We have developed a colorimetric assay for DNA detection based on the aggregation of unmodified metallic nanoparticles. Charge neutral peptide nucleic acids (PNA) are used as a “coagulant” of citrate ...anion-coated particles and as hybridization probe. In the absence of a complementary target DNA, free PNA molecules in solution induce aggressive particle aggregation because of the removal of charge repulsion as a result of PNA coating on nanoparticles. When a complementary DNA is present and PNA−DNA complexes are formed, the particles remain stable because the negative charges of the DNA strands in the complexes adsorbed on the particle surface ensure sufficient charge repulsions. In this method, no probe immobilization is needed and PNA−DNA hybridization occurs in a homogeneous phase. The assay results are displayed as rapidly as the changes in color and/or in UV−vis adsorption spectra of the colloidal solutions. We have validated the assay principle using gold- and silver-nanoparticles (AuNPs and AgNPs), with the involvement of a shorter (13 mer) and a longer (22 mer) probe sequences. A specific DNA can be detected in the presence of at least 10 times of interference DNA, and the detection limit is at a DNA/PNA ratio of 0.05. When NaCl is added to accelerate the particle aggregation, the selectivity is further improved, and single-base-mismatch discrimination is achieved. A two-component assay using a mixture of AuNPs and AgNPs has also been constituted, aiming to improve the result accuracy by making use of the multiple aggregation signatures from the two types of particles. For single-base-mismatch discrimination, the AgNPs offer a higher sensitivity than AuNPs by showing more obvious spectra and color alternation, and the two-component assay offers three parameters in the UV−vis adsorption spectra.
We have demonstrated that mixed-base PNA oligomers are effective coagulants of citrate ion-coated gold and silver nanoparticles (AuNPs and AgNPs), and PNA-induced particle aggregation can be ...disrupted by hybridization of PNA with a specific DNA. Using particles’ aggregation/dispersion as a measure, we have investigated how PNA and PNA−DNA complexes bind to AuNPs and AgNPs and modulate particles’ stability differently relative to their DNA counterparts. We have made the following original discoveries: (1) mix-base PNA oligomers can induce immediate particle aggregation in a concentration- and chain-length-dependent manner; (2) PNA oligomers have a higher affinity to AuNPs and AgNPs than its ssDNA counterpart; (3) PNA−DNA complexes, although having a stable double helix structure similar to dsDNA, can effectively protect the particles from salt induced aggregation, and the protection effect of different nucleic acids are in the order of PNA−DNA complex > ssDNA > dsDNA; (4) all the characteristics are identical for AuNPs and AgNPs; and (5) AgNPs is more sensitive in response to destabilization effect and is proven a more sensitive platform for colorimetric assays. The control of particle aggregation and dispersion by PNA and PNA−DNA complexes has been used to detect a specific DNA sequence with single-base-mismatch resolution. ζ potential measurements have been conducted to reveal how distinct backbone properties of PNA and PNA−DNA complexes relative to their DNA counterparts contribute to the distinct binding characteristics.
In aquaculture industry, fish, shellfish, and aquatic plants are cultivated in fresh, salt, or brackish waters. The increasing demand of aquatic products has stimulated the rapid growth of ...aquaculture industries. How to effectively monitor and control water quality is one of the key concerns for aquaculture industry to ensure high productivity and high quality. There are four major categories of water quality concerns that affect aquaculture cultivations, namely, (1) physical parameters, e.g., pH, temperature, dissolved oxygen, and salinity, (2) organic contaminants, (3) biochemical hazards, e.g., cyanotoxins, and (4) biological contaminants, i.e., pathogens. While the physical parameters are affected by climate changes, the latter three are considered as environmental factors. In this review, we provide a comprehensive summary of sensors, biosensors, and analytical technologies available for monitoring aquaculture water quality. They include low-cost commercial sensors and sensor network setups for physical parameters. They also include chromatography, mass spectrometry, biochemistry, and molecular methods (e.g., immunoassays and polymerase chain reaction assays), culture-based method, and biophysical technologies (e.g., biosensors and nanosensors) for environmental contamination factors. According to the different levels of sophistication of various analytical techniques and the information they can provide (either fine fingerprint, highly accurate quantification, semiquantification, qualitative detection, or fast screening), we will comment on how they may be used as complementary tools, as well as their potential and gaps toward current demand of real-time, online, and/or onsite detection.
Since the early 2000s, extensive research has been performed to address numerous challenges in biochip and biosensor fabrication in order to use them for various biomedical applications. These ...biochips and biosensor devices either integrate biological elements (e.g., DNA, proteins or cells) in the fabrication processes or experience post fabrication of biofunctionalization for different downstream applications, including sensing, diagnostics, drug screening, and therapy. Scalable lithographic techniques that are well established in the semiconductor industry are now being harnessed for large-scale production of such devices, with additional development to meet the demand of precise deposition of various biological elements on device substrates with retained biological activities and precisely specified topography. In this review, the lithographic methods that are capable of large-scale and mass fabrication of biochips and biosensors will be discussed. In particular, those allowing patterning of large areas from 10 cm2 to m2, maintaining cost effectiveness, high throughput (>100 cm2 h–1), high resolution (from micrometer down to nanometer scale), accuracy, and reproducibility. This review will compare various fabrication technologies and comment on their resolution limit and throughput, and how they can be related to the device performance, including sensitivity, detection limit, reproducibility, and robustness.
Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. ...Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.
