pH-responsive glycine functionalized magnetic iron oxide nanoparticles for SARS-CoV-2 RNA extraction from clinical...

The recent outbreak of the novel corona virus disease 2019 (COVID-19) has been made a serious global impact due to its high infectivity and severe symptoms. The Severe Acute Respiratory Syndrome (SARS-CoV-2) RNA extraction is considered as one of the most important steps in COVID-19 detection. Several commercially available kits and techniques are currently being used for specific extraction of SARS-CoV-2 RNA. However, such methods are time consuming and expensive due to the requirement of trained labors, and several chemical reagents. To overcome the mentioned limitations, magnetic RNA adsorption methodology of glycine functionalized iron oxide nanoparticles (GNPs) was established. It showed an efficient potential in SARS-CoV-2 RNA extraction due to pH responsive nature of GNPs. The highly magnetic pH responsive GNPs were synthesized by one-pot co-precipitation method. Random morphology and average 20 nm size of GNPs were denoted by Transmission Electron Microscopy (TEM). X-ray diffractometer (XRD) showed the crystalline magnetite nature. Fourier transform infrared spectroscopy (FT-IR) and UV-visible spectrometry confirmed the presence of glycine on the surface of magnetic nanoparticles. Furthermore, the magnetic nature and thermal properties of GNPs were examined by vibrating sample magnetometer (VSM) and thermo-gravimetric analysis (TGA), respectively. In this study, glycine performed the role of RNA adsorbent. The adsorption of RNA onto the surface of GNPs was achieved in acidic medium (pH 6). In contrary, the elution of RNA from the surface of GNPs was achieved in basic medium (pH 8). The purity of obtained RNA was analyzed by UV-visible spectrometry. Further, the obtained RNA was examined for the presence of SARS-CoV-2 specific Envelope (E), RNA dependent RNA polymerase (RDRP) and Nucleocapsid (N) genes using an RT-PCR analysis. It showed the sudden rise in amount of these genes after 25 cycles of RT-PCR and hence indicated the efficient RNA extraction by GNPs. Agarose gel electrophoresis was used for validation of the quantity and quality of RNA extracted from SARS-CoV-2 patient's sample. The reusability studies of GNPs were performed by monitoring the repeated use of GNPs for SARS-CoV-2 RNA extraction. This method possesses potential role in the field of disease diagnosis. The extraction results of RNA from SARS-CoV-2 patient's sample indicated that the GNPs have an outstanding property over the current existing extraction protocols. It leads to the new advancements in extraction and detection of RNA.

Graphical abstract of the pH responsive SARS-CoV-2 RNA extraction by using glycine functionalized magnetic iron oxide nanoparticles (GNPs) which were prepared by modified cost effective one pot chemical synthesis method. Prepared GNPs were characterized by XRD, FT-IR and UV-Visible spectrometry, Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM). Glycine present on the surface of nanoparticles (NPs) played an important role in pH responsive RNA extraction procedure. When nanoparticles added in acidic (pH < 7) medium, glycine gained positive surface charge hence overall surface charge of NPs became positive. Thereby SARS-CoV-2 RNA adsorption/binding occurred on the surface of GNP. Later, the RNA-GNP complex was separated by an external magnet. Separated complex was added in basic (pH > 7) medium to elute RNA from GNP. This phenomenon occurred due to surface negative charge of glycine that caused charge repulsion with RNA. Eluted RNA was examined qualitatively and quantitatively by RT-PCR, nanodrop technique and agarose gel electrophoresis. Results were compared with kit based extracted RNA.

The online version contains supplementary material available at 10.1007/s10853-022-07464-6.