Supplementary Information

Label-free Upconversion Nanoparticles-based Fluorescent

Probes for Sequential Sensing of Cu2+, Pyrophosphate and

Alkaline Phosphatase Activity

Fangfang Wang, Cuiling Zhang,* Qin Xue, Huaping Li, and Yuezhong Xian*

Shanghai Key Laboratory of Green Chemistry and Chemical Processes, Department

of Chemistry, School of Chemistry and Molecular Engineering, East China Normal

University, Shanghai 200241, People’s Republic of China

Material and methods

Chemicals and materialsEthylene glycol (EG, ≥ 99%) and NaCl (AR) were purchased from Sinopharm

Chemical Reagent Co., Ltd. (Shanghai, China). NH4F (AR, 98%), CuCl2·2H2O

(99.99% metals basis), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),

lysozyme, and sodium pyrophosphate were obtained from Aladdin Industrial Co. Ltd.

(Shanghai, China). Ln(NO3)3·xH2O (Ln = Gd, Yb, and Tm, 99.99% metals basis), PEI

(average MW ~25 000), sodium orthovanadate (Na3VO4), and glucose oxidase (GOD)

were purchased from Sigma-Aldrich. The Ln(NO3)3·xH2O were dissolved in EG to

form 0.5 mol/L Gd(NO3)3, Yb(NO3)3 and 0.1 mol/L Tm(NO3)3 solutions. ALP (Calf

intestine) was obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China).

Exonuclease I (EXO I) was purchased from Thermo Fisher Scientific Co. Ltd.

(Waltham, MA, USA). Bovine serum albumin (BSA) and trypsin were purchased

from Sangon Biotechnology Co., Ltd. (Shanghai, China). ALP enzyme-linked

immunosorbent assay (ELISA) kit was purchased from Beyotime Institute of

Biotechnology (Shanghai, China). All of these materials were used as received

without further purifications. HEPES buffer (10 mM, pH 7.4) was employed in the

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experiments. Deionized distilled water was used throughout the experiment.

ApparatusX-ray diffraction (XRD) pattern was characterized on a D8 Advance X-ray powder

diffractometer (Bruker, Karlsruhe, Germany). Transmission electron microscopy

(TEM) and high-resolution transmission electron microscopy (HRTEM) imaging was

performed using a HT-7700 transmission electron microscope (Hitachi, Tokyo,

Japan). Energy Dispersive X-ray Analysis (EDX) of the samples was performed

during the TEM determinations to obtain the elemental composition of the samples.

Fourier transform infrared (FTIR) spectra were recorded on a Thermo Scientific

Nicolet iS50 FTIR spectrometer. UV-vis absorption spectra were recorded by a UV-

vis spectrophotometer (UV-2550). Zeta potential and size distribution analysis were

tested on a Zetasizer Nano ZS90 (Malvern) instrument. Fluorescence spectra and

fluorescence lifetime were measured upon excitation with a 980 nm continuous-wave

(CW) laser (MDL-III-980 nm) using a FLS980 Fluorescence Spectrometer

(Edinburgh Instruments Ltd, UK). The cell images were obtained with a FV1000

confocal laser scanning upconversion luminescence microscope (CLSUCLM)

(OLYMPUS, Tokyo, Japan).

Synthesis of PEI-Capped NaGdF4:Yb/Tm UCNPsIn a typical experiment, PEI-capped NaGdF4:Yb/Tm UCNPs were synthesized by a

facile modified one-step solvothermal method with PEI as capping agent (Zhou et al.,

2011). In briefly, 0.4 g of PEI was dispersed in 18 mL of EG with vigorous stirring

and then 2.4 mmol of NaCl was added to the mixture. 1.2 mmol of lanthanide dopants

Gd(NO3)3, Yb(NO3)3, and Tm(NO3)3 with a molar ratio of 60 : 39.5 : 0.5 were added

to the above solution. The mixture was agitated for 30 min to form a transparent

solution. After that, 6.24 mmol of NH4F in 12 mL of EG was added to the above

mixture. Stirred for another 10 min, the solution was transferred into a 50 mL Teflon-

lined stainless steel autoclave and sealed. Then the autoclave was heated under 200

°C for 1.5 h and cooled to room temperature in air. After that, the as-synthesized

UCNPs were separated from the reaction mixture by centrifugation and washed

several times by ethanol and water. And then, the UCNPs were dried in vacuum at 60

°C for 12 h.

Fluorescent Detection of Cu2+

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Stock solutions of Cu2+ (0.2 mM) were prepared in deionized water. For

optimization the incubation time, 20 μL of Cu2+ (0.2 mM) was added into 2 mL of

UCNPs solution (100 μg/mL). Then, the fluorescence was recorded after different

shaking time. In a typical experimental procedure, a certain concentration of Cu2+ was

added to 2 mL UCNPs solution (100 μg/mL) using a micropipette. After 5 min

incubation, the fluorescence spectra were recorded at room temperature. All

experiments in this work were repeated three times.

