Ultrasensitive Capture, Detection, and Release of Circulating Tumor

Subscriber access provided by University of Sussex Library

Article

Ultrasensitive Capture, Detection and Release of Circulating Tumor Cells Using Nanochannel-Ionchannel Hybrid Coupled with Electrochemical Detection Technique Jing Cao, Xiao-Ping Zhao, Muhammad Rizwan Younis, Zhong-Qiu Li, Xing-Hua Xia, and Chen Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02765 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry 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.

Page 1 of 21

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

Analytical Chemistry

Ultrasensitive Capture, Detection and Release of Circulating Tumor Cells Using Nanochannel-Ionchannel Hybrid Coupled with Electrochemical Detection Technique Jing Cao,1,2 Xiao-Ping Zhao,2 Muhammad Rizwan Younis,1 Zhong-Qiu Li,1 Xing-Hua Xia,*1 Chen Wang*2

1

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative

Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China 2

Key Laboratory of Biomedical Functional Materials, School of Science, China

Pharmaceutical University, Nanjing, 211198, China *To whom correspondence should be addressed. E-mail: [email protected]; [email protected]

ABSTRACT With the growing demands of the early, accurate, and sensitive diagnosis for cancers, the development of new diagnostic technologies becomes increasingly important. In this study, we proposed a strategy for efficient capture and sensitive

detection

of

circulating

tumor

cells

(CTCs)

using

array

nanochannel-ionchannel hybrid coupled with electrochemical detection technique. The aptamer probe was immobilized on the ionchannel surface to couple with the protein overexpressed on CTCs membrane. Through this special molecular recognition, CTCs can be selectively captured. The trapped CTCs cover the ionchannel entrance efficiently, which will dramatically block the ionic flow through channels, resulting in a varied mass transfer property of the nanochannel-ionchannel hybrid. Based on the changed mass transfer properties, the captured CTCs can be sensitively detected using electrochemical linear sweep voltammetry technique. Furthermore, due to the amplified response of array channels compared to single

ACS Paragon Plus Environment


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

Page 2 of 21

channel, the detection sensitivity can be enhanced greatly. The results showed that acute leukemia CCRF-CEM concentration as low as 100 cells mL-1 can be successfully captured and detected. The present method provides a simple, sensitive, and label-free technique for CTCs capture, detection and release, which would hold great potential in the early clinical diagnosis and treatment of cancers. Keywords: nanochannel-ionchannel hybrid, circulating tumor cells, mass transfer property, electrochemical detection, cancer

Cancer has become one of the leading causes of death in the world for human beings.1 Cancer statistics show that 90% of the cancer-related deaths are caused by cancer metastasis.2 When tumor cells transfer into peripheral blood from the primary tumor site, they will become the circulating tumor cells (CTCs). As CTCs pass through the circulatory system, they can form metastasis in the resident organs, eventually leading to cancer-related deaths.3,4 Therefore, early discovery of the CTCs is of great significance towards effective treatment of cancers.5,6 However, the number of CTCs is extremely rare in the peripheral blood of early stage cancer patients (about one CTC in 106-107 leukocytes), which results in extreme difficulty for their isolation and detection.7,8 In addition, there are seldom probes found to have special binding capacity to CTCs. The conventional CTC capture technologies often suffer from the poor sensitivity, selectivity and efficiency. Therefore there is an urgent demand to develop simple and ultrasensitive approaches to efficiently capture and detect the trace amount of CTCs in blood samples. To enhance the detection sensitivity, a variety of methods regarding CTCs enrichment, capture and detection are emerging including immunomagnetic separation,9 polymerase chain reaction,10 fluorescent sensing and imaging,11

X-ray

radiography,12 spiral computed tomography,13 and flow cytometry.14 The rapid development of CTCs detection techniques enables us to understand the tumor metastasis mechanism in depth. However, most of these methods suffer from either time-consuming experiment procedures or sophisticated instruments, which ultimately


