Supporting Information

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1 Supporting Information Ultra-robust Biochips with Metal-Organic Framework Coating for Point-of-Care Diagnosis Congzhou Wang, Lu Wang, Sirimuvva Tadepalli, Jeremiah J. Morrissey, Evan D. Kharasch, Rajesh R. Naik*, and Srikanth Singamaneni* Dr. C. Wang, L. Wang, S. Tadepalli and Prof. S. Singamaneni Department of Mechanical Engineering and Materials Science, Institute of Materials Science and Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA Prof. J. J. Morrissey and Prof. E. D. Kharasch Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, 63110, USA Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA The Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University School of Medicine, St. Louis, MO, USA Dr. R. R. Naik 711 th Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH

2 Experimental Section: Chemicals: Chloroauric acid, sodium borohydride, ascorbic acid, cetyltrimethylammonium bromide (CTAB), (3-mercaptopropyl)-triethoxy-silane (MPTES), dimethylformamide (DMF), protease from Streptomyces griseus, sucrose, human serum albumin, hemoglogin, 2-methylimidazole and zinc acetate dihydrate were purchased from Sigma-Aldrich. Silver nitrate was purchased from VWR international. Rabbit IgG, goat anti-rabbit IgG (Mw = 150 kda), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Thermo scientific. SH-PEG-COOH (Mw = 5000 g/mol) was purchased from Jenkem Technology. Synthesis of gold nanorods (AuNRs): Gold nanorods were synthesized using a seed-mediated approach. Seed solution was prepared by adding ice-cold sodium borohydride solution (10 mm, 0.6 ml) into CTAB (100 mm, 9.75 ml) and chloroauric acid solution (0.25 mm, 0.25 ml) under vigorous stirring at room temperature. The color of the seed solution changed from yellow to brown. Growth solution was prepared by mixing CTAB (100 mm, 95 ml), silver nitrate (10 mm, 0.5 ml), chloroauric acid (10 mm, 5 ml), and ascorbic acid (100 mm, 0.55 ml) in the same order. The solution was mixed by gentle stirring. To the resulting colorless solution, freshly prepared seed solution (0.12 ml) was added and set aside in dark for 14 h. Prior to use, the AuNR solution was centrifuged twice at 10,000 rpm for 8 min to remove excess CTAB and re-dispersed in nanopure water (18.2 MΩ cm) each time. AuNR-IgG antibody conjugates preparation: To a solution of SH-PEG-COOH in water (20 µm, 37.5 µl), EDC and NHS, with the same molar ratio as SH-PEG-COOH, were added followed by shaking for 1 h. The ph of the above reaction mixture was adjusted to 7.4 by adding 10X phosphate buffered saline (PBS). Subsequently, rabbit IgG (75 µm, 10 µl) was added to the reaction mixture and was incubated for 2 h. Then the mixture was filtered to remove any byproduct during the reaction using centrifuge tube with 50 kda filter. The final SH-PEG-IgG conjugates solution (0.75 µm) was obtained after washing with PBS buffer (ph 7.4) through the filter twice. In order to make sure that the amount of IgG conjugated on the AuNR are 2

3 consistent for each batch, we used the same amount and concentration of IgG solution (0.75 µm, 20 µl) and AuNR solution (1 ml, UV extinction 2.0 a.u.). Also, we used LSPR shift to monitor each batch of conjugation to ensure the same amount of LSPR red shift (~ 9 nm). Adsorption of AuNR-IgG on glass surface: First, rectangular slides of glass (1 2 cm) were cleaned with piranha solution (3:1 (v/v) mixture of H 2 SO 4 and 30% H 2 O 2 ) followed by extensive rinsing with nanopure water (Caution: Piranha solution is extremely dangerous and proper care needs to be executed in handling and disposal). Then the cleaned glass slides were modified with MPTES to render thiol functionality by immersing the glass substrate into 1% MPTES ethanol (190 proof) solution for 1 h followed by ultrasonication in ethanol for 20 min and rinsing with nanopure water. AuNR-IgG conjugates were immobilized onto MPTES-functionalized glass substrates by exposing them to AuNR-IgG conjugates solution for 3 h, and then rinsing with water to remove the loosely bound AuNRs. By controlling the absorption time (3 h) and UV extinction of the substrates (0.1 a.u.), we make sure that the same amounts of AuNR-IgG conjugates deposited on the substrates. ZIF-8 film growth and removal: To form ZIF-8 films, 2-methylimidazole solution (160 mm or 1.6 M, 1 ml, in nanopure water) was mixed with zinc acetate dihydrate solution (40 mm, 1 ml, in nanopure water), and agitated for 10 s. The glass substrates were incubated in a freshly-made ZIF-8 precursor solution described above for 3 h, 12 h and 24 h to facilitate the growth of ZIF-8 layer. For removing the ZIF-8 coating, samples were gently rinsed for 5 min using nanopure water at ph 6. Silk and sucrose film coating and removal: Silk fibroin was reconstituted from Bombyx mori silkworm cocoon following a reported protocol. The degumming time was performed for 30 min, leading to 4% (w/v) of final concentration. To form silk coatings, silk fibroin solution (100 µl) was deposited onto glass adsorbed with AuNR-IgG conjugates and spun (model WS-400, Laurell Technologies Corporation) at 3000 rpm for 30 s. For removing the silk film, the silk-coated glass substrate was gently rinsed by nanopure water for 5 min. For sucrose coating, 15% sucrose in PBS (w/v) was prepared and the similar procedure as the silk coating was followed. 3

