17.Nanoarchitectured electrochemical cytosensors for selective

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Nanoarchitectured Electrochemical Cytosensors for Selective Detection of Leukemia Cells and Quantitative Evaluation of Death Receptor Expression on Cell Surfaces Tingting Zheng, Jia-Ju Fu, Lihui Hu, Fan Qiu, Minjin Hu, Jun-Jie Zhu, Zi-Chun Hua, and Hui Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400994p • Publication Date (Web): 26 Apr 2013 Downloaded from http://pubs.acs.org on May 6, 2013

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Analytical Chemistry

Nanoarchitectured Electrochemical Cytosensors for Selective Detection of Leukemia Cells and Quantitative Evaluation of Death Receptor Expression on Cell Surfaces

Tingting Zheng†,‡, Jia-Ju Fu†, Lihui Hu†, Fan Qiu§, Minjin Hu¶, Jun-Jie Zhu†,*, Zi-Chun Hua§,*, and Hui Wang‡,*

†

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

Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China ‡

Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street,

Columbia, South Carolina 29208, United States §

State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu

210093, China ¶

Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma

Laboratories Inc., Changzhou, Jiangsu 213164, China * Corresponding Authors. Emails: [email protected] (J.-J. Z.); [email protected] (Z.-C.H.); [email protected] (H.W.)

ACS Paragon Plus Environment

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ABSTRACT:

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The variable susceptibility to the tumor necrosis factor-related apoptosis-

inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nano-biotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ~ 40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor.

The high sensitivity and selectivity of this electrochemical

cytosensing approach also allow us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. By using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.

Key Words: nanoprobe, nano-biointerface, electrochemical cytosensing, leukemia, TRAIL, death receptor


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Leukemia is one of the most common fatal cancers that affect the bone marrow, the blood cells, and other parts of the lymphatic system.1 Innate and acquired resistance to chemotherapy and radiation therapy has been a major obstacle for leukemia treatment. One potential adjunct to the conventional treatments is direct induction of cell death by activation of death receptormediated apoptosis.2 Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a recently identified member of the growing TNF super-family, which specifically binds to its cognate death receptors, DR4 and DR5, with strong binding affinities.3 Binding of TRAIL to DR4/DR5 on the leukemia cell surfaces stimulates rapid apoptosis in a variety of leukemia cell lines independent of p53 status.4-9 Such TRAIL-stimulated apoptosis, however, does not occur in normal cell lines.

Recently finished Phase III clinic trails of recombinant human TRAIL

(rhTRAIL) in China have shown promising therapeutic outcomes against cancers without obvious toxicity. However, certain kinds of tumor cells are insensitive to TRAIL, which causes failure in TRAIL treatment. It is essentially the difference in the expression levels of DR4/DR5 on cell surfaces that gives rise to the variable susceptibility to TRAIL shown by various types of leukemia cells.10-14 Therefore, the quantitative characterization of DR4/DR5 expression on the cell surface has become an extremely important subject directly related to the development of diagnostic tools to guide DR4/DR5-based personalized leukemia treatment. Currently, the most commonly used methods for DR4/DR5 detection are the western blot assay15 and flow cytometry,16 which typically provide only the relative abundance or distribution, not the exact number or expression kinetics of DR4/DR5 on leukemia cell surfaces. In comparison to these methods, electrochemical approaches exhibit attractive advantages in terms of high sensitivity, inherent simplicity, and low cost, and can be readily implemented into quantitative bioassays for clinical applications.17-19 Utilization of multifunctional nanoprobes in


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electrochemical cytosensing processes makes it possible to obviate the need of cell lysis, cell labeling, or complicated instrumentation that other existing methods typically require.20, 21 Here we report a new nano-biotechnology-based electrochemical cytosensing platform for both selective detection of various types of leukemia cells and quantitative characterization of death receptor expression status on leukemia cell surfaces. The electrochemical cytosensor we have developed has two major components, multifunctional hybrid nanoprobes and nanoarchitectured electrode interfaces. The electrochemical nanoprobes used for the cytosensing were fabricated through co-immobilization of both rhTRAIL and horseradish peroxidase (HRP) on Au nanoparticle-decorated magnetic Fe3O4 beads. The as-fabricated nanoprobe integrated both the specific recognition of DR4/DR5 on leukemia cell surfaces by rhTRAIL and amplification of electrochemical signals based on the HRP-catalyzed oxidation of thionine by H2O2. Design and construction of biocompatible electrode interfaces that specifically target the analytes and significantly facilitate the electron transfers between the analytes and electrodes are also a key issue in the field of electrochemical biosensing. Using a layer-by-layer (LBL) assembly method, we assembled a hierarchically nanoarchitectured electrode interface for cytosensing which integrated the high biocompatibility of dentrimer-stabilized Au nanoparticles (Au DSNPs), the excellent conductivity of nitrogen-doped carbon nanotubes (CNx), and the high specificity of cell-targeting aptamers/antibodies. The leukemia cells being analyzed are sandwiched between the electrochemical nanoprobes and electrode interface. This sandwich-like geometry allows for both cell detection and DR4/DR5 quantification simply based on the amperometric electrochemical readout from the cytosensors.