Page 3 of 21

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


limit their application in real diagnostic cases. In comparison, electrochemical techniques have the overwhelming advantage for monitoring cell viability and proliferation because of their remarkable characteristics such as high sensitivity, low cost, convenient operation, rapid detection.15,16 Aptamers are single stranded nucleic acids with specific three dimensional structure, which are designed through systematic evolution of ligands by exponential enrichment (SELEX) system in vitro.17 They have high affinity and specificity toward their target molecules ranging from small molecules like ATP18and proteins19 to intact cells.20,21 Compared with other molecular probes like antibodies, aptamers possess remarkable features such as relatively smaller size, rapid synthesis process, high stability, low immunogenicity and special binding affinity for molecular recognition.22-24 As well known, porous anodic alumina (PAA) membrane has been widely adopted in the construction of bioanalysis and detection devices due to high density of nanochannel array structure.25-27 The detection response can be amplified by several orders of magnitude compared to single nanopore.28,29 However, up to now most of the work were focused on the porous layer with nanochannels. There are few studies making use of the barrier layer adjacent to the aluminum substrate. Using this hybrid structure, Xia et al. successfully achieved the enrichment of protein.30 It has been proved that there are ionchannels with size ranging from 0 to 1 nm in the barrier layer.30,31 Actually, the PAA membrane with the barrier layer is a kind of nanochannel-ionchannel hybrid structure, which has strong geometry-asymmetry and unique mass transfer properties. In this work, we for the first time make use of this nanochannel-ionchannel hybrid structure for CTCs capture, detection and release. First, the PAA membrane was clamped between two plastic poly(dimethylsiloxane) (PDMS) films, and placed between the two half cells of the home-made electrolyte cell (Scheme 1A). The aptamer probe sgc8c was then immobilized on the ionchannel side surface, which can selectively bind with the transmembrane receptor protein tyrosine kinase 7 highly expressed on CCRF-CEM cells membrane (one type of CTCs).32 Thus CCRF-CEM



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

Page 4 of 21

cells can be trrapped selectivity by tthis speciall molecularr recognitioon. The trap pped cells will efficciently cov ver the ionnchannel sid de surface, blocking the ionic flow throough the naanochannel--ionchannell. As a result, an obv vious variedd ionic tran nsfer behavior is exppected. Fin nally, under the action of benzonaase nucleasse, the capttured CCR RF-CEM cells was released from m PAA with h nearly no o damage, rresulting in n the recoovery of ioonic flow through t thee nanochann nel-ionchan nnel hybridd (Scheme 1B). Usinng the elecctrochemicaal linear sw weep voltam mmetry tech hnique, thee changed ionic i trannsfer behaviors can bee monitoredd in real-tiime by I-V V propertiess of the hy ybrid struucture, thuss enables an a accuratee, sensitivee and labell-free detecction of CTCs C (Schheme 1C).

Sch heme 1. (A)) Schematicc illustratioon of the seetup for CT TCs capturee, detection and releease. (B) Diiagrammaticcal illustrattion of CTC Cs capture and a release process on n the nanochannel-ioonchannel hybrid. (C C) Schematic repressentation oof the vaaried elecctrochemicaal responses towards CT TCs capturee and releasse.

EXP PERIMEN NTAL SECT TION Matterials and d Reagents. (3-aminoppropyl) trieethoxysilanee (APTES) was purch hased


Page 5 of 21

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


from Alfa Aesar. Glutaraldehyde (GA), Albumin from bovine serum (BSA) were from Sinopharm Chemical Reagent Co., Ltd. Aptamer sgc8c sequence: 5’-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTT TTT-3’-(CH2)6–NH2 was ordered from Shanghai Shenggong Biotechnology Co.. Acridine orange (AO), propidium iodide (PI) and RPMI-1640 culture medium were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Corning Co. (Manassas). Benzonase nuclease was from EMD Millipore (USA). The CCRF-CEM (CCL-119, T cell line, human, acute lymphoblastic leukemia, ALL) was obtained from Shanghai Institutes for Biological Sciences (China). The k562 (CCL-243, human CML) and the Ramos (CRL-1596, B cell line, human Burkitt lymphoma) were obtained from Beijing Xiehe Hospital. All reagents were of analytical grade. All solutions were prepared using ultrapure water (18.2 M Ω∙cm) from the Millipore Elix 5 Pure Water System. (Purelab Classic Corp., USA). Instrumentation. The morphology of the prepared nanochannel-ionchannel hybrid structure and the captured cells were characterized using a scanning electron microscope (SEM, S-4800, Japan). The presence of APTES was characterized by an X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Fisher Scientific, USA). UV-vis absorbance was recorded on a UV-1800 spectrophotometer. Fourier-transform infrared (FTIR) spectroscopy was performed on a Nicolet 6700 model 912A0637. The electrochemical detection was performed in 1 mM KCl solution on an electrochemical workstation (CHI 650A, Chenhua, China) with two Ag/AgCl electrodes as the anode and cathode. The captured cells were observed using a Fluorescence Inversion Microscope System (Nikon, TI-U). Cell Culture. Cells were cultured in flasks with RPMI-1640 medium containing penicillin (100 μg mL-1), streptomycin (100 μg mL-1), and 10% FBS at 37 °C under 5% CO2 in the cell incubator. Fabrication of Nanochannel-Ionchannel Hybrid. The nanochannel-ionchannel hybrid used in this work was prepared via a two-step aluminum oxidation process.33 The fabrication process was illustrated in Figure S1 in the supporting information.



