SUPPORTING INFORMATION

Transcription

1 SUPPORTING INFORMATION Wet NH 3 -Triggered NH 2 -MIL-125(Ti) Structural Switch for Visible Fluorescence Immunoassay Impregnated on Paper Shuzhen Lv, Yun Tang, Kangyao Zhang and Dianping Tang, * Key Laboratory for Analytical Science of Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou , P. R. China. Grinnell College, th Avenue, Grinnell, Iowa 50112, United State. CORRESPONDING AUTHOR INFORMATION Phone: ; fax: ; dianping.tang@fzu.edu.cn (D. Tang) S-1

2 TABLE OF CONTENTS Material and Reagent...S3 Preparation of MIL-125-NH 2...S3 Preparation of Gold Nanoparticles...S4 Preparation of GDH-AuNP-Ab 2...S4 Immunoreaction and Visual Fluorescence Measurement...S4 Optimization of Experimental Conditions...S6 Figure S1...S7 Figure S2...S7 Table S1...S8 References...S8 S-2

3 EXPERIMENTAL SECTION Material and Reagent. Human carcinoembryonic antigen (CEA) enzyme-linked immunosorbent assay (ELISA) kits (cat#: ab183365) and monoclonal mouse anti-human CEA capture antibody (Ab 1 ; cat#: ab130880) was purchased from Abcam (Shanghai, China). Polyclonal goat anti-human CEA antibody (Ab 2 ) was achieved from ImmunoReagents Inc. (Raleigh, NC). Sodium glutamate, bovine serum albumin (BSA) and NAD + were acquired from Sangon Biotech. Co., Ltd. (Shanghai, China). Glutamate dehydrogenase (GDH) was gotten from Sigma-Aldrich (USA). 2-Aminoterephthalic acid (H 2 ATA), methanol (MeOH), N,N-dimethylformamide (DMF) and tetra-n-butyl titanate [Ti(OC 4 H 9 ) 4 ], gold(iii) chloride trihydrate (HAuCl 4 3H 2 O) and sodium citrate dihydrate (Na 3 C 6 H 5 O 7 2H 2 O) were purchased from Aladdin (Shanghai, China). All other reagents were of analytical grade and were used without further purification. Ultrapure water obtained from a Millipore water purification system was used in all runs (18.2 MΩ cm, Milli-Q). Clinical serum samples were made available by Fujian Provincial Hospital, China. A ph 9.6 carbonate buffer (1.69 g Na 2 CO 3 and 2.86 g NaHCO 3 ), a ph 9.8 carbonate buffer (2.33 g Na 2 CO 3 and 2.35 g NaHCO 3 ), and a ph 7.4 phosphate-buffered saline (PBS, 0.01 M) (2.9 g Na 2 HPO 4 12H 2 O, 0.24 g KH 2 PO 4, 0.2 g KCl and 8.0 g NaCl) were prepared by adding the corresponding chemicals in 1000-mL distilled water, respectively. The blocking buffer and washing buffer were obtained by adding 1.0 wt % BSA and 0.05% Tween 20 (v/v) in PBS, respectively. Preparation of NH 2 -MIL-125(Ti). NH 2 -MIL-125(Ti)-based metal-organic framework (MOF) materials was prepared consulting to the previous report with minor modification. 1 NH 2 -MIL-125(Ti) consisted of Ti 8 O 8 (OH) 4 -(O 2 CC 6 H 5 -CO 2 -NH 2 ) 6 (as a basic unit) and six cyclic octamers Ti 8 O 20 (OH) 4 at the corner, which was connected by eight linkers. Initially, 1.35 g of H 2 ATA was dissolved in anhydrous DMF (22.5 ml) under vigorous stirring. Then, MeOH (2.5 ml) and Ti(OC 4 H 9 ) 4 (0.65 ml, 1.88 mmol) were added into the mixture in turn. After being stirred for 30 min at room temperature, the resulting mixture was transferred into a 50-mL Teflon-lined autoclave, and heated for 5 days at 150 C. Following that, the resultant yellow product was soaked into MeOH for one day, and washed for three time with MeOH. S-3