Experimental Section Reagents and Materials. Fourth generation (G = 4) poly(amidoamine) dendrimer with


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surface

terminated

amino

groups

(PAMAM,

10

wt

%

in

methanol),

poly(diallyldimethylammonium chloride) (PDDA, 20%, w/w in water, MW=200,000-350,000), bovine serum albumin (BSA), and melatonin were purchased from Sigma-Aldrich. Chloroauric acid (HAuCl4 ·4H2O), NaBH4, trisodium citrate, and glutaraldehyde (GLU) were obtained from Shanghai Chemical Reagent Co. (Shanghai, China).

rhTRAIL was provided by Jiangsu

TargetPharma Laboratories Inc. (Changzhou, China). Horseradish peroxidase (HRP), TRAILFITC and DR4 were purchased from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing, China).

Amino-group functionalized KH1C12 aptamer was synthesized and purified by

Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China).

Mouse anti-human anti-CD3

monoclonal antibody (mAb) was obtained from Invitrogen Corp. Thionine chloride (TH+) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The PDDAfunctionalized nitrogen-doped carbon nanotubes (PDCNx) were prepared following a previously reported protocol.22 Phosphate buffer saline (PBS, pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 87 mM Na2HPO4, and 14 mM KH2PO4. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). Apparatus. UV-vis extinction spectra were recorded on a UV-3600 spectrophotometer (Shimadzu, Kyoto, Japan). Scanning electron micrographs (SEM) were obtained using a Hitachi S4800 scanning electron microscope. Transmission electron micrographs (TEM) were obtained on a JEOLJEM 200CX transmission electron microscope using an accelerating voltage of 200 kV. Confocal laser scanning microscopy (CLSM) studies were performed using a Leica TCS SP5 microscope (Germany) with excitation at 405 nm. The static water contact angles were measured at 25 ℃ by a contact anglemeter (Rame-Hart-100) using drops of pure deionized water. The readings were stabilized and taken within 120 s after the addition. Electrochemical measurements were performed on a CHI 660C workstation (Shanghai Chenhua Apparatus Corporation, China)


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with a conventional three-electrode system composed of a platinum wire as the auxiliary, a saturated calomel electrode (SCE) as the reference, and a glassy carbon electrode (GCE) as the working electrode. Fabrication of the Electrochemical Nanoprobes. Fe3O4 beads terminated by carboxyl groups were prepared following a previously reported protocol with minor modification.23 Briefly, 1.35 g iron trichloride hexahydrate, 3.2 g sodium acetate anhydrous, and 0.5 mL polyacrylic acid were added to 38 mL of ethylene glycol with the help of ultrasound at room temperature. Then the mixture was maintained at 473 K for 6 hours in poly-tetrafluoroethylene reaction kettle. The black products were separated by magnetic field and washed with deionized water and ethanol alternately several times, and dried at 343 K in a vacuum drying oven. The HRP-TRAIL-Fe3O4@Au nanoprobes were prepared via a layer-by-layer assembly approach.24-26 A colloidal suspension of Fe3O4 (10.0 mg in 1 mL water) was added to 1 mL of PDDA solution (1%) and after sonication for 30 min, the suspension was collected by sedimentation with the help of an external magnetic field and washed three times with pure water. Au NPs with an average diameter of 13 nm were prepared according to the reported method via deoxidizing HAuCl4 aqueous solution with sodium citric acid.27 The as-prepared Au NPs was added to the Fe3O4/PDDA nanocomposites under mechanical stirring. Au NPs were attached to the surface of the Fe3O4/PDDA nanocomposites through electrostatic interactions. The wine-red colloidal suspension of Au NPs turned colorless after all the Au NPs were attached to the surface of Fe3O4/PDDA particles. Then the product was removed from the solution by applying an external magnetic field. This process was repeated multiple times until the color of added Au NPs no longer changed. The final product was separated using an external magnetic field and then redispersed in pure water.


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After the pH value of the Fe3O4@Au suspension was adjusted to 9.0 by 0.1 M NaOH, 50 µL of HRP (1 mg mL-1) and 50 µL of 20 µg mL-1 rhTRAIL (Jiangsu TargetPharma Laboratories Inc., Changzhou, Jiangsu, China) were added to the dispersion, and the mixture was stirred mechanically overnight at 4℃. The reaction mixture was washed with phosphate buffered saline (PBS) and the supernatant was discarded. To obtain the nanoprobes with optimized electrochemical responses, different ratios of HRP to TRAIL were used for preparation of the nanoprobes. Assembly of Electrode Interface for Cytosensing.

Au DSNPs were prepared by the

chemical reduction of a HAuCl4-dendrimer (generation=4.0) mixture with NaBH4 in an aqueous solution following a previously reported procedure.28 Briefly, gold-dendrimer complexes were prepared in aqueous solution by mixing 5mL of 4.86 mM aqueous solution of HAuCl4 with 5 mL aqueous solution of the PAMAM dendrimer. The yellow HAuCl4 solution lost its color immediately upon mixing the PAMAM, indicating the formation of complexes between the dendrimer terminal amines and the gold anions. Stable Au DSNPs were prepared by reducing the PAMAM-tetrachloroaurate complexes at room temperature with NaBH4 under vigorous magnetic stirring for another 30 min. Upon addition of NaBH4 to the PAMAM-tetrachloroaurate complexes, a color change from slightly yellow to deep red, indicating the formation of metallic Au NPs. A glassy carbon electrode (GCE, 3 mm diameter) was polished to a mirror using 0.3 and 0.05 µm alumina slurry (Buehler) followed by thorough rinse with deionized water. After successive sonication in 1:1 nitric acid, acetone, and deionized water, the electrode was rinsed with deionized water and allowed to dry at room temperature. 5 µL of 5.0 mg mL-1 PDCNx were first dropped on the pretreated GCE, dried in a silica gel desiccator, and then immersed into a Au DSNPs solution for 1h to obtain Au DSNPs/PDCNx/GCE. Conjugation of KH1C12 onto the


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electrode was achieved by using the classic glutaraldehyde coupling reaction between amine groups on the film surface and the amine-functionalized KH1C12 aptamer. The Au DSNPs/PDCNx/GCE was immersed in 2.5% glutaraldehyde solution for 1h at room temperature. Then it was rinsed with PBS (pH 7.4) carefully and dried in air. Following this step, 5 µL of amine-functionalized KH1C12 (5 µM), or 5 µL of anti-CD3 mAb (5 µM), was dropped onto the electrode surface and was incubated at 4℃ overnight. After incubation, the electrode was rinsed with PBS (pH 7.4) carefully, immersed in 1% BSA (w/v) for 1h at room temperature to block the nonspecific binding sites, and then washed with PBS (pH 7.4) thoroughly for subsequent cell capture. Cell Culture and Capture. HL-60 and Jurkat cells were cultured in a flask in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10 % fetal calf serum (FCS, Sigma), penicillin (100ug mL-1) and streptomycin (100 µg mL-1) in an incubator (5% CO2, 37℃). At the logarithmic growth phase, the cells were collected and separated from the medium by centrifugation at 1000 rpm for 2 min and then resuspended in the binding buffer (4.5 g L-1 glucose, 5 mM MgCl2, 0.1 mg mL-1 tRNA and 1mg mL-1 BSA, all dissolved in Dulbecco’s PBS with CaCl2 and MgCl2) to obtain a homogeneous cell suspension. The binding buffer was used to ensure the effective binding affinity between HL-60 cells and the KH1C12 aptamer. The cell number was determined using a Petroff-Hausser cell counter (U.S.A.). 60 µL of cell suspension at a certain concentration was dropped onto the KH1C12/Au DSNPs/PDCNx-modified GCE surface and incubated at 37 ℃ for 50 min. The cells were captured via the specific binding between the KH1C12 aptamer and HL-60 cells, or between anti-CD3 mAb and Jurkat cells. Then the electrodes were taken out and rinsed with incubation


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buffer to remove the noncaptured cells. The obtained cell-captured electrodes were used for subsequently immunoassay. Enzyme-Amplified Electrochemical Immunoassay. The cell-captured electrodes were incubated with 10 µL of the HRP-TRAIL-Fe3O4@Au nanoprobes at 37℃ for 60 min, and then washed thoroughly with pH 7.4 PBS to remove nonspecifically bound conjugates to minimize the background response. The Cyclic Voltammetry detection of DR4/DR5 was carried out from 50 mV to -550 mV (vs. SCE) with the pulse amplification of 50 mV in 0.01 M pH 7.4 PBS containing 1 mM H2O2 and 25 µM thionine under N2 atmosphere. In order to verify the signal amplification of the HRP-TRAIL-Fe3O4@Au nanoprobes, HRP-TRAIL-Au NPs (10 µL) was also employed to replace the nanoprobes to perform the same electrochemical analysis. Monitoring Dynamic DR4/DR5 Expression in Response to Melatonin. The KH1C12/Au DSNPs/PDCNx/GCE was incubated with melatonin-untreated and treated HL 60 cells (1.0×105 cells mL-1) for different times. After carefully rinsing with PBS (0.01 M, pH 7.4), the obtained electrodes were subjected to the nanoprobe-based electrochemical analysis. Flow Cytometric Analysis. HL-60 cells and HL-60 cells treated with melatonin for 24 h were collected by centrifugation, washed with sterile cold PBS, and resuspended in 500 µL binding buffer. Then the cells were stained with TRAIL-fluorescein isothiocyanate (FITC) according to the manufacturer’s instructions. The sample was immediately analyzed on the FACS Sort flow cytometer (Becton Dickinson, U.S.A.) and CLSM with excitation at 488nm. Unlabeled HL 60 cells were used as the negative control for estimation of autofluorescence.

Results and Discussions Fabrication

of

HRP-TRAIL-Fe3O4@Au

Nanoprobes.

