Interaction of Metal-Based Nanoparticles with Proteins: Relation to Structure, Function and Amyloid Forming Propensity of Lysozyme and α-Lactalbumin

Metal nanoparticles (NPs) such as gold (AuNP), silver (AgNP), and zinc oxide (ZnONP) demonstrate variety of applications including drug delivery, imaging, nanomedicine, and sensing. However, many of their applications involve biological systems, which might trigger their interaction with various biomolecules such as proteins. Proteins are highly sensitive to various stresses and ligand interaction due to the intimate correlation of their structure with biological function. Moreover, it is also a well-known fact that protein misfolding and aggregation process is the prime cause of a number of neurodegenerative disorders like Alzheimer’s disease, Parkinson disease, Prion disease, etc. The interaction of NP with the proteins can cause the change of structure as well as the function of proteins and can generate a new identity such as ‘nanoparticle-protein’ complex. Here, in our present study, we investigated the interaction of two small homologous proteins: bovine α-lactalbumin (BLA) and hen egg white lysozyme (HEWL) with three different nanoparticles (AuNP, AgNP, ZnONP) in vitro. The structure, function, stability and amyloid forming propensity of the proteins were studied during the interaction with three different NPs using various spectroscopic and microscopic techniques. We synthesized NPs by chemical as well as semi-green methods using non-toxic materials such as starch, PEG, and NaOH with precursor salts with a size of below 20 nm. Moreover, we synthesized the self-assembly of ZnONP of an average size of 163 nm. The NPs were further characterized using field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) as well as dynamic light scattering (DLS) zeta sizer for the analysis of size and shape and stability of NP....

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 (ZnONP_uncap), starch-capped (ZnONP_ST), and self-assembled (ZnONP_assmb) (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 ZnONP_ST. Such inhibition was also associated with a decrease in cross β-sheet amount, surface hydrophobicity as well as increase of stability of proteins. Furthermore, we observed that ZnONP_ST 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 ZnONP_ST 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

Femtomolar Level-Specific Detection of Lead Ions in Aqueous Environments, Using Aptamer-Derivatized Graphene Field-Effect Transistors

The detection of lead ion (Pb2+) contamination in aqueous media is relevant for preventing endemic health issues as well as damage to cognitive and physical health. Existing home kit tests are unable to achieve clinically relevant sensitivity and specificity. Here, a label-free graphene field-effect transistor sensor for detecting Pb2+ at the femtomolar (fM) level, discriminating between confounding ions, is reported. The sensing principle is based on electrically monitoring Pb2+-binding-mediated conformational changes of a specific aptamer tethered to graphene, modeled through the Hills–Langmuir mechanism. A record sensitivitythrough a limit of detection of ∼61 fM, for Pb2+ was demonstrated. For model verification, specific discrimination of Pb2+ from other ions at the 1 picomolar (pM) level was shown. The reported work provides motivation for development of portable, label-free, point-of-care devices with both high specificity and sensitivity

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

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

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

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