Spectrum-resolved Dual-color Electrochemiluminescence Immunoassay for Simultaneous Detection of Two Targets with Nanocrystals as Tags
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1 Supporting Information Spectrum-resolved Dual-color Electrochemiluminescence Immunoassay for Simultaneous Detection of Two Targets with Nanocrystals as Tags Guizheng Zou, *, Xiao Tan, Xiaoyan Long, Yupeng He, and Wujian Miao, * School of Chemistry and Chemical Engineering, Shandong University, Jinan, , China Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS 39406, USA Contents (a) Chemicals and Materials (b) Electrochemical characterization for fabrication procedures... 4 (c) Potential resolved ECL characterization for fabrication procedures... 6 (d) Calibration curves for simultaneous determination of CEA and AFP... 7 (e) References... 7 * To whom correspondence should be addressed. Tel: ; fax: ; zouguizheng@sdu.edu.cn; wujian.miao@usm.edu S-1
2 (a) Chemicals and Materials. All chemical reagents were of analytical grade or better and used as received, and all aqueous solutions were prepared with doubly distilled (DD) water. Polysorbate 20 (Tween-20) and p-aminobenzoic acid (ABA) were purchased from Kermel Chemical Reagent Co., Ltd. (Shanghai, China). Mercaptopropionic acid (MPA), bovine serum albumin (BSA), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) were purchased from Sigma-Aldrich Chemicals Co. LLC. (St. Louis, MO, U.S.A.). Sodium hexametaphosphate (HMP) and potassium phosphate monobasic (KH 2 PO 4 ) were purchased from Guangcheng Chemical Reagent Co., Ltd. (Tianjin, China). Sodium selenite pentahydrate (Na 2 SeO 3 5H 2 O) was purchased from Tianjin Chemical Reagent Research Institute (Tianjin, China). Cadmium chloride (CdCl 2 2.5H 2 O), ammonium persulfate ((NH 4 ) 2 S 2 O 8 ), hydrazine hydrate (N 2 H 4 H 2 O), and N-hydroxysuccinimide (NHS) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Potassium ferricyanide (K 3 [Fe(CN) 6 ]), potassium hexacyanoferrate (K 4 [Fe(CN) 6 ]), dipotassium hydrogen phosphate trihydrate (K 2 HPO 4 3H 2 O), and potassium chloride (KCl) were purchased from Xilong Chemical Co., Ltd. (Shantou, China). Preparation of dual-stabilizers-capped CdSe NCs. CdSe NCs were obtained via green one-pot synthesizing strategy reported previously. 1,2 Briefly, CdCl 2 (0.20 M, 0.80 ml), HMP (72.5 mg), and MPA (34.6 μl) were added and dissolved in 50 ml of DD water successively under thorough stirring. After 6 M NaOH was added to adjust its ph to 8.0, Na 2 SeO 3 (20.0 mm, 0.80 ml) was subsequently added to the mixture, followed by refluxing for 10 min. Then, 3.67 ml of N 2 H 4 H 2 O was added into this mixture and refluxed for 10 h. The resulting products CdSe NCs were centrifuged with isopropanol at 12,000 rpm for three times, then re-dispersed in DD water and stored at 4 C. The concentration of CdSe NCs stock solution was calculated to be 6.87 μm with an empirical equation. 2,3 Preparation of dual-stabilizers-capped CdTe NCs. Dual-stabilizers-capped CdTe NCs were prepared according to the previous reports, 4,5 which was similar to the way of preparing dual-stabilizers-capped CdSe NCs. Briefly, CdCl 2 (0.20 M, 0.80 ml), HMP (293.6 mg), and MPA (34.6 μl) were added and dissolved in 50 ml of DD water successively. After the solution ph was adjust to 8.0 with 6 M NaOH, 1.20 ml of 20.0 mm Na 2 TeO 3 was added to the mixture, followed by refluxing for 10 min. S-2