Fluorescent Detection of PPi In order to optimize the incubation time of the interaction between PPi and Cu2+, 90

μL of PPi (0.2 mM) and 20 μL of Cu2+ (0.2 mM) were added to HEPES buffer (10

mM, pH 7.4). Then, 100 μL 2 mg/mL UCNPs solution was added to the above

mixture after different shaking time. In a typical procedure, the fluorescence spectra

were recorded after 5 min incubation at room temperature. As for PPi detection, 2 μM

Cu2+ and PPi with different concentration were added to HEPES buffer (10 mM, pH

7.4). After incubation for 5 min at room temperature, 100 μL 2 mg/mL UCNPs

solution was added to the above mixture. Then, the mixture was further incubated at

room temperature for 5 min.

Fluorescent Detection of ALP80 μL of PPi (0.2 mM) and varying amounts of ALP were added to HEPES buffer

(10 mM, pH 7.4), followed by reaction for 90 min at 37 °C according to the reported

procedure with minor modification.(Chen et al., 2014) After deactivation of the

enzyme for 10 min at 90 °C, 20 μL of Cu2+ (0.2 mM) was added to the reaction

system, followed by incubation for 5 min at room temperature to enable sufficient

coordination between PPi and Cu2+. Finally, 100 μL 2 mg/mL UCNPs solution was

added to the above reaction solution. Then, the mixture was incubated at room

temperature for 5 min.

Detection of PPi in human urineAll human urine samples were collected from healthy volunteers. PPi in human

urine samples was determined through the standard addition method. The urine

samples were centrifuged at 6,000 rpm for 20 min. For the fluorescent sensing of PPi

in human urine, 40-fold dilution urine samples pretreated were added to the sensing

system and the detection procedure was the same as the experiment for PPi detection

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in buffer.

Detection of ALP in serum samplesALP with different concentration was spiked in 1% diluted bovine serum. For ALP

detection in bovine serum, the detection procedure was the same as the experiment for

ALP detection in buffer except 15 μM Cu2+ and 10 μM PPi were used. All human

serum samples were collected from healthy volunteers. Then, each serum sample was

divided into two parts. One was tested by our proposed biosensor, and the other was

assayed by ALP ELISA Kit. To concentrate ALP, the serum samples were pretreated

by centrifugal filtration devices (Molecular weight cutoff or MWCO 10 kDa,

Millipore Amico Ultra) to centrifuge at 10, 000 rpm for 20 min at 4 ºC. The

subsequent operation is the same as the procedure for ALP detection in bovine serum.

Detection of ALP using the ELISA kit assay was conducted according to the

instructions of the ELISA kit and 9 steps were needed.

Cell cytotoxicityHeLa cells (5×104 cells/well) were cultured in RPMI 1640 medium supplemented

with 10% FBS (fetal bovine serum) and 1% penicillin−streptomycin at 37 °C and 5%

CO2 for 24 h. In vitro cytotoxicity was evaluated by methyl thiazolyl tetrazolium

(MTT) assays. Cells were seeded into 96-well cell culture plates at 5 × 104 per well,

and were cultured at 37 °C and 5% CO2 for 24 h. Different concentrations of PEI-

capped UCNPs (0, 20, 60, 80, 100, 200 μg/mL) were added into the wells. The cells

were subsequently incubated for another 24 h at 37 °C with 5% CO2. Then, MTT (10

μL, 5 mg/mL) was added to each well and the plate was incubated for an additional 4

h under the same conditions. Next, 100 μL dimethyl sulphoxide (DMSO) was added

to dissolve the formazan crystals. The optical density (OD) at 490 nm of each well

was measured using a microplate reader. The following formula was used to calculate

the cell viability,

Cell bioimagingBefore the experiments, HeLa cells were washed with phosphate-buffered saline

(PBS) buffer for three times, and then UCNPs (100 μg/mL) were added to the cell

culture for incubation 6 h at 37 °C. After that, HeLa cells were washed three times

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with the medium to remove the remaining UCNPs. The Cu2+ uptake was performed in

the same medium supplemented with 140 μM Cu2+ for 15 min. After washed three

times with fresh PBS buffer, HeLa cells were observed using a CLSUCLM under the

excitation of a CW980 nm laser, and the emission was collected from 450 to 500 nm.

Then the HeLa cells were further treated with 80 μM Cu2+ for additional 10 min, and

the fluorescence image was collected.

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Fig. S1. Characterization of the as-prepared UCNPs (A) TEM image (inset: the size

distribution of UCNPs), (B) XRD pattern (the line in the bottom is the standard

diffraction pattern of cubic α-NaGdF4 crystals (JCPDS no. 27－0697)), (C) HRTEM

image (inset shows the SAED pattern), and (D) luminescent spectrum under the

excitation of 980 nm.

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Fig. S2. Hydrodynamic diameter of PEI-capped UCNPs in water.

Fig. S3. EDX patterns of the α-NaGdF4:Yb/Tm UCNPs

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Fig. S4. Zeta potential of PEI-capped UCNPs measured in deionized water.

