Photoelectrochemical Cytosensing of RAW264.7 Macrophage Cells

Subscriber access provided by UNIVERSITY OF CONNECTICUT

Article

Photoelectrochemical Cytosensing of RAW264.7 Macrophage Cell Based On TiO2 Nanoneedls@MoO3 Array Xuehui Pang, Hongjun Bian, Minhui Su, Yangyang Ren, Jianni Qi, Hongmin Ma, Dan Wu, Lihua Hu, Bin Du, and Qin Wei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01038 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017

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

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

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Photoelectrochemical Cytosensing of RAW264.7 Macrophage Cell Based On TiO2 Nanoneedls@MoO3 Array Xuehui Pang†, Hongjun Bian‡, Minhui Su†, Yangyang Ren†, Jianni Qi§, Hongmin Ma†, Dan Wu†, Lihua Hu†, Bin Du†, Qin Wei*† †

Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of

Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, China ‡

Department of Emergency, Shandong Provincial Hospital Affiliated to Shandong

University, Jinan, 250021, China §

Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong University,

Jinan, 250021, China

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

Abstract: We have developed a photoelectrochemical (PEC) cytosensor for ultrasensitive detection of RAW264.7 cells by the signal change of a TiO2 nanoneedles (NNs)@MoO3 array. For the first time, TiO2 NNs@MoO3 array was adopted for the fabrication of the cytosensor for the signal output. The well-matched alignment of TiO2 NNs and MoO3 efficiently suppresses the recombination of photo-generated electron and hole (e-/h+) pairs for improved photon-to-current conversion efficiency. RAW264.7 cell and F4/80 antibody could form the biocomplexes because of the specific recognition between each other. The constructed PEC cytosensor based on TiO2 NNs@MoO3 array displayed good PEC property for detection of RAW264.7 cell. The numbers of RAW264.7 cells are directly detected through the decrement of photocurrent intensity, due to the increased steric hindrance when RAW264.7 cells are captured. The PEC cytosensor showed an ultrasensitive response to RAW264.7 cell with a linear range of 50 cell/mL to 15000 cell/mL and a detection limit of 50 cell/mL. The designed cytosensor based on a TiO2 NNs@MoO3 array offers an ideal platform to detect RAW264.7 cells with excellent stability, reproducibility and selectivity, and served as a model for the fabrication of cytosensors for other cells. Keywords:

TiO2

nanoneedles@MoO3

array,

RAW264.7

cell,

macrophage,

photoelectrochemical cytosensor Monocyte-macrophage systems play a very important role in innate and adaptive immune response 1. For anti-infection and anti-tumor immunity, although several groups have reported that the RAW264.7 mouse cell line is a pivotal macrophage, the 2


Page 3 of 24

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


precise immunological mechanisms are not known so far. Hence, it is urgently necessary to detect RAW264.7 cells for early warning. This paper reports a PEC cytosensor to detect RAW264.7 cells based on visible light-activated TiO2 NNs@MoO3 array. MoO3, a well-known layered metal oxide semiconductor with high work function and good hole conductivity, has exhibited promising applications in many fields

2-6

. However, the wide band-gap energy of MoO3 (2.9 eV) leads to

recombination of photogenerated e−/h+ pairs and low conversion efficiency of photon energy. Several methods have been used to improve the utilization of MoO3, including preparation of nanocomposites or construction heterojunctions coupling MoO3 and another semiconductor. In this research, TiO2, which has attracted considerable attention in the biosensing

21-38

7-20

, especially

, is used as coupled semiconductor. But TiO2 can absorb only

UV photons due to its large band gap of about 3.2 eV

39

and low photo-catalytic

40,41

ability. By coupling of MoO3 with TiO2, a p-n heterojunction is formed. This theoretically hinders the recombination of photogenerated e−/h+ pairs by lengthening the lifetime of the charge carriers and enlarging the photocurrent response range

42,43

.

The TiO2/MoO3 combination produces a band offset of about 2.6 eV. To better utilize the band offset, the core-shell structure was employed in this work because the interfacial microstructure can be tuned. By electrodepositing MoO3 on the surfaces of as-prepared TiO2 NNs, we

3



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

developed a novel PEC cytosensor for ultrasensitive detection of RAW264.7 cells (Figure 1). The fabricated cytosensor shows ultrasensitive photocurrent response, a relatively wide linear range with a low detection limit. This PEC cytosensing method offers a fast and ultrasensitive strategy for detection of RAW264.7 cells.

