DNA Labeling Generates a Unique Amplification Probe. HIV-1 p24 Antigen

Transcription

1 Supporting Information DNA Labeling Generates a Unique Amplification Probe for Sensitive Photoelectrochemical Immunoassay of HIV-1 p24 Antigen Wei-Wei Zhao, Ying-Mei Han, Yuan-Cheng Zhu, Nan Zhang, Jing-Juan Xu* and Hong-Yuan Chen* State key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing , China * To whom correspondence should be addressed. *Tel/Fax: xujj@nju.edu.cn. *Tel/Fax: hychen@nju.edu.cn Experimental section, synthesis of TGA-Stabilized CdTe QDs, fabrication of CdTe QDs modified electrode, and the immunoassay development process are included in this Supporting Information. S1

2 EXPERIMENTAL SECTION Materials and Apparatus. Indium tin oxide (ITO) with type of N-STN-S1-10 was purchased from China Southern Glass Holding Co., Ltd. (Shenzhen, China), with coating thickness of 180±20 nm and sheet resistance of 8.1±0.6 Ωcm -2. HIV-1 p24(ag), Mouse anti HIV-1 p24 McAb (Ab 1 and Ab 2 ), prostate specific antigen (PSA), carcino-embryonic antigen (CEA) and thrombin were obtained from Shanghai Linc-Bio Science Co., Ltd (Shanghai,China). Poly[G]-NH 2 (NH 2 - gggggtgggggtgggggtgggggtg) was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). N-(3-Dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC), N- hydroxysuccinimide (NHS), bovine serum albumin (BSA), thioglycolic acid (TGA), (3- aminopropyl)triethoxysilane(aptes) and poly(diallyldimethylammonium chloride) (PDDA; 20%, w/w in water, MW = ) were purchased from Sigma-Aldrich (Shanghai, China). Tetradecanedioic acid (DDCA) was purchased from Adamas Reagent Co.Ltd. SiO 2 nanospheres (Average diameter: 20 nm) and glutaradehyde were purchased from Aladdin (Shanghai, China); Monoethanolamine and Te powder were purchased from Sinopharm Chemical Reagent Co.,Ltd (Shanghai, China); Cadmium Chloride (CdCl 2 2.5H 2 O) was purchased from Shanghai Jin Shanting chemical reagent factory (Shanghai, China). Sodium borohydride was purchased from Tianjin Chemical Reagent Research Institute (Tianjin, China). Other chemicals were of analytical reagent grade and used as received. Sodium Phosphate buffer(pbs) (0.01 M, ph 7.4) was used for the preparation of antigen, antibody and Poly[G] solutions. The blocking buffer was 0.01 M PBS (ph 7.4) containing 3% (w/v) BSA. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). PEC measurements were performed with a homemade PEC system equipped with a 500W Xe lamp and a monochromator. Photocurrent was measured on a CHI 660c electrochemical workstation (China) with a three-electrode system: a modified ITO electrode with a geometrical area of 0.25 ± 0.01 cm 2 as the working electrode, a Pt wire as the counter electrode and a saturated Ag/AgCl electrode as the reference electrode. All the photocurrent measurements were performed at a constant potential of 0 V S2

3 (versus Ag/AgCl). The assay buffer was 0.01 M PBS (ph7.4). Transmission electron microscopy (TEM) were performed with a JEM model 2000CX instrument operating at 200 kv accelerating voltage. X-ray photoelectron spectroscopy (XPS) was carried out on PHI5000 VersaProbe (ULVAC-PHI Co., Japan). The UV vis absorption spectra were obtained on a Shimadzu UV-3600 UV vis-nir photospectrometer (Shimadzu Co.). Synthesis of TGA-Stabilized CdTe QDs and Fabrication of CdTe QDs modified Electrode. The utilized CdTe QDs was synthesized according to the previous report 1 with a slight modification. Briefly, CdCl 2 2.5H 2 O ( g) was dissolved in 50mL deionized water ml TGA stabilizer was added under vigorous stirring and then the ph was adjusted to 11 by dropwise addition of 2 M NaOH solution. In the process, the solution was continuously stirred. After reaching the proper ph, the solution became optically clear. Then Te powder ( g) was placed in a 50mL three-necked flask with 10mL deionized water. Excessive sodium borohydride ( g) was added under magnetic stirring and the protection of N 2. Finally, the lavender solution of NaTeH was prepared after two hour s stirring. Then H 2 SO 4 (0.5 M) was introduced to NaHTe to produce H 2 Te gas, which passed through the cadmium precursor with a slow N 2 flow for 30 min. CdTe precursors were formed at this stage. The molar ratio of Cd 2+ /TGA/HTe - was fixed at 1:4.4:0.5. Then the resulting mixture was subjected to reflux at 100 for 4 h. Finally the desired TGA-stabilized CdTe QDs were obtained and then stored in a refrigerator at 4 for further use. PDDA/CdTe multilayer film was grown by alternately dipping of the freshly cleaned ITO slices into a solution of 2% PDDA containing 0.5 M NaCl and the as-obtained CdTe QDs solution for 10 min, 2 respectively and this process was repeated three times to obtain desired photocurrent intensity. The films were carefully washed with doubly distilled water after each dipping step. Immunoassay Development. 1. Preparation of poly[g]/s-nps biological label (i.e., SiO 2 -Poly[G]-Ab 2 nanocomposites) The schematic illustration of the preparation is demonstrated in Scheme S1. First, 20 mg SiO 2 nanospheres with an average diameter of about 20 nm were dispersed in 2 ml of ethanol and treated S3

