SARS-CoV-2 Variant Screening Using a Virus-Receptor-Based Electrical Biosensor.

SARS-CoV-2 variants are of particular interest because they can potentially increase the transmissibility and virulence of COVID-19 or reduce the effectiveness of available vaccines. However, screening SARS-CoV-2 variants is a challenge because biosensors target viral components that can mutate. One promising strategy is to screen variants via angiotensin-converting enzyme 2 (ACE2), a virus receptor shared by all known SARS-CoV-2 variants. Here we designed a highly sensitive and portable COVID-19 screening biosensor based on the virus receptor. We chose a dual-gate field-effect transistor to overcome the low sensitivity of virus-receptor-based biosensors. To optimize the biosensor, we introduced a synthetic virus that mimics the important features of SARS-CoV-2 (size, bilayer structure, and composition). The developed biosensor successfully detected SARS-CoV-2 in 20 min and showed sensitivity comparable to that of molecular diagnostic tests (∼165 copies/mL). Our results indicate that a virus-receptor-based biosensor can be an effective strategy for screening infectious diseases to prevent pandemics.

Stimuli-Responsive Adaptive Nanotoxin to Directly Penetrate the Cellular Membrane by Molecular Folding and Unfolding.

Biological nanomachines, including proteins and nucleic acids whose function is activated by conformational changes, are involved in every biological process, in which their dynamic and responsive behaviors are controlled by supramolecular recognition. The development of artificial nanomachines that mimic the biological functions for potential application as therapeutics is emerging; however, it is still limited to the lower hierarchical level of the molecular components. In this work, we report a synthetic machinery nanostructure in which actuatable molecular components are integrated into a hierarchical nanomaterial in response to external stimuli to regulate biological functions. Two nanometers core-sized gold nanoparticles are covered with ligand layers as actuatable components, whose folding/unfolding motional response to the cellular environment enables the direct penetration of the nanoparticles across the cellular membrane to disrupt intracellular organelles. Furthermore, the pH-responsive conformational movements of the molecular components can induce the apoptosis of cancer cells. This strategy based on the mechanical motion of molecular components on a hierarchical nanocluster would be useful to design biomimetic nanotoxins.

Magnesium Corrosion Triggered Spontaneous Generation of H2O2 on Oxidized Titanium for Promoting Angiogenesis.

Although the use of reactive oxygen species (ROS) has been extensively studied, current systems employ external stimuli such as light or electrical energy to produce ROS, which limits their practical usage. In this report, biocompatible metals were used to construct a novel electrochemical system that can spontaneously generate H2O2 without any external light or voltage. The corrosion of Mg transfers electrons to Au-decorated oxidized Ti in an energetically favorable process, and the spontaneous generation of H2O2 in an oxygen reduction reaction was revealed to occur at titanium by combined spectroscopic and electrochemical analyses. The controlled release of H2O2 noticeably enhanced in vitro angiogenesis even in the absence of growth factors. Finally, a new titanium implant prototype was developed by Mg incorporation, and its potential for promoting angiogenesis was demonstrated.

Universal antibody conjugation to nanoparticles using the Fcγ receptor I (FcγRI): quantitative profiling of membrane biomarkers.

Antibodies are a class of molecules widely used in bioengineering and nanomedicine for applications involving protein recognition and targeting. Here we report an efficient method for universal conjugation of antibodies to lipid-coated nanoparticles using radially oriented FcγRIs. This method is performed in physiological solution with no additional coupling reagents, thereby avoiding problems with antibody stability and functionality. Coupling to the Fc region of the antibody avoids aggregation and polymerization allowing high yield. In addition, the antibody is oriented perpendicular to the surface so that the binding sites are fully functional. Using this method we demonstrate quantitative profiling of a panel of four membrane-bound cancer biomarkers (claudin-4, mesothelin, mucin-4, and cadherin-11) on four cell lines (Panc-1, MIA PaCa-2, Capan-1, and HPDE). We show that by designing the lipid coating to minimize aggregation and nonspecific binding, we can obtain absolute values of biomarker expression levels as number per unit area on the cell surface. This method is applicable to a wide range of technologies, including solution based protein detection assays and active targeting of cell surface membrane biomarkers.

CRISPR/Cas12a antifouling nanocomposite electrochemical biosensors enable amplification-free detection of Monkeypox virus in complex biological fluids.

The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.

Facile solvothermal preparation of monodisperse gold nanoparticles and their engineered assembly of ferritin-gold nanoclusters.

Herein, we report a quick and simple synthesis of water-soluble gold nanoparticles using a HAuCl4 and oleylamine mixture. Oleylamine serves as a reduction agent as well as a stabilizer for nanoparticle surfaces. The particle sizes can be adjusted by modulating reaction temperature and time. Solvothermal reduction of HAuCl4 with oleylamine can be confirmed by measuring the product in Fourier transform infrared (FTIR) spectroscopy. The plasmon band shifting from yellow to red confirms a nanosized particle formation. Amide bonds on the surface of the nanoparticles formed hydrogen bonds with one another, resulting in a hydrophobic monolayer. Particles dispersed well in nonpolar organic solvents, such as in hexane or toluene, by brief sonication. Next, we demonstrated the transfer of gold nanoparticles into water by lipid capsulation using 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy polyethylene glycol)-2000 (DPPE-PEG2k), and 1,2-dioleoyl-sn-glycero-3-N-{5-amino-1-carboxypentyl}iminodiacetic acid succinyl nickel salt [DGS-NTA(Ni)]. The particle concentration can be obtained using an absorbance in ultraviolet-visible (UV-vis) spectra (at 420 nm). Instrumental analyses using transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) analysis, dynamic light scattering (DLS), and FTIR confirmed successful production of gold nanoparticles and fair solubility in water. Prepared gold particles were selectively clustered via engineered ferritin nanocages that provide multiple conjugation moieties. A total of 5-6 gold nanoparticles were clustered on a single ferritin nanocage confirmed in TEM. Reported solvothermal synthesis and preparation of gold nanoclusters may serve as an efficient, alternate way of preparing water-soluble gold nanoparticles, which can be used in a wide variety of biomedical applications.

Harnessing immunomagnetic separation and quantum dot-based quantification capacities for the enumeration of absolute levels of biomarker.

The field of biomarker quantification has experienced a growth parallel to the discovery of new materials. In this paper, we propose an innovative system for the separation and quantification of biomarkers using a simple magnetic bead (MB)-quantum dot (QD) sandwich assay. The basis of the system lies in the interaction between histidine residues on protein G and Ni ions on QDs, and the use of imidazole to selectively detach QDs bound to target biomarkers, in effect enumerating the absolute number of biomarker units. We used C-reactive protein (CRP) as a proof-of-concept and demonstrated a detection sensitivity of 82.5 fmoles in 50 µl of sample volume, a commonly used analytical volume (e.g. ELISA). Although CRP was used as a model to conduct this study, the sensitivity and simplicity of this detachable system make it a viable approach in the quantification of other target analytes.

Evaluation of novel pyrazol-4-yl pyridine derivatives possessing arylsulfonamide tethers as c-Jun N-terminal kinase (JNK) inhibitors in leukemia cells.

