‘Spotted Nanoflowers’: Gold-seeded Zinc Oxide Nanohybrid for Selective Bio-capture

Hybrid gold nanostructures seeded into nanotextured zinc oxide (ZnO) nanoflowers (NFs) were created for novel biosensing applications. The selected ‘spotted NFs’ had a 30-nm-thick gold nanoparticle (AuNP) layer, chosen from a range of AuNP thicknesses, sputtered onto the surface. The generated nanohybrids, characterized by morphological, physical and structural analyses, were uniformly AuNP-seeded onto the ZnO NFs with an average length of 2–3 μm. Selective capture of molecular probes onto the seeded AuNPs was evidence for the specific interaction with DNA from pathogenic Leptospirosis-causing strains via hybridization and mis-match analyses. The attained detection limit was 100 fM as determined via impedance spectroscopy. High levels of stability, reproducibility and regeneration of the sensor were obtained. Selective DNA immobilization and hybridization were confirmed by nitrogen and phosphorus peaks in an X-ray photoelectron spectroscopy analysis. The created nanostructure hybrids illuminate the mechanism of generating multiple-target, high-performance detection on a single NF platform, which opens a new avenue for array-based medical diagnostics.


Analytical performances of spotted nanoflower biosensor Sensitivity
The sensitivity of the proposed spotted nanoflower bioelecrode was investigated and a linear correlation of the differences in the charge transfer resistance were plot using equation (1): with respect to the logarithm of complementary DNA concentration are shown in Fig.   6a. It was observed that the ∆Rct linearly increases with increasing of complementary DNA concentration from 10 M-100 fM and thereafter saturated further. The difference value (∆Rct) between Rct at probe immobilized biolectrode and hybridized bioelectrode were found to be well proportional to the natural logarithm of t-DNA concentration with a linear equation of ∆Rct = 1.456E6x + 1.915E7, (R 2 = 0.99542). A detection limit of detection as 100 fM was estimated using signal to noise ratio of more than 3σ (where σ is the standard deviation of the blank solution, n=5). The detection limit is much lower than previously reported gold nanowire and ZnO nanowire using electrochemical impedance spectroscopy 18,45 .

Mis-matching and Specificity analyses
A high specificity is a necessity for a new designed biosensor with potential applications in complicated samples. In order to investigate the specificity of proposed It was observed that the value of Rct signal does not vary significantly in the presence of unrelated molecules indicating non-influence of the individual interferants.

Reproducibility and Response time
In the present work, the reproducibility of the proposed spotted nanoflower biosensor was also investigated by comparing five samples of the same batch and from different preparations. Fig. 6c

Stability and Regeneration
The stability of the proposed spotted nanoflower biosensor was examined by the shelflife study over a period of 14 weeks at a regular interval of 1 week and stored at 4C when not in use; the results are displayed in Fig. 6d. The bioelectrode was used to detect 1 nM of t-DNA concentration at room temperature. The stability results show that prepared bioelectrode is very stable and only loss 10% of its activity (Rct value) after 4 weeks. It was observed that the prepared bioelectrode retain more 70% of its activity even after 10 weeks and subsequently loss 50 % of its activity after 14 weeks.
Regeneration was accomplished by rinsing the DNA hybridized bioelectrode surfaces with hot double distilled H2O for 2 min, and subsequently rapid cooling in ice bath. The reusability of the biosensor was tested by repetitive hybridization with t-DNA, the Rct values of the regenerated biolectrode before and after hybridization of t-DNA were obtained ( Fig. 6d; inset). As shown in the inset, the Rct values for both the bioelectrode before and after regeneration were found to be same with negligible differences. The observed results suggest that thermal denaturation treatment can effectively break the hydrogen bond between the hybridized strands without desorbing the covalent Au thiol bond from DNA immobilization on the bioelectrode surface, indicates the stability of sensing surfaces under higher temperature. After consequent 10 regenerations and hybridizations, the electrode only loss about 9.3% of its original Rct signal value.
Therefore, the regeneration of the proposed DNA sensor possessed potential for repeatable monitoring of target DNA.