4 Finally, the obtained precipitate was dried in an oven at 50 C for further use. Preparation of Gold Nanoparticles. All glassware used in the following procedures was cleaned in a bath of freshly prepared solution (3 : 1 K 2 Cr 2 O 7 -H 2 SO 4 ), thoroughly rinsed with double distilled water, and dried prior to use. Firstly, 1.0 ml of HAuCl 4 3H 2 O aqueous solution (1.0 wt %) was mixed with 99-mL ultrapure water and heated to boiling under vigorously stirring. Thereafter, 0.75 ml of trisodium citrate (1.0 wt %) was quickly added to the mixture and kept heating for another 15 min. At the same time, the color of the solution changed from yellow-gray to red. Subsequently, the resulting product was purified by centrifugation (10 min, g). Finally, the obtained precipitate (gold nanoparticle: AuNP) were dispersed into ultrapure water, and stored at 4 C for use. Preparation of GDH-AuNP-Ab 2. Glutamate dehydrogenase (GDH) and polyclonal rabbit anti-human CEA detection antibody (Ab 2 )-labeled gold nanoparticle (GDH-AuNP-Ab 2 ) were prepared on the basis of the electrostatic and hydrophobic interactions between proteins and gold nanoparticles. 2 The process mainly consisted of the following steps: (i) gold colloids (5.0 ml, 60 nm in diameter, 5.0 ng ml -1 ) were adjusted to ph 9.5 by using 0.1 M Na 2 CO 3 aqueous solution; (ii) 200 μl GDH (0.5 mg ml -1 ) and 50 μl Ab 2 antibody (0.5 mg ml -1 ) were injected into colloidal gold nanoparticles and gently shaken for 60 min at room temperature on a shaker (MS, IKA GmbH, Staufen, Germany); (iii) 100 μl polyethylene glycol (1.0 wt %) was added into the suspension and the mixture was further incubated for 12 h at 4 C; and (iv) GDH-AuNP-Ab 2 conjugates were obtained by centrifugation at 4 C (10 min, g), and dispersed in 2.0-mL PBS, ph 7.4, containing 1.0 wt % BSA and 0.05% NaN 3 for the subsequent use. Immunoreaction and Visual Fluorescence Measurement. The immunoreaction was carried out in monoclonal mouse anti-human CEA capture antibody (Ab 1 )-coated microtiter plate with a sandwiched detection mode by using GDH-AuNP-Ab 2 as the detection antibody. Prior to measurement, paper-based analytical device (PAD) was constructed by immobilizing NH 2 -MIL-125(Ti) on paper. The chromatography paper (3.0 MM CHR, cm, cat# , Whatman, U.K.) was initially cut into many circular paper sheets with 6.0 mm in diameter by using a Deli hole puncher (No. 0102, Zhejiang, Chana) (note: The size was almost the same as that of the lid stopper). Thereafter, the round papers were immersed into S-4

5 the above-prepared NH 2 -MIL-125(Ti) suspension (5.0 mg ml -1 in alcohol) for 90 min under slight shaking under a shaker [note: For the sake of good reproducibility and precision, each circular paper was incubated with NH 2 -MIL-125(Ti) suspension alone]. Afterward, the yellow papers were taken out and dried in a vacuum at 37 C, followed by put into the plastic lid (stopper) for subsequent use. Next, the Ab 1 -coated microplates were prepared as follows. A high-binding polystyrene 96-well microtiter plates (Ref , Greiner, Frickenhausen, Germany) were coated overnight at 4 C with 50 µl per well of Ab 1 at a concentration of 10 μg ml -1 in 0.05 M sodium carbonate buffer (ph 9.6). The microplates were covered with adhesive plastics plate sealing film to prevent evaporation. On the following day, the plates were washed three time with the washing buffer, and then incubated with 300 µl per well of blocking buffer for 1 h at 37 C with shaking. The plates were then washed as before. Following that, 50 μl of CEA standards or samples with various concentrations were added into the microtiter plates, and incubated for 1 h at 37 C under shaking. After washing, 50 µl of the prepared-above GDH-AuNP-Ab 2 was added into the well and incubated for 1 h at 37 C with shaking. The plates were washed again, 100 µl of the mixture including sodium glutamate (10 mm) and NAD + (1.5 mm) was added to the wells, followed by the as-prepared plastic stoppers to seal the wells for construction of a closed system. After reaction for 100 min with GHD, 50 μl of NaOH (10 M) was injected into the wells by a sharp injector through the stopper and reacted for 40 min at 40 C. Finally, NH 2 -MIL-125(Ti)-impregnated papers were transferred to a UV test box to accomplish visual qualitative detection under a 365-nm UV light with a portable smartphone. Meanwhile, the fluorescence intensity of the resulting papers were quantitatively determined on a Hitachi F-4600 fluorescence spectrometer (Tokyo, Japan) via putting the paper in a solid sensing cell on a solid sample holder. The change of the fluorescence intensity relative to background signal were collected and registered as the sensing signal toward target CEA by using the Stern-Volmer equation (I 0 /I = K sv [Q] + C, where I 0 and I stand for the fluorescence intensity of QD-GOD-impregnated paper before and after reaction with the analyte, respectively, K SV for the Stern-Volmer quenching constant, and Q for the concentration of target CEA). All determinations were made at least in duplicate. The sigmoidal curves were calculated by mathematically fitting experimental points using the S-5