A series of 36 pyrazol-4-yl pyridine derivatives (8a-i, 9a-i, 10a-i, and 11a-i) was designed, synthesized, and evaluated for its antiproliferative activity over NCI-60 cancer cell line panel and inhibitory effect against JNK isoforms (JNK1, JNK2, and JNK3). All the synthesized compounds were tested against the NCI-60 cancer cell line panel. Compounds 11b, 11c, 11g, and 11i were selected to determine their GI50s and exerted a superior potency over the reference standard SP600125 against the tested cell lines. 11c showed a GI50 of 1.28 µM against K562 leukemic cells. Vero cells were used to assess 11c cytotoxicity compared to the tested cancer cells. The target compounds were tested against hJNK isoforms in which compound 11e exhibited the highest potency against JNK isoforms with IC50 values of 1.81, 12.7, and 10.5 nM against JNK1, JNK2, and JNK3, respectively. Kinase profiling of 11e showed higher JNK selectivity in 50 kinase panels. Compounds 11c and 11e showed cell population arrest at the G2/M phase, induced early apoptosis, and slightly inhibited beclin-1 production at higher concentrations in K562 leukemia cells relative to SP600125. NanoBRET assay of 11e showed intracellular JNK1 inhibition with an IC50 of 2.81 µM. Also, it inhibited CYP2D6 and 3A4 with different extent and its hERG activity showed little cardiac toxicity with an IC50 of 4.82 µM. hJNK3 was used as a template to generate the hJNK1 crystal structure to explore the binding mode of 11e (PDB ID: 8ENJ) with a resolution of 2.8 °A and showed a typical type I kinase inhibition against hJNK1. Binding energy scores showed that selectivity of 11e towards JNK1 could be attributed to additional hydrophobic interactions relative to JNK3.

Selective Cell-Cell Adhesion Regulation via Cyclic Mechanical Deformation Induced by Ultrafast Nanovibrations.

The adoption of dynamic mechanomodulation to regulate cellular behavior is an alternative to the use of chemical drugs, allowing spatiotemporal control. However, cell-selective targeting of mechanical stimuli is challenging due to the lack of strategies with which to convert macroscopic mechanical movements to different cellular responses. Here, we designed a nanoscale vibrating surface that controls cell behavior via selective repetitive cell deformation based on a poroelastic cell model. The vibrating indentations induce repetitive water redistribution in the cells with water redistribution rates faster than the vibrating rate; however, in the opposite case, cells perceive the vibrations as a one-time stimulus. The selective regulation of cell-cell adhesion through adjusting the frequency of nanovibration was demonstrated by suppression of cadherin expression in smooth muscle cells (fast water redistribution rate) with no change in vascular endothelial cells (slow water redistribution rate). This technique may provide a new strategy for cell-type-specific mechanical stimulation.

Quantitative characterization of the lipid encapsulation of quantum dots for biomedical applications.

The water solubilization of nanoparticles is key for many applications in biomedicine. Despite the importance of surface functionalization, progress has been largely empirical and very few systematic studies have been performed. Here we report on the water solubilization of quantum dots using lipid encapsulation. We systematically evaluate the monodispersity, zeta potential, stability, and quantum yield for quantum dots encapsulated with single and double acyl-chain lipids, pegylated double acyl-chain lipids, and single alkyl-chain surfactant molecules with charged head groups. We show that charged surfactants and pegylated lipids are important to obtain monodisperse suspensions with high yield and excellent long-term stability. FROM THE CLINICAL EDITOR: This study reports on solubilization of nanoparticles in water, a key, but often neglected aspect for biomedical applications. The authors demonstrate that charged surfactants and PEGylated lipids are important to obtain monodisperse suspensions with high yield and long-term stability.

Quantitative molecular profiling of biomarkers for pancreatic cancer with functionalized quantum dots.

Applications in nanomedicine, such as diagnostics and targeted therapeutics, rely on the detection and targeting of membrane biomarkers. In this article we demonstrate absolute quantitative profiling, spatial mapping, and multiplexing of cancer biomarkers using functionalized quantum dots (QDs). We demonstrate highly selective targeting molecular markers for pancreatic cancer with extremely low levels of nonspecific binding. We confirm that we have saturated all biomarkers on the cell surface, and, in conjunction with control experiments, extract absolute quantitative values for the biomarker density in terms of the number of molecules per square micron on the cell surface. We show that we can obtain quantitative spatial information of biomarker distribution on a single cell, important because tumors' cell populations are inherently heterogeneous. We validate our quantitative measurements (number of molecules per square micron) using flow cytometry and demonstrate multiplexed quantitative profiling using color-coded QDs. FROM THE CLINICAL EDITOR: This paper demonstrates a nice example for quantum dot-based molecular targeting of pancreatic cancer cells for advanced high sensitivity diagnostics and potential future selective therapeutic purposes.

