Synthesis of bioluminescent gold nanoparticle-luciferase hybrid systems for technological applications

Abstract

Bioluminescent gold nanoparticles (AuNPs) were synthesized in situ using dithiol-terminated polyethylene glycol (PEG(SH)₂) as reducer and stabilizing agents. Hybrid Au/F₃O₄ nanoparticles were also produced in a variation of synthesis, and both types of nanostructures had the polymer capping replaced by L-cysteine (Cys). The four types of nanoparticles, PEG(SH)₂AuNPs, PEG(SH)₂Au/F₃O₄NPs, CysAuNPs, and CysAu/F₃O₄NPs were associated with purified recombinant Pyrearinus termitilluminans green emitting click beetle luciferase (PyLuc) and Phrixotrix hirtus (RELuc) red-emitting railroad worm luciferase. Enzyme association with PEG(SH)₂ was also investigated as a control. Luciferases were chosen because they catalyze bioluminescent reactions used in a wide range of bioanalytical applications, including ATP assays, gene reporting, high-throughput screening, bioluminescence imaging, biosensors and other bioluminescence-based assays. The immobilization of PyLuc and RELuc promoted partial suppression of the enzyme luminescence activity in a functionalization-dependent way. Association of PyLuc and RELuc with AuNPs increased the enzyme operational stability in relation to the free enzyme, as evidenced by the luminescence intensity from 0 to 7 h after substrate addition. The stability of the immobilized enzymes was also functionalization-dependent and the association with CysAuNPs was the condition that combined more sustained luminescent activity with a low degree of luminescence quenching. The higher enzymatic stability and sustained luminescence of luciferases associated with nanoparticles may improve the applicability of bioluminescence for bioimaging and biosensing purposes.

Keywords: Bioluminescence; Dithiol-terminated polyethylene glycol; Gold nanoparticles; Luciferases; Magnetic nanoparticles; Nanotechnology.

Fig. 1

Schematic representation of AuNP synthesis in situ using PEG(SH)₂8000 as reducing and stabilizing agents. In the first step of the synthesis, Au³⁺ is reduced to Au¹⁺ assisted by HEPES. The oxidation of PEG(SH)₂8000 can produce sulfoxide and polymer crosslink. In the step 2, dismutation of Au⁺¹ produces gold atoms that form gold nanoclusters associated to the partially oxidized polymer. In the third step AuNPs are formed by nanocrystal growing

Fig. 2

Characterization of PEG(SH)₂AuNPs by UV–visible spectroscopy, FESEM and DLS. A SPR band of PEG(SH)₂AuNPs (black line) and PEG(SH)₂ (gray line). The inset shows the snapshot of the colloidal suspension; B hydrodynamic radius distribution obtained by dynamic light scattering indicating a mean value of 170 nm; C–E FESEM images of the PEG(SH)₂AuNPs in which it is possible to distinguish the corona of PEG(SH)₂8000 probably crosslinked by disulfide bonds

Fig. 3

Size, shape, and capping characterization of CysAuNPs. A FESEM images of the AuNPs with the inset showing the distribution of edge-to-edge distances obtained from FESEM images for CysAuNPs. B, C Shows the respective XPS spectra of Au 4f, and 1Os XPS of CysAuNPs. The XPS band were deconvoluted by using the software Microcal Origin 9.0 multi peak fit tool

Fig. 4

Characterization of PEG(SH)₂Au/Fe₃O₄ NPs by size, shape, elemental composition and SPR band. A FESEM images of the NPs with the inset showing the distribution of edge-to-edge distances obtained from FESEM images for PEG(SH)₂Au/Fe₃O₄ NPs; B SPR band of PEG(SH)₂Au/Fe₃O₄ NPs (black line) overlapped with the SPR band of PEG(SH)₂AuNPs (green line) and PEG(SH)₂ (red line). The inset shows the snapshot of the colloidal suspension of PEG(SH)₂Au/Fe₃O₄ NPs; C representative EDX graphic with elemental contributions of AuMα1 and FeKα. EDX maps showing co-localization of Fe and Au, and graphics are available in Supplemental material: Figure S1A and B

Fig. 5

Characterization of gold nanoparticle–luciferase hybrid system formation by FTIR. A FTIR spectra of free Py (green) and RE (wine) luciferases showing the contributions of typical protein amide I, amide II and amide III bands. B, C FTIR spectrum of PEG(SH)2AuNPs (black line) overlapped with the spectrum of AuNPs associated, respectively, with PyLuc and RELuc as blue lines. In these spectra, the region of 1600–1700 cm⁻¹ was decomposed using the multi-peak fit tool of Origin software to show the double overlap contributions (cyan and blue dotted lines). D PDB three-dimensional structure of Luciola cruciata luciferase in the presence of DLSA (PDB code 2D1S) with 56AEY58 amino-acid sequence that was aleatorily chosen to illustrate the vibrations responsible for amide I and amide II bands and a representation of PEG(SH)₂ structure. The polymer and amino acid sequence structures were constructed with ACD/ChemSketch software

Fig. 6

A–D Are representative FESEM images of PEG(SH)₂AuNPs associated with PyLuc. A–C Show representative images of the samples containing PyLuc compared with the PEG(SH)₂AuNPs (D)

Fig. 7

Effect of the association with PEG(SH)₂ and AuNPs on the stability and luminescence decays of luciferases. Graphics A and B refers to PyLuc results and C and D to RELuc results as indicated in the graphic legend in green and rose, respectively. A, C Effects of incubation time (aging) on the loss of PyLuc and RELuc activities, respectively, in different conditions: free in solution (grey), associated with PEGSH₂ (black), PEGSH₂AuNPs (navy), CysAuNPs (violet) PEGSH₂AuNPMag (orange) and CysAuNPMag (dark yellow). B, D Effects of the association with PEGSH₂ and different NPs on the luminescence decay of PyLuc and RELuc, respectively, after LH2 addition and after 2 h of incubation with Mg²⁺ and ATP in the absence of substrate. The normalized decays were obtained from the well densitometry of white color in the snapshots (insets of C and D, respectively), similarly to previously described by Yokomizo et al. [62]. The insets of B and D were composed by images of the first lines of wells as indicated in Figure S3 and S4 of Supplemental material