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

The high-purity (99.99%) aluminum sheet was cleaned in acetone and then chemically etched in 1M KOH solution. The first anodization process was performed in 0.3 M oxalic acid at 25 °C at a constant voltage of 50 V for 0.5 h. Then, the hybrid membrane was etched in an aqueous mixture solution of phosphoric acid (5 wt.%) and chromic acid (1.8 wt.%) at 60 °C for 40 min to remove the irregular oxide layer that formed in the first anodization. Subsequently, the second anodization under the same conditions as that in the first anodization was performed for 4 h. The formed nanochannel-ionchannel hybrid with an aluminum substrate was immersed in saturated CuCl2 solution to remove the aluminum substrate completely. Surface Modification of Nanochannel-Ionchannel Hybrid. The fabricated nanochannel-ionchannel hybrid was clamped between two thin poly(dimethylsiloxane) (PDMS) films and then placed between the two 2 mL homemade half cells for surface modification and electrochemical detection. The aptamer modification process was illustrated in Scheme 2. First, 2 mL ethanol solution containing 1% APTES was added on the ionchannel side of hybrid membrane for 4 h to graft aminopropyl functional groups. The excess silane solution was removed from the cell by rinsing with copious amount of ethanol, followed by deionized water, and then baked at 120 °C for 1 h to crosslink the silane layer. After that, the cell was added with 2 mL 2.5% GA solution for 4 h at 4 °C, following which the cell was immersed into 10 mg mL-1 of BSA solution at 4 °C overnight. After it was treated with 2 mL 2.5% GA solution again, 100 μL of sgc8c aptamer solution (10 μM) was dropped onto the ionchannel side surface at room temperature for 6 h. Finally, ethanol amine (1 M) was used to block the unreacted aldehyde group for 10 min. Each modification step was followed by complete rinsing by deionized water.


Page 6 of 21

Page 7 of 21

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


Sch heme 2. Thee illustratio on of surfacce modificattion process of aptameer probe on n the nanochannel-ioonchannel hybrid. h

Cell Capture and Electrrochemicall Measurem ments. Cellls were colllected from m the meddium by cenntrifugation at 1000 rpm m for 5 min n and redispersed to a ddensity of 2×105 cells mL-1 in PBS P (10 mM M, pH 7.4)). The denssity of the cells c was ddetermined by b a hem macytometerr. A 100 μL droplet of C CCRF-CEM M cells susp pension wass dropped on n the ioncchannel side surface of o PAA mem mbrane and d incubated at 37 °C w with 5% CO O2 in cell incubator. Then, the PAA P membbrane was carefully c waashed with PBS bufferr (10 mM M, pH 7.4).. After beiing washedd for three times, thee remainingg solution was colllected for cell c counting g and calcuulating captture yields of CCRF-C CEM cells. The elecctrochemicaal linear sw weep voltam mmetry from m -1.0 V to t +1.0 V in 1 mM KCl soluution was reecorded by an a electrochhemical worrkstation (C CHI 650A, C Chenhua, China) C withh two Ag/A AgCl electro odes as the aanode and cathode. c Ass a control, a 100 μL K562 K cells suspensioon and a 100 μL Raamos cells suspension n were droopped onto the ioncchannel sidde surface and a incubatted under th he same co onditions. T The cell den nsity wass the same as a CCRF-CE EM cells. Scaanning Elecctron Microscopy (SE EM) Obserrvation. The cells on PPAA memb brane werre fixed withh 2.5% GA solution buuffered in PBS P (10 mM M, pH 7.4) ffor 5 h and then dehyydrated through a seriies of alcohhol concentrrations (30% %, 50%, 700%, 90%, 95%, 9 1000%)

for 10

min subsequentlly.

The dehydrated cells

w were

hexamethyldisiilazane (HM MDS) and chharacterized d by SEM (S-4800, Jappan).


dried by


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

Cell Release and Viability Analysis. After CCRF-CEM cells were captured on PAA substrate, 200 μL PBS (10 mM, pH 7.4) solution containing benzonase nuclease (25 units mL-1, EMD Millipore) was dropped onto the membrane. Then, the device was placed in an incubator (37 °C, 5% CO2) for 5, 10, 20 min, respectively. After each incubating step, the I-V properties of the nanochahnel-ionchannel hybrid were investigated from -1.0 V to +1.0 V. Cell viability before and after cell capture were determined by Acridine orange (AO) / Propidium iodide (PI) staining assay.34 The cells suspensions before and after cell capture were respectively dropped into an AO/PI solution containing 1 μg mL-1 AO and 1 μg mL-1 PI in PBS (10 mM, pH 7.4) for 5 min. After the cells were released, the remaining cells solution were recollected and centrifuged at 1000 rpm for 5 min to redispersed in PBS (10 mM, pH 7.4) for AO/PI staining assay experiment. By AO/PI staining, dead cells and viable cells were observed using a fluorescence inversion microscope system (Nikon, TI-U).