4 with 0.4 ml of APTES to produce amino-group on the surface of silica nanospheres. After stirring for 6h, the suspension was centrifuged and washed with ethanol for four times. Second, the aminofunctionalized silica nanopheres were incubated with 500 µl of 2.5% glutaradehyde for 2 h. After centrifugation and washing three times with PBS (0.01M, ph7.4), the resulting nanoparticles were redispersed in a solution of 450 µl containing 25 mm poly[g] and 50 µl of 1.75 mg/ml Ab 2. The solution was gently stirred to react for 10h at room temperature. The unbound poly-g and Ab 2 were removed by successively washing the SiO 2 nanoparticles with PBS (0.01 M, ph 7.4). At last, the obtained SiO 2 -poly[g]-ab 2 nanocomposites were treated with blocking buffer (1% Monoethanolamine Solution) for 24 h to block any nonspecific binding site on the surface. After centrifugation and washing three times, the prepared SiO 2 -poly[g]-ab 2 nanocomposites were redispersed in 1 ml of PBS (0.01 M, ph 7.4) and store at 4 for later experiments. Scheme S1. Procedure for preparing poly[g]/s-nps biological label. 2. Immobilization of Ab 1 and SiO 2 -poly[g]-ab 2 onto ITO substrates a. ITO substrates were pretreated to ensure an active hydroxyl surface layer. Pretreatment involved immersion of the substrates in a mixture of 5:1:1 H 2 O/H 2 O 2 (30%)/NH 3 (25%) for 1 h at 70. The glass substrates were then washed with copious amount of water and blown dry with nitrogen. 3 b. Carboxylic acid monolayers were electrostatically bound by immersion of the pretreated ITO substrates in 5 mm DDCA in ethanol for h. The functionalized substrates were thoroughly rinsed in ethanol and PBS (0.01 M, ph 7.4) to remove weakly bound species. 3 The DDCA functionalized ITO substrates were activated by immersion in 0.7 ml solution containing 14 mg EDC and 7 mg NHS for 1 h at room temperature, followed by thoroughly rinsing with PBS (0.01 S4

5 M, ph 7.4) to wash off the excess EDC and NHS. Subsequently, 25 µl of 0.5 mg/ml Ab 1 was spread onto the resulting electrode surface at 4 in a moisture atmosphere to avoid evaporation of solvent. After incubation for 16 h, the electrode was rinsed with PBS (0.01 M, ph 7.4) to remove physically absorbed Ab 1. The electrodes were then block with 25 µl of blocking solution for 2 h at 4 to block nonspecific binding sites and washed with the PBS (0.01 M, ph 7.4) thoroughly. 2 c. Next, 25 µl of HIV p24-ag with different concentrations (0.01 µg/ml,0.1 µg/ml,1 µg/ml,10 µg/ml,100 µg/ml) was dropped onto the Ab 1 modified electrodes for an incubation of 60 min at 37 followed by washing with PBS (0.01 M, ph 7.4). 2 d. After binding reaction between Ab 1 and Ag, the electrodes were allowed for labeling by additional incubation with 25 µl of SiO 2 -Poly[G]-Ab 2 nanocomposites solution for 60 min at 37 and again the electrodes were washed thoroughly with PBS (0.01 M, ph 7.4) to remove nonspecifically bound conjugations. In control, we dropped the silicon nanoprobes on the electrode directly and then rinsed by PBS thoroughly, the final PEC test could not observe obvious signal change. 3. The detection of HIV-1 p24 Antigen Two Ab 1 -Ag-Ab 2 -poly[g] modified ITO electrodes were immersed in one tube with 0.5 ml 0.1 M NaOH in it in order to enhance the final electrical signal. The solution was heated to 80 to release the DNA maker. After 3 h, the electrodes were taken away from the solution and 0.5 ml 3 M H 2 SO 4 was added in the said solution. 4 The acid dipurinization proceeded by heating to 80 for 3 h. Then 6 M NaOH was added to neutralize the acid. Finally 0.01 M PBS (ph 7.4) was added to the solution until the total volume of the solution was 2 ml. The solution was used for the PEC measurement of the released purine nucleobases. S5

6 Specificity experiments The method is described as above. The deference is 2-c. 25 µl 10 µg/ml PSA, CEA, Thrombin, and HIV p24-ag were dropped onto the Ab 1 modified electrodes respectively for an incubation of 60 min at 37 followed by washing with 0.01 M PBS (ph7.4). S6

7 REFERENCES 1) Fan. X.Q.; Peng J.J.; Yan S.G.; Wang, L.; He Y.Q. J. Lumin. 2011, 131, ) Zhao, W. W.; Ma, Z. Y.; Yu, P. P.; Dong, X. Y.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, ) Hedges D. H. P.; Richardson D. J.; Russell D.A. Langmuir 2004, 20, ) Wang J.; Liu G.D.; Munge B.; Lin L.; Zhu Q.Y. Angew. Chem. Int. Ed. 2004, 43, S7