The

fabrication

of

the

electrochemical nanoprobes involves the co-immobilization of HRP and rhTRAIL on the


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surfaces of Fe3O4@Au nanocomposite particles. The HRP-TRAIL-Fe3O4@Au hybrid nanoprobes can be prepared using a layer-by-layer (LBL) assembly approach as schematically illustrated in Figure 1A. The as fabricated Fe3O4 beads had an average size of ~ 500 nm and were uniform in size and morphology as shown in Figure S1A in supporting information. The surface of the Fe3O4 spheres was terminated by carboxyl groups and thus was negatively charged at neutral pH with a ξ-potential of -37.3 mV. The subsequent absorption of positively charged PDDA on the colloidal Fe3O4 beads switched the ξ-potential to positive values (+34.60 mV). Then the as-fabricated Au NPs had negatively charged surfaces and could be electrostatically tethered on the surface of the Fe3O4/PDDA particles (see Figure S1B in supporting information). Multiple copies of HRP and rhTRAIL were subsequently immobilized onto the Fe3O4@Au nanocomposites through Au-thiol intercalations29 due to the presence of cysteine residues in HRP and rhTRAIL. The paramagnetic properties of the Fe3O4 cores allowed us to conveniently separate the hybrid particles from reaction mixtures by applying external magnetic fields in multiple steps during the nanoprobe fabrication procedure. This nanoprobe integrates both the specific recognition of DR4/DR5 on cell surfaces by rhTRAIL and electrochemical signal amplification based on the HRP-catalyzed oxidation of thionine by H2O2. Assembly of the Electrode Interface for Cell Recognition.

The construction of the

electrode interfaces for selective cell capture is schematically illustrated in Figure 1B. We first adsorbed a layer of PDCNx onto the surface of a glassy carbon electrode (GCE). The surface modification with PDCNx dramatically increased the surface area and electrical conductivity of the electrode.30, 31 Then negatively charged Au DSNPs were assembled on the surface of the PDCNx. The as-fabricated Au DSNPs, which were ~ 10 nm in diameter, displayed two extinction peaks at 283 nm and 513 nm in the UV-vis spectrum (Figure S2 in supporting information), corresponding to the absorption of the PAMAM dendrimer and the plasmon


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resonance of Au NPs, respectively. Au DSNPs are a novel class of biocompatible synthetic nanocomposites with well-defined composition, architectures, and surface properties32 and may serve as electron relay units in electrochemical biosensors.33, 34 Hierarchical assembly of both Au DSNPs and PDCNx on electrode surfaces can readily result in a new interfacial scaffold that greatly facilitates the electron transfers during the electrochemical cytosensing process. Finally, cell-targeting aptamers or antibodies were conjugated to the Au DSNPs to capture specific types of leukemia cells. For example, KH1C12 aptamer35 was conjugated to the Au DSNPs by glutaraldehyde cross-linking to specifically target HL-60 cells, a representative type of acute myeloid leukemia cells. Because of the specific recognition of cells by aptamers/antibodies and the excellent interfacial properties of Au DSNPs/PDCNx films, these electrode interfaces not only offer a conductive and biocompatible surface but also act as an analogue of extracellular matrix to induce cell adhesion effectively.


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Figure 1. Schemes of (A) fabrication of HRP-TRAIL-Fe3O4@Au hybrid nanoprobe, (B) assembly of the electrode interface, and (C) sandwich-like nanoarchitectured electrode geometry for the cytosensing of HL-60 cells.

The hydrophilicity of the electrode interface, which is directly related to its biocompatibility, was evaluated by surface contact angle measurements (Figure S3 in support information). The hydrophilicity of the electrode surface progressively increased during the multi-step assembly process, indicating that the biocompatibility of the electrode interface was significantly improved after modification of the electrode surface with the KH1C12/Au DSNPs/PDCNx composite nanoarchitectures. More than 95 % of the HL-60 cells captured on the electrode interface stayed alive in RPMI 1640 medium for over 36 h. Specificity of the Electrochemical Cytosensor. The leukemia cells of interest were sandwiched between the electrode interface and the nanoprobes (see Figure 1C). After specific types of leukemia cells were captured onto the electrode interfaces, the HRP-TRAIL-Fe3O4@Au hybrid nanoprobes were further anchored to the surfaces of the captured cells through specific interactions between rhTRAIL and DR4/DR5. The targeted cells, HL-60 cells, were selectively captured at the KH1C12/Au DSNPs/PDCNx-modified electrode interface (Figure 2A), while the nontarget cells, K562 cells, did not stick to the electrode surface (Figure 2B). A large number of HRP-TRAIL-Fe3O4@Au hybrid nanoprobes could be successfully anchored to the surface of each captured cell after stepwise incubation of the modified electrode with cell suspension and nanoprobes (Figure 2C) whereas the Fe3O4@Au nanocomposite particles did not attach to the cell surfaces (Figure 2D).