RESULTS AND DISCUSSION Characterization and Modification of Nanochannel-Ionchannel Hybrid. The morphology of the prepared nanochannel-ionchannel hybrid (PAA) was characterized using SEM images (Figure 1). As can be seen from Figure 1A, there are array nanochannels with diameters of ~50 nm in the porous layer of PAA. The barrier layer shows the regular hexagonal structure, in which there are many ionchannels (cannot be observed in SEM image due to the super small size). The thickness of the barrier layer is ~ 40 nm. The presence of APTES on the barrier layer of PAA membrane is characterized by XPS. As shown in Figure 1B, the bare PAA membrane does not show the Si2P peak (black curve), while a clear peak of Si2P appears (red curve) after the ionchannel side is treated with APTES, revealing successful immobilization of APTES on PAA membrane. BSA was used as an antifouling molecule to inhibit nonspecific adsorption of cells on PAA, which was immobilized by chemical coupling with the reactive aldehyde groups. There are plenty of Al-OH groups on PAA surface.35 After coupling with APTES and GA, the Al-OH groups would transform


Page 8 of 21

Page 9 of 21

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


into reactive aldehyde groups that can be used for the covalent binding of BSA.36 FTIR was used to probe the immobilization of BSA by monitoring the absorption of amide I and II vibrations using pure hybrid membrane as the reference. As shown in Figure 1C, the amide I band (1666 cm-1) is attributed to the C=O stretching vibration of the peptide linkage in the peptide background. The amide II band (1526 cm-1) is due to the N–H bending and C–N stretching. These results demonstrate BSA has been modified on PAA. In addition, XPS was also used to characterize the immobilization procedure of BSA. As shown in the inset of Figure 1C, the N1s peak did not appear for the bare PAA. However, it became significant after the PAA was modified with APTES. Further modification with glutaraldehyde and BSA lead to distinctly increased N1s peak due to the introduction of BSA containing a large amount of amino acid. Both the FTIR and XPS characterizations clearly show that BSA can be successfully immobilized on the surface of PAA. Figure 1D shows the UV-Vis spectrum of PAA modified with aptamer sgc8c. The black curve is the background response of bare PAA, and the blue curve is aptamer modified PAA. The inset shows the pure UV–Vis spectrum of aptamer subtracting the background response, clearly showing 260 nm characteristic absorption band of DNA strands immobilized on the PAA substrate.37



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

Page 10 of 21

Figu ure 1. (A) SEM images of PAA A (a: the porous p layeer; b: the ccross section of nanochannels; c: the barrrier layer; dd: the cross section off the ionchaannel layer)) (B) XPS S spectra off bare (blacck curve) annd APTES modified (rred curve) PPAA. (C) FTIR F specctrum of BS SA modified PAA. Ins et: XPS speectra of PAA A membranne without (bare ( PAA A) and wiith modificcation by A APTES (N NH2-PAA), glutaraldehhyde and BSA B (BS SA-PAA). (D D) UV–Vis spectrum oof bare (blacck curve) an nd aptamer sgc8c modified (bluue curve) PA AA. The inset is the U UV–Vis specctrum of blue curve suubtracting black b curvve (backgroound).

Cell Capture and a Electrochemical Detection. Before exp periment, thhe mass tran nsfer behavior of thee nanochan nnel-ionchannnel hybrid d was studieed in 1 mM M KCl solution. Thee result is inndicated as the t black cuurve in Figu ure 2A. An obvious ionnic rectificaation phennomenon

appears

due

to

the

stron ng

geomeetry-asymm metry

of

the

nanochannel-ioonchannel hybrid, h whicch is in goo od accordan nce with exppected theo ory.38 Theen, three diffferent typess of cells inccluding CC CRF-CEM cells, k562 ccells and Raamos cells were chossen as the model m CTCss to be trapp ped by sgc8c modified hybrid. Thee I-V


