ars.els-cdn.com · Web viewTin tetrachloride hydrate (SnCl 4 ·5H 2 O), Cerium (III) nitrate hexahydrate (Ce(NO 3) 3 ·6H 2 O) and thioaceTAMside were purchased from Sinopharm Chemical

Recognition of M2 Type Tumor-Associated Macrophages with

Ultrasensitive Photoelectrochemical Sensor Based on Ce Doped

SnO2/SnS2 Nano Heterostructure

Ruiqing Fenga, Kaixuan Tianb, Yifeng Zhanga, Wei Liu b, Jinglong Fanga, Malik

Saddam Khana, Qin Weia* Rongde Wua,b*

a Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of

Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan

250022, PR China

b Department of Pediatric Surgery, Shandong Provincial Hospital Affiliated to

Shandong University, Jinan, Shandong 250021, P.R. China.

* Corresponding author.

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Tel: +86 531 82767872

E-mail: sdjndxwq @ 163 . com ；[email protected]

Table of contents

Materials and Reagents…………………… ……………………… ……… ……S3

Apparatus…………………… …… ……… ………………………… ……………

S4

Immunofluorescence imaging…………………...…………………………………S4

Figure S1…………………………… ………………………………… ……………

S5

The experiments of photocurrent response assay……………………..……..……

S6

Table S1………………… ………… ………………………………… ……………S6

The experiments of energy levels determination………………………………..…

S6

Figure S2…………………………… ………………………………… ……………

S7

Table S2……………… …………… ………………………………… ……………S7

Table S3……………… …………… ………………………………… ……………S8

Table S4……………… …………… ………………………………… ……………S8

Figure S3…………………………… ………………………………… ……………

S8

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Figure S4…………………………… ………………………………… ……………

S9

Figure S5…………………………… ………………………………… ……………

S9

Table S5…………………………… ………………………… ……… ………...…

S10

Table S6…………………………… ………………………… ……… ………...…

S10

Materials and Reagents

Tin tetrachloride hydrate (SnCl4·5H2O), Cerium (III) nitrate hexahydrate

(Ce(NO3)3·6H2O) and thioaceTAMside were purchased from Sinopharm Chemical

Reagent Co., Ltd (Shanghai, China). 2-Mercaptoacetic acid (TGA),

methylthiazolyldiphenyl-tetrazolium bromide, anhydrous methanol and ascorbic acid

(AA) were purchased from J&K Chemical co. Ltd. (Beijing, China). Dimethyl

sulfoxide (DMSO) was purchased from innochem Co., Ltd (Beijing, China). N-

Hydroxysuccinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDC) was obtained from Nanjing kingsrui biotechnology co. Ltd.

(Nanjing, China). The Indium-Tin Oxides (ITO) was purchased from China Southern

Glass Holding Co., Ltd (Shenzhen, China). Phosphate buffer solution (PBS) is a

mixed solution of 0.1 mol·L-1 Na2HPO4 and 0.1 mol·L-1 KH2PO4, whose pH was

adjusted according to the ratio of Na2HPO4 to KH2PO4 and measured with pH meter.

PBS with the pH of 7.4 was used to prepare the solutions of antibody, antigen and

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washing buffer. All solutions were prepared by utilizing ultrapure water (18.25

MΩ·cm). All other chemicals were of analytical reagent grade and used without

further treatment.

THP-1 cells (human mononuclear cell lines in peripheral blood) and HeLa cells

(Human cervical cancer cell line) were purchased from the cell center of basic

institute of Chinese academy of medical sciences, Roswell Park Memorial Institute-

1640 (RPSI-1640), H-DMEM (Dulbecco’s Modified Eagle’s Medium, High Glucose)

and FBS (Fetal Bovine Serum) were purchased from Thermo Fisher Scientific Co.

