Rapid self-test of unprocessed viruses of SARS-CoV-2 and its variants in saliva by portable wireless graphene biosensor.

We have developed a DNA aptamer-conjugated graphene field-effect transistor (GFET) biosensor platform to detect receptor-binding domain (RBD), nucleocapsid (N), and spike (S) proteins, as well as viral particles of original Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus and its variants in saliva samples. The GFET biosensor is a label-free, rapid (≤20 min), ultrasensitive handheld wireless readout device. The limit of detection (LoD) and the limit of quantitation (LoQ) of the sensor are 1.28 and 3.89 plaque-forming units (PFU)/mL for S protein and 1.45 and 4.39 PFU/mL for N protein, respectively. Cognate spike proteins of major variants of concern (N501Y, D614G, Y453F, Omicron-B1.1.529) showed sensor response ≥40 mV from the control (aptamer alone) for fM to nM concentration range. The sensor response was significantly lower for viral particles and cognate proteins of Middle East Respiratory Syndrome (MERS) compared to SARS-CoV-2, indicating the specificity of the diagnostic platform for SARS-CoV-2 vs. MERS viral proteins. During the early phase of the pandemic, the GFET sensor response agreed with RT-PCR data for oral human samples, as determined by the negative percent agreement (NPA) and positive percent agreement (PPA). During the recent Delta/Omicron wave, the GFET sensor also reliably distinguished positive and negative clinical saliva samples. Although the sensitivity is lower during the later pandemic phase, the GFET-defined positivity rate is in statistically close alignment with the epidemiological population-scale data. Thus, the aptamer-based GFET biosensor has a high level of precision in clinically and epidemiologically significant SARS-CoV-2 variant detection. This universal pathogen-sensing platform is amenable for a broad range of public health applications and real-time environmental monitoring.

Toward the Ultimate Limit of Analyte Detection, in Graphene-Based Field-Effect Transistors.

The ultimate sensitivity of field-effect-transistor (FET)-based devices for ionic species detection is of great interest, given that such devices are capable of monitoring single-electron-level modulations. It is shown here, from both theoretical and experimental perspectives, that for such ultimate limits to be approached the thermodynamic as well as kinetic characteristics of the (FET surface)-(linker)-(ion-receptor) ensemble must be considered. The sensitivity was probed in terms of optimal packing of the ensemble, through a minimal charge state/capacitance point of view and atomic force microscopy. Through the fine-tuning of the linker and receptor interaction with the sensing surface, a record limit of detection as well as specificity in the femtomolar range, orders of magnitude better than previously obtained and in excellent accord with prediction, was observed.

MRI reporter gene MagA suppresses transferrin receptor and maps Fe2+ dependent lung cancer.

As a magnetic resonance imaging (MRI) reporter gene, MagA has become a powerful tool to monitor dynamic gene expression and allowed concomitant high resolution anatomical and functional imaging of subcellular genetic information. Here we establish a stably expressed MagA method for lung cancer MRI. The results show that MagA can not only enhance both in vitro and in vivo MRI contrast by specifically alternating the transverse relaxation rate of water, but also inhibit the malignant growth of lung tumor. In addition, MagA can regulate magnetic nanoparticle production in grafted tissues and also suppress transferrin receptor expression by acting as an iron transporter, and meanwhile can permit iron biomineralization in the presence of mammalian iron homeostasis. This work provides experimental evidence for the safe preclinical applications of MagA as both a potential inhibitor and an MRI-based tracing tool for iron ion-dependent lung cancer.

Graphene Oxide Quantum Dot Alters Amyloidogenicity of Hen Egg White Lysozyme via Modulation of Protein Surface Character.

