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Clinical available circulating tumor cell assay based on tetra(4-aminophenyl) porphyrin mediated reduced graphene oxide field effect transistor
Electrochimica Acta 313 (2019) 415e422
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Clinical available circulating tumor cell assay based on tetra(4aminophenyl) porphyrin mediated reduced graphene oxide field effect transistor Shihui Hu a, Zhongrong Wang a, Yajun Gu b, **, Yueguo Li c, ***, Yunfang Jia a, * a
College of Electronic Information and Optical Engineering, Nankai University, Tianjin, China School of Medical Laboratory, Tianjin Medical University, Tianjin, China Department of Clinical Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2018 Received in revised form 2 April 2019 Accepted 8 May 2019 Available online 12 May 2019
Circulating tumor cell (CTC) assay is one of the emerging “liquid biopsy” for cancer's diagnostic, an easyto-operate and state-of-the-art CTC sensor is the imperative need of clinical medical laboratory. A labelfree and solid-state sensor for clinical sample's CTC detection based on an aptamer (AS1411) functionalized graphene field effect transistor (GFET) by using tetra(4-aminophenyl) porphyrin mediated reduced graphene oxide as the channel material. Objects are CTCs of A549, MDA-MB-231, HeLa and HUVEC (as a control) in buffer solutions and EDTA anti-coagulant whole blood samples, as well as the anti-coagulant clinical blood samples from 49 breast cancer patients and 5 healthy persons. The sensitivities for CTCs in the range of 10 - 106 cells/ml are 3.14, 3.32, 2.60%/log10(cells/ml) for A549 (R2 ¼ 0.97), MDA-MB-231 (R2 ¼ 0.96) and HeLa (R2 ¼ 0.98), respectively. Meanwhile, the selectivity for CTCs is also confirmed by the low sensitivity and R2 for HUVEC which are 0.13%/log10(cells/ml) and 0.50. Furthermore, whole blood interference is found to make the sensing range narrow. At last, their applications in grading clinical blood samples are found to be in agreements with the parallel diagnostic conclusions of these patients, samples of adenofibroma and effect of chemotherapy can also be identified. The aptamer specific, directly electronic CTC detection is accomplished by the proposed GFET based CTC sensor, it can provide a novel analytical technique for cancers' clinical assay. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Circulating tumor cell Field effect transistor Graphene Porphyrin Aptamer
1. Introduction Circulating tumor cells (CTCs) are tumorigenic cells which shed from the lesion and disseminate in the peripheral blood. To date, based on the biomedical studies about the metastasis related over expressed biomolecules on CTCs' membrane (like EpCAM, nucleolin, CK etc.), CTCs can be specifically targeted [1] and converted to the measurable signals (optics [2] or electronics [3]). It has been well-accepted that, the CTC detection is a promising candidate for the emerging “liquid biopsy” assay [1,4], its application in a clinical setting will be helpful to cancers' diagnosis and therapy monitoring [4]. However, in the complicated blood environment, CTCs' scarcity,
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (Y. Gu), [email protected] (Y. Li), jiayf@ nankai.edu.cn (Y. Jia). https://doi.org/10.1016/j.electacta.2019.05.039 0013-4686/© 2019 Elsevier Ltd. All rights reserved.
short life-time, plasticity and heterogeneity are still frustrations for its real application [5]. Confronting with this challenge, ever since the publication of carbon nanotube field-effect-transistor (FET) based CTC sensors [6], there have been myriads of interdisciplinary studies for developing highly sensitive CTC capturing methods and efficient strategies to transduce the captured CTCs' information to the measurable data. The merits and drawbacks in these works have been systematically summarized in the recent reviews [1,3,5]. Amongst them, the aptamer based CTC “direct-capture” strategy [2,7e10], 3-dimensional (3D) porous micro/nano-environment for hosting cells [10,11] and aptamer functionalized graphene FET (apta-GFET) [12] are of great interest to us, because of aptamer's robustness and capability in precise-targeting, the large specificsurface-area in 3D-porous structure, graphene channel material's high mobility and biocompatibility, etc. Intrigued by the achievements in those works, we would like to develop a clinical available CTC sensor based on the apta-GFET. The main scheme of this work is illustrated in Fig. 1, in which the capability for CTC detection is examined in two steps (experimental tests and real applications).
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More explanations for the transducer (GFET) and the aptamer specific sensing interface on it are given below. As for GFET, 5,10,15,20 tetra-(4-aminophenyl) porphyrin (TAPP) mediated reduced graphene oxide (TAPP/rGO) is used as the channel material, which is distinct from the conventional surfacelinked porphyrin on graphene channels [13,14]. In this porphyrin and graphene hybridized compound, there is a hydrogen evolution reaction (HER) enhanced charge transfer (CT), which has been evidenced to improve the original rGO conductivity [15]. But, no report about its application as a channel material in FET has been found. In this work, it is exploited as a TAPP/rGO based FET (TAPP/ rGO-FET) for the purpose of increasing the mobility of rGO, for the first time. Furthermore, the sensing interface is established on the channel surface of TAPP/rGO-FET which is decorated by the porous silkfibroin (SF) mediated graphene oxide (pSF/GO), named as pSF/ GO-TAPP/rGO-FET. PSF/GO is an excellent integration of SF and GO and capable of accommodating cells [11] and biomedical reactions [16]. Similar 3D porous structures have been used for increasing aptamer electrochemical sensor's responding [17,18], but no report about pSF/GO decorated one has been found, up-to-now. Subsequently, AS1411 (a classical CTC aptamer) is used to functionalize the abovementioned pSF/GO-TAPP/rGO-FET, because of its specificity to the over-expressed nucleolin sites on CTCs [19]. Herein, in the first experimental stage, three kinds of CTCs which are human lung cancer cell line A549, breast cancer MDAMB-231 and cervical cancer HeLa are used as target cells, meanwhile the human umbilical vein endothelial cell (HUVEC) is used as a control. The current (IDS) of GFET is examined by a digital source meter (DSM). According to the CT mechanism between the nucleotides and graphene [20,21], more variations of IDS will be induced by more captured CTCs. So it is reasonable to believe the AS1411 captured CTCs can be identified by the currents responding of GFET, as depicted in Fig. 1. In the stage of real applications, the clinical blood samples from 49 breast cancer patients and 5 healthy people, which have been confirmed by the surgery or biopsy pathology, are tested. The comparisons of the measured GFET responding versus
the confirmed diagnostic conclusions are performed to demonstrate the correctness and clinical availability of the proposed CTC sensor. In general, an electronic and biochemical integration is accomplished in the proposed CTC detection method. 2. Materials and methods 2.1. Chemicals Graphite powder (Beijing HWRK Chem Co. Ltd., China), H2SO4(98.0%), hydrogen peroxide (H2O2, 30.0%), permanganate (KMnO4) 99.5% (Tianjin Chemical Reagent wholesale company, China), ammonia solution (25%, Tianjin wind boat chemical reagent Technology Co., Ltd.), hydrazine hydrate (80%, Tianjin Fuyu Fine Chemical Co., Ltd.), TAPP, 95%, Hangzhou Expo biotech Co., Ltd.) and lyophilized SF (Suzhou Simatech Inc., China) are the main chemicals for the synthesis of TAPP/rGO and pSF/GO. Other chemicals include the conductive silver paste (Shenzhen SYREO electronic paste Ltd., China), 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Sigma-Aldrich, USA), dimethylformamide (J&K Scientific, USA), 1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide (EDC) and N-hydroxysulfo-succinimide (NHS) (Thermo, USA), methanol (99.5%, Tianjin Jiang Tian Chemical Technology Co., Ltd.), magnesium chloride (MgCl2), potassium chloride (KCl), hydrochloric acid (HCl) (Tianjin wind boat chemical reagents Technology Co. Ltd., China). AS1411 is from Sangon Biotech Co. Ltd. (China), with sequence of 50 eNH2e(CH2)6-GGT-GGT-GGT-GGTTGT-GGT-GGT-GG-3'. The stock AS1411 solutions are prepared by diluting in 10 mM tris-buffer. (10 mM Tris, 2.5 mM MgCl2, 140 mM KCl, pH 7.4). 2.2. Materials syntheses GO and rGO dispersions are prepared by the modified Hummers' method, which is outlined at here: (1) Graphite oxide is prepared by the modified Hummers' method, that is: the ground graphite powder is dispersed in H2SO4 (98%) and stirred for 1 h,
Fig. 1. The main scheme. The structure of TAPP/rGO-FET based CTC sensor, which is fabricated on the GA and APTES modified glass slides, then decorated by pSF/GO and functionalized by AS1411, sequentially.
