Multiplexible Wash-Free Immunoassay Using. Colloidal Assemblies of Magnetic and. Photoluminescent Nanoparticles
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1 Supporting Information Multiplexible Wash-Free Immunoassay Using Colloidal Assemblies of Magnetic and Photoluminescent Nanoparticles Dokyoon Kim, Hyek Jin Kwon,, Kwangsoo Shin,, Jaehyup Kim, Roh-Eul Yoo, ǁ Seung Hong Choi,,ǁ Min Soh,, Taegyu Kang,, Sang Ihn Han,, and Taeghwan Hyeon *,, Center for Nanoparticle Research, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea, School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea, Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States, and ǁ Department of Radiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea. S1
2 Supporting Information Section S1 Figure S1. (a and b) TEM images of 18 nm-sized iron oxide nanoparticles. The inset is a highresolution TEM image. (c) Size distribution histogram. S2
3 Supporting Information Section S2 Figure S2. (a and b) TEM images of 4.8 nm-sized ZnS:Mn nanoparticles. The inset is a highresolution TEM image. (c) Size distribution histogram. (d, e, and f) EDS elemental mapping of Zn and Mn in ZnS:Mn nanoparticles (corresponds to the rectangular region in (d)). S3
4 Supporting Information Section S3 Figure S3. Schematic illustration of the colloidal nanoparticle assembly formation in oil-inwater microemulsion system. Nanoparticles dispersed in chloroform are emulsified with deionized water solution of DTAB. Chloroform in the DTAB-stabilized micelles is evaporated. The resulting DTAB-stabilized colloidal nanoparticle assemblies are coated with poly(acrylic acid). S4
5 Supporting Information Section S4 Figure S4. Plot showing the zeta potential difference before and after PAA coating. S5
6 Supporting Information Section S5 Figure S5. (a and b) TEM images of IONAs. (c) Size distribution histogram. (d) Hydrodynamic diameter measured by DLS. (e) Magnetic hysteresis loop of the IONAs measured at 300 K. S6
7 Supporting Information Section S6 Number of iron oxide nanoparticles in an IONA: [Average diameter of an IONA] = 190 nm [Diameter of an iron oxide nanoparticle] = 18 nm [Inter-particle distance of iron oxide nanoparticles in the superlattice structure] [reference 48] = 1.4 nm [Volume of an IONA] = 4 / 3 * π * (190 / 2)^3 = nm^3 [Volume of an iron oxide nanoparticle with surface capping layer] = 4 / 3 * π * (( ) / 2)^3 = nm^3 Assuming close-packed structure, the packing ratio is Therefore, [Number of iron oxide nanoparticles in an IONA] = * / = If we consider the size range of IONAs, which is 120 ~ 280 nm. Then, [Number range of iron oxide nanoparticles in IONAs] = ~ In a similar way, number of ZnS:Mn nanoparticles in an MZSNA can be calculated. [Average diameter of an MZSNA] = 323 nm [Diameter of a ZnS:Mn nanoparticle] = 4.8 nm [Volume of an MZSNA] = 4 / 3 * π * (323 / 2)^3 = nm^3 [Volume of an ZnS:Mn nanoparticle with surface capping layer] = 4 / 3 * π * (( ) / 2)^3 = nm^3 [Number of ZnS:Mn nanoparticles in an MZSNA] = * / = If we consider the size range of MZSNAs, which is 200 ~ 440 nm. Then, [Number range of ZnS:Mn nanoparticles in MZSNAs] = ~ The number of ZnS:Mn in an MZSNA can be different if a different packing ratio is used. S7
8 Supporting Information Section S7 Figure S7. (a and b) TEM images of MZSNAs. (c) Size distribution histogram. (d) Hydrodynamic diameter measured by DLS. (e) Normalized one-photon PLE (blue) and photoluminescence (red) spectra of the MZSNAs. (f) Comparison of the photoluminescence between ZnS:Mn nanoparticles and MZSNAs. S8
9 Supporting Information Section S8 Figure S8. (a) Plot showing the hydrodynamic diameter change after the conjugation of streptavidin and antibodies on IONAs. (b) Plot showing the hydrodynamic diameter change after the conjugation of antibodies on MZSNAs. (c) Bradford assay plot obtained using various concentrations of CRP antibodies. Horizontal line indicates the absorbance increase of IONAs after the antibody conjugation. S9