3 After being introduced in N 2 H 4 H 2 O (2.4 ml), the final mixture was refluxed for 25 h to obtain CdTe NCs that showed a PL peak at ~776 nm. The purification was done with centrifugation at 12,000 rpm for three times, then the NCs were re-dispersed in DD water and stored at 4 C before use. The concentration of CdTe NCs stock solution was calculated to be μm with an empirical equation. 5,6 Preparation of NCs Labeled Ab 2, i.e., Ab 2 NCs conjugates. The Ab 2 NCs conjugates were prepared with NHS and EDAC assisted labeling protocol. 5,7 For example, the CdSe NCs labeled Ab 2(CEA), i.e., Ab 2(CEA) CdSe, was prepared by the binding reaction between the carboxylic groups of CdSe NCs and amino groups of Ab 2 in the presence of EDAC and NHS. 7 The carboxylic groups of CdSe NCs were first activated with EDAC (100 mg/ml) and NHS (100 mg/ml) for 30 min at room temperature. Then, the excess EDAC and NHS were removed with centrifugation, and the resultant precipitates, i.e., the activated CdSe NCs, were re-dispersed in isovolumetric 10 mm ph 7.4 PBS and incubated with 10 μg/ml CEA-Ab 2 under a 30 ºC water bath for 3 h to form Ab 2 CdSe bio-conjugates. Finally, 10 μl of 0.1% (v/v) BSA solution (ph 7.4) was used to block the nonspecific binding sites of the Ab 2(CEA) CdSe conjugates, and the conjugates were separated using centrifugation at 12,000 rpm for 6 min. The CdTe NCs labeled Ab 2(AFP), i.e., Ab 2(AFP) CdTe were prepared in a way similar to that of Ab 2(CEA) CdSe. The obtained Ab 2(CEA) CdSe and Ab 2(AFP) CdTe were re-dispersed in 100 μl of 10 mm ph 7.4 PBS, which was stored at 4 C before directly used for sandwich typed immune-reaction. S-3
4 (b) Electrochemical characterization for fabrication procedures Figure S1. CV behavior of (a) bare GCE, (b) GCE ABA, (c) GCE ABA-{ Ab 1(CEA), (d) GCE ABA-{ Ab 1(CEA) < CEA and (e) < AFP GCE ABA-{Ab 1(CEA) < AFP > Ab 2(AFP) CdTe in 0.10 M ph 7.4 PBS containing 5.0 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6. The immune-complexes were formed with 20.0 L samples containing 10 ng/ml CEA and 100 pg/ml AFP. The formation of two sandwich-typed immuno-complexes on GCE, i.e., GCE ABA-{ Ab 1(CEA) < AFP > Ab 2(AFP) CdTe with Fe(CN) 6 3- /Fe(CN) 6 4-, was electrochemically characterized and confirmed redox couple as reported. 8 S-4 Figure S1 shows the cyclic voltammetry (CV) responses of the GCE modified at various stages. A well-defined CV with a peak separation of 70 mv was obtained on bare GCE (curve a). This value, accompanying the dramatic decrease in peak current, was increased after the electrodeposition of ABA layer (curve b) onto the GCE. These changes could be contributed to the electrostatic repulsion between negatively charged solution phase redox Fe(CN) 6 3- /Fe(CN) 6 4- species and negatively charged GCE ABA surface (ABA pk a = 4.80). 5,8 Whereas the increase of the current and the decrease of the peak potential separation values of GCE ABA-{ Ab 1(CEA) (curve c), with respect to GCE ABA (curve b), could be due to the partially restored electron transfer of