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use of B2900A (Agilent Technology Co., Ltd., USA). Bio-microscope WYS-CX23 (KJ Biotech, Tianjin, China) is used to take microphotographs of the captured cells. Digital source meter (DSM) used in the electronic examinations is B2900A (Agilent Technology Co., Ltd., USA).
KMnO4 is gradually added into the mixture under the ice bath condition and stirred for 1 h, H2O2 (30%) is added to remove the potential impurities, the graphite oxide powder is obtained by drying the precipitated solid substance in suspension; (2) GO solution is obtained by extracting supernatant from the centrifugalized graphite oxide suspension in DIW (about 0.25 mg/ml). (3) rGO dispersion is obtained by hydrazine-amino reducing system, which is dropping hydrazine hydrate in the product from (2) which is adjusted by ammonia to pH ¼ 10e11; (4) The solutions of (3) are placed in a water bath (98 C) and shaken continuously for 1 h; (5) After cooling naturally, the dispersion of rGO solutions are obtained by removing the precipitate in the centrifuged solutions of (4). TAPP/rGO is produced following the steps of: (1) The solution of TAPP and GO mixture is prepared by dispersing the mixture of them (with the mass ratio of GO: TAPP ¼ 4:1) in DIW, and the solution are divided to two parts: one is shaken evenly and the other is treated by ultrasonic for 1 h; (2) The hydrazine-ammonia reducing process is executed by the same method as in preparing rGO mentioned in the above paragraph; (3) The solutions of (2) are placed in a water bath (98 C) and shaken continuously for 1 h; (4) After natural cooling, the dispersion of TAPP/rGO solutions are obtained by removing the precipitate in the centrifuged solutions of (3). pSF/GO is prepared by cryodesiccation: (1) the precursor of SF/ GO is prepared by adding 20 mg of lyophilized SF and 10 mg of GO in 10 ml of deionized water and treating by ultrasonication for 2 h; (2) Solid state pSF/GO is obtain by three-step freeze-drying process, which are pre-chilling (20 C, 4 h), deep-freezing (80 C, overnight) and freeze-dry (80 C, 48 h); (3) Methanol treatment for freeze-dried pSF/GO is soaking the product of (2) in 80% (v/v) methanol aqueous solution; (4) pSF/GO stock solution is prepared by dispersing methanol treated pSF/GO in DIW followed by ultrasonic treatment (1 h), it is diluted by DIW to the concentrations of 0.5, 1, 2, 5, 10 mg/ml.
All the electronic measurements are conducted by the same measuring setup, as depicted in Fig. 1, after incubation the AS1411 functionalized GFET with the analyte (CTCs solutions or real clinical blood samples). In which, the electrode of G is a suspended Pt above the channel surface and immerged in the similar PBS during all the tests. The electrodes of D, S and G are connected to DSM, and S is grounded. The voltages between the electrodes of G and S (VGS), as well as S and D (VDS) are controlled at the optimized values. Meanwhile, the electronic measuring operations are separated from the cell's capturing procedure, for the purpose of avoiding the uncertain influences from different backgrounds of the analyte, in utmost.
2.3. Objects of CTC detection
3. Results
A549, MDA-MB-231, HeLa, and HUVEC were from the American Type Culture Collection (ATCC, Manassas, US), as described previously [18]. Briefly, they were independently cultured in ATCCformulated F-12 K Medium (Catalog No.30-2004), Leibovitz's L-15 Medium (Catalog No. 30-2008), Eagle's Minimum Essential Medium (Catalog No. 30-2003), and the Endothelial Cell Growth KitBBE (ATCC® PCS-100-040), respectively. All cell lines were incubated under a humid air with 5% CO2 containing 10% fetal bovine serum for cancer cells, while 2% fetal bovine serum for the normal cell line. Prior to each experiment, cells were harvested using 0.25% trypsin/EDTA and were enumerated by the cell counter. For the following experiments, cells were washed and re-suspended in PBS or anti-coagulant whole blood at a density of 10 ~ 106 cells/ml. The EDTA anti-coagulant clinical blood samples were obtained from patients of Tianjin medical university cancer institute & hospital (China), and all the cancer patients were confirmed by surgery or biopsy pathology.
3.1. Identification of TAPP/rGO and SF/GO
2.4. Apparatus Dimension Icon (Bruck, USA) and SEM S-3500 N (Hitachi Limited) are used for taking the images of AFM and SEM, respectively. XPS wide and core XPS spectra are achieved by Axis Ultra DLD (Kratos Analytical Ltd., UK). UV754 N (Shanghai Precision Science Instrument Co Ltd, China) is used for the UVevis examinations. FTIR examinations are conducted by Nicolet iS10 (Thermo Scientific, USA). Electronic measurements are executed with the
2.5. CTC sensors' preparation The structure of the GFET based CTC sensor is shown in Fig. 1, its preparation procedure is outlined at here: (1) On the APTES (10%, 50 C, 2 h) and GA (2.5%, room temperature, 1 h) treated glass slides, the prepared TAPP/rGO material are drop-coated; (2) the electrodes of source and drain (S and D) are made by painting the conductive silver paste; (3) the electrodes are encapsulated by the insulating silica gel to form a reservoir; (4) the pSF/GO layer is decorated by incubating it with GA (2.5%, room temperature, 1 h) and pSF/GO dispersions; (5) after being incubated with EDC (200 nM) and NHS (50 mM NHS), at room temperature for 15 min, the pSF/GO decorated device is functionalized by incubating with AS1411 solutions (in the thermostatic oscillator, 60 rpm, 37 C, 4 h). 2.6. Electronic measurements
UVevis analyses are performed to identify TAPP/rGO, as shown in Fig. 2A. The reducing effects are evidenced by the red shifts from about 234 nm in GO to 270 nm in TAPP/rGO, it is attributed to the pp* transition induced UVevis absorption. Whilst, it is also found there are shoulder peaks at about 300 nm in GO materials, which are caused by the absorbance of n-p* transition of C]O groups [22]. Furthermore, these shoulder peaks disappear in the TAPP/rGO. These phenomena demonstrate that the damaged sp2-conjugated domains are healed and the oxygen-containing groups are decreased. Moreover, the smaller peaks at about 451 and 439 nm are found on TAPP/rGO, but they are absent from the spectra of GO and rGO. These small peaks are attributed to the hybridized or attached TAPP molecules (named as TAPP-peak), because the peak at the similar position (~456 nm) is found in the spectrum of pure TAPP. In addition to the transformations induced by HER and the hybridized TAPP, the peak at ~270 nm is narrowed and enhanced by TAPP mediation, as shown by the peak of 270 nm from rGO (orange) to TAPP/rGO (blue). These are in accordance with the reported porphyrin enhanced GO reducing effects [15], because a sharper peak indicates more damaged or distorted hexatomic rings in GO are recovered, according to the UVevis and Raman studies of graphene materials [23]. FTIR spectra are provided in Fig. 2B to demonstrate pSF/GO, according to reference [11]. The typical absorption are located at 1725 and 1098 cm1 are contributed by the absorbance of
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Fig. 2. Identification of pSF/GO and TAPP/rGO. (A) The normalized UVevis spectra of TAPP/rGO. (B) The FTIR spectra for SF, GO, SF/GO and methanol-pSF/GO. (C) SEM photo of drop-coated TAPP/rGO films. (D) SEM photo of pSF/GO. (E) AFM height analysis of TAPP/rGO film. (F) XPS spectra of TAPP/rGO, after decorated by pSF/GO and AS1411.