EXPERIMENTAL Instruments. All scanning electron microscope (SEM) images and EDS mappings

were obtained with a JSM-6700F microscope (JEOL, Japan). The transmission electron microscope (TEM) images were collected from a TEM (JEOL, JEM-1400, Japan). XRD patterns were obtained with a D8 focus diffractometer (Bruker AXS, Germany). Photoluminescence (PL) experiments were carried out on a LS-45/55 PL spectrometer (Perkin Elmer, USA). UV-vis absorption spectra were obtained from a Lambda 35 UV-vis spectrometer (Perkin-Elmer, USA). The electrodeposition and PEC tests were performed on an electrochemical work station (Zahner Zennium PP211, Germany) with a three-electrode system. Fourier transform infrared (FT-IR) spectra were obtained using a Perkin-Elmer 580B spectrophotometer (Perkin-Elmer, USA). Synthesis of TiO2 NNs. The TiO2 NNs array was prepared according to a literature method 44. Twenty one mL HCl and 21.0 mL acetone were mixed with 0.5 ~ 5.5 mL tetrabutyl titanate. The mixture was vortexed at 25 °C and a clear solution was obtained. Then the obtained solution and a seeded carbon cloth (CC, the bare CC was placed in a TiCl4 alcohol solution (2.2 vol%) for 12 h and annealed for 1 h at 400 °C) were transferred to a Teflon-lined autoclave. The autoclave was heated for 2 h to 4


Page 4 of 24

Page 5 of 24

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


200 °C after being sealed. The TiO2 NNs array-covered CC was removed, cleaned 3 times with ethyl alcohol, dried at 60 °C in an oven, and heated again at 450 °C for 1 h.

Figure 1 Schematic illustration of PEC cytosensor fabrication procedure.

Preparation of TiO2 NNs@MoO3 array. MoO3 was electrodeposited to form the TiO2 NNs@MoO3 array according to a literature CV method

44

. The modified CC

electrode was used as the working electrode, a saturated calomel electrode (SCE) was used as the reference electrode and a platinum wire was used as the auxiliary electrode. Cyclic voltammetry was used with 0.05 mol/L (NH4)6Mo7O24 was the electrolyte solution. The potential range was 0 ~ -0.7 V with a sweep rate of 20 mV/s for 20 to 140 cycles. RAW264.7 cell culture and collection. The mouse macrophage cell lines 5



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

RAW264.7 were provided by Dr. Qi and cultured in DMEM (Gibco-BRL, Grand Island, NY, USA) containing 10% (vol/vol) fetal bovine serum (FBS, Gibco® Sera, AUS), 100

Figure 2 SEM images of TiO2 NNs arrays (A, B); TEM image of TiO2 NNs (C and the inset); EDS of TiO2 NNs (D); SEM images of TiO2 NNs@MoO3 arrays (E and F); TEM image of TiO2 NNs@MoO3 array (G) and EDS of TiO2 NNs@MoO3 arrays (H); EDS mapping of TiO2 NNs@MoO3 arrays (I), Ti element (J) and Mo element (K).

U/mL penicillin and 100 µg/mL streptomycin (Gibco, Grand Island, NY and Scotland, UK). The cell lines were put in a humidified incubator and maintained at 37 °C with 5% 6


Page 6 of 24

Page 7 of 24

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


CO2. When the cultured cells grew to 80% confluence, fresh complete medium was added and these cells were harvested by up and down pipetting. The cells were counted and the concentration was adjusted for later use. Fabrication of the PEC cytosensor. The fabrication procedure is shown in Figure 1. First, TiO2 NNs were grown with 4.5 mL tetrabutyl titanate on the CC. Then, MoO3 was electrodeposited on the TiO2 NN surfaces to form TiO2 NNs@MoO3 array in 0.10 mol/L (NH4)6Mo7O24 electrolyte solution with 110 cycles. Then, 10 µL of F4/80 (18 µmol/L) antibody was dropped on the TiO2 NNs@MoO3 array/electrode with EDC/NHS and then incubated for 5 h in a 4 ºC refrigerator. Finally, different concentrations of RAW264.7 cells were incubated with the F4/80/TiO2 NNs@MoO3 array/electrode surface. Photoelectrochemical detection. The PEC experiments were performed on the electrochemical work station described in the “Preparation of TiO2 NNs@MoO3

array” section in the similar three-electrode electrolytic tank in PB electrolyte solution containing 0.1 mol/L ascorbic acid (AA). PEC tests were operated at 25 °C, with 0.1 V bias voltage and 20 s Light duration.

