Supporting Information A Turn-On Fluorescent Sensor for Selective and Sensitive Detection of Alkaline Phosphatase Activity with Gold Nanoclusters Based on Inner Filter Effect Haijian Liu a, Ming Li a, Yining Xia b *, Xueqin Ren a * a Department of Environmental Science and Engineering, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China b Institute of Quality Standards and Testing Technology for Agro Products of Chinese Academy of Agricultural Sciences, Beijing 100081, China *Corresponding author: Dr. Yining Xia Institute of Quality Standards and Testing Technology for Agro-Products of Chinese Academy of Agricultural Sciences, Beijing 100081, China E-mail: ynxia010@hotmail.com Prof. Xueqin Ren Department of Environmental Sciences & Engineering, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, P. R. China Tel: 86-10-62733407; Fax: +86-10-62731016; E-mail: renxueqin@cau.edu.cn 1
Table of Contents: 1. Quantum yield measurement of AuNCs 2. Estimation of the concentration of the as-prepared AuNCs 3. Figure S1. The photostability of the AuNCs measured by fluorescence spectrophotometer every 5 min 4. Figure S2. Fluorescence intensity of AuNCs upon addition of various concentrations of PNPP from 0 to 1000 µm. 5. Figure S3. Effect of PNPP on the fluorescence lifetime of AuNCs 6. Figure S4. Detection of ALP in three adult volunteers serums by the clinic method and our method. 7. Table S1.Comparison of analytical performance of some assays for ALP detection 2
Quantum yield measurement The quantum yield (Φ) of AuNCs was determined by comparing the integrated fluorescent intensities (excitation at 330 nm) and absorbance values (at 330 nm) of the AuNC samples with those of rhodamine B. The quantum yield was calculated using equation (1), where Φ is the quantum yield, k is slope, η is the refractive index of the solvent, S is the standard and X is the sample. Φ Φ K K η η 1 Estimation of the concentration of the as-prepared AuNCs The number of Au in the HAuCl 4 solution: 0.005M 25 ml 6.02 10 23 = 7.525 10 22 According to previous literature 10, AuNCs stabilized with MUA consisted of 25 Au atoms. Therefore, mole of the as-prepared AuNCs can be estimated as below: 7.525 10 22 (total number of Au atom in solution) 25 (number per AuNC) 6.02 10 23 = 0.005 mole Molar concentration of the as-prepared AuNCs can be calculated as below: 0.005 mole 81.25 ml = 6.15 10-5 M Figure S1 The photostability of the AuNCs measured by fluorescence spectrophotometer every 5 min ( λ ex = 330 nm, λ em = 600 nm ) Figure S2 Fluorescence intensity of AuNCs upon addition of various concentrations of PNPP from 0 to 1000 µm. 3
Figure S3. Effect of PNPP on the fluorescence lifetime of AuNCs (1.2 10 6 mol L 1, ph 9.0). (a) AuNCs (τ = 5.11 µs); (b) AuNCs in the presence of PNPP (τ = 4.85 µs). Figure S4. Detection of ALP in three adult volunteers serums by the clinic method and our method. A 50 µl of serum sample was added to Tris-HCl buffer solution (10 mm, ph 9.0) containing 1 mm PNPP and 0.1 µm MgSO 4 and then incubated at 37 C for 50 min. The reaction solution was mixed with 1 ml of AuNCs. The fluorescence spectrum was measured with the excitation at 330 nm. Table S1.Comparison of analytical performance of some assays for ALP detection Analysis methods Linear LOD range (U/L) (U/L) References cerium oxide nanoparticles-based colorimetric assay 0-2 0.04 (1) CuInS 2 QDs-based fluorescence 8.4-168 3.6 nu/ml nu/ml (2) CDs-based fluorescence 16.7-782.6 1.1 (3) CDs-based fluorescence 4.6-383.3 1.4 (4) Gold nanoclusters-based fluorescent 0.1 - probe mu/ml (5) 4
Gold nanoclusters-based fluorescent probe CDs-based fluorescence 0.01-450 0.002 (6) 0.01-25 0.001 (7) fluorescent dye fluorescence 0-2.8 U/mL 0.06 U/mL (8) Betaine-modified PEI-based ratiometric fluorescent probe Gold nanoclusters-based fluorescent probe - 0.1 U/ml (9) 0.02-50 0.002 This work 5
References: (1) Hayat, A.; Bulbul, G.; Andreescu, S. Probing Phosphatase Activity Using Redox Active Nanoparticles: A Novel Colorimetric Approach for the Detection of Enzyme Activity. Biosens. Bioelectron. 2014, 56, 334-339. (2) Liu, S. Y.; Pang, S.; Na, W. D.; Su, X. G. Near-infrared Fluorescence Probe for the Determination of Alkaline Phosphatase. Biosens. Bioelectron. 2014, 55, 249-254. (3) Qian, Z. S.; Chai, L. J.; Huang, Y. Y.; Tang, C.; Shen, J. J.; Chen, J. R.; Feng, H. A Real-time Fluorescent Assay for the Detection of Alkaline Phosphatase Activity Based on Carbon Quantum Dots. Biosens. Bioelectron. 2015, 68, 675-680. (4) Qian, Z. S.; Chai, L. J.; Tang, C.; Huang, Y. Y.; Chen, J. R.; Feng, H. Carbon Quantum Dots-Based Recyclable Real-Time Fluorescence Assay for Alkaline Phosphatase with Adenosine Triphosphate as Substrate. Anal. Chem. 2015, 87, 2966-2973. (5) Chen, Y.; Li, W. Y.; Wang, Y.; Yang, X. D.; Chen, J.; Jiang, Y. N.; Yu, C.; Lin, Q. Cysteine-directed Fluorescent Gold Nanoclusters for the Sensing of Pyrophosphate and Alkaline Phosphatase. J. Mater. Chem. C. 2014, 2, 4080-4085. (6) Hu, X. L.; Wu, X. M.; Fang, X.; Li, Z. J.; Wang, G. L. Switchable Fluorescence of Gold Nanoclusters for Probing the Activity of Alkaline Phosphatase and Its Application in Immunoassay. Biosens. Bioelectron. 2016, 77, 666-672. (7) Li, G. L.; Fu, H. L.; Chen, X. J.; Gong, P. W.; Chen, G.; Xia, L.; Wang, H.; You, J. M.; Wu, Y. N. Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots Based on Inner Filter Effect. Anal. Chem. 2016, 88, 2720-2726. (8) Dong, L.; Miao, Q. Q.; Hai, Z. J.; Yuan, Y.; Liang, G. L. Enzymatic Hydrogelation-Induced Fluorescence Turn-Off for Sensing Alkaline Phosphatase in Vitro and in Living Cells. Anal. Chem. 2015, 87, 6475-6478. (9) Zheng, F. Y.; Guo, S. H.; Zeng, F.; Li, J.; Wu, S. Z. Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on Betaine-Modified Polyethylenimine via Excimer/Monomer Conversion. Anal. Chem. 2014, 86, 9873-9879. (10) Wu, Z, Suhan, J, Jin, R, One-Pot Synthesis of Atomically Monodisperse, Thiol-Functionalized Au25 Nanoclusters. J. Mater. Chem. 2009, 19, 622-626. 6