stretching vibrations of C]O, and CeOH, these oxygen-containing groups; meanwhile, the absorptions at 1447 and 1261 cm1 are assigned to the deformation vibration of carbon atoms' hexagon and the in-plane bending of hydrogen bonds; SF's typical absorption peaks at 1624, 1523, 1243 and 1060 cm1 in the random coiled SF film, belong to the amide I band, II band, III band and CeOH; in the pSF/GO, the typical peaks of GO and SF are integrated and identified by peaks at 1725 and 1447 cm1 which are contributed by the intertwined GO nano-flakes. Whilst, the absorption peaks at 1637 and 1533 cm1 are contributed by the amide I and amide II in SF which indicate there are both the ɑ-helical and b-sheet conformations [24]. Besides, in the band of 800e1050 cm1, an absorption peak at about 950 cm1 is found in pSF/GO, it may be attributed by the enhanced vibrations of Alarich segments [25]. The SEM photographs of TAPP/rGO films and pSF/GO are given in Fig. 2C and D, respectively, which indicate the smooth channel surface of TAPP/rGO and the porous structure of the pSF/GO. Meanwhile, the similar porous topography is also observed on the sensing surface of five pSF/GO-TAPP/rGO-FETs, as presented in Fig. S1. The height analysis of TAPP/rGO nano-flake is executed by AFM (Fig. 2E), which is about 3 nm, and estimated to be about 3.2
layers, since this AFM examination is conducted under the tapping mode, in which the thickness of one single graphene layer is about 0.95 nm. XPS full spectra and the quantification of the main elements in TAPP/rGO (named as #1), pSF/GO-TAPP/rGO and apta-pSF/GOTAPP/rGO presented in Fig. 2F. The main elements are C, N, O and P, their variations are in consistence with the experimental procedures, that is: with the coating of pSF/GO, the contents of C are lowered greatly from 84% (TAPP/rGO) to 63%, O elements are increased from 12% to 30% and N elements are slightly enhanced from 2.0% to 4.3%. These variations agree with the oxygencontaining groups in GO and amino groups in SF. With the immobilization of AS1411, the contents of C are lowered to 57%, N and O atoms are increased to 6.8% and 32%, respectively, which are consistent with the main elements in nucleotide bases. In particular, the emerging of P element in apta-pSF/GO-TAPP/rGO, as shown by the blue column in Fig. 2F, can demonstrate aptamer molecules (AS1411) are fixed on the pSF/GO-TAPP/rGO channel film, because the nucleic acid is the only source of P element. More core spectra analyses are executed in Supplementary Materials (Fig. S2), to have more understanding about the surface chemical
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Fig. 3. Basic electronic features of TAPP/rGO-FET. (A) Transfer curves of TAPP/rGO-FET. (B) The carriers mobilities of TAPP/rGO-FET and rGO-FET at different bias voltages.
states in each of the surface modifications. 3.2. Electronic characteristics The ambipolar transfer feature of the TAPP/rGO-FET is examined and compared with the traditional rGO based GFET, as presented in Fig. 3A. It is found that the charge neutral points (CNPs) are increased, which is deduced to be caused by TAPP induced nitrogen doping effect. Meanwhile, with the increasing of VDS from 0.1 to 0.5 V, the right-shifting of transfer curves are coincident with our previously reported work [26]. Furthermore, the transfer features of the pSF/GO decorated and AS1411 functionalized TAOO/rGO-FET are re-examined at the constant VDS (0.5 V), it indicates that the representative ambipolar characteristic is not changed by the modifications. The left-moved CNPs are induced by the coated pSF/ GO layer and the anchored AS1411 molecules. The injected electrons from pSF/GO and the nucleic acid degrade the holes' contents
in TAPP/rGO channel. Besides, the optimized VGS is selected based on the mobility of carriers in TAPP/rGO-FET, which are calculated and presented in Fig. 3B according to the theoretical and experimental studies of FET [27e30]. It indicates the carriers' mobilities in TAPP/rGO channel film are obviously increased comparing with rGO, which agrees with the porphyrin facilitated HER studies [13e15]. Since a higher mobility can be obtained at VGS ¼ 0.2 V, it is selected for the following tests. 3.3. Effects of pSF/GO modification The microscope of the captured CTC A549 is presented in Fig. 4A, in which the four photos indicate more CTCs are captured by the pSF/GO decorated GFET. This result has also been verified from the electrical signal. The output currents' responding (DIDS/IDS,0) of blank TAPP/rGO-FET and pSF/GO decorated ones are measured after being incubated with AS1411 solutions in different concentrations
Fig. 4. Effect of pSF/GO decoration, observed by microscopy and electrical signals. (A) The microscopy of the captured CTC A549 on TAPP/rGO coated glass slides with and without pSF/GO decoration. (B) The AS1411 immobilization induced currents' changing ratios (DIDS/IDS,0), in which DIDS(¼IDS-IDS,0) is the deviation of the current IDS with IDS,0, which are the currents after incubation in the selected AS1411 concentration (AS1411_Conc.) and before anchoring AS1411. (C) CTCs' responding (DIDS/IDS,0) of pSF/GO decorated TAPP/rGOFET and naked ones. The error bars are the standard deviations (STD) of the individually measured data (n ¼ 20).
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(AS1411_Conc.), as shown in Fig. 4B. In which, DIDS ¼ IDS-IDS,0 IDS,0 and IDS are the currents before and after the incubation with the selected concentrations of AS1411 (AS1411_Conc.), respectively. It is found the main stream of the two curves is downward, which is in agreement with the electron injection mechanism between nucleic acid and graphene derivatives [23]. That is to say, the electrons injected into the conjugated carbon network from AS1411's phosphate backbone can alter the balanced channel carriers. Meanwhile, under the selected working condition (VGS ¼ 0.2 V, VDS ¼ 0.5 V), the positive CNP point in Fig. 3A means holes are the major carriers; with the increasing of AS1411_Conc., then injected electrons will reduce hole's density, so the negatively increased DIDS/IDS,0 are measured, as shown in Fig. 4B. According to the variations of the curves in Figs. 4B and 2 mM AS1411 is used in the following experiments, more detailed discussion is provided in the supplementary material, “Discussions for Fig. 4B”. Furthermore, the influence of pSF/GO to devices' responding (-DIDS/IDS,0) for A549 is also measured, as shown in Fig. 4C. In Fig. 4C, though both pSF/GO decorated and blank TAPP/ rGO-FETs are sensitive to A549, more higher responding (-DIDS/ IDS,0) is obtained by the pSF/GO decorated ones. Meanwhile, the slope of the red fitted line (2.38%/log10[cell_Conc.]) is also bigger than the blue one's (1.60%/log10[cell_Conc.]), it indicates higher sensitivity is possessed by pSF/GO-TAPP/rGO-FETs. The
reproducibility of the method of pSF/GO decoration to improve the sensitivity of TAPP/rGO-FET based CTC-sensing platforms is evaluated by 20 individual measurements. As shown by Fig. 4C, the standard deviation is by “pSF/GO” is relatively lower than “blank”. 3.4. CTC assay The sensitivity, repeatability and selectivity of the proposed CTC sensors are examined by 12 devices after they are incubated with different concentrations of CTCs (HeLa, A549 and MDA-MB-231) and the control cell (HUVEC) in buffer solutions, respectively. Three devices are allocated to each of the cells and named as #1, #2 and #3. Their normalized data for each device are presented in Fig. 5A (A-1 to A-4) with symbols of D, , and B, in which the averages (star) are also calculated. It is found, there are almost linear responding curves in Fig. A-1 to A-3. In contrary, the randomly distributed data points in Fig. A-4 indicate there is almost no specific responding for HUVEC. Moreover, the linear fitting analyses for the average of measured DIDS/IDS,0 (Fig. S3) indicate the sensitivities for A549, MDA-MB-231 and HeLa are about 3.14, 3.32, 2.60%/log10(cells/ml) for A549 (R2 ¼ 0.97), MDA-MB-231 2 2 (R ¼ 0.96) and HeLa (R ¼ 0.98), respectively. Meanwhile, the selectivity for CTCs is also confirmed by the low slope and R2 for HUVEC which are 0.13%/log10(cells/ml) and 0.50.
Fig. 5. Responding for the CTCs in PBS buffer, whole blood and the real applications for clinical samples. (A) The normalized responding of 12 AS1411 functionalized pSF/GOTAPP/rGO-FETs for A549, MDA-MB-231, HeLa and HUVEC in PBS. The error bars are the standard deviations (STD) of the individually measured data (n ¼ 3). (B) The negatively increased changing rates in dependence on the denary logarithm of cells concentrations in the whole blood. The error bars are RSD and n ¼ 3. (C) The measured currents' responding for the clinical samples from 5 healthy persons and 49 patients with different diagnostic conclusions which are grouped as stages of I, II, III and IV, as well as adenofibroma (AFM) and after being treated by AC regimen chemotherapy (after_AC) for 3 and 6 cycles.