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A

B

C

D

Figure 2. Bright-field optical microscopy images of KH1C12/Au DSNPs/PDCNx-modified electrode interface interacting with (A) targeting cells (HL-60), (B) nontargeting cells (K562), and of HL-60 cells incubated with (C) HRP-TRAIL-Fe3O4@Au hybrid nanoprobes and (D) Fe3O4@Au nanocomposites. All the images share the same scale bar in panel A.

Signal Amplified Immunoassay Using HRP-TRAIL-Fe3O4@Au Nanoprobes. The electrochemical signals were generated through the HRP-catalyzed oxidation of thionine by H2O2. As shown in Figure 3A, upon the introduction of 25 µM thionine and 1 mM H2O2 to the phosphate buffered saline (PBS buffer, pH 7.4), the cyclic voltammogram (CV) of the HL60/KH1C12/Au DSNPs/PDCNx/GCE exhibited a pair of well-defined redox peaks at -0.231V and -0.287V at a scan rate of 50 mV/s, which corresponded to the electrochemical oxidation and reduction of thionine, respectively.36 After incubating the captured cells with HRP-TRAILfunctionalized Au nanoparticles for 1h, the reduction peak current significantly increased and the


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reduction peak shifted slightly toward a more negative potential while the oxidation peak currents decreased, indicating a typical enzymatic electrocatalytic process. This result indicated that the HRP immobilized on the nanoprobe surface could retain its high enzymatic catalytic activity. Interestingly, we observed a much more significant amplification of the reduction peak current and suppression of the oxidation current (Figure 3B) when replacing HRP-TRAILfunctionalized Au nanoparticles with the HRP-TRAIL-Fe3O4@Au hybrid nanoprobes.

It is

apparent that the multi-enzyme labeling strategy enhanced detection responses and the achieved amplification of electrochemical signals was ascribed to a large amount of enzymes loaded on each HRP-TRAIL-Fe3O4@Au hybrid nanoprobe.

A

B


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Figure 3. Cyclic voltammograms obtained on HL-60/KH1C12/Au DSNPs/PDCNx/GCE in the presence of 25 µM thionine and 1 mM H2O2 in PBS before (dash blue curves) and after (solid red curves) incubation with (A) HRP-TRAIL-Au NPs and (B) HRP-TRAIL-Fe3O4@Au hybrid nanoprobes for 1h. The insets show the cartoon illustrations of the cytosensor geometries.

Optimization of Detection Conditions. The ratio of rhTRAIL/HRP co-immobilized on each nanocarrier is an important factor that determines the overall electrochemical signals. Increasing the total amount of HRP per nanocarrier would enhance the signal amplification, while reducing the amount of rhTRAIL on the nanocarriers would decrease the immuno-coupling efficiency of the nanoprobes to the captured cells. As shown in Figure S4A in supporting information, the maximum electrochemical response was achieved at the TRAIL/HRP ratio of 1/50. The cell and nanoprobe incubation times are another important experimental parameter. As the cell incubation time (106 cells mL-1 of HL-60 cells) progressively increased, the catalytic current was observed to first increase and then decrease after 50 min (see Figure S4B), indicating an optimal incubation time of 50 min for the capture of HL-60 cells. When incubating the captured cells with HRPTRAIL-Fe3O4@Au hybrid nanoprobes, the maximum catalytic current was obtained after incubation for 60 min (see Figure S4C), which corresponded to saturation of the rhTRAIL binding to DR4/DR5. Longer nanoprobe incubation time resulted in decrease of the catalytic currents likely due to the cell apoptosis induced by the rhTRAIL immobilized on the nanoprobes. Detection of HL-60 Cells. Under the optimal sensing conditions, the catalytic current was found to be proportional to the logarithmic value of the cell concentration ranging from 1.0×103 to 1.0×106 cells mL-1 with a correlation coefficient R of 0.995 (n=7) (see Figure 4A). The detection limit for cell concentration was calculated to be 660 cells mL-1 at 3σ, which was comparable to that of 750 cells mL-1 obtained on a quartz crystal microbalance biosensor for detection of Escherichia Coli37 and much lower than that of 5.0×103 HL-60 cells mL-1 detected


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by a c-SWNTs-AuNPs-gelatin modified GCE with impedance measurements.38 Considering the fact that 60 µL of HL-60 cell suspension was used for incubation, the presented strategy achieved a detection limit of only 40 HL-60 cells. The low detection limit was attributed to the signal amplification enabled by the utilization of both the multifunctional hybrid nanoporbes and the nanoarchitectured electrode interface. Evaluation of DR4/DR5 on HL-60 Cell Surface. After incubating the KH1C12/Au DSNPs/PDCNx-modified electrode interface with a certain concentration of HL-60 cell suspension, the number of cells captured on the cytosensor, N, could be calculated by subtracting the number of free cells that remained in the solution after washing. The catalytic current was proportional to the number of captured HL-60 cells in the range from 800 to 8700 cells (R=0.993, n=7) (Figure 4B). The linear regression equation is ip (µA) = 2.54 + 2.37×10-4 N

(1).

