Supporting Information

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1 Supporting Information Enzyme-Induced Metallization as a Signal Amplification Strategy for Highly Sensitive Colorimetric Detection of Avian Influenza Virus Particles Chuan-Hua Zhou, Jing-Ya Zhao, Dai-Wen Pang and Zhi-Ling Zhang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan , P. R. China Wuhan Institute of Biotechnology, Wuhan , P. R. China S1 The sensing mechanism of the biometallization-based colorimetric assay Control experiments indicated that p-ap could reduce Ag + in the presence of AuNPs, which displayed a rapid and significant color change as shown in Table S1. No appreciable color change was obtained in the absence of ALP, p-app or Ag +. This indicated that sliver deposition was attributed to the enzyme-mediated generation of p-ap. The whole reaction could be described by the following equations. (1) (2) zlzhang@whu.edu.cn. Fax: Tel.: S1

2 Table S1 Control experiments of the biometallization-based colorimetric detection. The reagents added to each tube are indicated below the images. ALP (50 pm) AgNO 3 (2 mm) p-app (8 mm) AuNPs p-ap (0.1 mm) S2

3 The reduction reaction of Ag + by p-ap could be further demonstrated by the time-dependent plots as shown in Figure S1. In the presence of p-ap, A 370 increased dramatically in several seconds which indicated the fast reaction kinetics of the reduction reaction in the presence of AuNP seeds. Figure S1. Time-dependent plots of absorption intensity at 370 nm of AuNPs/Ag + solution in the presence and absence of 20 μm p-ap. The presence of AuNPs could greatly enhance the enzyme-induced metallization reaction. Figure S2. Absorption spectra in the presence or absence of AuNPs in the detection solution which containing 1 pm ALP, 8 mm p-app and 2 mm AgNO 3 at 37 C for 10 min, ph = 9.8. S3

4 The reduction reaction of Ag + by p-ap was also investigated in the presence of Na 3 VO 4 to confirm that this reaction could not be influence by Na 3 VO 4. As shown in Figure S3, Ag + could be reduced by p-ap in several seconds in the presence of 10 mm Na 3 VO 4, and under the same condition without p-ap, Ag + couldn t be reduced. This phenomenon was similar to the situation in the absence of Na 3 VO 4 (Figure S1). The photograph result (Figure S3B) also indicated that the presence of Na 3 VO 4 couldn t influence the reduction reaction of Ag +. Figure S3. (A) Time-dependent plots of absorption intensity at 370 nm of Na 3 VO 4 /AuNPs/Ag + solution in the presence and absence of 20 μm p-ap. (B) Photograph of AuNPs/Ag + solution in the presence and absence of p-ap and Na 3 VO 4. S4

5 Figure S4. Absorption intensity at 370 nm versus Na 3 VO 4 concentration. S5

6 S2 The influence parameters for the enzyme-induced silver deposition reaction. Figure S5. The effects of (A) p-app concentration, (B) Ag + concentration, (C) ph, (D) DEA concentration and (E) reaction temperature on absorbance of detection solution at 370 nm. S6

7 S3 Comparison with other assay method for ALP detection. Table S2 Brief summary of the results reported on several biosensors for the detection of ALP. Detection method Materials or method Detection range Detection limit Complex samples Colorimetric assay Gold nanoparticle 0.3~6.8 μm 0.3 μm - 1 Gold nanoparticle 16 nm~1μm 16 nm - 2 Reference Silver nanoparticle - 1 U/mL 1% fetal calf serum 3 Fluorescence Assay Conjugated Polyelectrolyte 5~300 nm 5 nm - 4 Tetracationic perylene probe U/mL A549 cell lysates 5 Double strand DNA template fluorescent copper nanoparticles 0.1~2.5 nm 0.1 nm 1% human serum 6 CdSe/ZnS Quantum Dots unit - 7 Anodic Electrochemiluminescence CdSe Nanoparticles 0.5~ 6.4 nm 0.5 nm 0.1% serum samples Electrochemical assay Tyrosinase and horseradish peroxidase bienzyme ~ M Surface-Enhanced Raman Spectroscopy Gold nanoparticles as a SERS material ~ M Colorimetric assay Ag/Au NPs 0.05~20 pm 12fM ~ fm fm - 10 U/mL 2.5% human serum This method 8 S7

8 S4 Detection of ALP in human serum samples. Table S3. Analytical results for the determination of ALP in human serum samples. The human serum samples were diluted with DEA buffer for 40 times. Added (pm) Found (pm) Mean recovery RSD (n=3) Sample % % 3.44% % 3.21% Sample % % 2.04% % 2.50% S8

9 S5 Comparison with other viral immunosensor proposed in literatures. Table S4 Brief summary of the results reported on several immunosensors for the detection of viral pathogens. Detection method Analyte Detection range Detection Complex samples Referen time ce ELISA H5N1 >1 ng ml -1 2h Tracheal swabs 11 Magnetic silica nanoparticles based resonance light scattering Electrochemical Magnetoimmunosenor Avian influenza virus 0.5~50 ng ml -1 80min Chicken serum and saliva H9N2 avian influenza virus >10 pg ml -1 1h Chicken dung Electrochemical immunoassay H1N1 avian influenza virus ~10-6 g ml -1 4h - 14 Electrochemical immunoassay Avian leukosis viruses to TCID 50 ml Optofluidic nanoplasmonic Biosensor PT-ebola and vaccinia virus 10 6 to 10 9 PFU ml -1 90min cell growth medium, 7% fetal calf serum Non-Faradic impedance immunosensor Surface plasmon resonance immunosensor Homogeneous fluorescence immunoassay Dual-color fluorescence and homogeneous immunosensor Japanese encephalitis virus 1~10 μg ml -1 20min - 17 Influenza virus 0.5~10 μg ml -1 - Serum 18 Enterovirus 71 (EV71) and Coxsackievirus B3 (CVB3) 1~15 ng ml 1 (EV71) 1~14 ng ml 1 (CVB3) 16 15min Human throat swabs 19 Enterovirus 71 (EV71) 1.8 ng ml -1 ~10 μg ml -1 15min 0.16 mg ml -1 BSA 20 Piezoelectric immunochip Dengue virus >1.727μg ml -1 (E protein) >0.740μg ml -1 (NS-1 protein) diluted serum with pretreatment Colorimetric magnetoimmunosensor H9N2 avian influenza virus 20 pg ml -1 to 1 ng ml h Chicken heart, chicken dung and chicken serum 21 This method S9

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