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Though the acceptable responses have been obtained for CTCs detection in PBS environment, in the real blood samples, there are large amount of fibrinogen, granulocyte and erythrocyte, which will make a negative interference on the performance of the CTC sensor. So an interference examination is conducted, the fictitious CTC samples are prepared by spiking cells into anti-coagulant blood. After being incubated with these samples, the device's responding is measured under the same condition as in the abovementioned experiments, the results are presented in Fig. 5B. In comparisons with the data in Fig. S3, bigger deviations are observed, we deduce they are caused by the interference of the components in blood. Nevertheless, the responses for cancer cells (A549, MDA-MB-231 and HeLa) are still noticeably higher than the normal cell (HUVEC), and grow with the increasing of cells' concentrations (Cell_Conc.). So, it is reasonable to believe that CTCs' content in the whole blood can be recognized by the varied currents. Finally, the real applications are conducted for the blood samples from 5 healthy persons and 49 patients with breast cancer and adenofibroma, their demographic and clinical characteristics are shown in Table 1. The correlations of the measured data by the proposed clinical available-CTC sensors and these samples' diagnostic conclusions are presented in Fig. 5C. It is found that the responding of IDS is growing up with the developing of the cancer stages from I to IV. In comparisons with the measured results in Fig. 5A and B, these data points indicate more CTCs exist in the blood samples in higher stages. Besides, the blood samples of adenofibroma patients and breast cancer patients after being treated by AC regimen (the liposomes of doropepirubicin were 50 mg and cyclophosphamide 800 mg) chemotherapy for 3 and 6 cycles, can also be identified by the proposed CTC sensors.
4. Discussion In conclusion, the proposed pSF/GO-TAPP/rGO-FET based CTC sensor based CTC assay is an ideal exploitation and integration of chemically synthesized graphene derivatives with aptamer sensing strategy. Comprehensive studies are provided in this work, including material and device preparation, characterizations, device evaluations and clinical assay. The typical ambipolar electronic feature and TAPP enhanced mobility are demonstrated to be possessed by the TAPP/rGO-FET, after being decorated by pSF/GO and functionalized by AS1411. Based on this novel TAPP/rGO-FET, CTC assay is fulfilled in two steps. The real clinical samples' assays indicate the pSF/GO-TAPP/rGO-FET based CTC sensor is a promising candidate for clinical assay.
Table 1 Demographic and clinical characteristics of patients with breast cancer and adenofibroma n (%). Variabes
Cancer (n ¼ 47)
Adenofibroma (n ¼ 2)
Age (yr) Median (min, max)
53 (22, 74)
Min ¼ 32, Max ¼ 58
32 (68.09%) 15 (31.91%)
2(100%) 0 (0)
59 >59 Clinical stage I II III IV Unidentified Metastasis Yes No
14 (29.79%) 25 (53.19%) 5 (10.64%) 2 (4.26%) 1 (2.13%) 3 (6.38%) 44 (93.62%)
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Acknowledgments This work is supported by the National Natural Science Foundation of China (Grant no. 61771260, 81601820, 81871719). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.electacta.2019.05.039. References [1] Y. Ming, Y. Li, H. Xing, M. Luo, Z. Li, J. Chen, J. Mo, S. Shi, Circ Circulating tumor cells: from theory to nanotechnology-based detection, Front. Pharmacol. 8 (2017) 35. [2] J. Liu, Y. Zhang, Q. Zhao, B. Situ, J. Zhao, S. Luo, B. Li, X. Yan, P. Vadgama, L. Su, W. Ma, W. Wang, L. Zheng, Bifunctional aptamer-mediated catalytic hairpin assembly for the sensitive and homogenous detection of rare cancer cells, Anal. Chim. Acta 1029 (2018) 58. [3] F. Zhao, Electrochemical techniques for the detection of cancer cells and cellsurface glycan expression: a mini review, Int. J. Electrochem. Sci. 12 (8) (2017) 7580e7596. [4] Y.J. Jan, J. Chen, Y. Zhu, Y.T. Lu, S.H. Chen, H. Chung, M. Smalley, Y.W. Huang, J. Dong, L.C. Chen, H.H.Y., J.S. Tomlinson, S. Hou, V.G. Agopian, E.M. Posadas, H.R. Tseng, Clinical applications of NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells, Theranostics 6 (9) (2016) 1425. [5] A. Kowalik, M. Kowalewska, S. Go zd z, Current approaches for avoiding the limitations of circulating tumor cells detection methods-implications for diagnosis and treatment of patients with solid tumors, Transl. Res. 185 (2017) 58e84. [6] N. Shao, E. Wickstrom, B. Panchapakesan, Nanotube-antibody biosensor arrays for the detection of circulating breast cancer cells, Nanotechnology 19 (46) (2008) 465101. [7] D. Xiang, S. Shigdar, G. Qiao, T. Wang, A.Z. Kouzani, S.F. Zhou, L. Kong, Y. Li, C. Pu, W. Duan, Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine, Theranostics 5 (1) (2015) 23e42. [8] X. Zhao, R. Fang, Y. Zhang, Development of electrochemical aptasensor for label-free glioma cell detection, Int. J. Electrochem. Sci. 12 (6) (2017) 9301e9310. [9] L. Feng, Y. Chen, J. Ren, X. Qu, A graphene functionalized electrochemical aptasensor for selective label-free detection of cancer cells, Biomaterials 32 (11) (2011) 2930e2937. [10] K. Urmann, S. Arshavskygraham, J.G. Walter, T. Scheper, E. Segal, Whole-cell detection of live lactobacillus acidophilus on aptamer-decorated porous silicon biosensors, Analyst 141 (18) (2016) 5432e5440. [11] S.D. Wang, Q. Ma, K. Wang, P.B. Ma, Strong and biocompatible threedimensional porous silk fibroin/graphene oxide scaffold prepared by phase separation, Int. J. Biol. Macromol. 111 (2018) 237e246. [12] M.S. Mansouri, A. Salimi, Ultrasensitive flexible FET-type aptasensor for CA 125 cancer marker detection based on carboxylated multiwalled carbon nanotubes immobilized onto reduced graphene oxide film, Anal. Chim. Acta 1000 (2018) 273. [13] S. Seo, K. Lee, M. Min, Y. Cho, M. Kim, H. Lee, A molecular approach to an electrocatalytic hydrogen evolution reaction on single-layer graphene, Nanoscale 9 (11) (2017) 3969e3979. [14] T. Kawata, T. Ono, Y. Kanai, Y. Ohno, K. Maehashi, K. Inoue, K. Matsumoto, Improved sensitivity of a graphene FET biosensor using porphyrin linkers, Jpn. J. Appl. Phys. 57 (6) (2018), 065103. [15] J. Geng, H.T. Jung, Porphyrin functionalized graphene sheets in aqueous suspensions: from the preparation of graphene sheets to highly conductive graphene films, J. Phys. Chem. C 114 (18) (2010) 8227e8234. [16] C. Zhang, H. Shao, J. Luo, X. Hu, Y. Zhang, Structure and interaction of silk fibroin and graphene oxide in concentrated solution under shear, Int. J. Biol. Macromol. 107 (2018) 2590e2597. [17] N. Hao, R. Hua, S. Chen, Y. Zhang, Z. Zhou, J. Qian, Q. Liu, K. Wang, Multiple signal-amplification via Ag and TiO2 decorated 3D nitrogen doped graphene hydrogel for fabricating sensitive label-free photoelectrochemical thrombin aptasensor, Biosens. Bioelectron. 101 (2017) 14e20. [18] R. Zhang, Y. Gu, Z. Wang, Y. Li, Q. Fan, Y. Jia, Aptamer cell sensor based on porous graphene oxide decorated ion-selective-electrode: double sensing platform for cell and ion, Biosens. Bioelectron. 117 (2018) 303e311. [19] J. Mosafer, A. Mokhtarzadehc, Cell surface nucleolin as a promising receptor for effective AS1411 aptamer-mediated targeted drug delivery into cancer cells, Curr. Drug Deliv. 15 (2018) 1e7. [20] H. Mousavi, M. Grabowski, Nonlinear electron transport across short DNA segment between graphene leads, Solid State Commun. 279 (2018) 30e33. [21] S. Kundu, A. Pyne, R. Dutta, N. Sarkar, Unveiling the interaction of duplex DNA with graphene oxide in the presence of two diverse binders: a detailed photophysical study, J. Phys. Chem. C 122 (12) (2018). [22] J.H. Kang, T. Kim, J. Choi, J. Park, Y.S. Kim, M.S. Chang, H. Jung, K.T. Park,
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Clinical available circulating tumor cell assay based on tetra(4aminophenyl) porphyrin mediated reduced graphene oxide field effect transistor Shihui Hu a, Zhongrong Wang a, Yajun Gu b, **, Yueguo Li c, ***, Yunfang Jia a, * a
College of Electronic Information and Optical Engineering, Nankai University, Tianjin, China School of Medical Laboratory, Tianjin Medical University, Tianjin, China Department of Clinical Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2018 Received in revised form 2 April 2019 Accepted 8 May 2019 Available online 12 May 2019
