Chemicals and materials
Fluorescamine was purchased from Invitrogen (Carlsbad, CA). HS-PEG5000-NH2 (M.W. ≈ 5,000) and HS-PEG20000-NH2 (M.W. ≈ 20,000) were purchased from Laysan Bio (Arab, AL), HS-PEG3000-NH2 (M.W. ≈ 3,000) was obtained from Rapp Polymere GmbH (Tübingen, Germany). 1,4,7,10-Tetraazacyclodocecane-1,4,7,10-tetreaacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS, ≥90%) was obtained from Macrocyclics (Dallas, TX). The Kaiser test kit was obtained from Fluka (Buchs, Switzerland). Fluorescein isothiocyanate (FITC, ~98%), Chelex 100 resin (50–100 mesh), copper(II) chloride (CuCl2, ~99.9%), gold(III) chloride trihydrate (HAuCl4·3H2O, ≥99.0%), sodium borohydride (NaBH4, 99%), L-ascorbic acid (>99%), silver nitrate (AgNO3, >99%), poly(vinyl pyrrolidone) (PVP, M.W. ≈ 55,000), cetyltrimethylammonium bromide (CTAB, ≥99%), cetyltrimethylammonium chloride (CTAC, ≥98%), and 1,10-phenanthroline ethylenediaminetetraacetic acid (EDTA, ≥99%) were all obtained from Sigma-Aldrich (St. Louis, MO). All the chemical reagents were used as received. Water with a resistivity of 18 MΩ·cm was prepared using a E-Pure filtration system from Barnstead International (Dubuque, IA). The water and buffer solutions used for Cu2+ labeling were treated with Chelex 100 overnight prior to use.
Synthesis of Au Nanostructures
The 30-, 50-, and 60-nm AuNCs covered by PVP were synthesized using a published procedure.51
The 40-nm AuNPs capped by CTAC were prepared using a recently reported, two-step procedure.52
Synthesis of the 42-nm AuNPs capped by citrate ions was conducted using the citrate reduction method.53
The AuNRs capped by CTAB were prepared using the procedure reported by El-Sayed and coworkers.54
PEGylation of Au Nanostructures with HS-PEG-NH2
In a typical process, 1.2 mL of a 0.25 mM aqueous HS-PEG-NH2 solution was added to 3 mL of an aqueous suspension of Au nanostructures (1.7 nM), followed by addition of 1.8 mL H2O to a total volume of 6 mL. The final concentration of Au nanostructures and HS-PEG-NH2 were 0.85 nM and 50 µM, respectively. The reaction mixture was vortexed immediately and then incubated at 4 °C overnight, followed by centrifuging at 14,000 rpm for 5 min. The supernatant was carefully collected, and centrifuged two more times to remove the remaining Au nanostructures. The resultant supernatant solution was subjected to the fluorescamine- and ninhydrin-based assays to determine the number of unreacted HS-PEG-NH2 molecules. The HS-PEG-NH2-functionalized Au nanostructures were washed three more times and then used for conjugation with FITC or DOTA-NHS for corresponding measurements.
Quantification of –S-PEG-NH2 Chains on Au Nanostructures Using Fluorescamine-Based Assay
Prior to analysis, a calibration curve was obtained from a series of HS-PEG-NH2 solutions with known concentrations. Briefly, to each 3 mL of HS-PEG-NH2 standard phosphate buffered (PB, pH = 10) solution, 0.25 mL of 2 µM fluorescamine solution in acetone was added. After 15 min, fluorescence spectra (λex ≈ 390 nm, λem ≈ 480 nm) were recorded. The fluorescent intensities at 480 nm for each solution were plotted as a function of the concentration of HS-PEG-NH2 to generate a calibration curve. For sample measurements, 100 µL of the supernatant solution was diluted into 3 mL with the PB buffer and treated using a procedure similar to what was used for the calibration curve. By comparing the fluorescence intensities with the calibration curve and multiplying the dilution factors, we obtained the concentrations of HS-PEG-NH2 in the supernatant solutions. The number of HS-PEG-NH2 on the Au nanostructures was obtained by subtracting the number of HS-PEG-NH2 in the supernatant from the total number of HS-PEGNH2 added into the suspension of Au nanostructures. This number was then converted to the coverage density by taking into account the total number of Au nanostructures and their total surface area. Each data point represents an average of three replicas.