4 Characterization: UV vis extinction spectra of glass substrates were collected in air using a Shimadzu UV-1800 UV vis spectrometer. Transmission electron microscopy (TEM) micrographs were collected by a JEM-2100F (JEOL) field emission instrument. For TEM sample preparation, a drop of the solution was casted and dried on a glow-discharged carbon-coated grid. AFM images were collected by Dimension 3000 AFM (Bruker) in light tapping mode. The Raman spectra were obtained using a Renishaw invia confocal Raman spectrometer mounted on a Leica microscope with a 50 objective and a 514 nm wavelength diode laser as an illumination source. The X-ray diffraction (XRD) measurements of the samples were recorded on a Bruker D8-Advance X-ray powder diffractometer using Cu Kα radiation (λ = Å) with scattering angles (2θ) of Dynamic light scattering (DLS) measurements were performed using Malvern Zetasizer (Nano ZS). 4

5 1.0 Normalized Intensity AuNR 50.7 nm AuNR-IgG 58.7 nm Hydrodynamic size (nm) Figure S1. Hydrodynamic size obtained from DLS showing that the average hydrodynamic size increased by 8 nm following SH-PEG-IgG conjugation. 5

6 LSPR shift (nm) µg/ml anti-igg 240 µg/ml HSA 240 µg/ml Hb Figure S2. Control experiments showing the small nonspecific binding of human serum albumin (HSA) or hemoglobin (Hb) on AuNR-IgG. Error bars represent standard deviations from three independent samples. 6

7 Normalized Extinction (a.u.) A AuNR-IgG (PEG-conjugated IgG) 32 nm shift +Anti-IgG Normalized Extinction (a.u.) B AuNR-IgG (Physically-absorbed IgG) 14 nm shift +Anti-IgG Wavelength (nm) Wavelength (nm) Figure S3. (A) Extinction spectra of AuNR-IgG conjugates (PEG-conjuated IgG) on the glass substrate before (red) and after binding with 24 µg/ml of anti-igg (blue). The λ red shifts by 32 nm. (B) Extinction spectra of AuNR-IgG conjugates (physically-absorbed IgG) on the glass substrate before (red) and after binding with 24 µg/ml of anti-igg (blue). The λ red shifts by 14 nm. 7

8 Figure S4. AFM scratch experiments showing an increase of ZIF-8 film thickness with the increase of growth time. The film thickness is determined by measuring the difference of average heights near the edge of the scratch. 3 h, 12 h and 24 h growth time of ZIF-8 (4:1molar ratio) result in ~13 nm, ~16 nm and ~19 nm of film thickness (Figure S1A, B and C). 24 h growth time of ZIF-8 (40:1molar ratio) results in ~28 nm of film thickness (Figure S1D). Scale bars: 5 µm. 8

9 100 Retained Recognition (%) o C 60 o C 40 o C Thickness (nm) Figure S5. Correlation between the thickness of ZIF-8 film and retained activity of biochips at three different temperatures. Error bars represent standard deviations from three independent samples. 9

10 (011) 3 h ZIF-IgG 24 h ZIF-IgG Intensity (a.u.) (002) (022) (013) (222) (114) (233) (224) (134) (044) (344) (244) (112) θ (deg) Figure S6. XRD spectra of ZIF-8 encapsulated IgG after 3 and 24 h growth. 10

11 Normalized Extinction A Before 24 h of incubation at 120 o C After 24 h of incubation at 120 o C B Wavelength (nm) Figure S7. (A) Extinction spectra of AuNR-IgG conjugates on the glass substrate before (black) and after incubation at 120 C for 1 day (red). (B) AFM images of AuNR-IgG conjugates on the glass substrate before and after incubation at 120 C for 1 day. Scale bar: 400 nm. 11

12 100 Retained Recogniton (%) MOF-coated biochips at 80 o C Uncoated biochips at 4 o C Uncoated biochips at 80 o C Time (days) Figure S8. Retained recognition capability of MOF-coated or uncoated IgG-AuNR conjugates on glass substrates stored at 80 C or 4 C for different durations. Error bars represent standard deviations from three independent samples 12

13 Figure S9. AFM scratch experiments showing the complete coverage of the biochip surface with 100 nm thick silk film (A, scale bar :10 µm) and sucrose film (B, scale bar: 5 µm). 13