A series of neurodegenerative disorders are caused by intracellular or extracellular amyloid deposition, including Alzheimer's disease, Parkinson's disease, Prion disease, and so on. To prevent the progress of such amyloid-mediated disorders, various agents have been tested including nanoparticles. Among different nanomaterials, graphene oxide shows unique electrochemical properties, which have potential applications in various biomedical fields. In our present investigation, we explored the effect of graphene oxide quantum dots (GOQDs) in amyloid ß-fibrillation of hen egg white lysozyme (HEWL) under various conditions. Electron microscopy imaging showed that administration of GOQD inhibited HEWL amyloid ß-fibrillation via producing thin and small fragments of fibrils. ζ-Potential measurement and 8-anilino-1-naphthalenesulfonic fluorescence study of lysozyme amyloid demonstrated a significant drop in surface hydrophobicity and an increase of surface charge of protein molecules. The reduced hydrophobic interaction and enhanced surface charge inhibit the hydrophobic assembly and colloidal stability of the protein. Circular dichroism and thioflavin-T fluorescence demonstrated that GOQD also interfered at the secondary structure level and prevented amyloid ß-sheet formation and assembly of a protein by reducing the amount of amyloid ß-sheet formation. Further, cellular toxicity analysis with HaCaT and 3T3 cells showed reduced toxicity of amyloid samples prepared with GOQD. Therefore, GOQD might be used to be a potential amyloid-preventive agent in various neurodegenerative diseases.

Graphene and Two-Dimensional Materials for Biomolecule Sensing.

An ideal biosensor material at room temperature, with an extremely large surface area per unit mass combined with the possibility of harnessing quantum mechanical attributes, would be comprised of graphene and other two-dimensional (2D) materials. The sensing of a variety of sizes and types of biomolecules involves modulation of the electrical charge density of (current through) the 2D material and manifests through specific components of the capacitance (resistance). While sensitive detection at the single-molecule level, i.e., at zeptomolar concentrations, may be achieved, specificity in a complex mixture is more demanding. Attention should be paid to the influence of inevitably present defects in the 2D materials on the sensing, as well as calibration of obtained results with acceptable standards. The consequent establishment of a roadmap for the widespread deployment of 2D material-based biosensors in point-of-care platforms has the potential to revolutionize health care.

Direct DNA Methylation Profiling with an Electric Biosensor.

DNA methylation is one of the principal epigenetic mechanisms that control gene expression in humans, and its profiling provides critical information about health and disease. Current profiling methods require chemical modification of bases followed by sequencing, which is expensive and time-consuming. Here, we report a direct and rapid determination of DNA methylation using an electric biosensor. The device consists of a DNA-tweezer probe integrated on a graphene field-effect transistor for label-free, highly sensitive, and specific methylation profiling. The device performance was evaluated with a target DNA that harbors a sequence of the methylguanine-DNA methyltransferase, a promoter of glioblastoma multiforme, a lethal brain tumor. The results show that we successfully profiled the methylated and nonmethylated forms at picomolar concentrations. Further, fluorescence kinetics and molecular dynamics simulations revealed that the position of the methylation site(s), their proximity, and accessibility to the toe-hold region of the tweezer probe are the primary determinants of the device performance.

DNA Nanotweezers and Graphene Transistor Enable Label-Free Genotyping.

Electronic DNA-biosensor with a single nucleotide resolution capability is highly desirable for personalized medicine. However, existing DNA-biosensors, especially single nucleotide polymorphism (SNP) detection systems, have poor sensitivity and specificity and lack real-time wireless data transmission. DNA-tweezers with graphene field effect transistor (FET) are used for SNP detection and data are transmitted wirelessly for analysis. Picomolar sensitivity of quantitative SNP detection is achieved by observing changes in Dirac point shift and resistance change. The use of DNA-tweezers probe with high-quality graphene FET significantly improves analytical characteristics of SNP detection by enhancing the sensitivity more than 1000-fold in comparison to previous work. The electrical signal resulting from resistance changes triggered by DNA strand-displacement and related changes in the DNA geometry is recorded and transmitted remotely to personal electronics. Practical implementation of this enabling technology will provide cheaper, faster, and portable point-of-care molecular health status monitoring and diagnostic devices.

Protein functionalization of ZnO nanostructure exhibits selective and enhanced toxicity to breast cancer cells through oxidative stress-based cell death mechanism.