Quantification of –S-PEG-NH2 Chains on Au Nanostructures Using Ninhydrin-Based Assay
All reagents used for the assay were prepared according to the literature.55–57
Typically, 6% ninhydrin ethanol solution was prepared by dissolving 2.5 g ninhydrin in 50 mL anhydrous ethanol. The KCN pyridine solution and 80% phenol solution in ethanol from the Kaiser test kit were combined at a 1:1 volume ratio to give a KCN/phenol solution. Prior to assay, a calibration curve was obtained from a series of HS-PEG-NH2
standard solutions with known concentrations. Briefly, to each 250 µL of HS-PEG-NH2
standard solutions, 100 µL 6% ninhydrin ethanol solution and 200 µL KCN/phenol solution were added, followed by heating at 100 °C for 4 minutes. After cooling down in an ice bath, 200 µL 60% wt. ethanol in water was added. UV-vis spectra were then recorded. The calibration curve was generated by plotting the absorbance at 565 nm as a function of the HS-PEG-NH2
concentration. 250 µL samples (the supernatant solution) were treated using same procedure as that was used for standard solutions. The number of overall HS-PEG-NH2
on the Au nanoparticle was calculated as same as the method of fluorescamine-based assay. Each data point represents an average of three replicas.
Quantification of Active –NH2 Groups on Au Nanostructures by Dye-Labeling Assay
To 500 µL Au-S-PEG-NH2 (with a known concentration), 2 µL of 50 mM FITC in dimethyl sulfoxide (DMSO) was added. The reaction mixture was incubated at room temperature for 15 min, followed by five times washing with water. The pellet was re-dispersed in 500 µL water. To the pellet suspension, 2.5 mL of 40 mM KCN aqueous solution and 2.0 mL water was added to dissolve the Au nanoparticles at room temperature for 30 min. After that, fluorescent spectra (λex ≈ 488 nm, λem ≈ 520 nm) were taken from the sample. The concentration of FITC in each sample was calculated by comparing with the calibration curve obtained for free FITC aqueous solution. The number of active –NH2 groups per Au nanoparticle was calculated from the numbers of FITCs and Au nanoparticles. Each data point represents an average of three replicas.
Quantification of Active –NH2 Groups on Au Nanostructures by Cu2+-Labeling Assay
500 µL of 0.4 nM Au-S-PEG-NH2 was washed 3 times with chelexed water and re-suspended in 500 µL chelexed water. Then, 500 µL of 1 mM DOTA-NHS in 0.1 M PB buffer (pH = 7.4) was added. The reaction mixture was incubated at room temperature for 1 h, followed by washing five times with chelexed water. After the final round of centrifugation, 1 mL of 0.1 M NaOAc buffer (pH = 5.5) was added to re-suspend the pellet, followed by the addition of 10 µL CuCl2 aqueous solution (50 mM). After incubation at 37 °C for 1 h, 500 µL of 1 mM EDTA aqueous solution was added to chelate the unbound Cu2+ ions for 15 min. The mixture was washed five times with chelexed water to remove the EDTA-Cu2+, resulting in Au-PEG-DOTA-Cu2+. For Cu2+ analysis, 5 µL of the sample solution was dissolved in 0.5 mL of concentrated aqua regia and further diluted to 30 mL using 1% HNO3 aqueous solution prior to ICP-MS measurement. The number of active –NH2 groups per Au nanoparticle could be estimated from the ratio of Cu2+ ions to Au nanoparticles from ICP-MS analysis. Each data point represents an average of three replicas.
TEM images of the Au nanostructures were obtained with a Technai G2 Spirit microscope operated at 120 kV (FEI). Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian). UV-vis spectra were taken with a Cary 50 UV-vis spectrophotometer (Varian). The hydrodynamic diameter of the Au nanostructures was measured in deionized water (pH ≈ 6.7) using dynamic light scattering (Malvern, NanoZS) which was equipped with a zeta-potential analyzer. The concentrations of Au, Ag and Cu elements were determined using ICP-MS (Perkin Elmer, Elan DRC II). The concentration of Au element was then converted to the concentration of Au nanostructures once the particle size and morphology had been determined by TEM.