Quantum dots (QDs) with unique quantum confinement effect and electro-optical properties are attractive for biomedical applications. Toxic effects of traditional semiconductor QDs made of heavy metal ...ions have serious safety concerns for their undesired environmental or healthy impact. Recent reports on core-shell structure modification of QDs by using biocompatible ligands or polymers is one way to be effectively minimizing toxicity effects of traditional QDs. Furthermore, designs of heave metal-free and metal-free QDs formulations are more promising alternatives, due to the non-toxic and eco-friendly nature of the starting materials. In article, we will review the recent designs of non-toxic or less toxic QDs, including carbon-based, biomolecules-based, silicon-based, and ternary I-III-VI QDs, and their biological applications (bio-imaging, drug delivery, gene therapy and immunoassay).
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•Toxic effect of traditional semiconductor QDs made of metal ions posts serious concerns.•This review summarized the toxicity of conventional QDs, recent designs of non-toxic QDs.
Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfaces for target DNA hybridization. Two immobilization strategies ...are involved for constructing streptavidin films, namely, (1) physical adsorption on biotin-containing thiol treated surfaces through biotin−streptavidin links and (2) covalent attachment to 11-mercaptoundecanoic acid (MUA) treated surfaces through amine coupling. To understand the structural properties of the streptavidin films, a quartz crystal microbalance with energy dissipation monitoring (QCM-D) is used to monitor the streptavidin immobilization procedures. The simultaneously measured frequency (Δf) and dissipation factor (ΔD) changes, together with the SPR angle shifts (Δθ), suggest that the streptavidin film assembled on the biotin-containing surface is highly rigid with a well-ordered structure while the streptavidin film formed through amine coupling is highly dissipative and less structured. The subsequent biotinylated DNA (biotin−DNA) assembly and target hybridization results show that the streptavidin film structure has distinct effects on the biotin−DNA binding amount. On the streptavidin matrix, not only the probe DNA density but also the strand orientation mediated by the streptavidin films has distinct effects on hybridization efficiency. Particularly, the molecularly ordered streptavidin films formed on the biotin-containing surfaces ensure a well-ordered DNA assembly, which in turn allows for a higher efficiency in target DNA capture and for a higher sensitivity in the hybridization analysis when compared to the biotin−DNA assembled on the less structured streptavidin films formed through amine coupling.
Biosensors incorporating nanomaterials have demonstrated superior performance compared to their conventional counterparts. Most reported sensors use nanomaterials as a single transducer of signals, ...while biosensor designs using dual transducing elements have emerged as new approaches to further improve overall sensing performance. This review focuses on recent developments in nanomaterial-based biosensors using dual transducing elements for solution phase detection. The review begins with a brief introduction of the commonly used nanomaterial transducers suitable for designing dual element sensors, including quantum dots, metal nanoparticles, upconversion nanoparticles, graphene, graphene oxide, carbon nanotubes, and carbon nanodots. This is followed by the presentation of the four basic design principles, namely Förster Resonance Energy Transfer (FRET), Amplified Fluorescence Polarization (AFP), Bio-barcode Assay (BCA) and Chemiluminescence (CL), involving either two kinds of nanomaterials, or one nanomaterial and an organic luminescent agent (
e.g.
organic dyes, luminescent polymers) as dual transducers. Biomolecular and chemical analytes or biological interactions are detected by their control of the assembly and disassembly of the two transducing elements that change the distance between them, the size of the fluorophore-containing composite, or the catalytic properties of the nanomaterial transducers, among other property changes. Comparative discussions on their respective design rules and overall performances are presented afterwards. Compared with the single transducer biosensor design, such a dual-transducer configuration exhibits much enhanced flexibility and design versatility, allowing biosensors to be more specifically devised for various purposes. The review ends by highlighting some of the further development opportunities in this field.
This review describes the design principles of dual-transducer nanosensors and the recent advances in this field ("T" = target; "Trans." in CL = Transducer).
This article examines three spatiotemporal methods used for analyzing of infectious diseases, with a focus on COVID-19 in the United States. The methods considered include inverse distance weighting ...(IDW) interpolation, retrospective spatiotemporal scan statistics and Bayesian spatiotemporal models. The study covers a 12-month period from May 2020 to April 2021, including monthly data from 49 states or regions in the United States. The results show that the spread of COVID-19 pandemic increased rapidly to a high value in winter of 2020, followed by a brief decline that later reverted into another increase. Spatially, the COVID-19 epidemic in the United States exhibited a multi-centre, rapid spread character, with clustering areas represented by states such as New York, North Dakota, Texas and California. By demonstrating the applicability and limitations of different analytical tools in investigating the spatiotemporal dynamics of disease outbreaks, this study contributes to the broader field of epidemiology and helps improve strategies for responding to future major public health events.
CD4 T cell deficiency or defective IFNγ signaling render humans and mice highly susceptible to Mycobacterium tuberculosis (Mtb) infection. The prevailing model is that Th1 CD4 T cells produce IFNγ to ...activate bactericidal effector mechanisms of infected macrophages. Here we test this model by directly interrogating the effector functions of Th1 CD4 T cells required to control Mtb in vivo. While Th1 CD4 T cells specific for the Mtb antigen ESAT-6 restrict in vivo Mtb growth, this inhibition is independent of IFNγ or TNF and does not require the perforin or FAS effector pathways. Adoptive transfer of Th17 CD4 T cells specific for ESAT-6 partially inhibited Mtb growth while Th2 CD4 T cells were largely ineffective. These results imply a previously unrecognized IFNγ/TNF independent pathway that efficiently controls Mtb and suggest that optimization of this alternative effector function may provide new therapeutic avenues to combat Mtb through vaccination.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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