6 Rodbard s four parameter function with Origin 6.0 software. Graphs were plotted in the form of absorbance against the logarithm of CEA concentration. PARTIAL RESULTS AND DISCUSSION Optimization of Experimental Conditions. To acquire optimum analytical properties, some experimental conditions including the concentrations of sodium glutamate concentration and NAD + for catalytic reaction should be investigated. In this case, 1.0 ng ml -1 CEA was used as an example to evaluate the optimal experimental conditions. Typically, the change in the fluorescence signal through the as-produced wet NH 3 mainly depends on the amount of glutamic acid. Therefore, the sodium glutamate concentration for the GDH-based catalytic reaction plays a very important role in the sensitivity of the immunoassay. As shown in Figure S2-A, the fluorescence intensity initially increased with the increment of sodium glutamate concentrations, and then slightly decreased when the concentration was more than 10 Mm. The reason was mainly attributed to the inhibition of enzymatic activity under relatively high substrate concentration. Therefore, 10 mm of sodium glutamate was used for the catalytic reaction of GDH. As described above, the visible fluorescence signal originated from the structural change of NH 2 -MIL-125(Ti)-based MOF materials after interaction with the wet NH 3. Generally spoken, it takes some time for the reaction of NH 2 -MIL-125(Ti) with wet NH 3. A short incubation time was unfavorable for the generation of fluorescence signal. As seen from Figure S2-B, the fluorescence intensity (I 0 /I) increased with the increasing reaction time, and tended to level off after 40 min. A longer incubation time did not significantly cause the increase in the signal. To save the assay time, 40 min was selected for the interaction of NH 2 -MIL-125(Ti) with wet NH 3 in this work. S-6

7 Figure S1. TEM image of the as-synthesized gold nanoparticles. Figure S2. Effects of (A) sodium glutamate concentration, and (B) reaction time of the NH 2 -MIL-125(Ti) with wet NH 3 on the fluorescence intensity of visible fluorescence immunoassay (1.0 ng ml -1 CEA used as an example in this case) S-7

8 Table S1. Comparison of the assayed results for human serum specimens by using NH 2 -MIL-125(Ti)-based visible fluorescence immunoassay and commercial human CEA ELISA kits method; concn [mean ± SD (RSD), ng ml -1, n = 3] sample no. PDA fluorescence immunoassay CEA ELISA kit t exp ± 0.17 (5.12%) 3.38 ± 0.12 (3.55%) ± 0.13 (8.61%) 1.63 ± 0.06 (3.68%) ± 1.84 (10.29%) ± 1.96 (10.76%) ± 0.10 (4.03%) 2.40 ± 0.08 (3.33%) ± 0.06 (6.32%) 0.88 ± 0.07 (7.95%) ± 0.90 (8.35%) ± 1.02 (9.60%) ± 0.29 (3.47%) 8.17 ± 0.37 (4.53%) 0.66 References (1) Li, X.; Pi, Y.; Hou, Q.; Yu, H.; Li, Z.; Li, Y.; Xiao, Y. Amorphous TiO 2 -MIL-125(Ti) homologous MOF-encapsulated heterostructures with enhanced photocatalytic activity. Chem. Commun. 2018, 54, (2) Gao, Z.; Xu, M.; Hou, L.; Chen G.; Tang, D. Magnetic bead-based reverse colorimetric immunoassay strategy for sensing biomolecules. Anal. Chem. 2013, 85, S-8