RESULTS AND DISCUSSION Characterization of TiO2 NNs and TiO2 NNs@MoO3 array. Figure 2A shows

the SEM morphology of an as-synthesized TiO2 NNs array. It can be seen that TiO2 NNs grew as orderly arranged needles. Figure 2B shows that theTiO2 NNs were independent of each other, indicating good dispersity of nanoneedles. Figure 2C displays a TEM image of TiO2 NNs, revealing the structure of the prepared materials 7



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

(TiO2 NNs and TiO2 NNs@MoO3 array). Figure 2C and its inset show that TiO2 was nanoneedle with homogeneous size. EDS results in Figure 2D show that all the peaks belonged only to Ti and O, indicating that TiO2 NNs preparation achieved the desired dispersity. Figure 2E and F show the macroscopic and microscopic SEM images of as-prepared TiO2 NNs@MoO3 array, which were also well-dispersed. However, the electrodeposition of MoO3 made the TiO2 nano-needle thicker and stronger, indicating that the hybridization of TiO2 NNs and MoO3 was compact. At the same time, the diameter of the TiO2 NNs@MoO3 array was average, showing that the preparation craft was well controlled. Figure 2G shows the TEM image of a single TiO2 NN@MoO3 rod, with distinct contrast between the middle and border. The TiO2 nano-needle located in the center of the rod is clearly visible, and the electrodeposited MoO3 around the TiO2 nano-needle is also clear. Four elements (Ti, O, C and N) appear in the EDS results. In addition, the EDS mapping results showed the compact location of Ti (Figure 2J) and Mo (Figure 2K) distributed homogeneously on TiO2 surface according to Figure 2I.

Figure 3 XRD pattern (A) of TiO2 NNs@MoO3 arrays; FTIR spectra (B) of TiO2 NNs (a) and TiO2 NNs@MoO3 arrays (b). 8


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The XRD pattern of the TiO2 NNs@MoO3 array is shown in Figure 3A. The diffraction peaks 45 located at 27.6°, 36.2°, 39.2°, 41.5°, 44.2°, 54.5°, 56.9°, 69.1° and 69.9° and marked with heart-shapes corresponds to rutile TiO2 according to JCPDS Card No. 21-1276. The diffraction peaks located at 12.8°, 23.4°, 25.9°, 27.6°, 29.8°, 33.3°, 34.0°, 37.9°, 46.3°, 48.2°, 49.5°, 53.0° and 64.2° marked as starts can be assigned to the orthorhombic MoO3 based on JCPDS Card No. 05-0508

45-47

. All the

above results show that the TiO2 NNs@MoO3 array was well prepared as expected. Figure 3B shows FT-IR spectra of TiO2 NNs (curve a) and TiO2 NNs@MoO3 array (curve b) from 1000 to 4000 cm-1. The peaks at 1404 (a) and 1408 (b) cm-1 indicate the characteristic bending vibration of C=O. The peaks of 1630 (a) and 1649 (b) cm-1 correspond to the stretching vibrations of the C-O. The peak at 2365 cm-1 results from a C=O stretching vibration. The wide weak peaks in 3140-3391 cm-1 in both curves are related to C-H and O-H. These data suggested that the TiO2 NNs@MoO3 array contains many carboxyl and amino groups on their surfaces to improve the combination ability and hydrophilicity to detect targets in aqueous systems. The mechanism about PEC performance improvement. To demonstrate the improvement of PEC activity of the TiO2 NNs@MoO3 array and illustrate the energy band structure, a postulated schematic diagram was proposed as shown in Figure 4A. Since the band gap of TiO2 NNs is close to 3.2 eV 39 and that of MoO3 is 2.9 eV 48,49, both absorbed only in the UV light region. However, the conduction band (CB) and the valence band (VB) of MoO3 was higher than those of TiO2 as shown in the Figure 4A. Thus, TiO2 and MoO3 can form type ΙΙ p−n heterojunctions 50. In addition, visible 9



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

light can be absorbed due to the band offset of 2.61 eV between the VB of TiO2 and the CB of MoO3

