Search

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.

Aβ deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aβ1-40 and Aβ1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aβ variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aβ4-42 exhibits a high tendency to form β-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aβ4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aβ4-42. An Aβ variant with a double truncation at phenylalanine-4 and leucine 34, (Aβ4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aβ species in AD pathophysiology.

In high-efficiency smart biomanufacturing, continuous monitoring of products, byproducts, and reagents is crucial for optimizing production processes, improving yield, and ensuring product quality. This monitoring maintains optimal conditions, reduces waste, and enables swift corrective actions, minimizing the risk of producing out-of-specification products. With advancements in cell-free biomanufacturing, the importance of in-line sensing technologies has increased, as they provide real-time tracking of biochemical processes, allowing immediate adjustments to keep manufacturing efficient and consistent. In this study, we successfully developed a graphene field-effect transistor (gFET) sensor to monitor key parameters such as glucose, pH, and immunoglobulin G antibody levels in cell culture media from a CHO cells bioreactor. The gFET sensor accurately detected pH levels between 6.8 and 8.2, glucose concentrations from 5 to 30 mM, and antibody levels ranging from 25 to 100 µg/mL, highlighting the potential of graphene sensors for inline sensing in advanced biomanufacturing. [ABSTRACT FROM AUTHOR]

Establishing an accurate prognosis for women diagnosed with breast cancer (BC) is extremely challenging. Axillary lymph node (ALN) evaluation is considered of major prognostic value. The one‐step nucleic acid amplification (OSNA) assay is currently used for assessing axillary sentinel lymph node (SLN) status in BC. Additionally, total tumor load (TTL) may help predict further metastatic axillary involvement beyond the SLN. The prognostic value of primary BC location remains controversial due to lack of consensus on the biological differences among tumors at various sites. Evidence suggests that tumors in the internal quadrants (INLs) have worse prognosis compared to those in the external quadrants. Furthermore, ALN involvement is believed to be mainly associated with external quadrant tumors, mainly due to the lymphatic drainage system of the breast. This pilot observational study, despite lacking a control group and having a relatively small sample size, is the first to evaluate the potential relationship between primary BC location and ALN metastasis using the OSNA assay. A sample of consecutive BC patients undergoing axillary staging with the OSNA assay were included. Tumors were categorized into three groups based on primary location: external quadrants and axillary tail (EXL), INLs, and nipple and areola location (NAL). Although not statistically significant, the INL group exhibited a higher mean TTL. Additionally, no significant differences were observed between groups concerning SLN detection techniques, SLN status, number of metastatic SLN, or mean TTL. These findings support the use of the innovative tracer superparamagnetic iron oxide regardless of tumor site. This study underscores the importance of understanding the relationship between BC location and ALN status, which may improve prognostic stratification and targeted therapies based on tumor site. If these observations are confirmed in larger, multicentric studies, the potential conclusions may shift the paradigm of INL tumor treatment, significantly impacting clinical practice and research. [ABSTRACT FROM AUTHOR]

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.

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.

The SARS‐CoV‐2 pandemic caused a public health crisis throughout the world and highlighted the need for rapid and sensitive testing as a countermeasure. A sensitive and specific biosensor platform is developed for the detection of antigen and RNA of SARS‐CoV‐2, and its variant (B1.1.529). The demonstrated biosensor platform combines unique protein catalyzed capture bioreceptors (PCCs) for antigen capture and a chimeric (RNA‐DNA) probe for RNA detection using LwaCas13a collateral cleavage activity atop graphene field effect transistors (gFETs). The reported biosensor is able to differentiate unprocessed 104 pfu m−1 samples of SARS‐CoV‐2 from Influenza and Rhinovirus. The limit of detection (LOD) calculated for SARS‐CoV‐2 antigen is 103 in buffer and 104 PFU mL−1 in 10% saliva, while LOD of ≈65 am calculated for viral RNA isolate without amplification. To provide a high reliability of detection, the role of internal and external factors with respect to gate voltage is further analyzed by Principal Component Analysis (PCA). Based on PCA analysis, the authors are able to classify the samples as pathogen positive or negative (Y > 0: Positive for pathogen, Y < 0: Negative for pathogen). The reported platform can be quickly adapted for multi‐omics and multiplexed diagnosis of continuously evolving biothreats and global pandemics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 sensitivitythrough 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. [ABSTRACT FROM AUTHOR]

Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.

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.

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 β-fibrillation of hen egg white lysozyme (HEWL) under various conditions. Electron microscopy imaging showed that administration of GOQD inhibited HEWL amyloid β-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 β-sheet formation and assembly of a protein by reducing the amount of amyloid β-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.

Hybrid nanocarriers with multifunctional properties have wide therapeutic and diagnostic applications. We have constructed hollow silica nanogolf balls (HGBs) and gold-embedded hollow silica nanogolf balls (Au@SiO 2 HGBs) using the layer-by-layer approach on a symmetric polystyrene (PS) Janus template; the template consists of smaller PS spheres attached to an oppositely charged large PS core. ζ Potential measurement supports the electric force-based template-assisted synthesis mechanism. Electron microscopy, UV-vis, and near-infrared (NIR) spectroscopy show that HGBs or Au@SiO 2 HGBs are composed of a porous silica shell with an optional dense layer of gold nanoparticles embedded in the silica shell. To visualize their cellular uptake and imaging potential, Au@SiO 2 HGBs were loaded with quantum dots (QDs). Confocal fluorescent microscopy and atomic force microscopy imaging show reliable endocytosis of QD-loaded Au@SiO 2 HGBs in adherent HeLa cells and circulating red blood cells (RBCs). Surface-enhanced Raman spectroscopy of Au@SiO 2 HGBs in RBC cells show enhanced intensity of the Raman signal specific to the RBCs' membrane specific spectral markers. Au@SiO 2 HGBs show localized surface plasmon resonance and heat-induced HeLa cell death in the NIR range. These hybrid golf ball nanocarriers would have broad applications in personalized nanomedicine ranging from in vivo imaging to photothermal therapy.

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.