The overall catalytic currents were determined by both the number of captured HL-60 cells and the number of DR4/DR5 on each cell. To quantify the DR4/DR5 expressed on cell surfaces, we used a binding competition method39 to partially block the DR4/DR5-TRAIL binding sites on the nanoprobes with DR4. Because the DR4-blocked TRAIL on the nanoprobe surfaces could not conjugate to the DR4/DR5 on the cell surfaces, the peak currents obtained on cytosensors incubated with the DR4-treated nanoprobes were lower than those of the cytosensors incubated with the non-blocked nanoprobes. The plot of decrease in catalytic current (∆ip) versus the amount of DR4 used to block the TRAIL on nanoprobe surfaces (m) showed a linear relationship in the range of 1.5-7.5 fmol (R=0.999, n=5) (Figure 4C): ∆ip (µA) = -0.34m (fmol) -0.03

(2).


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The average number of DR4/DR5 per captured HL-60 cell was calculated to be 8.64×105 using equations (1) and (2).

Figure 4. (A) Cyclic voltammograms of HRP-TRAIL-Fe3O4@Au/HL-60/KH1C12/Au DSNPs/PDCNx/GCE obtained with HL-60 cell concentrations of (a) 1×103, (b) 1×104, (c) 1×105, and (d) 1×106 cells mL-1. Inset: plot of catalytic peak current (ip) vs. logarithm of HL-60 cell concentration. (B) Plot of ip vs. number of captured HL-60 cells (N) on the cytosensor. (C) Effect of the DR4 concentration (m) on the decrease of catalytic peak current (∆ip).


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Monitoring Dynamic DR4/DR5 Expression on Cells in Response to Melatonin. The high sensitivity and selectivity of this cytosensing strategy allowed us to further evaluate the dynamic alteration of DR4/DR5 surface expression on living cells in response to drugs, using melatonin as a model drug system. Melatonin, which has recently been reported to induce apoptosis in some haematological cancer cell lines, can enhance the expression of DR4/DR5 on the cell surface.40 The catalytic current obtained from melatonin-treated HL-60 cells progressively increased as the melatonin incubation time increased and reached a plateau after 24h (Figure 5A). This result indicated that the melatonin treatment significantly stimulated expression of DR4/DR5 on the HL-60 cell surfaces within 24 h. To validate the observed change, the melatonin-treated HL-60 cells were stained with fluorescein isothiocyanate (FITC)conjugated rhTRAIL and assayed using flow cytometry (inset in Figure 5A), which also showed an increased binding of FITC-TRAIL to the melatonin-treated HL-60 cells than the untreated HL60 cells. Figure5B displays the melatonin concentration-dependence of DR4/DR5 expression. The standard calibration curve for melatonin detection is shown in inset of Figure 5B. The catalytic currents increased proportionally with the melatonin concentrations between 0.15 mM and 1.0 mM.


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A

B

Figure 5. (A) Catalytic peak current obtained from HL-60 cells treated with 1.0 mM melatonin over different incubation time. Inset: flow cytometric analysis of TRAIL-binding sites on HL-60 cells after being treated with melatonin for 24 h (blue), untreated HL-60 cells (red), and autofluorescence of unlabeled HL-60 cells (dark). (B) Cyclic voltammetry measurements after the HL-60 cells were incubated with 0, 0.15, 0.32, 0.49, 0.66, 0.83, and 1.0 mM melatonin for 24h (from a to g). Inset: Relationship between the catalytic peak current and melatonin concentration.

Evaluation of DR4/DR5 on Jurkat cell surface. To further confirm the role of DR4/DR5 motif in TRAIL-induced cell apoptosis signaling, we did parallel experiments on Jurkat cells, another acute leukemia cell line. We conjugated a monoclonal antibody, cluster of differentiation


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3 (CD 3),41, 42 to the Au DSNPs to construct an electrode interface for specific recognition of Jurkat cells through the high-affinity interactions between antigens and antibodies (Figure 6A). After successive incubation of Jurkat cells and HRP-TRAIL-Fe3O4@Au nanoprobes, the catalytic current obtained on the cytosensors showed a linear relation to the number of captured Jurkat cells in the range from 800 to 8900 cells (R=0.999, n=7) (Figure 6B). The linear regression equation is ip (µA) =2.63 + 9.20×10-4 N

(3).

After partially blocking the TRAIL sites on the nanoprobes with DR4, the decrease in catalytic current (∆ip) versus the amount of DR4 (m) showed a linear relation in the range of 1.5-9.0 fmol (R=0.998, n=6) (Figure 4C ): ∆ip (µA) = -0.35m (fmol) +0.03

(4).

Figure 6. (A) Scheme of electrochemical cytosensor for Jurkat cells. (B) Plot of catalytic peak current vs. number of captured Jurkat cells on the cytosensor. (C) Effect of the amount of DR4 used to block HRP-TRAIL-Fe3O4@Au nanoprobes on the decrease in peak current.