Page 11 of 21

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


properties after cells capture were investigated (Figure 2A). It is found that only in the case of CCRF-CEM cells capturing, the I-V property varies obviously (red curve in Figure 2A), while no significant changes occurrs for k562 cells and Ramos cells (pink and blue curves in Figure 2A), which nearly overlapped with the bare hybrid (refer to the enlarged part in the inset of Figure 2A). To clearly show the change of the ionic transfer behaviors after cells capture, the ionic current values at – 1.0 V potential were highlighted in Figure 2B, confirming that only capture of CCRF-CEM cell could result in sharp drop of ionic current. The decreased ionic current suggests a low mass transfer rate within the nanochannel-ionchannel hybrid. In case of CCRF-CEM cells capture, the special recognition reaction occurs between aptamer sgc8c and transmembrane receptor protein on CCRF-CEM cells membrane,31 enabling the efficient trap of CCRF-CEM cells on the hybrid surface. As a result, the trapped cells efficiently coveres the hybrid membrane surface (the SEM image in Figure 2C), blocking the ionic flow through the nanochannel-ionchannel hybrid, and thus resulting in a varied I-V property. In comparison, no special recognition occurs between aptamer sgc8c and k562 cells or Ramos cells. Therefore k562 cells or Ramos cells cannot be captured using the present platform, and accordingly the I-V properties kept very similar as the bare hybrid membrane. The capture yield was calculated using the trapped cell number divided by the total cell number per surface (Figure 2D). The capture yield of CCRF-CEM cells was calculated up to 70.1% ± 4.5%, which is approximately ten times more than that of k562 and Ramos cells. To further investigate the resistance to interference caused by other abundant cells, these three types of cells were mixed together, and the cell capture experiments were performed. The results were added in the supporting information as Figure S2. The result agrees well with that in Figure 2A and B, indicates the excellent selectivity of the present method for cell capture.



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

Page 12 of 21

Figu ure 2. (A) The T I-V properties of sggc8c modiffied PAA before and aft fter cells cap pture. blacck curve: bared PAA; blue line: R Ramos cap ptured PAA; pink line:: k562 capttured PAA A; red line: CCRF-CEM M captured PAA. Insett: the enlarg ged part of tthe curves from f -0.885 to -1.0 V. V (B) The absolute a vallues of ioniic current at a -1.0 V forr different cells captture. (C) SEM characterizationn of the captured c CCRF-CEM C M cells on the ioncchannel sidde surface of o PAA. Thhe scale barr is 2 μm. (D) ( Cell caapture yield ds of diffe ferent cells. Cell concen ntration: 2× 105 cells mL m -1.

Thee Effect of o the Apttamer Con ncentration n. To achiieve efficieent capturee of CCR RF-CEM cells, c the effect e of thhe aptamer concentrattion on ceell capture was inveestigated (F Figure 3A). The ionnic currentt values of o –1.0 V versus sg gc8c concentration are a shown in i Figure 33B. It is fou und that thee ionic currrent values first he aptamer cconcentratio on increasess from 1 μM M to 10 μM,, and decrreases sharpply when th thenn levelled off at hig gher concenntrations. To T make clear c the rreason for this phennomenon, the bright-field microoscope wass used to take the im mages after cell captture (Figuree 3C). It can n be seen thhat the sgc8 8c modified hybrid couuld capture cells


Page 13 of 21

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


efficciently, andd the capturred cells nuumber increeases with the t aptamerr concentration. When the aptaamer concen ntration reaaches 10 μM M, the trapp ped cells neearly coverss the whoole substraate surface,, and therre is no space s to accommodat a ate more cells. c Theerefore, the similar ionic transfer pproperties are a expected d when incrreasing aptaamer concentration from f 10 μM M to 100 μ μM. The miicroscope im mages agreee well with h the resuults achieved in Figure 3B.

Figu ure 3. (A) The I-V prroperties off different hybrid h mem mbranes. Blaack curve: bare hybbrid membraane; Pink cu urve: 1 μM sgc8c mod dified hybrid d; Red curvve: 10 μM sg gc8c moddified hybriid; Blue currve: 100 μM M sgc8c mod dified hybriid. (B) The absolute vaalues of ionic currennt at -1.0 V versus sgcc8c concentration. (C) Bright-fieldd images off the capttured cells on hybrid substrate s m modified witth different concentratiions of aptaamer sgc88c. (a-c: 1 μM, μ 10 μM, 100 μM). C Cell concen ntration: 2×1 105 cells mL L-1.

Thee Effect of Capture Time. In ordder to achiev ve the optim mum cell caapture time, 100 µL of CCRF-C CEM cells solution s waas dropped onto the io onchannel siide surface and incuubated for different d tim mes (10, 200, 30, 45, 60 0, 80, 100 min) m at 37 °C. The results werre shown in i Figure 4A. It is noteworth hy that thee I-V propperties drop pped conttinuously inn the first 30 3 min incuubation tim me, then leveeled off aftter 45 min. The



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

constant ionic current in ndicates thee complete cell capturre after 45 min. The cell captture yield is i calculateed and show wn in Figu ure 4B, wh hich proves to be in good g accoordance witth the resultt in Figure 44A.