Circulating tumor cell (CTC) assay is one of the emerging “liquid biopsy” for cancer's diagnostic, an easyto-operate and state-of-the-art CTC sensor is the imperative need of clinical medical laboratory. A labelfree and solid-state sensor for clinical sample's CTC detection based on an aptamer (AS1411) functionalized graphene field effect transistor (GFET) by using tetra(4-aminophenyl) porphyrin mediated reduced graphene oxide as the channel material. Objects are CTCs of A549, MDA-MB-231, HeLa and HUVEC (as a control) in buffer solutions and EDTA anti-coagulant whole blood samples, as well as the anti-coagulant clinical blood samples from 49 breast cancer patients and 5 healthy persons. The sensitivities for CTCs in the range of 10 - 106 cells/ml are 3.14, 3.32, 2.60%/log10(cells/ml) for A549 (R2 ¼ 0.97), MDA-MB-231 (R2 ¼ 0.96) and HeLa (R2 ¼ 0.98), respectively. Meanwhile, the selectivity for CTCs is also confirmed by the low sensitivity and R2 for HUVEC which are 0.13%/log10(cells/ml) and 0.50. Furthermore, whole blood interference is found to make the sensing range narrow. At last, their applications in grading clinical blood samples are found to be in agreements with the parallel diagnostic conclusions of these patients, samples of adenofibroma and effect of chemotherapy can also be identified. The aptamer specific, directly electronic CTC detection is accomplished by the proposed GFET based CTC sensor, it can provide a novel analytical technique for cancers' clinical assay. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Circulating tumor cell Field effect transistor Graphene Porphyrin Aptamer
1. Introduction Circulating tumor cells (CTCs) are tumorigenic cells which shed from the lesion and disseminate in the peripheral blood. To date, based on the biomedical studies about the metastasis related over expressed biomolecules on CTCs' membrane (like EpCAM, nucleolin, CK etc.), CTCs can be specifically targeted [1] and converted to the measurable signals (optics [2] or electronics [3]). It has been well-accepted that, the CTC detection is a promising candidate for the emerging “liquid biopsy” assay [1,4], its application in a clinical setting will be helpful to cancers' diagnosis and therapy monitoring [4]. However, in the complicated blood environment, CTCs' scarcity,
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (Y. Gu), [email protected] (Y. Li), jiayf@ nankai.edu.cn (Y. Jia). https://doi.org/10.1016/j.electacta.2019.05.039 0013-4686/© 2019 Elsevier Ltd. All rights reserved.
short life-time, plasticity and heterogeneity are still frustrations for its real application [5]. Confronting with this challenge, ever since the publication of carbon nanotube field-effect-transistor (FET) based CTC sensors [6], there have been myriads of interdisciplinary studies for developing highly sensitive CTC capturing methods and efficient strategies to transduce the captured CTCs' information to the measurable data. The merits and drawbacks in these works have been systematically summarized in the recent reviews [1,3,5]. Amongst them, the aptamer based CTC “direct-capture” strategy [2,7e10], 3-dimensional (3D) porous micro/nano-environment for hosting cells [10,11] and aptamer functionalized graphene FET (apta-GFET) [12] are of great interest to us, because of aptamer's robustness and capability in precise-targeting, the large specificsurface-area in 3D-porous structure, graphene channel material's high mobility and biocompatibility, etc. Intrigued by the achievements in those works, we would like to develop a clinical available CTC sensor based on the apta-GFET. The main scheme of this work is illustrated in Fig. 1, in which the capability for CTC detection is examined in two steps (experimental tests and real applications).
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More explanations for the transducer (GFET) and the aptamer specific sensing interface on it are given below. As for GFET, 5,10,15,20 tetra-(4-aminophenyl) porphyrin (TAPP) mediated reduced graphene oxide (TAPP/rGO) is used as the channel material, which is distinct from the conventional surfacelinked porphyrin on graphene channels [13,14]. In this porphyrin and graphene hybridized compound, there is a hydrogen evolution reaction (HER) enhanced charge transfer (CT), which has been evidenced to improve the original rGO conductivity [15]. But, no report about its application as a channel material in FET has been found. In this work, it is exploited as a TAPP/rGO based FET (TAPP/ rGO-FET) for the purpose of increasing the mobility of rGO, for the first time. Furthermore, the sensing interface is established on the channel surface of TAPP/rGO-FET which is decorated by the porous silkfibroin (SF) mediated graphene oxide (pSF/GO), named as pSF/ GO-TAPP/rGO-FET. PSF/GO is an excellent integration of SF and GO and capable of accommodating cells [11] and biomedical reactions [16]. Similar 3D porous structures have been used for increasing aptamer electrochemical sensor's responding [17,18], but no report about pSF/GO decorated one has been found, up-to-now. Subsequently, AS1411 (a classical CTC aptamer) is used to functionalize the abovementioned pSF/GO-TAPP/rGO-FET, because of its specificity to the over-expressed nucleolin sites on CTCs [19]. Herein, in the first experimental stage, three kinds of CTCs which are human lung cancer cell line A549, breast cancer MDAMB-231 and cervical cancer HeLa are used as target cells, meanwhile the human umbilical vein endothelial cell (HUVEC) is used as a control. The current (IDS) of GFET is examined by a digital source meter (DSM). According to the CT mechanism between the nucleotides and graphene [20,21], more variations of IDS will be induced by more captured CTCs. So it is reasonable to believe the AS1411 captured CTCs can be identified by the currents responding of GFET, as depicted in Fig. 1. In the stage of real applications, the clinical blood samples from 49 breast cancer patients and 5 healthy people, which have been confirmed by the surgery or biopsy pathology, are tested. The comparisons of the measured GFET responding versus
the confirmed diagnostic conclusions are performed to demonstrate the correctness and clinical availability of the proposed CTC sensor. In general, an electronic and biochemical integration is accomplished in the proposed CTC detection method. 2. Materials and methods 2.1. Chemicals Graphite powder (Beijing HWRK Chem Co. Ltd., China), H2SO4(98.0%), hydrogen peroxide (H2O2, 30.0%), permanganate (KMnO4) 99.5% (Tianjin Chemical Reagent wholesale company, China), ammonia solution (25%, Tianjin wind boat chemical reagent Technology Co., Ltd.), hydrazine hydrate (80%, Tianjin Fuyu Fine Chemical Co., Ltd.), TAPP, 95%, Hangzhou Expo biotech Co., Ltd.) and lyophilized SF (Suzhou Simatech Inc., China) are the main chemicals for the synthesis of TAPP/rGO and pSF/GO. Other chemicals include the conductive silver paste (Shenzhen SYREO electronic paste Ltd., China), 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Sigma-Aldrich, USA), dimethylformamide (J&K Scientific, USA), 1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide (EDC) and N-hydroxysulfo-succinimide (NHS) (Thermo, USA), methanol (99.5%, Tianjin Jiang Tian Chemical Technology Co., Ltd.), magnesium chloride (MgCl2), potassium chloride (KCl), hydrochloric acid (HCl) (Tianjin wind boat chemical reagents Technology Co. Ltd., China). AS1411 is from Sangon Biotech Co. Ltd. (China), with sequence of 50 eNH2e(CH2)6-GGT-GGT-GGT-GGTTGT-GGT-GGT-GG-3'. The stock AS1411 solutions are prepared by diluting in 10 mM tris-buffer. (10 mM Tris, 2.5 mM MgCl2, 140 mM KCl, pH 7.4). 2.2. Materials syntheses GO and rGO dispersions are prepared by the modified Hummers' method, which is outlined at here: (1) Graphite oxide is prepared by the modified Hummers' method, that is: the ground graphite powder is dispersed in H2SO4 (98%) and stirred for 1 h,
Fig. 1. The main scheme. The structure of TAPP/rGO-FET based CTC sensor, which is fabricated on the GA and APTES modified glass slides, then decorated by pSF/GO and functionalized by AS1411, sequentially.