51

, and an internal electric channel in the band gap alignment

between TiO2 and MoO3 can be built. The core-shell structure shortens the distance for the separation of photogenerated charge carriers in its hetero-structure of TiO2@MoO3. When exposed to visible light irradiation, TiO2 and MoO3 were both excited, followed by injection of the generated electrons in the VB of TiO2 into the CB of MoO3 52 and the holes in the VB of MoO3 into the VB of TiO2 through the internal electric channel. Thus, the holes are transferred to the electrode interface and reduced by AA

53

as illustrated in Figure 4A. Therefore, the charge separation

efficiency, the transfer process and the photocatalytic activity of TiO2 NNs@MoO3 array are thermodynamically increased because of the internal electric channel formed by band offset alignment and the core-shell structure of the TiO2 NNs@MoO3 array. The improved PEC process was verified by the following results in PL, PEC and UV-vis tests. To verify the improvement for the PEC property of the TiO2 NNs@MoO3 array, a PL method was utilized

54

. As shown in Figure 4B, curve a displayed a relative

stronger PL emission intensity of TiO2 NNs. When MoO3 was electrodeposited, PL intensity became weaker as shown in curve b. This verified that the recombination efficiency between e− and h+ became lower and the electron injection efficiency became higher because of the alignment between TiO2 NNs and MoO3. Time-based photocurrent responses were also tested as shown in Figure 4C. As described in the above mechanism, TiO2 NNs absorbed visible light poorly as shown 10


Page 10 of 24

Page 11 of 24

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


in curve a, due to the wide band gap. The electrolytic deposition of MoO3 increased the photocurrent response apparently. This also verified that the formation of the TiO2 NN@MoO3 array allowed electron injection through the forbidden band and efficiently suppressed recombination of e−/h+ pairs. UV-vis absorption spectra were collected to further verify the PEC enhancement process, as shown in Figure 4D. Curve a did not exhibit light absorption of TiO2 NNs because of the wide band gap. However, curve b (TiO2 NN@MoO3 array) displayed a slight obvious absorption hill. This further verified that enhanced PEC properties can be ascribed to the combination of TiO2 NNs and MoO3.

Figure 4 (A) Energy band diagram of TiO2 NNs@MoO3 arrays; (B) PL emission spectra, (C) time-based photocurrent responses and (D) UV-vis spectra of (a) TiO2 11



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

NNs and (b) TiO2 NNs@MoO3 arrays.

Fabrication of the PEC cytosensor. The fabrication process was monitored by the photocurrent responses of PEC cytosensor in Figure 5A and B. As for curve a, there almost no electric current appeared on the bare CC electrode. For curve b, the photocurrent response of TiO2 NNs improved than that of the bare CC electrode. For curve c, the photocurrent of TiO2 NNs@MoO3 array was obviously improved as a result of good dispersity of the array, the short distance between TiO2 NNs and MoO3, and enhanced visible-light absorption ability. Subsequently, the photocurrent signal decreased after incubation with F4/80 antibody (curve d) due to the specific binding of antibody on the TiO2 NNs@MoO3 array/CC electrodes, which depressed electron transfer and partially obstructed the reaction of electrons with the photogenerated holes on the modified electrode surface. All of these observations suggest that the fabrication of the cytosensing interface occurred as desired.

Figure 5 (A) Time-based photocurrent responses and (B) histogram of naked CC (a), TiO2 NNs/CC (b), TiO2 NNs@MoO3 arrays/CC (c), F4/80 antibody/TiO2 NNs@MoO3 arrays/CC (d). 12


Page 12 of 24

Page 13 of 24

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


Optimization of experimental conditions. To achieve better detection performance, we optimized the experiment conditions and achieved the results shown in Figure 6. TiO2 NNs served as the basic material to output the signal. As shown in Figure 6A, the photocurrent signal gradually enhanced with increasing Ti source volume from 0.5 to 4.5 mL, and the photocurrent signal declined beyond 4.5 mL. Thus, 4.5 mL was selected as the optimal volume of Ti source.

Figure 6 The optimization of experimental conditions by PEC of (A) the volume of Ti source in the preparation of TiO2 NNs, (B) different electrodeposition cycles of MoO3 arrays, (C) different F4/80 concentrations (pH=7) and (D) different pH value after being modified TiO2 NNs@MoO3 arrays.