Zinc oxide nanostructure (ZnONS) was chemically synthesized and functionalized (FZnONSBLA) with a small protein bovine α-lactalbumin (BLA) by chemical cross-linking methods. Both nano-structures were characterized using various techniques such as electron microscopy, dynamic light scattering (DLS), UV-Vis spectroscopy, FT-IR, photo-luminescence and X-ray diffraction. Electron microscopy and DLS analysis revealed their (ZnONS and FZnONSBLA) average size of 200nm and 450nm, respectively. When cytotoxicity of both the nanostructures were assessed in breast cancer cells MCF-7 and MDAMB231 by MTT assay and PI/Annexin V staining (FACS), FZnONSBLA demonstrated higher cell death than ZnONS primarily due to generation of intracellular reactive oxygen species (ROS). Our experimental results also suggested that such enhanced toxicity was due to the lethal structural variant of BLA in FZnONSBLA as well as higher cellular uptake than ZnONS by cancer cells. The death kinetics study with time in cancer cells further proved that FZnONSBLA caused toxicity much faster than ZnONS, thus suggested a strong role of lethal variant of BLA in FZnONSBLA as a cytotoxic agent in cancer cells. Furthermore, FZnONSBLA demonstrated excellent cytocompatibility (normal cells) and hemocompatibility compared to ZnONS. Hence, considering the biodegradable nature of ZnO nonmaterial, our results demonstrated that BLA functionalized ZnONS could be used to develop a suitable therapeutic strategy in cancer.

Multifunctional stimuli responsive polymer-gated iron and gold-embedded silica nano golf balls: Nanoshuttles for targeted on-demand theranostics.

Multi-functional nanoshuttles for remotely targeted and on-demand delivery of therapeutic molecules and imaging to defined tissues and organs hold great potentials in personalized medicine, including precise early diagnosis, efficient prevention and therapy without toxicity. Yet, in spite of 25 years of research, there are still no such shuttles available. To this end, we have designed magnetic and gold nanoparticles (NP)-embedded silica nanoshuttles (MGNSs) with nanopores on their surface. Fluorescently labeled Doxorubicin (DOX), a cancer drug, was loaded in the MGNSs as a payload. DOX loaded MGNSs were encapsulated in heat and pH sensitive polymer P(NIPAM-co-MAA) to enable controlled release of the payload. Magnetically-guided transport of MGNSs was examined in: (a) a glass capillary tube to simulate their delivery via blood vessels; and (b) porous hydrogels to simulate their transport in composite human tissues, including bone, cartilage, tendon, muscles and blood-brain barrier (BBB). The viscoelastic properties of hydrogels were examined by atomic force microscopy (AFM). Cellular uptake of DOX-loaded MGNSs and the subsequent pH and temperature-mediated release were demonstrated in differentiated human neurons derived from induced pluripotent stem cells (iPSCs) as well as epithelial HeLa cells. The presence of embedded iron and gold NPs in silica shells and polymer-coating are supported by SEM and TEM. Fluorescence spectroscopy and microscopy documented DOX loading in the MGNSs. Time-dependent transport of MGNSs guided by an external magnetic field was observed in both glass capillary tubes and in the porous hydrogel. AFM results affirmed that the stiffness of the hydrogels model the rigidity range from soft tissues to bone. pH and temperature-dependent drug release analysis showed stimuli responsive and gradual drug release. Cells' viability MTT assays showed that MGNSs are non-toxic. The cell death from on-demand DOX release was observed in both neurons and epithelial cells even though the drug release efficiency was higher in neurons. Therefore, development of smart nanoshuttles have significant translational potential for controlled delivery of theranostics' payloads and precisely guided transport in specified tissues and organs (for example, bone, cartilage, tendon, bone marrow, heart, lung, liver, kidney, and brain) for highly efficient personalized medicine applications.

Nano Zinc Oxide Inhibits Fibrillar Growth and Suppresses Cellular Toxicity of Lysozyme Amyloid.