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use of B2900A (Agilent Technology Co., Ltd., USA). Bio-microscope WYS-CX23 (KJ Biotech, Tianjin, China) is used to take microphotographs of the captured cells. Digital source meter (DSM) used in the electronic examinations is B2900A (Agilent Technology Co., Ltd., USA).
KMnO4 is gradually added into the mixture under the ice bath condition and stirred for 1 h, H2O2 (30%) is added to remove the potential impurities, the graphite oxide powder is obtained by drying the precipitated solid substance in suspension; (2) GO solution is obtained by extracting supernatant from the centrifugalized graphite oxide suspension in DIW (about 0.25 mg/ml). (3) rGO dispersion is obtained by hydrazine-amino reducing system, which is dropping hydrazine hydrate in the product from (2) which is adjusted by ammonia to pH ¼ 10e11; (4) The solutions of (3) are placed in a water bath (98 C) and shaken continuously for 1 h; (5) After cooling naturally, the dispersion of rGO solutions are obtained by removing the precipitate in the centrifuged solutions of (4). TAPP/rGO is produced following the steps of: (1) The solution of TAPP and GO mixture is prepared by dispersing the mixture of them (with the mass ratio of GO: TAPP ¼ 4:1) in DIW, and the solution are divided to two parts: one is shaken evenly and the other is treated by ultrasonic for 1 h; (2) The hydrazine-ammonia reducing process is executed by the same method as in preparing rGO mentioned in the above paragraph; (3) The solutions of (2) are placed in a water bath (98 C) and shaken continuously for 1 h; (4) After natural cooling, the dispersion of TAPP/rGO solutions are obtained by removing the precipitate in the centrifuged solutions of (3). pSF/GO is prepared by cryodesiccation: (1) the precursor of SF/ GO is prepared by adding 20 mg of lyophilized SF and 10 mg of GO in 10 ml of deionized water and treating by ultrasonication for 2 h; (2) Solid state pSF/GO is obtain by three-step freeze-drying process, which are pre-chilling (20 C, 4 h), deep-freezing (80 C, overnight) and freeze-dry (80 C, 48 h); (3) Methanol treatment for freeze-dried pSF/GO is soaking the product of (2) in 80% (v/v) methanol aqueous solution; (4) pSF/GO stock solution is prepared by dispersing methanol treated pSF/GO in DIW followed by ultrasonic treatment (1 h), it is diluted by DIW to the concentrations of 0.5, 1, 2, 5, 10 mg/ml.
All the electronic measurements are conducted by the same measuring setup, as depicted in Fig. 1, after incubation the AS1411 functionalized GFET with the analyte (CTCs solutions or real clinical blood samples). In which, the electrode of G is a suspended Pt above the channel surface and immerged in the similar PBS during all the tests. The electrodes of D, S and G are connected to DSM, and S is grounded. The voltages between the electrodes of G and S (VGS), as well as S and D (VDS) are controlled at the optimized values. Meanwhile, the electronic measuring operations are separated from the cell's capturing procedure, for the purpose of avoiding the uncertain influences from different backgrounds of the analyte, in utmost.
2.3. Objects of CTC detection
3. Results
A549, MDA-MB-231, HeLa, and HUVEC were from the American Type Culture Collection (ATCC, Manassas, US), as described previously [18]. Briefly, they were independently cultured in ATCCformulated F-12 K Medium (Catalog No.30-2004), Leibovitz's L-15 Medium (Catalog No. 30-2008), Eagle's Minimum Essential Medium (Catalog No. 30-2003), and the Endothelial Cell Growth KitBBE (ATCC® PCS-100-040), respectively. All cell lines were incubated under a humid air with 5% CO2 containing 10% fetal bovine serum for cancer cells, while 2% fetal bovine serum for the normal cell line. Prior to each experiment, cells were harvested using 0.25% trypsin/EDTA and were enumerated by the cell counter. For the following experiments, cells were washed and re-suspended in PBS or anti-coagulant whole blood at a density of 10 ~ 106 cells/ml. The EDTA anti-coagulant clinical blood samples were obtained from patients of Tianjin medical university cancer institute & hospital (China), and all the cancer patients were confirmed by surgery or biopsy pathology.
3.1. Identification of TAPP/rGO and SF/GO
2.4. Apparatus Dimension Icon (Bruck, USA) and SEM S-3500 N (Hitachi Limited) are used for taking the images of AFM and SEM, respectively. XPS wide and core XPS spectra are achieved by Axis Ultra DLD (Kratos Analytical Ltd., UK). UV754 N (Shanghai Precision Science Instrument Co Ltd, China) is used for the UVevis examinations. FTIR examinations are conducted by Nicolet iS10 (Thermo Scientific, USA). Electronic measurements are executed with the
2.5. CTC sensors' preparation The structure of the GFET based CTC sensor is shown in Fig. 1, its preparation procedure is outlined at here: (1) On the APTES (10%, 50 C, 2 h) and GA (2.5%, room temperature, 1 h) treated glass slides, the prepared TAPP/rGO material are drop-coated; (2) the electrodes of source and drain (S and D) are made by painting the conductive silver paste; (3) the electrodes are encapsulated by the insulating silica gel to form a reservoir; (4) the pSF/GO layer is decorated by incubating it with GA (2.5%, room temperature, 1 h) and pSF/GO dispersions; (5) after being incubated with EDC (200 nM) and NHS (50 mM NHS), at room temperature for 15 min, the pSF/GO decorated device is functionalized by incubating with AS1411 solutions (in the thermostatic oscillator, 60 rpm, 37 C, 4 h). 2.6. Electronic measurements
UVevis analyses are performed to identify TAPP/rGO, as shown in Fig. 2A. The reducing effects are evidenced by the red shifts from about 234 nm in GO to 270 nm in TAPP/rGO, it is attributed to the pp* transition induced UVevis absorption. Whilst, it is also found there are shoulder peaks at about 300 nm in GO materials, which are caused by the absorbance of n-p* transition of C]O groups [22]. Furthermore, these shoulder peaks disappear in the TAPP/rGO. These phenomena demonstrate that the damaged sp2-conjugated domains are healed and the oxygen-containing groups are decreased. Moreover, the smaller peaks at about 451 and 439 nm are found on TAPP/rGO, but they are absent from the spectra of GO and rGO. These small peaks are attributed to the hybridized or attached TAPP molecules (named as TAPP-peak), because the peak at the similar position (~456 nm) is found in the spectrum of pure TAPP. In addition to the transformations induced by HER and the hybridized TAPP, the peak at ~270 nm is narrowed and enhanced by TAPP mediation, as shown by the peak of 270 nm from rGO (orange) to TAPP/rGO (blue). These are in accordance with the reported porphyrin enhanced GO reducing effects [15], because a sharper peak indicates more damaged or distorted hexatomic rings in GO are recovered, according to the UVevis and Raman studies of graphene materials [23]. FTIR spectra are provided in Fig. 2B to demonstrate pSF/GO, according to reference [11]. The typical absorption are located at 1725 and 1098 cm1 are contributed by the absorbance of
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Fig. 2. Identification of pSF/GO and TAPP/rGO. (A) The normalized UVevis spectra of TAPP/rGO. (B) The FTIR spectra for SF, GO, SF/GO and methanol-pSF/GO. (C) SEM photo of drop-coated TAPP/rGO films. (D) SEM photo of pSF/GO. (E) AFM height analysis of TAPP/rGO film. (F) XPS spectra of TAPP/rGO, after decorated by pSF/GO and AS1411.