Figu ure 4. (A) The T effect of o the CCRF F-CEM cellls incubation time on II-V propertiees of PAA A. (B) Captture yields of o CCRF-C CEM cells at a different incubation i ttimes. The Cell concentration is i 2×105 cells mL-1.

The high sensitivity plays p an im mportant rolee for the eaarly stage caancer diagnosis. In vview of this,, we use thee present meethod to quaantify trace amounts off cancer cellls in PBS S buffer. Figgure 5A sho ows the I-V V properties of the nanochannel-ionnchannel hy ybrid afteer capture off different concentratio c on of CCRF F-CEM cellss (from botttom to top: 102, 103, 104, 105 annd 106 cells mL-1). Thee inset show ws the enlarg ged part of tthe overlapping curvves in Figurre 5A from -0.8 - V to -11.0 V. The difference d off the ionic ccurrent values at -1.00 V before and after cell c capture is denoted d as the currrent drop, and the currrent dropp value verrsus cell concentrationn is shown in Figure 5B. 5 It can bbe seen thatt the CCR RF-CEM ceells in a wid de concentraation range from 1×102 to 2×106 ccells mL-1caan be succcessfully deetected usin ng the pressent metho od. Moreover, there iss a good liinear corrrelation betw ween the cu urrent drop value and the cell con ncentration in the rang ge of 1000−1000 cellls mL-1 (th he inset of Figure 5B B, R=0.9976 6). The dettection lim mit is calcculated as 10 1 cells 100 0 μL-1, whicch was low wer than man ny previouss reports (T Table 1).144,38-40


Page 14 of 21

Page 15 of 21

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


Table 1.

Com mparison of Analytical P Performancce for CCRF F-CEM Cyttosensors

Cytosensoor

detectioon

lin near range

methood

detecction

Ref R

lim mit

aptaamer/APBA A-MWCNT Ts

EIS

1.0×103-1.0× ×107

10000

14

HA-MN NPs

M QCM

8.0×103-1.0× ×105

80000

39

flow cytoometry

7.5×103-6.25×105

7550

40

aaptamer-miccrofluidic

fluoresccence

2 4×10 4 -5.0×1 106

4000

41

aptamerr/PAA

LSV V

1×102-1.0×106

1000

this work w

aptamer/A Ag NCs

APB BA-MWCN NTs: 3-amiinophenylbooronic acid d-functionallized multiiwalled carrbon nanotubes; HA A-MNPs: hy yaluronic aciid-coated magnetic m nan noparticles; Ag NCs: silver nanoclusters.

Figu ure 5. (A) The I-V prroperties off nanochann nel-ionchan nnel hybrid after captu uring diffe ferent conccentrations of CCRF--CEM cells. Inset: the enlargeed part of the overrlapping cuurves from -0.8 V to -1..0 V. (B) Th he current drop values aat -1.0 V veersus the CCRF-CEM M cells co oncentrationn. Inset: the linear caalibration pplot at low cell concentrations..

Cell Release and Viabilitty Analysis ole in The succeessful releasse of CTCs from substrrate after ceell capture pplays key ro subssequent stuudy of metaastasis, muttation of CTCs C and cancer theraapy. Herein, we usess the benzoonase nucleease for ennzyme-induced CCRF--CEM cellss `release.422 To



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

achieve the proper time for effective cell release, we treated the substrates with the same concentration of enzyme solution (25 units mL-1) for 5 min, 10 min, 20 min, respectively. As shown in Figure 6A, the I-V property varies continuously with the enzyme incubation time. The ionic current first increases with the enzyme incubation time from 0 to 10 min, then becomes nearly constant after 10 min. The final ionic plateau demonstrates the nearly complete release of the captured CCRF-CEM cells. Release yield of 98.5% can be calculated using the value of the released cell number divided by the captured cell number. The cell viability variation before capture and after release was performed by the AO/PI staining assay method. AO is a membrane permeable dye with green fluorescence when binding with DNA in cells. PI is a red fluorescent dye that cannot pass through intact cell membranes, but can easily bind with DNA when the cell membranes are damaged. The results in Figure 6C suggested a nearly 100% cells activity before being captured. After release, the viability decreased to ~ 94% (the ratio of alive cells to total cells) due to the little amount of dead cells (red dot in Figure 6D). The released CCRF-CEM cells from PAA by the enzyme digestion were further cultured, and the fluorescent microscope images were shown in Figure S3. These results demonstrates that the chosen enzyme has only little damage to the cells.


