cODN-functionalized beads with another MP, tOmpA, trapped with the same 1:1 OligAPol/
FAPol AF647 mixture (Le Bon et al. 2014a).
The presence of the MPs at the surface of gold NPs was confirmed by incubation with Alexa
Fluor 488- or Alexa Fluor 647-labeled antibodies directed against BR (Fig. 13.17C) or against tOmpA
(Le Bon et al. 2014a). Several control experiments were designed to confirm the specificity of the
interaction between cODN-coated gold NPs and OligAPol-trapped MPs. No binding was observed, for
instance, when gold NPs were coated with thiolated polycytosine, which is not complementary to the
ODN (Control 1, Fig. 13.17E, G), nor onto cODN-coated gold NPs that had been preincubated with an
excess of free, Cy3-labeled ODN (Control 2, Fig. 13.17F, G), hybridization of the free ODN being
confirmed by the Cy3 fluorescence signal. The FAPol AF647 signals measured in these control
experiments were much lower ($10%) and barely emerged from the background noise (Fig. 13.17G).
Immobilization of MPs via OligAPols offers the advantage of being reversible. Indeed, most
complexes were released from gold NPs that had been functionalized with cODN and BR trapped in
OligAPol/FAPol AF647 when the beads were exposed for 10 min to 8 M urea or when they were brought
for 10 min under shaking at 72
C, that is above the estimated melting temperature of the duplex
(Le Bon et al. 2014a).
OligAPols are highly complementary to biotinylated or polyhistidine-tagged APols: their binding is strong, but it is reversible, the melting temperature can be modulated at will by playing on the
sequence of the ODN and/or the cODN, and it features an exceptional flexibility in choosing
complementary partners: by hybridizing OligAPol-trapped MPs with di-block oligonucleotides, one
part of which recognizes the OligAPol and the other has any desirable sequence, each individual MP
Fig. 13.17 Immobilization of bacteriorhodopsin (BR) via OligAPol hybridization with a thiol-tagged
cODN adsorbed onto gold nanoparticles (NPs) and specific recognition by an anti-BR antibody. (A)
Differential interference contrast image of gold NPs (100 nm diameter) deposited on a polylysine-coated
glass slide and washed with 10 mM potassium phosphate buffer, pH 7.2 (PPB). (B) Fluorescence image
(λ exc ¼ 620 Æ 30 nm, detection at 700 Æ 37 nm) of gold NPs preincubated with cODN, washed with PPB,
and incubated for 2 h with BR trapped in a 1:1 OligAPol/ FAPol AF647 mixture. (C) and (D) Fluorescence
images (λ exc ¼ 470 Æ 20 nm, detection at 525 Æ 25 nm) of gold NPs preincubated with cODN, washed
with PPB, and incubated for 2 h either with BR trapped in a 1:1 OligAPol/ FAPol AF647 mixture (C) or, as a
control, with the OligAPol/FAPol AF647 mixture alone (D), after which both samples were washed with
PPB and incubated for 1 h with a BR-specific antibody labeled with Alexa Fluor 488. The fluorescence
image showed a much smaller signal when BR/OligAPol/FAPol AF647 complexes were incubated with
particles coated with noncomplementary poly-C DNA strands ((E); Control 1) or with gold NPs
functionalized with the cODN, but hybridized with an excess of Cy3-labeled ODN prior to the addition
of the BR/OligAPol/FAPol AF647 complexes ((F); Control 2). Brightness and contrast settings were the
same for each set of images taken at the same wavelength (B, E, F, on the one hand, and C, D, on the
other). Scale bar is 5 μm. (G) Line scans extracted from Panels B, E, and F. Each scan went through two
representative NPs (From Le Bon et al. 2014, # 2014 Oxford University Press).
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13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
FAPol AF647 mixture (Le Bon et al. 2014a).
The presence of the MPs at the surface of gold NPs was confirmed by incubation with Alexa
Fluor 488- or Alexa Fluor 647-labeled antibodies directed against BR (Fig. 13.17C) or against tOmpA
(Le Bon et al. 2014a). Several control experiments were designed to confirm the specificity of the
interaction between cODN-coated gold NPs and OligAPol-trapped MPs. No binding was observed, for
instance, when gold NPs were coated with thiolated polycytosine, which is not complementary to the
ODN (Control 1, Fig. 13.17E, G), nor onto cODN-coated gold NPs that had been preincubated with an
excess of free, Cy3-labeled ODN (Control 2, Fig. 13.17F, G), hybridization of the free ODN being
confirmed by the Cy3 fluorescence signal. The FAPol AF647 signals measured in these control
experiments were much lower ($10%) and barely emerged from the background noise (Fig. 13.17G).
Immobilization of MPs via OligAPols offers the advantage of being reversible. Indeed, most
complexes were released from gold NPs that had been functionalized with cODN and BR trapped in
OligAPol/FAPol AF647 when the beads were exposed for 10 min to 8 M urea or when they were brought
for 10 min under shaking at 72
C, that is above the estimated melting temperature of the duplex
(Le Bon et al. 2014a).
OligAPols are highly complementary to biotinylated or polyhistidine-tagged APols: their binding is strong, but it is reversible, the melting temperature can be modulated at will by playing on the
sequence of the ODN and/or the cODN, and it features an exceptional flexibility in choosing
complementary partners: by hybridizing OligAPol-trapped MPs with di-block oligonucleotides, one
part of which recognizes the OligAPol and the other has any desirable sequence, each individual MP
Fig. 13.17 Immobilization of bacteriorhodopsin (BR) via OligAPol hybridization with a thiol-tagged
cODN adsorbed onto gold nanoparticles (NPs) and specific recognition by an anti-BR antibody. (A)
Differential interference contrast image of gold NPs (100 nm diameter) deposited on a polylysine-coated
glass slide and washed with 10 mM potassium phosphate buffer, pH 7.2 (PPB). (B) Fluorescence image
(λ exc ¼ 620 Æ 30 nm, detection at 700 Æ 37 nm) of gold NPs preincubated with cODN, washed with PPB,
and incubated for 2 h with BR trapped in a 1:1 OligAPol/ FAPol AF647 mixture. (C) and (D) Fluorescence
images (λ exc ¼ 470 Æ 20 nm, detection at 525 Æ 25 nm) of gold NPs preincubated with cODN, washed
with PPB, and incubated for 2 h either with BR trapped in a 1:1 OligAPol/ FAPol AF647 mixture (C) or, as a
control, with the OligAPol/FAPol AF647 mixture alone (D), after which both samples were washed with
PPB and incubated for 1 h with a BR-specific antibody labeled with Alexa Fluor 488. The fluorescence
image showed a much smaller signal when BR/OligAPol/FAPol AF647 complexes were incubated with
particles coated with noncomplementary poly-C DNA strands ((E); Control 1) or with gold NPs
functionalized with the cODN, but hybridized with an excess of Cy3-labeled ODN prior to the addition
of the BR/OligAPol/FAPol AF647 complexes ((F); Control 2). Brightness and contrast settings were the
same for each set of images taken at the same wavelength (B, E, F, on the one hand, and C, D, on the
other). Scale bar is 5 μm. (G) Line scans extracted from Panels B, E, and F. Each scan went through two
representative NPs (From Le Bon et al. 2014, # 2014 Oxford University Press).
614
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications