off. The concentration of curve (a-g) is 0, 5, 50, 1


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× 102, 1 × 103, 1 × 104, 1 × 105 cells/mL respectively. (B) The relationship between the current change (ΔI) and the logarithm value of cells concentration from 5 to 1×105 cells/mL. (C) CVs after CCRF-CEM cells capturing in the dark. The curves (a-f) of different cells concentrations are respectively: 5, 50, 1 × 102, 1 × 103, 1 × 104, 1 × 105 cells/mL. (D) The linear relationship of the peak current values and the logarithm of cells concentration.

Detection Selectivity. To investigate the selectivity of the constructed method, CCRF-CEM cells, K562 cells, Raji cells, Ramos cells and the mixture of these four types of cells were used to do the same experiments. The I-t curves after cells capture were in Figure 6A. For CCRF-CEM cells, the current response upon light irradiation decreased obviously. In comparison, no obvious changes occur for K562 cells, Raji cells and Ramos cells. Moreover, the current value of a mixture solution (1 × 105 cell/mL of CCRF-CEM cells, K562 cells, Raji cells and Ramos cells) was almost the same with 1 × 105 cell/ mL of CCRF-CEM cells. For capture of CCRF-CEM cells, due to the interaction between aptamer sgc8c and PTK-7 overexpressed on the cell membrane,45 efficient trap of CCRF-CEM cells can be achieved via special biomolecular recognition. To verify the selectivity of the present method towards cells detection, the recognition of aptamer (FAM-sgc8c) with different cells including K562 cells, Raji cells and CCRF-CEM cells was investigated using flow cytometry analysis.54-58 From Figure S6, a large shift in the flow cytometry analysis occurs before and after CCRF-CEM cell incubation with FAM-sgc8c, while no obvious changes appear in the cases of K562 and Raji cells. The results suggest that only CCRF-CEM cells can be specially recognized by FAM-sgc8c, inducing the change of fluorescence intensity in the flow cytometry analysis. In addition, the confocal microscopy images by incubating different cells with FAM-sgc8c were taken (Figure S7). The CCRF-CEM cells show obvious fluorescence, while K562 and Raji cells show little green fluorescence compared to CCRF-CEM cells, proving the overexpression of PTK-7 mainly occurs on CCRF-CEM cells.59-62 The results indicate that the proposed method displays an excellent specificity and selectivity toward



CCRF-CEM cells. To ensure the light irradiation will not hurt the cell activity, the captured cell viability variation after light irradiation was also investigated by the well-established AO/PI staining method.26 After AO/PI staining, viable cells and dead cells were observed as blue dots and red dots respectively under fluorescence inversion microscope (Figure 6C). Compared to that before 20 min light irradiation (Figure S8), it can be found that a nearly 100% cells activity could be achieved, demonstrating no hurt on cells by the light irradiation in our experiments.

Figure 6. (A) I−t curves of various cells with light on and off (the cell concentrations are 1 × 105 cells/mL). (B) The current values with light on in response to various cells (the cell concentrations are 1 × 105 cells/mL). (C) Fluorescence microscope image of CCRF-CEM cells with 808 nm laser irradiation (200 mW) for 20 min. (D) The detection CTCs in different samples. Results were obtained from the PBS buffer solution and human blood samples.

Detection of CTCs in Blood Samples. To demonstrate the potential application of


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the present method in complicated real sample, CCRF-CEM cells were spiked in 10% human serum at concentrations of 50, 1 × 102 and 1 × 104 cells/mL. The results are shown in Figure S9A and S9B. The current value displays a little variation in different mediums of buffer and serum, confirming the feasibility of proposed method for detection of CTCs in serum samples. In addition to the serum sample, different concentrations of CCRF-CEM cells (50, 1×102, 1×103, 1×104 cells mL-1) were spiked into the blood samples and detected using the same method. A little variation of current values is observed in the buffer and blood (Figure 6D). The recoveries ranged from 92.68% to 116.00% for the four different concentrations of cells, and a relative standard deviation (RSD) ranged from 3.68 to 6.88 was obtained, confirming the feasibility of proposed method for detection of CTCs in the complicated blood matrix (Table S3). Compared to PBS buffer, there are a lot of cells in the whole blood. To investigate the response of the blood cells on the aptamer-immobilized AuNSs and confirm our experimental results, the confocal microscopy images and flow cytometry analysis were performed. The results were shown in Figure S10, S11. It could be seen that nearly no fluorescence dots in the confocal microscopy image occurs, and only a minor shift in the flow cytometry analysis appears. These experimental results indicate ignorable binding of blood cells by the used aptamer, which agree well with the previous work. However, it is worthy to note that if the cell concentration is relative low, this influence would become obvious, which can be seen in the case of spiking 50 CCRF-CEM cells in PBS and blood (Figure 6D). The similar results have also been reported in previous references.27,62,63 The result confirms the practical value of the present DPEE based electrochemical biosensor for CTCs detection.

CONCLUSION In summary, we have developed an elelctrochemical method for ultrasensitive and label-free detection of CTCs in blood based on DPEE mechanism. Owing to the efficient hot electron transfer by light irradiation, AuNSs can dramatically enhance the current response of AA electrocatalysis. The captured cells on AuNSs surface will



influence the hot electrons transport efficiency from AuNSs to the external circuit, leading to a decreased current of photocurrent. It was found that the changed I-t curves upon light irradiation varied with the CTCs concentration, based on which, an ultrasensitive and label-free electrochemical strategy for CTCs detection was constructed. The results show that the CCRF-CEM cells with the concentration ranging from 5 to 1 × 105 cells /mL could be successfully detected. The detection limit can be achieved as low as 5 cells/mL. This research shows the promising application of LSPR effect in the construction of biosensors for bioanalysis and early clinical diagnosis.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the grants from the National Natural Science Foundation of China (21874155, 21635004, 21575163) and the National Key Research and Development Program of China (2017YFA0206500). Supporting Information Supporting Information Available: Supporting Information is available free of charge from the Analytical Chemistry home page (http://pubs.acs.org/journal/ancham). Supplementary Figure S1, SEM images of the AuNSs deposited on GC substrate before and after experiment; Supplementary Figure S2, CV curves of the AuNSs/GC electrode in the presence of 1 mM AA at 1st circle and 100th circle; Supplementary Figure S3, the magnification of I-t curves of Figure 2B; Supplementary Figure S4, the changed current versus the concentrations of CCRF-CEM cells; Supplementary Figure S5, the detection of MCF-7 cells; Supplementary Figure S6, flow cytometry analysis of CCRF-CEM, K562, Raji cells; Supplementary Figure S7, confocal microscopy images of CCRF-CEM, K562, Raji cells incubated with FAM-Sgc8c;


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Supplementary Figure S8, fluorescence microscope images of CCRF-CEM cells after and before 808 nm laser irradiation (200 mW); Supplementary Figure S9, the detection CTCs in different samples in PBS buffer solution and diluted human serum samples; Supplementary Figure S10, confocal microscopy images of blood cells incubated with FAM-Sgc8c; Supplementary Figure S11, flow cytometry analysis of blood cells. Supplementary Table S1, values of Rct for the stepwise construction of the cytosensor; Supplementary Table S2, comparison of cytosensors performance for CCRF-CEM; Supplementary Table S3, determination of CCRF-CEM cells in human blood (n=5) with the cytosensors.

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for TOC only


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