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The average number of DR4/DR5 sites on each captured Jurkat cell was calculated to be ~2.04×106, which was more than 2-fold of that on each HL 60 cell. This result suggests that the Jurkat cells may be more sensitive to TRAIL treatment than the HL 60 cells. To further examine whether the number of DR4/DR5 sites is associated with the TRAIL-susceptibility, the cells were treated with serial dilutions of rhTRAIL stained with Annexin V-FITC and Propidium Iodide (PI) followed by flow cytometry measurements. The EC50 vales (concentration at which 50% cell appeared apoptotic) for TRAIL on the HL 60 and Jurkat cells were 4.886×105 and 6.7 ng/mL respectively. Jurkat cells turned out to be significantly more susceptible to TRAIL treatment than HL-60 cells essentially because of their higher surface expression level of DR4/DR5.

Conclusions We have developed a new electrochemical cytosensing platform based on which selective detection of various types of leukemia cells and quantitative characterization of DR4/DR5 expression status on cell surfaces can be both achieved. This nano-biotechnology-based cytosensing strategy provides a useful tool for profiling the evolution of death receptor expression on cell surfaces in the process of leukemia treatment and can be miniaturized for high throughput clinical applications. This versatile electrochemical cytosensing platform is believed to be of great clinical value for diagnosis and treatment of human leukemia, especially for evaluation of individualized therapeutic effects on leukemia patients after chemo-/radiation therapy or drug treatment.

ASSOCIATED CONTENT


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Supporting Information. Additional figures as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION * Corresponding Author Emails: [email protected] (J.-J. Z.); [email protected] (Z.-C.H.); [email protected] (H.W.)

ACKNOWLEDGMENT H.W. acknowledges University of South Carolina for Start-up support. J.-J.Z. acknowledges the funding provided by National Basic Research Program of China (Grant 2011CB933502) and the NSFC (21020102038, 21121091). Z.-C.H. acknowledges the funding provided by the Ministry of Science and Technology of China (2012CB967004), NSFC (81121062) and the Jiangsu Provincial Nature Science Foundation (BZ2011048, BZ2012050). T. Z. acknowledges the Fellowship for Oversea Visiting Students provided by the Chinese Scholarship Council, Ministry of Education of China.


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REFERENCES (1) Nordlund, J.; Milani, L.; Lundmark, A.; Lonnerholm, G.; Syvanen, A.-C. PLoS ONE 2012, 7, e34513. (2) Ghobrial, I. M.; Witzig, T. E.; Adjei, A. A. Ca-a Cancer Journal for Clinicians 2005, 55, 178-194. (3) Lee, H. W.; Lee, S. H.; Ryu, Y. W.; Kwon, M. H.; Kim, Y. S. Biochemical and Biophysical Research Communications 2005, 330, 1205-1212. (4) Mitsiades, C. S.; Treon, S. P.; Mitsiades, N.; Shima, Y.; Richardson, P.; Schlossman, R.; Hideshima, T.; Anderson, K. C. Blood 2001, 98, 795-804. (5) Newsom-Davis, T.; Prieske, S.; Walczak, H. Apoptosis 2009, 14, 607-623. (6) Bouralexis, S.; Findlay, D. M.; Evdokiou, A. Apoptosis 2005, 10, 35-51. (7) Plasilova, M.; Zivny, J.; Jelinek, J.; Neuwirtova, R.; Cermak, J.; Necas, E.; Andera, L.; Stopka, T. Leukemia 2002, 16, 7. (8) Gonzalvez, F.; Ashkenazi, A. Oncogene 2010, 29, 4752-4765. (9) Spencer, S. L.; Gaudet, S.; Albeck, J. G.; Burke, J. M.; Sorger, P. K. Nature 2009, 459, 428-432. (10) Cheng, J. R.; Hylander, B. L.; Baer, M. R.; Chen, X.; Repasky, E. A. Molecular Cancer Therapeutics 2006, 5, 1844-1853. (11) Mahalingam, D.; Szegezdi, E.; Keane, M.; de Jong, S.; Samali, A. Cancer Treatment Reviews 2009, 35, 280-288. (12) Pan, G. H.; Ni, J.; Wei, Y. F.; Yu, G. L.; Gentz, R.; Dixit, V. M. Science 1997, 277, 815818. (13) Pan, G. H.; Orourke, K.; Chinnaiyan, A. M.; Gentz, R.; Ebner, R.; Ni, J.; Dixit, V. M. Science 1997, 276, 111-113.