stretching vibrations of C]O, and CeOH, these oxygen-containing groups; meanwhile, the absorptions at 1447 and 1261 cm1 are assigned to the deformation vibration of carbon atoms' hexagon and the in-plane bending of hydrogen bonds; SF's typical absorption peaks at 1624, 1523, 1243 and 1060 cm1 in the random coiled SF film, belong to the amide I band, II band, III band and CeOH; in the pSF/GO, the typical peaks of GO and SF are integrated and identified by peaks at 1725 and 1447 cm1 which are contributed by the intertwined GO nano-flakes. Whilst, the absorption peaks at 1637 and 1533 cm1 are contributed by the amide I and amide II in SF which indicate there are both the ɑ-helical and b-sheet conformations [24]. Besides, in the band of 800e1050 cm1, an absorption peak at about 950 cm1 is found in pSF/GO, it may be attributed by the enhanced vibrations of Alarich segments [25]. The SEM photographs of TAPP/rGO films and pSF/GO are given in Fig. 2C and D, respectively, which indicate the smooth channel surface of TAPP/rGO and the porous structure of the pSF/GO. Meanwhile, the similar porous topography is also observed on the sensing surface of five pSF/GO-TAPP/rGO-FETs, as presented in Fig. S1. The height analysis of TAPP/rGO nano-flake is executed by AFM (Fig. 2E), which is about 3 nm, and estimated to be about 3.2
layers, since this AFM examination is conducted under the tapping mode, in which the thickness of one single graphene layer is about 0.95 nm. XPS full spectra and the quantification of the main elements in TAPP/rGO (named as #1), pSF/GO-TAPP/rGO and apta-pSF/GOTAPP/rGO presented in Fig. 2F. The main elements are C, N, O and P, their variations are in consistence with the experimental procedures, that is: with the coating of pSF/GO, the contents of C are lowered greatly from 84% (TAPP/rGO) to 63%, O elements are increased from 12% to 30% and N elements are slightly enhanced from 2.0% to 4.3%. These variations agree with the oxygencontaining groups in GO and amino groups in SF. With the immobilization of AS1411, the contents of C are lowered to 57%, N and O atoms are increased to 6.8% and 32%, respectively, which are consistent with the main elements in nucleotide bases. In particular, the emerging of P element in apta-pSF/GO-TAPP/rGO, as shown by the blue column in Fig. 2F, can demonstrate aptamer molecules (AS1411) are fixed on the pSF/GO-TAPP/rGO channel film, because the nucleic acid is the only source of P element. More core spectra analyses are executed in Supplementary Materials (Fig. S2), to have more understanding about the surface chemical
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Fig. 3. Basic electronic features of TAPP/rGO-FET. (A) Transfer curves of TAPP/rGO-FET. (B) The carriers mobilities of TAPP/rGO-FET and rGO-FET at different bias voltages.
states in each of the surface modifications. 3.2. Electronic characteristics The ambipolar transfer feature of the TAPP/rGO-FET is examined and compared with the traditional rGO based GFET, as presented in Fig. 3A. It is found that the charge neutral points (CNPs) are increased, which is deduced to be caused by TAPP induced nitrogen doping effect. Meanwhile, with the increasing of VDS from 0.1 to 0.5 V, the right-shifting of transfer curves are coincident with our previously reported work [26]. Furthermore, the transfer features of the pSF/GO decorated and AS1411 functionalized TAOO/rGO-FET are re-examined at the constant VDS (0.5 V), it indicates that the representative ambipolar characteristic is not changed by the modifications. The left-moved CNPs are induced by the coated pSF/ GO layer and the anchored AS1411 molecules. The injected electrons from pSF/GO and the nucleic acid degrade the holes' contents
in TAPP/rGO channel. Besides, the optimized VGS is selected based on the mobility of carriers in TAPP/rGO-FET, which are calculated and presented in Fig. 3B according to the theoretical and experimental studies of FET [27e30]. It indicates the carriers' mobilities in TAPP/rGO channel film are obviously increased comparing with rGO, which agrees with the porphyrin facilitated HER studies [13e15]. Since a higher mobility can be obtained at VGS ¼ 0.2 V, it is selected for the following tests. 3.3. Effects of pSF/GO modification The microscope of the captured CTC A549 is presented in Fig. 4A, in which the four photos indicate more CTCs are captured by the pSF/GO decorated GFET. This result has also been verified from the electrical signal. The output currents' responding (DIDS/IDS,0) of blank TAPP/rGO-FET and pSF/GO decorated ones are measured after being incubated with AS1411 solutions in different concentrations
Fig. 4. Effect of pSF/GO decoration, observed by microscopy and electrical signals. (A) The microscopy of the captured CTC A549 on TAPP/rGO coated glass slides with and without pSF/GO decoration. (B) The AS1411 immobilization induced currents' changing ratios (DIDS/IDS,0), in which DIDS(¼IDS-IDS,0) is the deviation of the current IDS with IDS,0, which are the currents after incubation in the selected AS1411 concentration (AS1411_Conc.) and before anchoring AS1411. (C) CTCs' responding (DIDS/IDS,0) of pSF/GO decorated TAPP/rGOFET and naked ones. The error bars are the standard deviations (STD) of the individually measured data (n ¼ 20).
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(AS1411_Conc.), as shown in Fig. 4B. In which, DIDS ¼ IDS-IDS,0 IDS,0 and IDS are the currents before and after the incubation with the selected concentrations of AS1411 (AS1411_Conc.), respectively. It is found the main stream of the two curves is downward, which is in agreement with the electron injection mechanism between nucleic acid and graphene derivatives [23]. That is to say, the electrons injected into the conjugated carbon network from AS1411's phosphate backbone can alter the balanced channel carriers. Meanwhile, under the selected working condition (VGS ¼ 0.2 V, VDS ¼ 0.5 V), the positive CNP point in Fig. 3A means holes are the major carriers; with the increasing of AS1411_Conc., then injected electrons will reduce hole's density, so the negatively increased DIDS/IDS,0 are measured, as shown in Fig. 4B. According to the variations of the curves in Figs. 4B and 2 mM AS1411 is used in the following experiments, more detailed discussion is provided in the supplementary material, “Discussions for Fig. 4B”. Furthermore, the influence of pSF/GO to devices' responding (-DIDS/IDS,0) for A549 is also measured, as shown in Fig. 4C. In Fig. 4C, though both pSF/GO decorated and blank TAPP/ rGO-FETs are sensitive to A549, more higher responding (-DIDS/ IDS,0) is obtained by the pSF/GO decorated ones. Meanwhile, the slope of the red fitted line (2.38%/log10[cell_Conc.]) is also bigger than the blue one's (1.60%/log10[cell_Conc.]), it indicates higher sensitivity is possessed by pSF/GO-TAPP/rGO-FETs. The
reproducibility of the method of pSF/GO decoration to improve the sensitivity of TAPP/rGO-FET based CTC-sensing platforms is evaluated by 20 individual measurements. As shown by Fig. 4C, the standard deviation is by “pSF/GO” is relatively lower than “blank”. 3.4. CTC assay The sensitivity, repeatability and selectivity of the proposed CTC sensors are examined by 12 devices after they are incubated with different concentrations of CTCs (HeLa, A549 and MDA-MB-231) and the control cell (HUVEC) in buffer solutions, respectively. Three devices are allocated to each of the cells and named as #1, #2 and #3. Their normalized data for each device are presented in Fig. 5A (A-1 to A-4) with symbols of D, , and B, in which the averages (star) are also calculated. It is found, there are almost linear responding curves in Fig. A-1 to A-3. In contrary, the randomly distributed data points in Fig. A-4 indicate there is almost no specific responding for HUVEC. Moreover, the linear fitting analyses for the average of measured DIDS/IDS,0 (Fig. S3) indicate the sensitivities for A549, MDA-MB-231 and HeLa are about 3.14, 3.32, 2.60%/log10(cells/ml) for A549 (R2 ¼ 0.97), MDA-MB-231 2 2 (R ¼ 0.96) and HeLa (R ¼ 0.98), respectively. Meanwhile, the selectivity for CTCs is also confirmed by the low slope and R2 for HUVEC which are 0.13%/log10(cells/ml) and 0.50.
Fig. 5. Responding for the CTCs in PBS buffer, whole blood and the real applications for clinical samples. (A) The normalized responding of 12 AS1411 functionalized pSF/GOTAPP/rGO-FETs for A549, MDA-MB-231, HeLa and HUVEC in PBS. The error bars are the standard deviations (STD) of the individually measured data (n ¼ 3). (B) The negatively increased changing rates in dependence on the denary logarithm of cells concentrations in the whole blood. The error bars are RSD and n ¼ 3. (C) The measured currents' responding for the clinical samples from 5 healthy persons and 49 patients with different diagnostic conclusions which are grouped as stages of I, II, III and IV, as well as adenofibroma (AFM) and after being treated by AC regimen chemotherapy (after_AC) for 3 and 6 cycles.
S. Hu et al. / Electrochimica Acta 313 (2019) 415e422
Though the acceptable responses have been obtained for CTCs detection in PBS environment, in the real blood samples, there are large amount of fibrinogen, granulocyte and erythrocyte, which will make a negative interference on the performance of the CTC sensor. So an interference examination is conducted, the fictitious CTC samples are prepared by spiking cells into anti-coagulant blood. After being incubated with these samples, the device's responding is measured under the same condition as in the abovementioned experiments, the results are presented in Fig. 5B. In comparisons with the data in Fig. S3, bigger deviations are observed, we deduce they are caused by the interference of the components in blood. Nevertheless, the responses for cancer cells (A549, MDA-MB-231 and HeLa) are still noticeably higher than the normal cell (HUVEC), and grow with the increasing of cells' concentrations (Cell_Conc.). So, it is reasonable to believe that CTCs' content in the whole blood can be recognized by the varied currents. Finally, the real applications are conducted for the blood samples from 5 healthy persons and 49 patients with breast cancer and adenofibroma, their demographic and clinical characteristics are shown in Table 1. The correlations of the measured data by the proposed clinical available-CTC sensors and these samples' diagnostic conclusions are presented in Fig. 5C. It is found that the responding of IDS is growing up with the developing of the cancer stages from I to IV. In comparisons with the measured results in Fig. 5A and B, these data points indicate more CTCs exist in the blood samples in higher stages. Besides, the blood samples of adenofibroma patients and breast cancer patients after being treated by AC regimen (the liposomes of doropepirubicin were 50 mg and cyclophosphamide 800 mg) chemotherapy for 3 and 6 cycles, can also be identified by the proposed CTC sensors.
4. Discussion In conclusion, the proposed pSF/GO-TAPP/rGO-FET based CTC sensor based CTC assay is an ideal exploitation and integration of chemically synthesized graphene derivatives with aptamer sensing strategy. Comprehensive studies are provided in this work, including material and device preparation, characterizations, device evaluations and clinical assay. The typical ambipolar electronic feature and TAPP enhanced mobility are demonstrated to be possessed by the TAPP/rGO-FET, after being decorated by pSF/GO and functionalized by AS1411. Based on this novel TAPP/rGO-FET, CTC assay is fulfilled in two steps. The real clinical samples' assays indicate the pSF/GO-TAPP/rGO-FET based CTC sensor is a promising candidate for clinical assay.
Table 1 Demographic and clinical characteristics of patients with breast cancer and adenofibroma n (%). Variabes
Cancer (n ¼ 47)
Adenofibroma (n ¼ 2)
Age (yr) Median (min, max)
53 (22, 74)
Min ¼ 32, Max ¼ 58
32 (68.09%) 15 (31.91%)
2(100%) 0 (0)
59 >59 Clinical stage I II III IV Unidentified Metastasis Yes No
14 (29.79%) 25 (53.19%) 5 (10.64%) 2 (4.26%) 1 (2.13%) 3 (6.38%) 44 (93.62%)
421
Acknowledgments This work is supported by the National Natural Science Foundation of China (Grant no. 61771260, 81601820, 81871719). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.electacta.2019.05.039. References [1] Y. Ming, Y. Li, H. Xing, M. Luo, Z. Li, J. Chen, J. Mo, S. Shi, Circ Circulating tumor cells: from theory to nanotechnology-based detection, Front. Pharmacol. 8 (2017) 35. [2] J. Liu, Y. Zhang, Q. Zhao, B. Situ, J. Zhao, S. Luo, B. Li, X. Yan, P. Vadgama, L. Su, W. Ma, W. Wang, L. Zheng, Bifunctional aptamer-mediated catalytic hairpin assembly for the sensitive and homogenous detection of rare cancer cells, Anal. Chim. Acta 1029 (2018) 58. [3] F. Zhao, Electrochemical techniques for the detection of cancer cells and cellsurface glycan expression: a mini review, Int. J. Electrochem. Sci. 12 (8) (2017) 7580e7596. [4] Y.J. Jan, J. Chen, Y. Zhu, Y.T. Lu, S.H. Chen, H. Chung, M. Smalley, Y.W. Huang, J. Dong, L.C. Chen, H.H.Y., J.S. Tomlinson, S. Hou, V.G. Agopian, E.M. Posadas, H.R. Tseng, Clinical applications of NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells, Theranostics 6 (9) (2016) 1425. [5] A. Kowalik, M. Kowalewska, S. Go zd z, Current approaches for avoiding the limitations of circulating tumor cells detection methods-implications for diagnosis and treatment of patients with solid tumors, Transl. Res. 185 (2017) 58e84. [6] N. Shao, E. Wickstrom, B. Panchapakesan, Nanotube-antibody biosensor arrays for the detection of circulating breast cancer cells, Nanotechnology 19 (46) (2008) 465101. [7] D. Xiang, S. Shigdar, G. Qiao, T. Wang, A.Z. Kouzani, S.F. Zhou, L. Kong, Y. Li, C. Pu, W. Duan, Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine, Theranostics 5 (1) (2015) 23e42. [8] X. Zhao, R. Fang, Y. Zhang, Development of electrochemical aptasensor for label-free glioma cell detection, Int. J. Electrochem. Sci. 12 (6) (2017) 9301e9310. [9] L. Feng, Y. Chen, J. Ren, X. Qu, A graphene functionalized electrochemical aptasensor for selective label-free detection of cancer cells, Biomaterials 32 (11) (2011) 2930e2937. [10] K. Urmann, S. Arshavskygraham, J.G. Walter, T. Scheper, E. Segal, Whole-cell detection of live lactobacillus acidophilus on aptamer-decorated porous silicon biosensors, Analyst 141 (18) (2016) 5432e5440. [11] S.D. Wang, Q. Ma, K. Wang, P.B. Ma, Strong and biocompatible threedimensional porous silk fibroin/graphene oxide scaffold prepared by phase separation, Int. J. Biol. Macromol. 111 (2018) 237e246. [12] M.S. Mansouri, A. Salimi, Ultrasensitive flexible FET-type aptasensor for CA 125 cancer marker detection based on carboxylated multiwalled carbon nanotubes immobilized onto reduced graphene oxide film, Anal. Chim. Acta 1000 (2018) 273. [13] S. Seo, K. Lee, M. Min, Y. Cho, M. Kim, H. Lee, A molecular approach to an electrocatalytic hydrogen evolution reaction on single-layer graphene, Nanoscale 9 (11) (2017) 3969e3979. [14] T. Kawata, T. Ono, Y. Kanai, Y. Ohno, K. Maehashi, K. Inoue, K. Matsumoto, Improved sensitivity of a graphene FET biosensor using porphyrin linkers, Jpn. J. Appl. Phys. 57 (6) (2018), 065103. [15] J. Geng, H.T. Jung, Porphyrin functionalized graphene sheets in aqueous suspensions: from the preparation of graphene sheets to highly conductive graphene films, J. Phys. Chem. C 114 (18) (2010) 8227e8234. [16] C. Zhang, H. Shao, J. Luo, X. Hu, Y. Zhang, Structure and interaction of silk fibroin and graphene oxide in concentrated solution under shear, Int. J. Biol. Macromol. 107 (2018) 2590e2597. [17] N. Hao, R. Hua, S. Chen, Y. Zhang, Z. Zhou, J. Qian, Q. Liu, K. Wang, Multiple signal-amplification via Ag and TiO2 decorated 3D nitrogen doped graphene hydrogel for fabricating sensitive label-free photoelectrochemical thrombin aptasensor, Biosens. Bioelectron. 101 (2017) 14e20. [18] R. Zhang, Y. Gu, Z. Wang, Y. Li, Q. Fan, Y. Jia, Aptamer cell sensor based on porous graphene oxide decorated ion-selective-electrode: double sensing platform for cell and ion, Biosens. Bioelectron. 117 (2018) 303e311. [19] J. Mosafer, A. Mokhtarzadehc, Cell surface nucleolin as a promising receptor for effective AS1411 aptamer-mediated targeted drug delivery into cancer cells, Curr. Drug Deliv. 15 (2018) 1e7. [20] H. Mousavi, M. Grabowski, Nonlinear electron transport across short DNA segment between graphene leads, Solid State Commun. 279 (2018) 30e33. [21] S. Kundu, A. Pyne, R. Dutta, N. Sarkar, Unveiling the interaction of duplex DNA with graphene oxide in the presence of two diverse binders: a detailed photophysical study, J. Phys. Chem. C 122 (12) (2018). [22] J.H. Kang, T. Kim, J. Choi, J. Park, Y.S. Kim, M.S. Chang, H. Jung, K.T. Park,
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