Structural optimization of 4-(imidazol-5-yl)pyridine derivatives affords broad-spectrum anticancer agents with selective B-RAFV600E/p38α kinase inhibitory activity: Synthesis, in vitro assays and in silico study.

In the current article, we introduce design of a new series of 4-(imidazol-5-yl)pyridines with improved anticancer activity and selective B-RAFV600E/p38α kinase inhibitory activity. Based on a previous work, a group of structural modifications were applied affording the new potential antiproliferative agents. Towards extensive biological assessment of the target compounds, an in vitro anticancer assay was conducted over NCI 60-cancer cell lines panel representing blood, lung, colon, CNS, skin, ovary, renal, prostate, and breast cancers. Compounds 7c, 7d, 8b, 9b, 9c, 10c, 10d, and 11b exhibited the highest potency among the tested compounds and demonstrated sub-micromolar or one-digit micromolar GI50 values against the majority of the employed cell lines. Compound 10c emerged as the most potent agent with nano-molar activity over most of the cells and incredible activity against melanoma (MDA-MB-435) cell line (GI50 70 nM). It is much more potent than sorafenib, the clinically used anticancer drug, against almost all the NCI-60 cell lines. Further cell-based mechanistic assays showed that compound 10c induced cell cycle arrest and promoted apoptosis in K562, MCF-7 and HT29 cancer cell lines. In addition, compound 10c induced autophagy in the three cancer cell lines. Kinase profiling of 10c showed its inhibitory effects and selectivity towards B-RAFV600E and p38α kinases with IC50 values of 1.84 and 0.726 µM, respectively. Docking of compound 10c disclosed its high affinity in the kinases pockets. Compound 10c represent a promising anticancer agent, that could be optimized in order to improve its kinase activity aiming at developing potential anticancer agents. The conformational stability of compound 10c in the active site of B-RAFV600E and p38α kinases was studied by applying molecular dynamic simulation of the compound in the two kinases for 600 ns in comparison to the native ligands.

Noninvasive Precision Screening of Prostate Cancer by Urinary Multimarker Sensor and Artificial Intelligence Analysis.

Screening for prostate cancer relies on the serum prostate-specific antigen test, which provides a high rate of false positives (80%). This results in a large number of unnecessary biopsies and subsequent overtreatment. Considering the frequency of the test, there is a critical unmet need of precision screening for prostate cancer. Here, we introduced a urinary multimarker biosensor with a capacity to learn to achieve this goal. The correlation of clinical state with the sensing signals from urinary multimarkers was analyzed by two common machine learning algorithms. As the number of biomarkers was increased, both algorithms provided a monotonic increase in screening performance. Under the best combination of biomarkers, the machine learning algorithms screened prostate cancer patients with more than 99% accuracy using 76 urine specimens. Urinary multimarker biosensor leveraged by machine learning analysis can be an important strategy of precision screening for cancers using a drop of bodily fluid.

Electrical Cartridge Sensor Enables Reliable and Direct Identification of MicroRNAs in Urine of Patients.