23


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 24 of 27

(14) Sheridan, J. P.; Marsters, S. A.; Pitti, R. M.; Gurney, A.; Skubatch, M.; Baldwin, D.; Ramakrishnan, L.; Gray, C. L.; Baker, K.; Wood, W. I.; Goddard, A. D.; Godowski, P.; Ashkenazi, A. Science 1997, 277, 818-821. (15) Kurita, S.; Mott, J. L.; Almada, L. L.; Bronk, S. F.; Werneburg, N. W.; Sun, S. Y.; Roberts, L. R.; Fernandez-Zapico, M. E.; Gores, G. J. Oncogene 2010, 29, 4848-4858. (16) Park, S. J.; Wu, C. H.; Choi, M. R.; Najafi, F.; Emami, A.; Safa, A. R. Biochemical Pharmacology 2006, 72, 293-307. (17) Liu, T.; Zhu, W.; Yang, X.; Chen, L.; Yang, R. W.; Hua, Z. C.; Li, G. X. Analytical Chemistry 2009, 81, 2410-2413. (18) Chen, D.; Zhang, H.; Li, X.; Li, J. H. Analytical Chemistry 2010, 82, 2253-2261. (19) Du, D.; Zou, Z. X.; Shin, Y. S.; Wang, J.; Wu, H.; Engelhard, M. H.; Liu, J.; Aksay, I. A.; Lin, Y. H. Analytical Chemistry 2010, 82, 2989-2995. (20) Zhang, J. J.; Zheng, T. T.; Cheng, F. F.; Zhang, J. R.; Zhu, J. J. Analytical Chemistry 2011, 83, 7902-7909. (21) Zheng, T. T.; Zhang, R.; Zou, L. F.; Zhu, J. J. Analyst 2012, 137, 1316-1318. (22) Zhang, J. J.; Cheng, F. F.; Zheng, T. T.; Zhu, J. J. Analytical Chemistry 2010, 82, 35473555. (23) Liu, S. H.; Lu, F.; Jia, X. D.; Cheng, F. F.; Jiang, L. P.; Zhu, J. J. CrystEngComm 2011, 13, 2425-2429. (24) Quinn, J. F.; Johnston, A. P. R.; Such, G. K.; Zelikin, A. N.; Caruso, F. Chemical Society Reviews 2007, 36, 707-718. (25) Hong, X.; Li, J.; Wang, M. J.; Xu, J. J.; Guo, W.; Li, J. H.; Bai, Y. B.; Li, T. J. Chemistry of Materials 2004, 16, 4022-4027. (26) Li, J.; Zhao, X.-W.; Zhao, Y.-J.; Gu, Z.-Z. Chemical Communications 2009, 2329-2331.


24

Page 25 of 27

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


(27) Ding, C. F.; Ge, Y.; Zhang, S. S. Chemistry-a European Journal 2010, 16, 10707-10714. (28) Shi, X. Y.; Ganser, T. R.; Sun, K.; Balogh, L. P.; Baker, J. R. Nanotechnology 2006, 17, 1072-1078. (29) Wang, M. J.; Wang, L. Y.; Wang, G.; Ji, X. H.; Bai, Y. B.; Li, T. J.; Gong, S. Y.; Li, J. H. Biosensors & Bioelectronics 2004, 19, 575-582. (30) Tang, Y.; Allen, B. L.; Kauffman, D. R.; Star, A. Journal of the American Chemical Society 2009, 131, 13200-13201. (31) Xu, X.; Jiang, S.; Hu, Z.; Liu, S. ACS Nano 2010, 4, 4292-4298. (32) Esumi, K.; Hayakawa, K.; Yoshimura, T. Journal of Colloid and Interface Science 2003, 268, 501-506. (33) Zhang, H.; Hu, N. F. Journal of Physical Chemistry B 2007, 111, 10583-10590. (34) Lu, X.; Imae, T. Journal of Physical Chemistry C 2007, 111, 2416-2420. (35) Sefah, K.; Tang, Z. W.; Shangguan, D. H.; Chen, H.; Lopez-Colon, D.; Li, Y.; Parekh, P.; Martin, J.; Meng, L.; Phillips, J. A.; Kim, Y. M.; Tan, W. H. Leukemia 2009, 23, 235-244. (36) Yu, H.; Yan, F.; Dai, Z.; Ju, H. X. Analytical Biochemistry 2004, 331, 98-105. (37) Shen, Z. H.; Huang, M. C.; Xiao, C. D.; Zhang, Y.; Zeng, X. Q.; Wang, P. G. Analytical Chemistry 2007, 79, 2312-2319. (38) Zhang, J. J.; Gu, M. M.; Zheng, T. T.; Zhu, J. J. Analytical Chemistry 2009, 81, 66416648. (39) Ding, L.; Cheng, W.; Wang, X. J.; Ding, S. J.; Ju, H. X. Journal of the American Chemical Society 2008, 130, 7224-7225. (40) Casado-Zapico, S.; Martin, V.; Garcia-Santos, G.; Rodriguez-Blanco, J.; SanchezSanchez, A. M.; Luno, E.; Suarez, C.; Garcia-Pedrero, J. M.; Menendez, S. T.; Antolin, I.; Rodriguez, C. Journal of Pineal Research 2011, 50, 345-355.


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(41) Takahashi, M.; Yoshino, T.; Takeyama, H.; Matsunaga, T. Biotechnology Progress 2009, 25, 219-226. (42) Song, E. Q.; Hu, J.; Wen, C. Y.; Tian, Z. Q.; Yu, X.; Zhang, Z. L.; Shi, Y. B.; Pang, D. W. ACS Nano 2011, 5, 761-770.


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17.Nanoarchitectured electrochemical cytosensors for selective

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