Page 16 of 21

Page 17 of 21

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


Figu ure 6. (A) The I-V prroperties off PAA for different d enzzyme incubbation time.. (B) Thee absolute values v of io onic currentt at -1.0 V versus enzzyme incubbation time. (C) Fluoorescent miicroscope images of C CCRF-CEM M cells in solution s staained by PII/AO befoore captureed. (D) Flu uorescent m microscope images off CCRF-CE EM cells after releeased. The scale bar is 50 5 μm. The red dots are indicated by red circlles.

CO ONCLUSIO ON In summaary, we havee demonstraated an ultraasensitive and a label-freee CTCs seensor baseed on nanoochannel-io onchannel hhybrid cou upled with electrochem mical detecction techhnique. Duee to the uniique mass ttransfer pro operty of th he nanochannnel-ionchaannel hybbrid and the special bin nding capaciity of aptam mer to cells, the presentt method sh hows exceellent selecctivity and high senssitivity tow ward CCRF F-CEM celllls capture and deteection. At thhe same tim me, the cap tured cells can be releeased by ennzyme digesstion withh little daamage. Thee experimeent results show thaat the CCR RF-CEM cells concentration ranging r from 1×102 too 2×106 cellls mL-1 can n be successsfully deteected usinng the pressent approaach. The deetection lim mit is 100 cells mL-1. The proposed strattegy in thee work prov vides a new w promising g platform for label-frree detectio on of tracce amount of o CTCs in n a real-tim me format, showing s go ood applicabbility and great g



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

potential in early clinical diagnosis and cancer therapy.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] ACKNOWLEDGMENT This work was supported by the grants from the National Natural Science Foundation of China (21327902, 21575163, 21635004) and the Natural Science Foundation of Jiangsu Province (BK20151437). References (1) Buck, C. B.; Lowy, D. R., J. Clin. Oncol. 2011, 29, 1506-1508. (2) Yoon, H. J.; Kozminsky, M.; Nagrath, S., ACS Nano 2014, 8, 1995-2017. (3) Chambers, A. F.; Groom, A. C.; MacDonald, I. C., Nat. Rev. Cancer 2002, 2, 563-572. (4) Fidler, I. J., Nat. Rev. Cancer 2003, 3, 453-458. (5) Abts, H.; Emmerich, M.; Miltenyi, S.; Radbruch, A.; Tesch, H. J., Immunol. Methods 1989, 125, 19-28. (6) Mocellin, S.; Keilholz, U.; Rossi, C. R.; Nitti, D., Trends. Mol. Med. 2006, 12, 130-139. (7) Hayes, D. F.; Cristofanilli, M.; Budd, G. T.; Ellis, M.; Stopeck, A.; Matera, J.; Miller, M. C.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W., J Clin. Oncol. 2004, 22, 509-509. (8) Wang, S.; Liu, K.; Liu, J.; Yu,Z. T.F.; Xu, X. Zhao, L.; Lee, T.; Lee, E. K.; Reiss, J.; Lee, Y. K.; Chung, L. W. K.; Huang, J.; Rettig, M.; Seligson, D.; Duraiswamy, K. N.;


Page 18 of 21

Page 19 of 21

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


Shen, C. K. F.; Tseng, H. R., Angew. Chem. Int. Ed. 2011, 50, 3084-3088. (9) Naume, B.; Borgen, E.; Beiske, K.; Herstad, T. K.; Ravnas, G.; Renolen, A.; Trachsel, S.; ThraneSteen, K.; Funderud, S.; Kvalheim, G., J. Hematother 1997, 6, 103-114. (10) Schamhart, D.; Swinnen, J.; Kurth, K. H.; Westerhof, A.; Kusters, R.; Borchers, H.; Sternberg, C., Clin. Chem. 2003, 49, 1458-1466. (11) Kang, C. C.; Chang, C. C.; Chang, T. C.; Liao, L. J.; Lou, P. J.; Xie, W.; Yeung, E. S., Analyst 2007, 132, 745-749. (12) Liu, J., Qin, Y., Li, D., Wang, T., Liu, Y., Wang, J., Wang, E. Biosens. Bioelectron. 2013, 41, 436-441. (13) Sutedja, G., Eur. Respir. J. 2003, 21, 57s-66s. (14) Paredes-Aguilera, R.; Romero-Guzman, L.; Lopez-Santiago, N.; Burbano-Ceron, L.; Camacho-Del Monte, O.; Nieto-Martinez, S., Am. J. Hematol. 2001, 68, 69-74. (15) Zhong, X.; Bai, H. J.; Xu, J. J.; Chen, H. Y.; Zhu, Y. H., Adv. Funct. Mater. 2010, 20, 992-999. (16) Zhu, Y.; Chandra, P.; Shim, Y. B., Anal. Chem. 2013, 85, 1058-1064. (17) Tuerk, C.; Gold, L., Science 1990, 249, 505-510. (18) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C., J. Am. Chem. Soc. 2007, 129, 1042-1043. (19) Cheng, A. K.; Ge, B.; Yu, H. Z., Anal. Chem. 2007, 79, 5158-5164. (20) Shangguan, D.; Li, Y.; Tang, Z.; Cao, Z. C.; Chen, H. W.; Mallikaratchy, P.; Sefah, K.; Yang, C. J.; Tan, W., Proc. Natl. Acad. Sci. USA 2006, 103, 11838-11843. (21) Shangguan, D.; Meng, L.; Cao, Z. C.; Xiao, Z.; Fang, X.; Li, Y.; Cardona, D.; Witek, R. P.; Liu, C.; Tan, W., Anal. Chem. 2008, 80, 721-728. (22) Jiang, Y.; Zhu, C.; Ling, L.; Wan, L.; Fang, X.; Bai, C., Anal. Chem. 2003, 75, 2112-2116. (23) Li, J. J.; Fang, X.; Tan, W., Biochem. Biophys. Res. Commun. 2002, 292, 31-40. (24) Bagalkot, V.; Farokhzad, O. C.; Langer, R.; Jon, S., Angew. Chem. Int. Ed. 2006, 45, 8149 -8152. (25) Wang, C.; Liu, H. L.; Li, Y.-Q.; Cao, J.; Zheng, B.; Xia, X.-H.; Feng, F.,



