Ethylene Glycol-Manipulated Syntheses of Calcium Carbonate Particles and DNA Capsules toward Efficient ATP-Responsive Cargo Release.

Cargo molecule-encapsulated DNA capsules synthesized with a solid sacrificial template have elicited significant interest in the last decade and have been used for active materials in applications ranging from biosensors to drug delivery. However, the correlation between template properties and the subsequent assembly and triggered release behavior of the resultant carriers remain uninvestigated. In this study, ethylene glycol (EG) was added during the CaCO3 precipitation synthesis to yield particles of various sizes and surface properties, and the adenosine triphosphate (ATP)-responsive release characteristics of the fabricated DNA capsules affected by these particle properties were investigated. The geometry, crystallization, and surface morphology of the CaCO3 particles co-precipitated at various EG concentrations were characterized. We discuss the integrity of cross-linking hybridization, fluorescent molecule internalization, degree of leakage, and release efficiency of the resulting DNA capsules and their relevance brought by particle properties. To achieve efficient encapsulation and cargo release, the surface roughness of the CaCO3 particles was explored and was deemed a key determinant of the compactness of the DNA shell after template removal. This effect was particularly strong in CaCO3 particles in connection with high EG concentrations. The DNA capsules fabricated using 83% EG exhibited low leakage, high loading, and moderate release efficiencies as well as a greater apparent association constant with ATP due to their small particle size and the high-integrity DNA shells.

Engineered multivalent DNA capsules for multiplexed detection of genotoxicants via versatile controlled release mechanisms.

Assessing the risks associated with genotoxic compounds is challenging because of their complex genotoxicity and the difficulty in the dynamic monitoring of coexisting hazards. In this paper, DNA-assembly-based multistimulus responsive capsules that can detect multiple genotoxic agents simultaneously are presented. By exploiting the sequence- and reactivity-editable properties of DNA, DNA sequences in a DNA shell are designed to exhibit multivalent susceptibility against ultraviolet B radiation, aflatoxin B1, and styrene oxide. Upon exposure to genotoxicants, the developed DNA capsules dissociate because of the production of DNA adducts or aptamer-ligand complex-activated dehybridization, which results in the release of encapsulated fluorophores for a measure of the genotoxicant level. The fluorophore release kinetics for each genotoxicant is investigated. Moreover, the destruction behaviors of the developed capsules are evaluated in binary and ternary toxin mixtures. Multiple linear regression indicates the existence of a strong relationship between the fluorescent response and the genotoxicant level; the result highlights the significance of particular genotoxicant and the antagonistic effect of interacting genotoxic substances on capsule destruction. This DNA architecture allows the monitoring of human exposure to genotoxic agents, which enables the timely adoption of remedial measures, and benefits development of an endogenous genotoxin-responsive drug delivery system.

High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination.

For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.

Unraveling cardiolipin-induced conformational change of cytochrome c through H/D exchange mass spectrometry and quartz crystal microbalance.

Cardiolipin (CL), a crucial component in inner mitochondrial membranes, interacts with cytochrome c (cyt c) to form a peroxidase complex for the catalysis of CL oxidation. Such interaction is pivotal to the mitochondrial regulation of apoptosis and is affected by the redox state of cyt c. In the present study, the redox-dependent interaction of cyt c with CL was investigated through amide hydrogen/deuterium exchange coupled with mass spectrometry (HDXMS) and quartz crystal microbalance with dissipation monitoring (QCM-D). Ferrous cyt c exhibited a more compact conformation compared with its ferric form, which was supported by the lower number of deuterons accumulated and the greater amplitude reduction on dissipation. Upon association with CL, ferrous cyt c resulted in a moderate increase in deuteration, whereas the ferric form caused a drastic increase of deuteration, which indicated that CL-bound ferric cyt c formed an extended conformation. These results were consistent with those of the frequency (f) - dissipation (D) experiments, which revealed that ferric cyt c yielded greater values of |ΔD/Δf| within the first minute. Further fragmentation analysis based on HDXMS indicated that the effect of CL binding was considerably different on ferric and ferrous cyt c in the C-helix and the Loop 9-24. In ferric cyt c, CL binding affected Met80 and destabilized His18 interaction with heme, which was not observed with ferrous cyt c. An interaction model was proposed to explain the aforementioned results.

Studies on the substrate-dependent photocatalytic properties of Cu2O heterojunctions.