Fig. S5. FTIR spectrum of PEI-capped UCNPs.

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Fig. S6. The dynamic quenching of the fluorescence of UCNPs-based probe (0.1 mg/mL) in the presence of 2.0 μM Cu2+.

Fig. S7. The fluorescence spectra in the range of 400~900 nm with various

concentration of Cu2+.

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Table S1. Comparison of the performance of different nanomaterial-based fluorescent

nanopbrobes for Cu2+, PPi, and ALP detection

Nanomaterials Analyte LOD Liner range Reference

carbon quantum dots Cu2+ 10 nM 1-100 μM Qu et al. (2012)UCNPs Cu2+ 0.82 μM / Xu et al. (2016)

hybrid quantum dots Cu2+ 0.36 nM 0-100 nM Wang et al. (2016)PEI-capped UCNPs Cu2+ 57.8 nM 0.1-2 μM This work

Ag nanoclusters PPi 112.69 nM 0.25-10 μM Ma et al. (2016)carbon quantum dots PPi 2.56 μM 8.53-700 μM Qian et al. (2015)

Au nanoclusters PPi 2 μM 0-30 μM Chen et al. (2014)UCNPs PPi 184 nM 0.5-8 μM This workAgNPs ALP 1 U/mL / Wei et al. (2008)

CuInS2 quantum dots ALP 3.6 nU/mL 8.4-168 nU/mL Liu et al. (2014)Polymer NPs ALP 0.01 U/mL 0.025-0.2 U/mL Deng et al. (2015)

UCNPs ALP 0.019 U/mL 0.0625-0.875 U/mL This work

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Fig. S8. (A) UV-Vis absorption spectra of PEI-capped UCNPs (black line) and Cu2+-

PEI-UCNPs (red line). (B) UV-Vis absorption spectra (solid lines) of Cu2+, PEI, Cu2+

+PEI, and PL spectrum (dot line) of PEI-capped UCNPs.

Fig. S9. Fluorescence lifetimes of PEI-capped UCNPs (2 mg/mL) at 796 nm in the

absence and presence of Cu2+ with different concentrations, respectively.

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Fig. S10. The fluorescence spectra of (A) UCNPs, (B) UCNPs + Cu2+, (C) UCNPs +

Cu2+ + PPi, and (D) UCNPs + Cu2+ + PPi + ALP. [UCNPs] = 0.1 mg/mL, [Cu2+] = 2.0

μM, PPi = 9.0 μM, ALP = 3.0 U/mL.

Fig. S11. Optimization the incubation time for the interaction between PPi (9 μM) and

Cu2+ (2 μM).

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Fig. S12. Optimization of Cu2+ concentration in 1% diluted bovine serum. (A)

fluorescence spectra of NaGdF4:Yb/Tm UCNPs with various concentrations of Cu2+

(from top to bottom: 0, 1, 2, 4, 6, 8, 10, 12, 15, and 16 μM, respectively). (B)

fluorescence quenching efficiency (1－F/F0, F and F0 are the fluorescence intensities

at 796 nm in the presence and absence of Cu2+, respectively) against Cu2+

concentration (1−16 μM). Inset is the plot of linear region from 1 to 15 μM. The error

bars were calculated from three independent experiments.

Fig. S13. Optimization of PPi concentration for ALP detection in 1% diluted bovine

serum. (A) The fluorescence spectra of the sensing system containing different

concentrations of PPi in presence of 15 μM Cu2+. (B) fluorescence change ratio (F/F0

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－1) responses to the different concentrations of PPi. F and F0 are the fluorescence

intensities at 796 nm in the presence and absence of PPi, respectively. The error bars

were calculated from three independent experiments.

Table S2. Determination of PPi in urine samples

urine samplesNo.

Added

(μM)

Found (μM)

Recovery(%)

RSD(%)

1 01

1.2912.430

--106.07

5.264.07

2 02

1.0123.077

--102.16

6.156.04

3 0 1.538 -- 4.92

4 5.319 96.05 4.65

When PPi level was determined by using this probe, the urine samples were 40-fold

diluted for measurement; the values herein represent PPi levels in the diluted urine

samples.

Table S3. Recovery experiment for measurement ALP in 1% diluted bovine serum

Spiked amount (U/mL) Detected amount (U/mL) Recovery(%) RSD(%)

0.250 0.247 98.80 3.240.500 0.526 105.27 2.930.750 0.785 104.69 1.55

1.500 1.406 93.70 3.97

Table S4. Determination of ALP in human serum

serum

samplesNo.

ALP (U/mL) byELISA kit assay

ALP (U/mL) by this probe

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No. 1 0.092±0.005 0.095±0.006No. 2 0.182±0.008 0.163±0.066No. 3 0.107±0.006 0.113±0.008

Mean ± SD were used to describe the data (n = 3).

Fig. S14. Cell viabilities of HeLa cells incubated with PEI-capped UCNPs at different

concentration (0, 20, 60, 80, 100, 200 μg·mL−1) for 24 h.

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