Ltd. (America). Bovine serum albumin (BSA, 96-99%) was purchased from

innochem Co., Ltd (Beijing, China). Phorbol ester (PMA), Interleukin-4 (IL-4),

Interleukin-13 (IL-13) and Interferon-γ (IFN-γ) were purchased from Pepro Tech Co.

Ltd. (America). Anti-CD163, Anti-CD80, Alexa Fluor 488 goat anti-rabbit IgG (H+L)

and Alexa Fluor 594 goat anti-rabbit IgG (H+L) were purchased from Abcam Co.

Ltd. (England). DAPI was purchased from J&K Chemical co. Ltd. (Beijing, China).

Normal goat serum, Glycine, Triton X-100 and Trypsin were purchased from Beijing

Dingguo Changsheng Biotechnology Co. Ltd. (Beijing, China).

Apparatus

X-ray power diffraction (XRD) was obtained using a D8 advance X-ray

diffractometer (Bruker AXS, Germany) with CuKα radiation (λ = 1.5406 Å), in the

range of 10° - 80° (2θ). Scanning electron microscope (SEM) and energy dispersive

spectrometer (EDS) images were obtained from a field emission SEM (FEI Quanta

250, USA). X-ray photoelectron spectroscopy (XPS) analysis was performed on

ESCALAB 250 X-ray photoelectron spectrometer with an Al Kα radiation source

(1486.6 eV). JEOL JEM-2100F TEM (Japan) was used to get HRTEM, Energy

dispersive spectrometer (EDS) and element mapping images. Diffuse reflectance

ultraviolet-visible light spectra were measured on a UV-vis spectrophotometer (TU-

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1901, Beijing Purkinje General Instrument Co Ltd.,). PEC measurements and

electrochemical impedance spectroscopy (EIS) performed on an electrochemical

workstation (Zahner Zennium Pro, Germany) with a light excitation source of 450 nm

(Zahner, Germany). A three-electrode system was used through the PEC

measurements, and composed of an ITO as the working electrode, a Pt wire electrode

as a counter electrode and a saturated calomel electrode (SCE) as the reference

electrode. The 450 nm excitation light sources were provided by the LED lamp and

switched “off-on-off” for 20-20-20 s with 0 V applied potential. Deionized (DI) water

(18.25 MΩ cm) was obtained from YouPu water purification system (UPR-11-10T,‧

Chengdu Youpu Equipment Co Ltd., China) and was used throughout the experiment.

Wide-field fluorescence images were acquired with an Olympus IX71 inverted

microscope coupling with a CCD and display controller software.

Immunofluorescence imaging

The experimental methods of M2 immunofluorescence imaging are as follows: The

cells were 100% methanol fixed (5 min), permeabilized with 0.1% Triton X-100 for 5

minutes and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in

0.1% PBS-Tween for 1h to block nonspecific protein-protein interactions. The cells

were then incubated with the primary antibody (Anti-CD163, 18 μg/ml) for 8 h at

4°C. The secondary antibody (green) was Alexa Fluor 488 goat anti-rabbit IgG (H+L)

used at 2 μg/ml for 1 h at 37°C. DAPI was used to stain the cell nuclei (blue) at a

concentration of 2 μM. Then, the fluorescence imaging experiments were carried out.

As shown in Fig. S1 a and c, green fluorescence was detected in almost all cells,

which was emitted by Alexa Fluor 488 goat anti-rabbit IgG (H+L). This result proves

that M2 has been successfully induced.

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Fig. S1 Widefield fluorescent images of M2 stained with Alexa Fluor 488 goat anti-rabbit IgG

(H+L) and DAPI, (a) Images of Alexa Fluor 488 goat anti-rabbit IgG (H+L), λ ex = 460-490 nm,

λem = 510-540 nm; (b) Images of DAPI, λex = 330-385 nm, λem = 420-460 nm; (c) Overlay images

of a and b.

In addition, we do not know whether the M2 were used in the study are 100%.