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The electrodeposition of MoO3 shell can enhance the photocurrent response of TiO2 NNs (Figure 6B). The photocurrent intensity strengthened along increasing numbers of electrodeposition cycles. However, with more 110 cycles, the signal decreased. Thus, 110 electrodeposition cycles were chosen as the optimal electrodeposition time. The concentration of capture probe also has a significant effect on the analytical performance and selectivity. In Figure 6C, the photocurrent signal decreased with increasing antibody concentration. We chose 18 µmol/L as the optimal concentration of the antibody to balance the photocurrent intensity and the best specific recognition of target cells. Figure 6D displays the influence of pH on the photocurrent responses. The experiment was performed at different pHs in the range of 5.0 to 9.0. The maximum signal was achieved at pH 7.0, thus the optimal pH was 7.0 in subsequent experiments. Analytical performance characteristics. Quantitative detection process was performed under the optimal experimental conditions by incubating different concentrations of RAW264.7 cells with the fabricated electrode. Time-based photocurrent response is displayed in Figure 7A and B. The capture probe anchored RAW264.7 cells by the specific recognition between RAW264.7 cells and the antibody, and the concentration of RAW264.7 cells determined the photocurrent signal. The photocurrent intensity decreased along with increasing concentration of RAW264.7 cells, suggesting that the proposed cytosensor could be used for 14


Page 14 of 24

Page 15 of 24

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


quantitative determination of the target cells. A plot of photocurrent response change (∆I) versus the logarithm of RAW264.7 cell concentration (ccell) in Figure 7C is linear from 50 to 15000 cell/mL. The linear equation is I (µA) =4.8757 + 0.8936 lg(cRAW264.7, cell/mL) (R2= 0.9956) with a detection limit of 50 cell/mL. The performance is relatively better than that achieved by the other methods (see Supporting Information, Table S1, S2). The low detection limit is attributed to the sensitive detection signal of this PEC cytosensor and the separated excitation energy. The TiO2 NNs@MoO3 array nanomaterials may be endowed large surface area to effectively capture target cells. Stability, reproducibility and selectivity. Because stability is a very significant aspect influencing the application of a cytosensor, the stability of the cytosensor was assessed. No obvious variation was observed in the reproducible photocurrent responses after more than 37 on/off irradiation cycles, as shown in Figure 8A. The inter-assay and intra-assay relative standard deviations (RSD) were used to estimate the reproducibility of the PEC cytosensor. According to the test data, the intra-assay RSDs were 3.62%, 2.89% and 2.05%, corresponding respectively to 100, 3000 and 8000 cell/mL of target RAW264.7 cells. The inter-assay RSDs were 3.54%, 1.98% and 2.03% respectively according to results for the same sample concentration using five independently fabricated electrodes under the same experimental conditions. The above results illuminated that the reproducibility was acceptable.

15



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

Figure 7 (A)Time-based photocurrent of the electrode with different RAW264.7 cell concentrations; (B) photocurrent changes (∆I) versus increasing RAW264.7 cell concentrations; (C) The plot of ∆I vs concentrations logarithm of RAW264.7 cells.

Figure 8 (A) Signal stability under 37 on/off irradiation cycles; (B) selectivity toward different analytes: (1) 100 cell/mL RAW264.7 cells; (2) 100 cell/mL RAW264.7 cells + 1000 cell/mL THP-1 cells; (3) 100 cell/mL RAW264.7 cells +1000 cell/mL BEL-7402 cells; (4) 1000 cell/mL THP-1 cells; (5) 1000 cell/mL BEL-7402 cells; (C) selectivity of the cytosensor (1) 18 µmol/L F4/80+100 cell/mL RAW264.7 cells; (2) 18 µmol/L IgG+100 cell/mL RAW264.7 cells; (3) 18 µmol/L IgG+100 cell/mL THP-1 cells; (4) 18 µmol/L IgG+100 cell/mL BEL-7402 cells; (5) 18 µmol/L CD95+100 cell/mL BEL-7402 cells; (6) 18 µmol/L CD95+100 cell/mL THP-1 cells; (7) 18 µmol/L CD95+100 cell/mL BEL-7402 cells.