5 Fe(CN) 6 3- /Fe(CN) 6 4- after the electrode was immobilized with Ab 1 and the activated carboxyl groups of ABA were blocked with BSA, which significantly reduced the negative charges of the GCE ABA surface. Finally, the formation of bulky immune complexes on the electrode can certainly inhibit the electron transfer of the redox probe, therefore further increased the peak separation potential values as shown in curve d for GCE ABA-{ Ab 1(CEA) < CEA Ab and curve e for 1(AFP) < AFP GCE ABA-{Ab 1(CEA) respectively., < AFP > Ab 2(AFP) CdTe Figure S2. EIS plots of (a) bare GCE, (b) GCE ABA, (c) GCE ABA-{ Ab 1(CEA), (d) GCE ABA-{ Ab 1(CEA) < CEA, and (e) < AFP GCE ABA-{Ab 1(CEA) < AFP > Ab 2(AFP) in 0.10 M ph 7.4 CdTe PBS containing 5.0 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6. The immune-complexes were formed with 20.0 L samples containing 10 ng/ml CEA and 100 pg/ml AFP. Electrochemical impedance spectroscopy (EIS) technique was also used to investigate the fabrication of immunoassay (Figure S2). GCE displayed a small semicircle at the high frequency section (curve a), while GCE ABA showed a dramatic increase in R et (curve b), implying that the electron transfer of the Fe(CN) 6 3- /Fe(CN) 6 4- redox probe was inhibited by ABA. The BSA blocked GCE ABA-{ Ab 1(CEA) < CEA < AFP displayed a decreased R et than that of GCE ABA (curves b and c, respectively), because covalently immobilized Ab 1 on GCE ABA and the blocking of active carboxylic groups with BSA reduced the negative charges of the electrode S-5
6 surface. Both GCE ABA-{ Ab 1(CEA) < CEA (curve d) and < AFP GCE ABA-{Ab 1(CEA) < AFP > Ab 2(AFP) CdTe (curve e) displayed increased R et in sequence as compared to GCE ABA-{ Ab 1(CEA) < CEA < AFP, which further confirmed the successful immobilizing of huge bio-molecule and Ab 2 -NCs conjugates onto the GCE surface. (c) Potential resolved ECL characterization for fabrication procedures Figure S3. (A) ECL-potential and (B) CV profiles of (a) bare GCE, (b) GCE ABA, (c) GCE ABA-{ Ab 1(CEA) < < AFP > Ab 2(AFP) CdTe formed with 20.0 L sample only containing 100 pg/ml AFP, (d) GCE ABA-{ Ab 1(CEA) formed with 20.0 L sample only containing 10 ng/ml CEA, and (e) GCE ABA-{ Ab 1(CEA) < AFP > Ab 2(AFP) formed with CdTe 20.0 L sample containing both 10 ng/ml CEA and 100 pg/ml AFP. All experiments were conducted in 0.10 M ph 7.4 PBS containing 0.10 M (NH 4 ) 2 S 2 O 8 at a scan rate of 50 mv/s for one cycle. S-6
7 (d) Calibration curves for simultaneous determination of CEA and AFP Figure S4. Calibration curves for simultaneous determination of (A) CEA and (B) AFP with the spectrum-resolved dual-color ECL immunoassay strategy. The measurements were conducted under the same conditions as those in Figure 4 of the main manuscript. In other words, the concentrations of the second antigen were the same as those shown in Figure 4 when the ECL signal of the target antigen was measured at different concentrations. (e) References (1) Liu, S.; Zhang, X.; Yu, Y.; Zou, G. Biosens. Bioelectron. 2014, 55, (2) Liu, S.; Zhang, X.; Yu, Y.; Zou, G. Anal. Chem. 2014, 86, (3) Yu, W. W.; Qu, L. H.; Guo, W. Z.; Peng, X. G. Chem. Mater. 2003, 15, (4) Liang, G.; Shen, L.; Zhang, X.; Zou, G. Eur. J. Inorg. Chem. 2011, (5) Liang, G.; Liu, S.; Zou, G.; Zhang, X. Anal. Chem. 2012, 84, (6) Zhou, J.; He, Y.; Zhang, B.; Sun, Q.; Zou, G. Talanta 2017, 165, (7) Zhang, X.; Zhang, B.; Miao, W.; Zou, G. Anal. Chem (8) Tan, X.; Zhang, B.; Zhou, J.; Zou, G. Z. ChemElectroChem 2017, 4, S-7
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