But, In “PEC analysis of M2-TAMs” section, the different concentrations samples of

M2 were adjusted with PBS solution, which were diluted the same batch of cells. So,

the proportion of M2 in these different concentration samples is consistent. Moreover,

the fabricated cytosensor could more accurately recognize M2. Therefore, the

fabricated PEC cytosensor can be used for recognition and regular analysis of M2.

The experiments of photocurrent response assay

Photocurrent response tests were carried out in ascorbic acid (AA) solution (0.1

mol·L-1, pH = 7.4). The data of photocurrent were acquired with the PEC workstation

at 180 W·m-2 intensity light whose wavelength is 450 nm at room temperature (0 V

bias voltage).

Table S1. Photocurrents of the as-synthesized materials with different amount of

feedstock

SnCl4·5H2O

Thioacetamide

(mol)

Ce(NO3)3·6H2O

Photocurrent-

1 (µA)

Photocurrent-

2 (µA)

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(mol) (mol)

Compound-a 0.005 0.0025 0.00025 4.86 5.13

Compound-b 0.005 0.005 0.00025 15.05 16.75

Compound-c 0.005 0.00625 0.00025 30.4 39.71

Compound-d 0.005 0.0075 0 34.39 36.511

Compound-e 0.005 0.0075 0.0001 59.77 56.93

Compound-f 0.005 0.0075 0.00015 56.67 53.83

Compound-g 0.005 0.0075 0.00025 69.99 69.41Compound-h 0.005 0.0075 0.0005 37.45 39.46

Compound-i 0.005 0.0082 0.00025 24.46 27.41

Compound-j 0.005 0.009 0.00025 6.63 7.94

Compound-k 0.005 0.0095 0.00025 14.679 18.534

Compound-m 0.005 0 0 0.182 0.254

The experiments of energy levels determination

As shown in Fig. S2, the energy levels of SnO2 and SnS2 were determined by flat-

band potential and band gap. The flat-band potentialwas obtained from Mott-Schottky

plots (Fig. S2A). For SnO2 and SnS2, the flat-band potentials were -0.16 eV and -0.23

eV (NHE, NHE = SCE + 0.24eV, SnO2: -0.40eV + 0.24eV = -0.16V, SnS2: -0.47eV +

0.24eV = -0.23eV), respectively. The positive slope in the linear region demonstrated

that SnO2 and SnS2 were both n-type semiconductors (SnO2/SnS2: n-n type

heterojunction). In general, the flat-band potential is 0.1-0.3 eV higher than the

conduction band (CB) potential in the n-type semiconductor. Therefore, the

conduction band (CB) potential of SnO2 and SnS2 were calculated to be -0.06 eV

and -0.13 eV. According to the UV-vis diffuse reflection spectra of SnO2 and SnS2

(Fig. S2B), the band gaps of SnO2 and SnS2 were estimated by the following formula

proposed by Tauc, Davis, and Mott:

(αhμ)1/2=A (hμ−Eg)

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where α, h, ν, Eg, and A are the absorption coefficient, Planck's constant, frequency of

light, the band gap, and a constant, respectively. According to the above equation, by

extrapolating the tangent line to the X-coordinate, the band gaps of SnO2 and SnS2

are measured to be 3.51 eV and 2.12 eV (Fig. S2C), respectively. Therefore, the

valence band (VB) potentials of SnO2 and SnS2 were calculated to be +3.49 eV and

+1.99 eV.

Fig. S2 (A) Mott-Schottky curves of SnO2 and SnS2; (B) UV-vis DRS diffuse-reflectance spectra

of SnO2 and SnS2; (C) The band gap width of SnO2 and SnS2.

Table S2. Cytotoxicity Data (HeLa cells, Incubate concentration: 5 μM)a of

Ce:SnO2/SnS2 .

Incubate time (h) 2 8 12 24

DMI (% cell survival) 93±2 85±5 79±5 65±7a Cell viability was quantified by the MTT assay (mean±SD)

Table S3. Cytotoxicity Data (HeLa cells)a of fabricated electrode

(ITO/Ce:SnO2/SnS2/TAG/(EDC/NHS)/Ab1/BSA, under the experimental condition.) .