Urinary miRNAs are biomarkers that demonstrate considerable promise for the noninvasive diagnosis and prognosis of diseases. However, because of background noise resulting from complex physiological features of urine, instability of miRNAs, and their low concentration, accurate monitoring of miRNAs in urine is challenging. To address these limitations, we developed a urine-based disposable and switchable electrical sensor that enables reliable and direct identification of miRNAs in patient urine. The proposed sensing platform combining disposable sensor chips composed of a reduced graphene oxide nanosheet and peptide nucleic acid facilitates the label-free detection of urinary miRNAs with high specificity and sensitivity. Using real-time detection of miRNAs in patient urine without pretreatment or signal amplification, this sensor allows rapid, direct detection of target miRNAs in a broad dynamic range with a detection limit down to 10 fM in human urine specimens within 20 min and enables simultaneous quantification of multiple miRNAs. As confirmed using a blind comparison with the results of pathological examination of patients with prostate cancer, the sensor offers the potential to improve the accuracy of early diagnosis before a biopsy is taken. This study holds the usefulness of the practical sensor for the clinical diagnosis of urological diseases.

Drug resistance-free cytotoxic nanodrugs in composites for cancer therapy.

Drug resistance is a major cause of treatment failure for small-molecule cancer chemotherapies, despite the advances in combination therapies, drug delivery systems, epigenetic drugs, and proteolysis-targeting chimeras. Herein, we report the use of a drug resistance-free cytotoxic nanodrug as an alternative to small-molecule drugs. The present nanodrugs comprise 2 nm core gold nanoparticles (AuNPs) covered completely with multivalent hydrocarbon chains to a final diameter of ∼10 nm as single drug molecules. This hydrophobic drug-platform was delivered in composite form (∼35 nm) with block-copolymer like other small-molecular drugs. Upon uptake by cells, the nanodrugs enhanced the intracellular levels of reactive oxygen species and induced apoptosis, presumably reflecting multivalent interactions between aliphatic chains and intracellular biomolecules. No resistance to our novel nanodrug was observed following multiple treatment passages and the potential for use in cancer therapy was verified in a breast cancer patient-derived xenograft mouse model. These findings provide insight into the use of nano-scaled compounds as agents that evade drug resistance to cancer therapy.

Ionic contrast across a lipid membrane for Debye length extension: towards an ultimate bioelectronic transducer.

Despite technological advances in biomolecule detections, evaluation of molecular interactions via potentiometric devices under ion-enriched solutions has remained a long-standing problem. To avoid severe performance degradation of bioelectronics by ionic screening effects, we cover probe surfaces of field effect transistors with a single film of the supported lipid bilayer, and realize respectable potentiometric signals from receptor-ligand bindings irrespective of ionic strength of bulky solutions by placing an ion-free water layer underneath the supported lipid bilayer. High-energy X-ray reflectometry together with the circuit analysis and molecular dynamics simulation discovered biochemical findings that effective electrical signals dominantly originated from the sub-nanoscale conformational change of lipids in the course of receptor-ligand bindings. Beyond thorough analysis on the underlying mechanism at the molecular level, the proposed supported lipid bilayer-field effect transistor platform ensures the world-record level of sensitivity in molecular detection with excellent reproducibility regardless of molecular charges and environmental ionic conditions.

Design, synthesis, biological evaluation, and docking studies of novel (imidazol-5-yl)pyrimidine-based derivatives as dual BRAFV600E/p38α inhibitors.

The synergistic effect of dual inhibition of serine/threonine protein kinases that are involved in the same signalling pathway of the diseases can exert superior biological benefits for treatment of these diseases. In the present work, a new series of (imidazol-5-yl)pyrimidine was designed and synthesized as dual inhibitors of BRAFV600E and p38α kinases which are considered as key regulators in mitogen-activated protein kinase (MAPK) signalling pathway. The target compounds were evaluated for dual kinase inhibitory activity. The tested compounds exhibited nanomolar scale IC50 values against BRAFV600E and low to sub-micromolar IC50 range against p38α. Compound 20h was identified as the most potent dual BRAFV600E/p38α inhibitor with IC50 values of 2.49 and 85 nM, respectively. Further deep investigation revealed that compound 20h possesses inhibitory activity of TNF-α production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 value of 96.3 nM. Additionally, the target compounds efficiently frustrated the proliferation of LOX-IMVI melanoma cell line. Compound 20h showed a satisfactory antiproliferative activity with IC50 value of 13 µM, while, compound 18f exhibited the highest cytotoxicity potency with IC50 value of 0.9 µM. Compound 18f is 11.11-fold more selective toward LOX-IMVI melanoma cells than IOSE-80PC normal cells. The newly reported compounds represent therapeutically promising candidates for further development of BRAFV600E/p38α inhibitors in an attempt to overcome the acquired resistance of BRAF mutant melanoma.