Deposition of amyloid fibers has been a common pathological event in many neurodegenerations, such as Alzheimer's disease, Parkinson's disease, and Prion disease. Although various therapeutic interventions have been reported, nanoparticles have recently been considered as possible inhibitors of amyloid fibrillation. Here, we reported the effect of three different forms of zinc oxide nanoparticles (ZnONP): uncapped (ZnONPuncap), starch-capped (ZnONPST), and self-assembled (ZnONPassmb) (average sizes of 10, 30, and 163 nm, respectively), having a core size of 10-15 nm, in the amyloid growth of hen egg white lysozyme (HEWL). We monitored the amyloid growth by electron microscopy as well as Thioflavin-T (ThT) measurement. We observed that ZnONP demonstrated a dose-dependent inhibition of fibrillar amyloid growth of HEWL, with the greatest effect being exhibited by ZnONPST. Such inhibition was also associated with a decrease in cross ß-sheet amount, surface hydrophobicity as well as increase of stability of proteins. Furthermore, we observed that ZnONPST prolonged the nucleation phase and shortened the elongation phase of HEWL amyloid growth. Although pure amyloid caused profound cellular toxicity in both mouse carcinoma N2a and normal cells such as human keratinocytes HaCaT cells, amyloid formed in the presence of ZnONP showed much reduced cellular toxicity. We also observed that the inhibition of amyloid growth was effective when ZnONP was administered during the lag phase. When our amyloid inhibition results were compared with a well-known inhibitor curcumin, we observed that ZnONPST demonstrated a better inhibitory effect than curcumin. Overall, here, we reported the inhibitory activity of three different forms of ZnONP to amyloid fibrillation of HEWL and amyloid-mediated cytotoxicity to different extents, while starch-capped ZnONP showed the highest fibrillation inhibitory effect.

Protein corona over silver nanoparticles triggers conformational change of proteins and drop in bactericidal potential of nanoparticles: Polyethylene glycol capping as preventive strategy.

Here, we demonstrated that starch-capped silver nanoparticles (AgNPST) with a size range of 10-15nm could readily interact with a small protein bovine α-lactalbumin (BLA) through the formation of protein corona. We further observed that such phenomena not only caused structural change of BLA but drastic drop in the bactericidal potential of AgNP. To design a strategy towards minimizing protein adsorption and maximizing the retention of bactericidal potential of AgNP, we developed stable polyethylene glycol (PEG)-capped AgNP (AgNPPEG) that clearly demonstrated reduced conformational changes of protein and retention of substantial bactericidal potential of AgNPPEG, compared to AgNPST. Moreover, AgNPPEG also showed excellent hemocompatibility. A relatively larger protein bovine serum albumin (BSA) and human blood serum solution containing serum proteins were also used in this study to validate our hypotheses. Overall, our study established that protein coated AgNP losses its inherent bactericidal potential substantially; however, when functionalized with a suitable material such as PEG, it could reduce such drop in substantial amount. Moreover, it achieved improved biocompatibility in actual physiological condition that might find a better therapeutic avenue in many bacteria-mediated disorders.

Zinc oxide nanoparticles modulates the production of ß-glucosidase and protects its functional state under alcoholic condition in Saccharomyces cerevisiae.

In the present investigation, we have investigated the effect of zinc oxide nanoparticles (ZnONP) on the production of ß-glucosidase (BGL) in Saccharomyces cerevisiae under various conditions. ZnONP was synthesized chemically and characterized using various standard techniques. The results revealed that yeast culture administered with 5 mM ZnONP enhanced the intracellular BGL activity up to 28 % compared to control with simultaneous growth of cells. However, at a higher dose of ZnONP (10 and 15 mM), both the activity of the enzyme and yeast growth was dropped. When yeast cells were grown in alcoholic medium (2, 5, and 10 % ethanol), the growth was found inhibited with substantial reduction of intracellular BGL activity. Interestingly, the administration of ZnONP further inhibited the cell growth, however, suppressed the alcoholic effect on enzyme activity. Moreover, under the same condition, ZnONP enhanced the biological activity of the enzyme in cells, indicated a higher yield of BGL production. When the mechanism of ZnONP-mediated cell growth inhibition was investigated, N-acetylcysteine (NAC)-based cell growth study proved that reactive oxygen species (ROS) was not the sole cell death mechanism induced by ZnONP, indicating a second mechanism of cell death. Our findings provide a new insight on the potential application of ZnONP as an external supplement to enhance the active production of BGL like important industrial enzyme in S. cerevisiae in both normal and alcohol stressed condition as well as to produce baker's yeast in higher amount.