Cu2O is a promising material for photocatalysis because of its absorption ability in the ultraviolet (UV)-visible light range. Cu2O deposited on conductive Ti and fluorine-doped tin oxide (FTO) substrates behaves as a photocathode. Cu2O deposited on an n-type semiconductor such as TiO2 nanotube arrays (TNA)/Ti behaves as a photoanode and has demonstrated better photocatalytic activity than that of TNA/Ti. The substrate-dependent photocatalytic properties of Cu2O heterojunctions are not well studied. In this work, the photocatalytic properties of a Cu2O/TNA/Ti junction as a photoanode and of Cu2O/Ti and Cu2O/FTO junctions as photocathodes without bias were systematically studied to understand their performance. The Cu2O/TNA/Ti photoanode exhibited higher photocurrent spectral responses than those of Cu2O/Ti and Cu2O/FTO photocathodes. The photoanodic/photocathodic properties of those junctions were depicted in their energy band diagrams. Time-resolved photoluminescence indicated that Cu2O/TNA/Ti, Cu2O/Ti, and Cu2O/FTO junctions did not enhance the separation of photogenerated charges. The improved photocatalytic properties of Cu2O/TNA/Ti compared with TNA/Ti were mainly attributed to the UV-visible light absorption of Cu2O.

Polymerase-assisted fluorescence resonance energy transfer (FRET) assay for simultaneous detection of binary viral sequences.

The analysis of a specific sequence of nucleic acids enables identification of pathogens and the diagnosis of human genetic disorders. This emphasises the need to develop methods of detecting nucleic acids, particularly in a multiplex format, that yield a decisive conclusion for clinical interpretation. Herein, we introduce a polymerase-assisted fluorescence resonance energy transfer (FRET) assay to simultaneously analyse binary viral genes that are characteristic of hemagglutinin and neuraminidase in influenza A virus. The approach takes advantage of the formation of an X-shaped four-way DNA junction (4WJ), thus enabling a selective response to the binary targets of specific sequences. Polymerase drives the cycling of the target complex and incorporates 2'-deoxyuridine-5-triphosphate (dUTP) labelled with Texas Red (TR-dUTP) into the double-stranded DNA (dsDNA) that is produced, which induces FRET to produce a sensing output. Crucially, the mechanism relies on a DNA hairpin instead of a molecular beacon, which substantially increases the simplicity of the assay and reduces its cost. The results revealed that the lowest detectable concentration was approximately 2 pM. The donor-acceptor distance was approximately 7 Šand independent of the ratio of TR-dUTP to 2'-deoxythymidine-5-triphosphate (dTTP). An 'off-on' assay operating in AND gate mode was demonstrated and is potentially useful for the fast diagnosis and subtyping of influenza viruses.

Simultaneous detections of genetic fragment and single nucleotide mutation with a three-tiered output for tuberculosis diagnosis.

Tuberculosis (TB) remains one of the major infectious diseases worldwide. The pathogenic bacterium, Mycobacterium tuberculosis (M.tb), continuously evolves strains carrying drug-resistance genes, thus posing a growing challenge to TB prevention and treatment. We report a diagnostic system that uses a molecular beacon probe and an assistant strand as the core to simultaneously interact with an M.tb-specific fragment (in IS6110) and a single nucleotide substitution (SNS)-encoded segment (in rpoB) associated with drug resistance. A single fluorescent output in three-tiered levels was produced for combinatorial interpretations based on formation of a four-way DNA junction (4WJ). The SNS caused the 4WJ to partially dissociate, thus resulting in medium-level fluorescence. By contrast, high- and low-level fluorescence, represented the complete complementary complex and absence of either targeted fragments, respectively. Manipulating the length of the analyte-binding arm realized the medium output. The thermodynamics and kinetics of 4WJ construction were investigated to maximize the tiered-output performance. Biocatalytic amplification driven by the Klenow Fragment and Nt.AlwI was incorporated into the method to enhance the signal 64-fold and ensure long-term stability of the three-tiered output. The detection accuracy of the sensing system was verified using unpurified amplicons with templates of extracted DNA and boiled bacterial solutions. The tiered-output mechanism was usable at bacterial loads ranging from 4 × 100 to 4 × 103 CFU per reaction. The interference caused by nontuberculous mycobacteria was minimal. The results demonstrated the integrity of the sensing method as an alternative strategy for rapid screening of M.tb and detecting rifampin-resistance.

A mutation-resistant deoxyribozyme OR gate for highly selective detection of viral nucleic acids.

Highly selective probes hybridize only to fully complementary DNA or RNA sequences and, therefore, often fail to recognize mutated viral genomes. Here we designed a probe that possesses two seemingly incompatible properties: it tolerates some point mutations in genome, while it remains selective towards others. An OR deoxyribozyme logic gate was designed to fluorescently report the sequences of enterovirus 71 (EV71) covering ∼90% of all known EV71 strains. Importantly, sequences of closely related coxsackieviruses that differed by single nucleotides were reliably differentiated in 7 out of 8 cases.

Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.

Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.

Implantable Graphene-based Neural Electrode Interfaces for Electrophysiology and Neurochemistry in In Vivo Hyperacute Stroke Model.

Implantable microelectrode arrays have attracted considerable interest due to their high temporal and spatial resolution recording of neuronal activity in tissues. We herein presented an implantable multichannel neural probe with multiple real-time monitoring of neural-chemical and neural-electrical signals by a nonenzymatic neural-chemical interface, which was designed by creating the newly developed reduced graphene oxide-gold oxide (rGO/Au2O3) nanocomposite electrode. The modified electrode on the neural probe was prepared by a facile one-step cyclic voltammetry (CV) electrochemical method with simultaneous occurrence of gold oxidation and GOs reduction to induce the intimate attachment by electrostatic interaction using chloride ions (Cl(-)). The rGO/Au2O3-modified electrode at a low deposition scan rate of 10 mVs(-1) displayed significantly improved electrocatalytic activity due to large active areas and well-dispersive attached rGO sheets. The in vitro amperometric response to H2O2 demonstrated a fast response of less than 5 s and a very low detection limit of 0.63 µM. In in vivo hyperacute stroke model, the concentration of H2O2 was measured as 100.48 ± 4.52 µM for rGO/Au2O3 electrode within 1 h photothrombotic stroke, which was much higher than that (71.92 µM ± 2.52 µM) for noncoated electrode via in vitro calibration. Simultaneously, the somatosensory-evoked potentials (SSEPs) test provided reliable and precise validation for detecting functional changes of neuronal activities. This newly developed implantable probe with localized rGO/Au2O3 nanocomposite electrode can serve as a rapid and reliable sensing platform for practical H2O2 detection in the brain or for other neural-chemical molecules in vivo.

Recognition of dual targets by a molecular beacon-based sensor: subtyping of influenza A virus.

A molecular beacon (MB)-based sensor to offer a decisive answer in combination with information originated from dual-target inputs is designed. The system harnesses an assistant strand and thermodynamically favored designation of unpaired nucleotides (UNs) to process the binary targets in "AND-gate" format and report fluorescence in "off-on" mechanism via a formation of a DNA four-way junction (4WJ). By manipulating composition of the UNs, the dynamic fluorescence difference between the binary targets-coexisting circumstance and any other scenario was maximized. Characteristic equilibrium constant (K), change of entropy (ΔS), and association rate constant (k) between the association ("on") and dissociation ("off") states of the 4WJ were evaluated to understand unfolding behavior of MB in connection to its sensing capability. Favorable MB and UNs were furthermore designed toward analysis of genuine genetic sequences of hemagglutinin (HA) and neuraminidase (NA) in an influenza A H5N2 isolate. The MB-based sensor was demonstrated to yield a linear calibration range from 1.2 to 240 nM and detection limit of 120 pM. Furthermore, high-fidelity subtyping of influenza virus was implemented in a sample of unpurified amplicons. The strategy opens an alternative avenue of MB-based sensors for dual targets toward applications in clinical diagnosis.

Biomolecular logic gate for analysis of the New Delhi metallo-ß-lactamase (NDM)-coding gene with concurrent determination of its drug resistance-encoding fragments.

We herein exploit a newly schemed logic gate to superiorly facilitate analysis of long and highly structured nucleic acids. This strategy uniquely enables the identification of NDM-specific genes and concurrent screening of two active site-encoded fragments, which is promising for evaluating microbial drug resistance.

Biosensors with built-in biomolecular logic gates for practical applications.

Molecular logic gates, designs constructed with biological and chemical molecules, have emerged as an alternative computing approach to silicon-based logic operations. These molecular computers are capable of receiving and integrating multiple stimuli of biochemical significance to generate a definitive output, opening a new research avenue to advanced diagnostics and therapeutics which demand handling of complex factors and precise control. In molecularly gated devices, Boolean logic computations can be activated by specific inputs and accurately processed via bio-recognition, bio-catalysis, and selective chemical reactions. In this review, we survey recent advances of the molecular logic approaches to practical applications of biosensors, including designs constructed with proteins, enzymes, nucleic acids, nanomaterials, and organic compounds, as well as the research avenues for future development of digitally operating "sense and act" schemes that logically process biochemical signals through networked circuits to implement intelligent control systems.

Electrochemical sensor for multiplex screening of genetically modified DNA: identification of biotech crops by logic-based biomolecular analysis.

Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO.

Multifunctional microelectrode array (mMEA) chip for neural-electrical and neural-chemical interfaces: characterization of comb interdigitated electrode towards dopamine detection.

Microelectrode array platforms have attracted considerable interest owing to their ability to facilitate interactive communications between investigators and neuronal network. We herein present an integrated multifunctional microelectrode array (mMEA) chip harnessed with multiple measurement modalities of both neural-electrical and neural-chemical recordings to enable simultaneous monitoring of action potential and the level of the specific neurotransmitter. A dopamine sensor modality fabricated in interdigitated electrodes (IDE) fashion was realized and characterized, subsequently applied to trace dopamine exocytosis in PC12 cells cultured on such mMEA chip. Facile fabrication process leveraging electroplating technique to implement the regulation of gap width was investigated and resulted in preferred IDE configuration. Collection efficiency and amplification effect were systematically evaluated. The as-fabricated sensing device exhibited a favorable diffusion-determining behavior reflected by the steady state current output, and in virtue of this feature, to detect dopamine in connection with limit of detection at 0.62 µM. The current signal was observed linear against the level of dopamine over the investigated concentration range with a resulting sensitivity of 0.096 nA µM(-1).

Nanotheranostics--a review of recent publications.

Theranostics is referred to as a treatment strategy that combines therapeutics with diagnostics, aiming to monitor the response to treatment and increase drug efficacy and safety, which would be a key part of personalized medicine and require considerable advances in predictive medicine. Theranostics associates with both a diagnosis that tests patients for possible reactions to taking new medication and targeted drug delivery based on the test results. Emerging nanotechnology provides a great deal of opportunity to design and develop such combination agents, permitting the delivery of therapeutics and concurrently allowing the detection modality to be used not only before or after but also throughout the entire treatment regimen. The introduction of nanotheranostics into routine health care has still a long way to go, since evaluations on cytotoxicity, genotoxicity, and immunotoxicity of prospective nanotheranostics, demonstration of cost-effectiveness, and availability of appropriate accessible testing systems are still required. An extensive review, from a chemistry point of view, of the recent development of nanotheranostics and its in vitro and in vivo applications are herein presented.

Synthesis, characterization, and electrochemical applications of carbon nanoparticles derived from castor oil soot.

A simple procedure for the modification of carbon nanoparticles (CNPs) from castor oil soot using acid treatment was described herein. Characterization studies revealed the presence of edge plane sites and surface carbon-oxygen functionalities at the surface of the CNP material. Voltammetric studies revealed the increased electrochemical activity of the CNP-modified electrode toward various biologically important molecules, including dopamine, uric acid, dihydronicotinamide adenine dinucleotide, tyrosine, and serotonin, relative to those obtained using the unmodified electrode. The improved electro-oxidation potentials for these compounds-and, thereby, the enhanced sensitivity of related sensors-was due directly to the presence of surface C(δ+)O(δ-) functional groups and the greater number of edge plane sites developed after acid treatment of the soot sample.

Microneedle array-based carbon paste amperometric sensors and biosensors.

The design and characterization of a microneedle array-based carbon paste electrode towards minimally invasive electrochemical sensing are described. Arrays consisting of 3 × 3 pyramidal microneedle structures, each with an opening of 425 µm, were loaded with a metallized carbon paste transducer. The renewable nature of carbon paste electrodes enables the convenient packing of hollow non-planar microneedles with pastes that contain assorted catalysts and biocatalysts. Smoothing the surface results in good microelectrode-to-microelectrode uniformity. Optical and scanning electron micrographs shed useful insights into the surface morphology at the microneedle apertures. The attractive performance of the novel microneedle electrode arrays is illustrated in vitro for the low-potential detection of hydrogen peroxide at rhodium-dispersed carbon paste microneedles and for lactate biosensing by the inclusion of lactate oxidase in the metallized carbon paste matrix. Highly repeatable sensing is observed following consecutive cycles of packing/unpacking the carbon paste. The operational stability of the array is demonstrated as well as the interference-free detection of lactate in the presence of physiologically relevant levels of ascorbic acid, uric acid, and acetaminophen. Upon addressing the biofouling effects associated with on-body sensing, the microneedle carbon paste platform would be attractive for the subcutaneous electrochemical monitoring of a number of physiologically relevant analytes.

High-fidelity determination of security threats via a Boolean biocatalytic cascade.

The ability to assess diverse security threats using a biochemical logic network system is demonstrated. The new biocatalytic cascade, emulating a NOR logic gate, is able to identify the presence of explosive compounds and nerve agents by providing a simple and rapid 'YES'/'NO' alert.