10 Supporting Information Section S9 Figure S9. (a) Concentration-dependent PL intensity of MZSNAs. (b) Magnified view of the low concentration region in (a). For a measurable PL intensity, it is estimated that ~7.5 pg [Zn]/ml of MZSNAs is required (detection limit shown in Figure S9b). [Density of ZnS] = 4.09 * 10^6 g/m^3 [Volume of an ZnS:Mn nanoparticle] = 4 / 3 * π * (4.8 / 2)^3 = 57.9 nm^3 [Mass of an ZnS:Mn nanoparticle] = (57.9 * 10^-27) * (4.09 * 10^6) = 2.37 * 10^-19 g [Mass of Zn in an ZnS:Mn nanoparticle] = * 2.37 * 10^-19 = 1.59 * 10^-19 g According to the Supplementary Section 6, it is estimated that the number of ZnS:Mn nanoparticles in an MZSNA is [Mass of Zn in an MZSNA] = 1.59 * 10^-19 * = 1.66 * 10^-14 g [Number of MZSNAs corresponding to 7.5pg [Zn]/ml] = 7.5 * 10^-12 / 1.66 * 10^-14 = particles/ml S10
11 Supporting Information Section S10 Figure S10. (a-d) TEM images of the colloidal nanoparticle assemblies obtained after the magnetic isolation of IONAs and target protein-bound MZSNAs. S11
12 Supporting Information Section S11 Figure S11. (a) ELISA standard curve measured for CRP. (b) Magnified view of low concentration region in (a). S12
13 Supporting Information Section S12 ELISA was performed according to the manufacturer s instructions. Capture antibody concentration: 2 µg/ml Detection antibody concentration: 90 ng/ml Since 100 µl volume was used for each sample, [Amount of capture antibody used] = 2000 * 0.1 = 200 ng [Amount of detection antibody used] = 90 * 0.1 = 9 ng [Total amount of antibodies used] = = 209 ng In the colloidal nanoparticle assembly immunoassay, 500 µg of capture antibody was used to functionalize 1 mg [Zn] of MZSNAs, and 50 µg of detection antibody was used to functionalize 1 mg [Fe] of IONAs. The concentrations of MZSNAs and IONAs used in the measurements were 400 ng [Zn]/ml and 400 ng [Fe]/ml for MZSNAs and IONAs, respectively. The same 100 µl volume was used for each sample. [Amount of capture antibody used] = 500 / 1000 * 400 * 0.1 = 20 ng [Amount of detection antibody used] = 50 / 1000 * 400 * 0.1 = 2 ng [Total amount of antibodies used] = = 22 ng S13
14 Supporting Information Section S13 Figure S13. Correlation plot showing the CRP concentrations in spiked PBS samples measured using ELISA (y-axis) and the colloidal nanoparticle assembly immunoassay (x-axis). R 2 value is S14
15 Supporting Information Section S14 Figure S14. (a and b) TEM images of Cd 1-x Zn x S/ZnS core/shell nanoparticles. (c) Size distribution histogram. S15
16 Supporting Information Section S15 Figure S15. (a and b) TEM images of CZSNAs. (c) Size distribution histogram. (d) Hydrodynamic diameter measured by DLS. (e) Comparison of the photoluminescence between the Cd 1-x Zn x S/ZnS core/shell nanoparticles and CZSNAs. S16
17 Supporting Information Section S16 Figure S16. CRP standard curve was measured using MZSNAs and IONAs (Figure 4a in the main text). EpCAM standard curve was measured using CZSNAs and IONAs (Figure 5e in the main text). Dynamic ranges of each standard curve are shown as black (for CRP) and red (for EpCAM) arrowed lines. (a) CRP concentration (black column) in a sample was higher than the dynamic range of the CRP standard curve, preventing the accurate assessment. (b) The sample in (a) was diluted 100 times to enable the CRP concentration measurement. Then, EpCAM concentration (red column) became lower than the dynamic range of EpCAM standard curve, so the EpCAM concentration could not be measured accurately. (c) The sample in (a) was diluted 5 times. EpCAM concentration could be measured, but CRP concentration was still too high and could not be measured correctly. (d) The sample in (a) was diluted 5 times and the amount of MZSNAs was increased 8 times to adjust the dynamic range of the CRP standard curve. Then, both the concentrations of CRP and EpCAM in the sample could reside in the dynamic ranges of CRP and EpCAM standard curves, respectively, allowing the reliable measurement. S17
18 Supporting Information Section S17 Figure S17. Cylindrical magnets (6 mm diameter 5 mm height), are inserted in column-2 wells of the plate. Solutions are added to column-1 and 3 wells for magnetic isolation (black squared region). An enlarged image of the red region (bottom right) shows the isolated IONAs and MZSNAs near top and bottom of the magnet (blue circles). S18
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