Incubate time (h) 2 2.5 3 4

DMI (% cell survival) 97±1 96±2 94±2 93±3a Cell viability was quantified by the MTT assay (mean±SD)

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Table S4. Cytotoxicity Data (HeLa, Incubate time: 4 h)a of Ce:SnO2/SnS2 .

Incubate concentration (µM) 2 5 10 20

DMI (% cell survival) 97±1 94±3 85±3 79±5a Cell viability was quantified by the MTT assay (mean±SD)

Fig. S3 Optimization of experimental parameters: (A) influence of AA concentrations on the immunosensor, (B) Effect of pH of detection solution containing 0.1 mol·mL -1, (C) The experiment of capture antibody concentration (Incubation time: After that dried to the film state), (D) The experiment of incubation time (Antibody concentration: 6 μL, Canti-CD163=18μg/mL). (Error bars=SD, n=3 ).

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Fig. S4 (A) The selectivity of the immunosensor: 5 × 102 cells·mL-1 M2-TAMs(a), 5 × 102

cells·mL-1 M2-TAMs and 1 × 103cells·mL-1 interfering substance HeLa(b), SiHa(c), A549 cells(d), THP-1(e) or M1-TAMs(f), Error bars=SD, n=5. (B) The photocurrent-time test of the cytosensor under several on/off irradiation cycles for 500 s, cM2-TAMs = 5 × 104 cells·mL-1.

Fig. S5. (A): Reproducibility test of the fabricated cytosensor; (B): Reproducibility test of material

(Ce Doped SnO2/SnS2) preparati

Table S5. Detection Results of M2 Sample

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sample content

(cells∙mL-1)

Addition content

(cells∙mL-

1)

The detection content

(cells∙mL-1)

Averagevalue

(cells∙mL-

1)

RSD(%, n=5)

Recovery(%, n=5)

5001470, 1520,

1570, 1460, 15201508 2.94 101.6

10002000

2960, 3040,

2940., 3020,

2940

2980 1.74 99

50006110, 6010,

5940, 6030, 59606010 1.13 100.2

Table S6. Comparison of different methods in cell detection

Ref. MethodLinear range

(cell∙mL−1)

Detection limit

(cell∙mL−1)Detection target

(Cheng et al. 2008)Cheng,

2008 #103

DifferentialPulse Voltammetry

1.0 × 103 ~ 1.0 ×107

620 BGC cell

(Zhang et al. 2009)

Electrochemical Impedance Spectroscopy

1.0 × 104 ~ 1.0 ×107 5000 HL-60 cell

(Shen et al. 2007)

Quartz Crystal Microbalance

7.5 × 102 ~ 7.5 ×107 750 Escherichia coli.

(Jia et al. 2016)

Surface Plasmon Resonance

5.0 × 102 ~ 4.0 ×104 136 MCF-7 cell

(Cai et al. 2015)

Resonance Rayleigh

Scattering4.0 × 102 ~

2.0 ×103 12 MCF-7 cell

(Arya et al. 2013)

Cyclic Voltammetry1.0 × 104 ~

1.0 ×108 10000 MCF-7 cell

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(Hua et al. 2013)

Fluorescence5.0 × 102 ~

4.0 ×104 201 MCF-7 cell

(Wu et al. 2013)

Electrochemiluminescence1.0 × 100 ~

7.0 ×103 20 MCF-7 cell

(Pang et al. 2017)

Photoelectrochemical5.0 × 101 ~

5.0 ×104 50 RAW264.7 cell

(Zhang et al. 2020)

Plasmonic Metasurface1.0 × 104~ 1.0 ×105 10000

A549 or HepG2 cell

This work PEC5.0 × 101 ~

1.0 ×105 50M2-Tumor-associated

macrophages

References

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