Names / Sequences (5’-3’)
Oligo 1 / SH-ATCGCAAGACCG
Oligo 2 / TTTTTTTTTTTTCGGTCTTGCGAT
Oligo 3 / amino-AAAAAAAAAAAA
Oligo 4 / biotin-CGGTCTTGCGAT
Characterization of modified GNPs
Agarose gel electrophoresis results demonstrate that ds-DNA of different numbers were conjugated to GNPs successfully (Supplementary Fig. 1). This result demonstrated the more conjugates move at approximately the higher mobility rate (lane 0 in Fig. S1). The numbers was important in the conjugation.
The gold with more DNA will change its amount of charge, space and molecular weight. The amount of dsDNAs on the surface of GNPs would increase the structure complexity. The charge-mass ratio could be change and the pattern on gel will change, also. Considering the size effect of DNA, the molecular weight should be the dominant factor for the mobility, though negatively charged DNA should increase the charge volume of the conjugated GNPs. The increase of DNA will decrease the charge mass ratio of gold and the distance of movement will decrease. More primers led to slower mobility (lanes 1 to 4 in Supplementary Fig. 1).
The modification need modify the maximum amount of DNA on the surface. The GNP, which ds-DNA has covered all surface of it, exhibits almost the same mobility in gels (lanes 5 to 7 in Supplementary Fig. 1). When the DNA to Gold is over 500 to 1, the distance was stable. The reaction of thiol to gold is dynamic. That means the real number of DNA on 18nm GNPs was less than 500.
Supplementary Fig.1: Agarose gel electrophoresis of GNPs with ds-DNA
The accurate amount of dsDNA on the GNPs was confirmed by UV spectrometer. Use a bench top or high-speed centrifuge to centrifuge the suspension to form a red oil of nanoparticles beneath a clear solution of excess oligonucleotide. Carefully remove the clear supernatant and re-suspend the oil in the same volume of (0.025% by weight) sodium citrate buffer. The unreacted ds-DNA was then characterized by UV/visible spectroscopy. The definite ratio of DNA to gold is 303. The area of 18nm gold surface is 324π (=4*π*92). The diameter of double helix DNA is 2nm, so the area of ds-DNA is π (=π*12). So the theory calculate of the ratio is 324. The density of DNA on GNPs is 93.5% (=303/324). The surface of gold could be almost full covered.
Limits of detection
Supplementary Fig.2 Detection limit of SA on PVDF with biotin GNPs . A: schematic diagram of detecting streptavidin (SD). SA and BSA were absorbed on PVDF films. Biotin on ds-DNA GNPs could interact with SA and dye it with the color of GNPs. BSA was negative control without interacting with biotin and was colorless. B, C: SA detected by modified GNPs and Coomoss Blue. (B) Because Coomoss Blue is a non-specific detecting method for protein, both SA (objective molecule) and BSA (negative control) were blue. (C) ds-DNA-modified GNPs dyed SA specifically with colorless negative control. D: detection limit of modified GNPs was 25 ng. E: detection limit of modified Coomoss Blue was 50 ng.
Preparation of GNPs
· The GNPs preparation followed a classic method (Grabar et al. 1995; Elghanian et al. 1997). Because of the reliability of this protocol, the labs could synthesize GNPs by themselves easily. The diameters of GNPs can be regulated by the concentrations of reducing agent and gold precursor salt used.
· Solution: reducing agent (sodium citrate, 1% by weight) and gold precursor salt (HAuCl4, 0.01% by weight)
· Boil 100ml HAuCl4 under stirring and reflux
· Add 1.36 ml of sodium citrate quickly to the boiling solution
· Boil the mixed solution for 15min
· Stop heating and keep stirring to cool from boiling to room temperature.
· Store the wine red GNPs in room temperature for usage.
· The concentration was tested with UV spectrum at 530nm, ɛ=8.6*108 L/mol.
Protocol 1: modification of GNPs with protein
Step 1 was to prepare dsDNA contained Oligo 1 and 2.
· Dilute Oligo 1 and 2 respectively with water to 0.01mM
· Mix Oligo 1 and 2 with same volumes
· Boil the mixture in 100°C water for 1min
· Cool the mixture to room temperature during a leisurely speed in order to form ds-DNA.
· Add ds-DNA to GNPs and mix (molar ratio is 500:1).
· Stay the mixture for night to modify dsDNA on GNPs surface.
· Centrifuge the mixture with 16000rpm for 10min.
· Disperse the deposition with PBS (0.01M pH: 7.3).
Step 2 was to conjugate Oligo 3 and protein (such as: IgG)
· Attention: the solution must avoid free amino, so Tris buffer should be avoided (Hermanson 2008)
· Solute Oligo 3 with water to 0.01mM
· Solute protein with PBS (0.01M pH: 7.3) to 0.05mM.
· solute EDC in water to 100mg/ml
· Mix oligo 3 and protein (molar ratio is 1:10)
· Add 10ul EDC and mix.
· Stay at room temperature for 4h
· Centrifuge the mixture with ultrafiltration tube (50kDa).
· Attention: EDC could destroy thiol group, so it must be clear with multi-ultrafiltration (Hermanson 2008).
· Solute conjugation with PBS (0.01M pH: 7.3) buffer.
Step 3: modification of GNPs with proteins
· Mix conjugation in stage 2 and dsDNA coated GNPs in stage 1 (molar ratio> 5:1) at room temperature for at least 12 hours.
· Centrifuge the mixture with 16000rpm for 10min.
· Disperse the deposition with PBS (0.01M pH: 7.3) buffer.
Protocol 2: modification of GNPs with biotin or SA
Step 1: preparation of dsDNA
· Dilute Oligo 1 and 4 respectively with water to 0.01mM.
· Mix Oligo 1 and 4 with same volumes.
· Boil the mixture in 100°C water for 1min.
· Cool the mixture to room temperature during a leisurely speed in order to form ds-DNA.
Step 2: preparation of biotin-GNPs
· Add ds-DNA to GNPs and mix (molar ratio is 500:1).
· Stay the mixture for night to modify biotin on GNPs surface.
· Centrifuge the mixture with 16000rpm for 10min.
· Disperse the deposition with PBS (0.01M pH: 7.3).
· The biotin GNPs is completed and could be used in lab.
· The modification can be identified by one of agarose gel electrophoresis, UV spectrophotometer and laser particle size analyzer.
Step 3: preparation of SA-GNPs
· Solute SA in water to 0.076mM
· Add SA to biotin modified GNPs. The molar ratio of SA to DNA on GNPs is 1:10.
· Stay for 30min in room temperature.
· Centrifuge the mixture with 16000rpm for 10min.
· Disperse the deposition with PBS buffer (0.01M pH: 7.3)
Detection on PVDF film with biotin-GNPs
l The target molecule (streptavidin) was adsorbed on the PVDF film. To ensure the specificity of this method, BSA was selected as negative control.
l The film was immersed in the block solution (0.5% BSA in PBS) for 30min.
l Then it was soaked in biotin-GNPs solution for 10 min
l Wash the film with PBS buffer (0.01M pH: 7.3) for 1min.
Detection on Nylon film with SA-GNPs
l The target molecule was adsorbed on the Nylon film. To ensure the specificity of this method, ordinary DNA without biotin was selected as negative control.
l The film was immersed in the block solution (1mg/ml fish sperm DNA in PBS) for 30min.
l Then it was soaked in SA-GNPs solution for 10 min
l Wash the film with PBS buffer (0.01M pH: 7.3) for 1min.
Detection on NC film with IgG-GNPs
l The target molecule was adsorbed on the NC film. To ensure the specificity of this method, BSA was selected as negative control.
l The film was immersed in the block solution (BSA 0.5% in PBS) for 30min.
l Then it was soaked in the working GNPs solution for 1min.
l Wash the film with PBS buffer (0.01M pH: 7.3) for 1min.
References
Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA (1997) Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles. Science 277 (5329):1078-1081
Grabar KC, Freeman RG, Hommer MB, Natan MJ (1995) Preparation and Characterization of Au Colloid Monolayers. Analytical Chemistry 67 (4):735-743
Hermanson GT (2008) Bioconjugate Techniques. 2nd Edition edn. Academic Press,
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