(C-110) Inorganic metal-DNA hybrid microstructure for colorimetric cell detection

Introduction::

The recent development of DNA nanotechnology, DNA-based materials as a novel platform for biosensing have been regarded for biosensing probe materials with their programmability and feasible nature. Among them, the hybridization of DNA materials with inorganic materials such as gold, silver, or iron oxide was also drawn attention for exploiting their exceptional functionality. However, the conjugation of inorganic molecules with biomolecules was expensive and laborious. Here we introduce the fabricating method of iron oxide nanoparticle (IONP) hybridized DNA structures via electrostatic interaction. Template DNA microstructure manufactured with rolling circle amplification (RCA) method was highly porous and negatively charged and could act as active sites for co-precipitation reaction of iron cations. DNA/IONP hybrid was core-shell structured with covering iron oxide particles on a surface of single DNA microstructure. Rationally designed DNA template with targeting aptamer sequence, DNA/IONP exhibited cancer cell targeting ability and was easily observed by colorimetric dye staining iron oxide.

Materials and Methods:: Synthesis of the DNA microsponge (DMS) by rolling circle amplification (RCA). The circular DNA was incubated with a dNTP mix, Phi 29 polymerase and reaction buffer at 30 ◦C for 20 h. The final reactant was sonicated briefly to break up any connections between the particles. The solution was centrifuged at 6000 rpm for 3 min and washed with nuclease-free water several times to collect the particles to remove any none-reacted reactants. 

Fabrication of DNA/IONP hybrid. Ferric ammonium sulfate and ferrous ammonium sulfate were prepared in 2:1 molar ratio and mixed with DMS solution, and the mixture was incubated for 15 min at 60 ◦C. Then the solution was added drop by drop into the KOH solution. The solution was washed with nuclease-free water to remove any residual particles and centrifuged to collect DNA/IONP. For further growth, co-precipitation steps with additional iron salt solution were repeated.

Cancer cell binding assay. MDA-MB-231 cells were seeded in a 24 well plate and incubated for 24 h. DNA/IONP was treated and incubated at 37 ◦C with 5% CO2 for 2 h. Then the cells were washed for 2 times with DPBS to remove unbound DNA/IONP and fixed with 4% paraformaldehyde and stained with Prussian blue for visualization. The ratio of bound/unbound DIONPs to the cells was calculated from bright field image using ImageJ software. For quantification, trypsinized cells were lysed and stained with Prussian blue. UV–vis absorbance spectrum of sample was obtained using Nano Drop.

Results, Conclusions, and Discussions:: DNA microsponge (DMS) is fabricated via rolling circle amplification method. DNA polymerase called Phi 29 continuously produces long ssDNA strands along the circularized DNA. The resulting ssDNA strands then make self-assembly bound with Mg2PPi crystals to form DNA microsponges. Moreover, rationally designed template DNA containing complement sequence of desiring aptamer, Apt-DMS could functionalized with targeting ability. Then, owing to the negatively charged DNA backbone enabling electrostatic interaction with cationic Fe ions, nanoparticles were generated evenly on the surface of the microsponge via DNA-templated co-precipitation. SEM and TEM analysis results showing the iron oxide nanoparticles evenly covered the surface of the DNA microsponge (DMS) after the in situ co-precipitation reaction. Due to the negatively charged phosphate backbone, iron cations easily deposited on the surface of the DMS and homogeneously grown into the iron oxide nanoparticles. EDX and SAED pattern analysis exhibit further characteristics of produced iron oxide nanoparticles. The surface-defined IONP growth had been confirmed by controlling the synthetic condition of IONP. Post-deposited IONPs were heterogeneously covered the DMS particle. By adjusting the synthetic conditions, the growth of IONPs on the DNA-based template is feasible for preparing DNA-metal conjugated probes. When co-incubated with the cells with overexpressing target protein named nucleolin, AS1411 aptamer-templated DIONP bound to target cells and expressing noticeable colorimetric signals compared to DIONP with scrambled DNA sequence.

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