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. 2024 Sep 20;25(18):10108.
doi: 10.3390/ijms251810108.

Freeze-Driven Adsorption of Oligonucleotides with polyA-Anchors on Au@Pt Nanozyme

Nikita E Lapshinov  1 , Svetlana M Pridvorova  1 , Anatoly V Zherdev  1 , Boris B Dzantiev  1 , Irina V Safenkova  1
Affiliations

Freeze-Driven Adsorption of Oligonucleotides with polyA-Anchors on Au@Pt Nanozyme

Nikita E Lapshinov et al. Int J Mol Sci. .

Abstract

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze-thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5'-polyadenine anchor (An, with n = 3, 5, 7, 10; triple-branched A3, or triple-branched A5), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3'-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A7 > A5 > 3A3 > 3A5 > A10 > A3 anchors. These [Au@Pt]NP-oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A3, A5, A7, and 3A3 anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems.

Keywords: freeze–thaw method; gold nanoparticles; lateral flow test; nanozymes; oligonucleotide conjugation; peroxidase-like activity; platinum nanoparticles; spherical nucleic acids.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schemes of the experiments. (A) Oligonucleotide (An-ssDNA) for immobilization consisting of 5′-polyadenine anchor (An, with n = 3, 5, 7, 10; triple-branched A3 and triple-branched A5), random sequence, polythymine block, fluorescein; (B) syntheses of conjugates and testing of their binding capacity using lateral flow assay (1—lateral flow test strip with anti-FAM immobilized in the binding zone before assay; 2—test strip with red line due to the binding of [Au]NP conjugate in the binding zone; 3—test strip with gray line due to the binding of [Au@Pt]NP conjugate in the binding zone; 4—test strip with brown line due to the peroxidase-like catalytic properties of [Au@Pt]NP conjugate in the binding zone). FAM—fluorescein; anti-FAM—monoclonal antibodies specific to fluorescein; DAB—3,3′-diaminobenzidine.
Figure 2
Figure 2
Characterization of the synthesized nanoparticles. Absorption spectra of [Au]NPs (A) and [Au@Pt]NPs (B); TEM micrographs of [Au]NPs (C) and [Au@Pt]NPs (D); hydrodynamic diameter distributions obtained by DLS for [Au]NPs (E) and [Au@Pt]NPs (F).
Figure 2
Figure 2
Characterization of the synthesized nanoparticles. Absorption spectra of [Au]NPs (A) and [Au@Pt]NPs (B); TEM micrographs of [Au]NPs (C) and [Au@Pt]NPs (D); hydrodynamic diameter distributions obtained by DLS for [Au]NPs (E) and [Au@Pt]NPs (F).
Figure 3
Figure 3
Characterization of synthesized conjugates. Absorption spectra of [Au]NP–An-ssDNA (A) and [Au@Pt]NP–An-ssDNA (B); TEM micrographs of [Au]NP–A10-ssDNA (C) and [Au@Pt]NP–A7-ssDNA (D); hydrodynamic diameter distributions obtained by DLS for [Au]NP–A10-ssDNA (E) and [Au@Pt]NP–A7-ssDNA (F). Arrow 1 shows the nanoparticle; arrow 2 shows the immobilized oligonucleotide.
Figure 3
Figure 3
Characterization of synthesized conjugates. Absorption spectra of [Au]NP–An-ssDNA (A) and [Au@Pt]NP–An-ssDNA (B); TEM micrographs of [Au]NP–A10-ssDNA (C) and [Au@Pt]NP–A7-ssDNA (D); hydrodynamic diameter distributions obtained by DLS for [Au]NP–A10-ssDNA (E) and [Au@Pt]NP–A7-ssDNA (F). Arrow 1 shows the nanoparticle; arrow 2 shows the immobilized oligonucleotide.
Figure 4
Figure 4
Test strips with binding zones stained by binding of [Au@Pt]NP–An-ssDNA conjugates and the dependencies of binding zone coloration. (A) Test strips colored due to the colorimetric properties of nanozyme for [Au@Pt]NP–A7-ssDNA conjugates (the numbers indicate the dilution factor; C shows the control experiment without conjugate), and (B) dependencies of binding zone coloration on conjugate dilution (for each dilution, two replicates were made; the figure shows the mean values and standard deviations as error bars); (C) test strips with enhanced coloration due to catalytic properties of nanozyme for [Au@Pt]NP–A7-ssDNA (the numbers indicate the dilution factor; C shows the control experiment without the conjugate), and (D) dependencies of binding zone coloration on conjugate dilution (for each dilution, two replicates were made; the figure shows the mean values and standard deviations as error bars); (E) comparison of test strips for six conjugates at 9-fold dilution without catalytic enhancement; (F) comparison of test strips for six conjugates at 9-fold dilution after catalytic enhancement.
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