Selectivity is another important criterion for a cytosensor. To make certain that 16


Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the change in photocurrent signal was a result of specific recognition, two representative interfering cells including THP-1 cells and BEL-7402 cells were used for interference tests by measuring the change in photocurrent intensity of 100 cell/mL RAW264.7 cell solution containing 1000 cell/mL of the two interfering cells, respectively. As seen in Figure 8B (samples 2 and 3), these interfering agents did not change the signal. To further evaluate the selectivity of the cytosensor, RAW264.7 cell-free cytosensors were tested. Two RAW264.7 cell-free sensors containing 1000 cell/mL THP-1 cells and BEL-7402 cells, respectively, were tested (Figure 8B samples 4 and 5). It can be seen that there was no conspicuous photocurrent signal. Further, the capture probe F4/80 antibody was replaced by IgG and CD95 antibodies, respectively, to test the selectivity of the cytosensor. As shown in Figure 8C, when the replaced cytosensor was respectively used to detect RAW264.7 cells, THP-1 cells and BEL-7402 cells, there almost no photocurrent signal, suggesting that IgG or CD95 cannot specifically recognize these three kinds of cells (Figure 8C 2-7). The above results demonstrated that the selectivity was satisfactory for the quantitative detection of the target cells.

Table 1 Detection results of RAW264.7 samples Sample

Added

(cell/mL) (cell/mL)

content Detected content (cell/mL)

RSD

Recovery

(%, n= 8) (%)

205

400

600, 615, 598, 595, 608

1.35

99.5

308

600

905, 910, 880, 910, 896

1.40

98.7

17



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

Standard addition recovery was also evaluated (Table 1). The cytosensor presented a relative standard deviation (RSD) of 1.35% and 1.40% and a recovery of 99.5% and 98.7% respectively. Thus, the proposed cytosensor showed the potential for application to detect the RAW264.7 cells.

CONCLUSION

In this work, a novel PEC cytosensor based on TiO2 NNs@MoO3 array was developed and demonstrated as a novel promising method for RAW264.7 cell detection. As for TiO2 NNs@MoO3 array, the composited mode of TiO2 NNs and MoO3 improved the photoelectrochemical performance of the cytosensor. The electrodeposition of MoO3 shell significantly increased the separation and transfer performance of the charge under visible light. The proposed cytosensor achieved a low detection limit of 50 cell/mL along with a broad linear range between 50 and 15000 cell/mL for RAW264.7 cell detection by the specific binding effect. This proposed cytosensor serves not only as a promising method, but also as a prototype cytosensor, with excellent sensitivity, selectivity, reproducibility and stability.

AUTHOR INFORMATION

Corresponding author. Tel.: +86 531 8276 7872. Fax: +86 531 8276 7367. E-mail address: [email protected] Author Contributions The manuscript has written through contributions of all authors. All authors have given approval to the final version of the manuscript. 18


Page 18 of 24

Page 19 of 24

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


ACKNOWLEDGEMENTS

This research was supported by China Postdoctoral Science Foundation (No. 2016M592125), Key Research and Development Program of Shandong Province, China (No. 2015GGH301001), the Technology Research Project of Shandong Provincial Education Department (No. J15LC07), National Key Scientific Instrument and Equipment Development Project of China (No.21627809), National Natural Science Foundation of China (Nos. 21405059, 81600469), Graduate Innovation Foundation of University of Jinan (No. YCXB15004) and Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).

REFERENCES

(1) El-Serag, H. B. New Eng. J. Med. 2011, 365, 1118-1127. (2) Miyauchi, M.; Nakajima, A.; Watanabe, T.; Hashimoto, K. Chem. Mater. 2002, 14, 2812-2816. (3) Shakir, I.; Shahid, M.; Kang, D. J. Chem. Commun. 2010, 46, 4324-4326. (4) Cao, X.; Zheng, B.; Shi, W.; Yang, J.; Fan, Z.; Luo, Z.; Rui, X.; Chen, B.; Yan, Q.; Zhang, H. Adv. Mater. 2015, 27, 4695-4701. (5) Brezesinski, T.; Wang, J.; Tolbert, S. H.; Dunn, B. Nat. Mater. 2010, 9, 146-151. (6) Wen, X.; Yang, W.; Ding, Y.; Niu, S.; Wang, Z. L. Nano Res. 2014, 7, 180-189. (7) Kumar, R.; Govindarajan, S.; Siri Kiran Janardhana, R. K.; Rao, T. N.; Joshi, S. V.; Anandan, S. ACS Appl. Mater. Inter. 2016, 8, 27642-27653. (8) Li, Y.; Selloni, A. ACS Catal. 2016, 6, 4769-4774. (9) Choi, J.; Song, S.; Hörantner, M. T.; Snaith, H. J.; Park, T. ACS Nano 2016, 10, 19



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

6029-6036. (10) Dong, R.; Zhang, Q.; Gao, W.; Pei, A.; Ren, B. ACS Nano 2015, 10, 839-844. (11) Liu, J.; Wang, J.; Ku, Z.; Wang, H.; Chen, S.; Zhang, L.; Lin, J.; Shen, Z. X. ACS Nano 2015, 10, 1007-1016. (12) Kupfer, B.; Majhi, K.; Keller, D. A.; Bouhadana, Y.; Rühle, S.; Barad, H. N.; Anderson, A. Y.; Zaban, A. Adv. Energy Mater. 2015, 5, 1401007. (13) Roose, B.; Gödel, K. C.; Pathak, S.; Sadhanala, A.; Baena, J. P. C.; Wilts, B. D.; Snaith, H. J.; Wiesner, U.; Grätzel, M.; Steiner, U. Adv. Energy Mater. 2016, 6, 1501868. (14) Tahir, M. N.; Oschmann, B.; Buchholz, D.; Dou, X.; Lieberwirth, I.; Panthöfer, M.; Tremel, W.; Zentel, R.; Passerini, S. Adv. Energy Mater. 2016, 6, 1501489. (15) Chen, J.; Yang, H. B.; Tao, H. B.; Zhang, L.; Miao, J.; Wang, H. Y.; Chen, J.; Zhang, H.; Liu, B. Adv. Funct. Mater. 2016, 26, 456-465. (16) Cheng, Y.; Chen, Z.; Wu, H.; Zhu, M.; Lu, Y. Adv. Funct. Mater. 2016, 1338-1346. (17) Cui, M.; Ng, W. S.; Wang, X.; Darmawan, P.; Lee, P. S. Adv. Funct. Mater. 2015, 25, 401-408. (18) Lin, H.; Wang, X. Adv. Funct. Mater. 2016, 26, 1580-1589. (19) Naoi, K.; Kurita, T.; Abe, M.; Furuhashi, T.; Abe, Y.; Okazaki, K.; Miyamoto, J.; Iwama, E.; Aoyagi, S.; Naoi, W. Adv. Mater. 2016, 28, 6751-6757. (20) Wang, N.; Bai, Z.; Qian, Y.; Yang, J. Adv. Mater. 2016, 28, 4126-4133. (21) Chang, H.; Lv, J.; Zhang, H.; Zhang, B.; Wei, W.; Qiao, Y. Biosens. Bioelectron. 20