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

Electrochem. Commun. 2016, 66, 25-28. (26) Gao, H. L.; Wang, M.; Wu, Z. Q.; Wang, C.; Wang, K.; Xia, X. H., Anal. Chem. 2015, 87, 3936-3941. (27) Zhao, X. P.; Cao, J.; Nie, X.-G.; Wang, S. S.; Wang, C.; Xia, X. H., Electrochem. Commun. 2017, 81, 5-9. (28) Li, C. Y.; Wu, Z. Q.; Yuan, C. G.; Wang, K.; Xia, X. H., Anal. Chem. 2015, 87, 8194-8202. (29) Yu, J.; Zhang, L.; Xu, X.; Liu, S., Anal. Chem. 2014, 86, 10741-10748. (30) Chen, W.; Jin, B.; Hu, Y. L.; Lu, Y.; Xia, X. H., Small 2012, 8, 1001-1005. (31) Chen, W.; Wu, J, S.; Xia, X. H., ACS Nano 2008, 2, 959-965. (32) Shangguan, D.; Cao, Z. H.; Meng, L.; Mallikaratchy, P.; Sefah, K.; Wang, H.; Li, Y.; Tan, W.H., J. Proteome Res. 2008, 7, 2133-2139. (33) Yuan, J. H.; He, F. Y.; Sun, D. C.; Xia, X. H., Chem. Mater. 2004, 16, 1841-1844. (34) Ouyang, J.; Chen, M.; Bao, W. J.; Zhang, Q.-W.; Wang, K.; Xia, X. H., Adv. Funct. Mater. 2015, 25, 6122-6130. (35) Gao, H. L.; Li, C. Y.; Ma, F. X.; Wang, K.; Xu, J. J.; Chen, H. Y.; Xia, X. H., Phys. Chem. Chem. Phys. 2012, 14, 9460-9467. (36) Vlassiouk, I.; Krasnoslobodtsev, A.; Smirnov, S.; Germann, M., Langmuir, 2004, 20, 9913-9915. (37) Deiminiat, B.; Rounaghi, G. H.; Arbab-Zavar, M. H.; Razavipanah, I., Sens. Actuators B 2017, 242, 158-166. (38) Liang, Y.; Liu, Z., Langmuir 2016, 32, 13349-13357. (39) Zhou, Y.; Xie, Q., Sens. Actuators B 2016, 223, 9-14. (40) Yin, J.; He, X.; Wang, K.; Xu, F.; Shangguan, J.; He, D.; Shi, H., Anal. Chem. 2013, 85, 12011-12019. (41) Zhu, J.; Nguyen, T.; Pei, R.; Stojanovic, M.; Lin, Q., Lab Chip, 2012, 12, 3504-3513. (42) Shen, Q.; Xu, L.; Zhao, L.; Wu, D.; Fan, Y.; Zhou, Y.; Ouyang, W. H.; Xu, X.; Zhang, Z.; Song, M.; Lee, T.; Garcia, M. A.; Xiong, B.; Hou, S.; Tseng, H. R.; Fang, X., Adv. Mater. 2013, 25, 2368-2373.


Page 20 of 21

Page 21 of 21

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


Schematic illustration of the principle for CTCs capture, detection and release 269x197mm (150 x 150 DPI)


Ultrasensitive Capture, Detection, and Release of Circulating Tumor

Recommend Documents