Electronic Supplementary Material
An ELISA for the determination of human IgG based on the formation of a colored iron(II) complex and photometric or visual read-out
Ke Zeng1, Shiyu Tian1, Zixiao Wang, Congcong Shen, Junjun Luo, Minghui Yang*, You-Nian Liu
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China,
*Email: (M. Yang), Tel: (+86) 731 88836954
1 These author contributed equally to the work
Optimization of method
The maximal loading capacity of the synthesized MSN and optimal GOx/Ab2 ratio were studied. The loading of GOx as well as Ab2 and the ratio of GOx to Ab2 on the MSN surface will significantly affect the sensitivity of the assay. To test the capacity of MSN, the largest GOx loading capacity was determined. The GOx dosage used to react with MSN-CHO was changed from 250 μg.mL-1 to 2000μg.mL-1 (MGOx:MMSN = 1.15:1-9.23:1), and then the catalytic activity of these prepared GOx-MSN to glucose was measured by recording the amount of Fe(II) consumed with the produced H2O2. Typically, 10 μL of GOx-MSN solution was mixed with 200 μL of Fe(II)/glucose solution and reacted for 3 min at room temperature. Phen was then added immediately, and the OD490nm change was monitored by microplate reader. As shown in Figure S3a, with GOx/MSN ratio increased from 1.15 to 4.61, the UV-Vis absorption changes at 490 nm (λ490nm) increased continually from 0.14-0.73. When GOx ratio was higher than 4.61, the changes of λ490nm show no obvious changes at around 0.73, which means that the maximal GOx loading amount is about 4.6 mg per mg MSN.
Then we lowered the GOx amount while increased Ab2 amount to investigate the effect of GOx/Ab2 ratio on the sensitivity of the assay. We set weight ratio used for GOx/Ab2-MSN preparation at 50:1, 75:1, 150:1, 450:1(MGOx:MAb2), abbreviated as Ab2/GOx50-MSN Ab2/GOx75-MSN, Ab2/GOx150-MSN and Ab2/GOx450-MSN. The catalytic and binding ability of these series of Ab2/GOx-MSN was investigated and the data was shown in Figure S3b. For the detection of 100 ng.mL-1 of human IgG, Ab2/GOx450-MSN displays the lowest absorption changes which indicated it oxidize much fewer amount of Fe(II) to Fe(III) compared with other MSN, while Ab2/GOx75-MSN possess the highest absorption changes. The reason for this result might be that Ab2/GOx450-MSN had few number of Ab2 on the surface of MSN which was not enough to ensure efficient binding with the antigen. However, the number of Ab2 on the Ab2/GOx50-MSN and Ab2/GOx150-MSN surface was enough for recognizing and binding with antigens, but there was no sufficient GOx for catalytic process. Thus, Ab2/GOx75-MSN was selected for the following study.
The optimal pH and incubation time were also need to be taken into consideration. It is well known that GOx has good catalytic activity between pH range from 3-7, and rapidly become inactivated under the situation of pH < 3 or pH > 8. So the catalytic activity of GOx at different pH (from 4-7) is measured. The pH changes of the buffer solution due to the GOx catalyzed production of gluconic acid are also investigated. Experimental results indicated no obvious pH changes of the reaction solution were observed due to the production of gluconic acid (Figure S4a). Experimental data demonstrated the highest catalytic activity of GOx was obtained at pH = 5 (Figure S4b). So we continued the following experiment under the condition of pH = 5. The optimal time for Ab2/GOx150-MSN incubation with antigens was also identified (Figure S5). The results show that with the incubation time increasing from 1-4 h, the binding amount of Ab2/GOx150-MSN to antigen reached the maximum when incubated for 4 h.
Figure S1. UV-vis absorption of different concentration of [Fe(phen)3]2+ in acetic acid buffer (a) and calibration curve for [Fe(phen)3]2+ at λ490nm with Fe(II) concentration range from 5-150 μM (b). y= 0.00503x + 0.09073, R-Square=0.996.
Figure S2. UV-vis absorption of 0.15 mM of [Fe(phen)3]3+ in acetic acid buffer shows nearly no absorption at 490 nm.
Figure S3. The pore size distribution of as prepared NH2-MSN
Figure S4. The effect of different GOx : MSNmass ratio used for the preparation of GOx-MSN towards the catalytic activity of the resulted MSN to glucose (a) and the optimal GOx/Ab2 ratio on the sensitivity of the ELISA (b).
Figure S5. The effect of produced gluconic acid on the pH changes of acetic acid buffer (a) and the influence of pH to the catalytic ability of GOx immobilized on MSN(b).
Figure S6. The optimal incubation time determined by the absorption change of the assay after incubation with 1ng/mL of human IgG for 1h, 2h, 3h and 4h, respectively.
Figure S7. The total detective concentration of human IgG range from 1 pg/mL to 10 μg/mL, error bar = RSD (n = 3).
Figure S8. The monodispersity stability (a) line 1 for 0 day and line 2 for 2 weeks later and the catalytic ability (b) of Ab2/GOx-MSN, error bar = RSD (n = 3).
Table S1. The hydrodynamic size and PDI of NH2-MSN, CHO-MSN and GOx/Ab2-MSN.
Samples / Size by DLS / PDINH2-MSN / 158.8 ± 2.1 / 0.123 ± 0.009
CHO-MSN / 171.3 ± 2.8 / 0.196 ± 0.015
GOx/Ab2-MSN / 205.3 ± 4.3 / 0.250 ± 0.021
DLS measurement demonstrated that the as-prepared NH2-MSN with hydrodynamic size of 158.8 ± 2.1 nm. After the modification, the hydrodynamic size of GOx/Ab2-MSN was 205.3 ± 4.3 nm. PDI data revealed that both NH2-MSN and GOx/Ab2-MSN possess good monodispersity in solution.