Page 20 of 24

Page 21 of 24

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


2017, 87, 579-586. (22) Fan, D.; Ren, X.; Wang, H.; Wu, D.; Zhao, D.; Chen, Y.; Wei, Q.; Du, B. Biosens. Bioelectron. 2017, 87, 593-599. (23) Liu, H.; Guo, K.; Duan, C.; Dong, X.; Gao, J. Biosens. Bioelectron. 2017, 87, 473-479. (24) Xin, Y.; Li, Z.; Wu, W.; Fu, B.; Wu, H.; Zhang, Z. Biosens. Bioelectron. 2017, 87, 396-403. (25) Jia, Z.; Xiu, P.; Li, M.; Xu, X.; Shi, Y.; Cheng, Y.; Wei, S.; Zheng, Y.; Xi, T.; Cai, H. Biomaterials 2016, 75, 203-222. (26) Fan, G.; Han, L.; Zhang, J.; Zhu, J.-J. Anal. Chem. 2014, 86, 10877-10884. (27) Fan, G.; Han, L.; Zhu, H.; Zhang, J.; Zhu, J. Anal. Chem. 2014, 86, 12398-12405. (28) Fan, G.; Zhu, H.; Du, D.; Zhang, J.; Zhu, J.; Lin, Y. Anal. Chem. 2016, 88, 3392-3399. (29) Li, X.; Zhu, L.; Zhou, Y.; Yin, H.; Ai, S. Anal. Chem. 2017, 89, 2369-2376. (30) Lu, J.; Li, H.; Cui, D.; Zhang, Y.; Liu, S. Anal. Chem. 2014, 86, 8003-8009. (31) Ma, W.; Wang, L.; Zhang, N.; Han, D.; Dong, X.; Niu, L. Anal. Chem. 2015, 87, 4844-4850. (32) Tu, W.; Dong, Y.; Lei, J.; Ju, H. Anal. Chem. 2010, 82, 8711-8716. (33) Wang, L.; Ma, W.; Gan, S.; Han, D.; Zhang, Q.; Niu, L. Anal. Chem. 2014, 86, 10171-10178. (34) Zhao, W.; Chen, R.; Dai, P.; Li, X.; Xu, J.; Chen, H. Anal. Chem. 2014, 86, 11513-11516. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(35) Zhao, W.; Ma, Z.-Y.; Yan, D.; Xu, J.; Chen, H. Anal. Chem. 2012, 84, 10518-10521. (36) Zhao, W.; Shan, S.; Ma, Z.; Wan, L.; Xu, J.; Chen, H. Anal. Chem. 2013, 85, 11686-11690. (37) Zhou, Y.; Wang, H.; Zhuo, Y.; Chai, Y.; Yuan, R. Anal. Chem. 2017, DOI: 10.1021/acs.analchem.1027b00090. (38) Zhu, Y.; Zhang, N.; Ruan, Y.; Zhao, W.; Xu, J.; Chen, H. Anal. Chem. 2016, 88, 5626-5630. (39) Huang, Q.; Tian, S.; Zeng, D.; Wang, X.; Song, W.; Li, Y.; Xiao, W.; Xie, C. ACS Catal. 2013, 3, 1477-1485. (40) Friedmann, D.; Mendive, C.; Bahnemann, D. Appl. Catal. B: Environ. 2010, 99, 398-406. (41) Liu, G.; Zhao, Y.; Sun, C.; Li, F.; Lu, G. Q.; Cheng, H. M. Angew. Chem. Int. Ed. 2008, 47, 4516-4520. (42) Wu, L.; Xing, J.; Hou, Y.; Xiao, F. Y.; Li, Z.; Yang, H. G. Chem. Eur. J. 2013, 19, 8393-8396. (43) Weng, B.; Zhang, X.; Zhang, N.; Tang, Z.-R.; Xu, Y.-J. Langmuir 2015, 31, 4314-4322. (44) Wang, C.; Wu, L.; Wang, H.; Zuo, W.; Li, Y.; Liu, J. Adv. Funct. Mater. 2015, 25, 3524–3533. (45) Pang, X.; Pan, J.; Gao, P.; Wang, Y.; Wang, L.; Du, B.; Wei, Q. Biosens. Bioelectron. 2015, 74, 49-58. 22


Page 22 of 24

Page 23 of 24

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


(46) Chen, Y.; Lu, C.; Xu, L.; Ma, Y.; Hou, W.; Zhu, J. J. Crystengcomm 2010, 12, 3740-3747. (47) Chen, J. S.; Cheah, Y. L.; Madhavi, S.; Lou, X. W. J. Phys. Chem. C 2010, 114, 8675-8678. (48) Liu, H.; Lv, T.; Zhu, C.; Zhu, Z. Sol. Energ. Mat. Sol. C 2016, 153, 1-8. (49) Mahmood, H.; Habib, A.; Mujahid, M.; Tanveer, M.; Javed, S.; Jamil, A. Mater. Sci. Semicon. Pro. 2014, 24, 193-199. (50) Hamwi, S.; Meyer, J.; Winkler, T.; Riedl, T.; Kowalsky, W. Appl. Phys. Lett. 2009, 94, 253307. (51) Elder, S.; Cot, F.; Su, Y.; Heald, S.; Tyryshkin, A.; Bowman, M.; Gao, Y.; Joly, A.; Balmer, M.; Kolwaite, A. C. J Am. Chem. Soc. 2000, 122, 5138-5146. (52) Lu, B.; Li, X.; Wang, T.; Xie, E.; Xu, Z. J. Mater. Chem. A 2013, 1, 3900-3906. (53) Gao, P.; Ma, H.; Yang, J.; Wu, D.; Zhang, Y.; Du, B.; Fan, D.; Wei, Q. New J. Chem. 2015, 1483-1487. (54) Cheng, C.; Amini, A.; Zhu, C.; Xu, Z.; Song, H.; Wang, N. Sci. reps. 2014, 4, 4181.

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


Page 24 of 24

Photoelectrochemical Cytosensing of RAW264.7 Macrophage Cells

Recommend Documents