Discovery of New Imidazo[2,1-b]thiazole Derivatives as Potent Pan-RAF Inhibitors with Promising In Vitro and In Vivo Anti-melanoma Activity.

BRAF is an important component of MAPK cascade. Mutation of BRAF, in particular V600E, leads to hyperactivation of the MAPK pathway and uncontrolled cellular growth. Resistance to selective inhibitors of mutated BRAF is a major obstacle against treatment of many cancer types. In this work, a series of new (imidazo[2,1-b]thiazol-5-yl)pyrimidine derivatives possessing a terminal sulfonamide moiety were synthesized. Pan-RAF inhibitory effect of the new series was investigated, and structure-activity relationship is discussed. Antiproliferative activity of the target compounds was tested against the NCI-60 cell line panel. The most active compounds were further tested to obtain their IC50 values against cancer cells. Compound 27c with terminal open chain sulfonamide and 38a with a cyclic sulfamide moiety showed the highest activity in enzymatic and cellular assay, and both compounds were able to inhibit phosphorylation of MEK and ERK. Compound 38a was selected for testing its in vivo activity against melanoma. Cellular and animal activities are reported.

Interfacial charge regulation of protein blocking layers in transistor biosensor for direct measurement in serum.

Ion-sensitive field-effect transistor (ISFET) as a biosensor facilitates a process of data-acquisition through label-free and real-time monitoring. Direct quantification of a biomarker in serum is challenging in ISFET biosensor since charged proteins in serum interfere transduction to electrical signals. Here, we report the fabrication of protein blocking layers (PBLs) with intended interfacial charges to minimize non-specific protein bindings on ISFET. Use of charged protein precursors enables to regulate the interfacial charge of PBLs, preserving their intrinsic electric features (neutral: hemoglobin, positively charged: lysozyme, negatively charged: BSA). The effect of this interfacial charge on the signal was demonstrated through PSMA (prostate cancer biomarker) sensing using a dual-gate ISFET biosensor. The neutral PBL showed the minimum noise compared to the negatively and positively charged PBLs, enabling the ISFET to exhibit the same detection range in untreated serum as with pre- or post-treatment (1 fg/ml to 100 ng/ml). The introduction of neutral PBLs to ISFET biosensors would allow the application of the ISFET biosensor as a point-of-care device.

Combinatorial Inhibition of Cell Surface Receptors Using Dual Aptamer-Functionalized Nanoconstructs for Cancer Treatment.

Membrane receptors overexpressed in diseased states are considered novel therapeutic targets. However, the single targeting approach faces several fundamental issues, such as poor efficacy, resistance, and toxicity. Here, we report a dual-targeting strategy to enhance anti-cancer efficacy via synergistic proximity interactions between therapeutics and two receptor proteins. Importantly, we report the first finding of an interaction between c-Met and nucleolin and demonstrate the therapeutic value of targeting the interaction between them. Bispecific nanocarriers densely grafted with anti-c-Met and -nucleolin aptamer increased the local concentration of aptamers at the target sites, in addition to inducing target receptor clustering. It was also demonstrated that the simultaneous targeting of c-Met and nucleolin inhibited the cellular functions of the receptors and increased anti-cancer efficacy by altering the cell cycle. Our findings pave the way for the development of an effective combinatorial treatment based on nanoconstruct-mediated interaction between receptors.