DNA-directed formation of plasmonic core-satellite nanostructures for quantification of hepatitis C viral RNA.

Hepatitis C virus (HCV) continues to be a significant public health challenge, affecting an estimated 71 million people globally and posing risks of severe liver diseases. Despite advancements in treatments, diagnostic limitations hinder the global elimination efforts targeted by 2030. This study introduces an innovative diagnostic approach, integrating catalytic hairpin assembly (CHA) with plasmonic core-satellite gold nanoparticle (AuNP) assemblies, to enable sensitive and specific detection of HCV RNA. We optimized the stoichiometry of DNA hairpins to form highly stable three-way junctions (3WJs), minimizing non-specific reactions in an enzyme-free, isothermal amplification process. The resulting dual-transduction biosensor combines colorimetric and surface-enhanced Raman spectroscopy (SERS) techniques, utilizing the Raman reporter malachite green isothiocyanate (MGITC) for signal generation. Our system targets a conserved 23-nucleotide sequence within the HCV 5'-UTR, essential for RNA replication, facilitating pan-genotypic HCV detection that complements direct-acting antiviral strategies. We evaluated the biosensor's efficacy using fluorescence spectroscopy, native PAGE, AFM, and TEM. Findings indicate that the 60 nm core AuNPs surrounded by 20 nm satellite AuNPs achieved a ten-fold increase in sensitivity over the 10 nm satellites, detecting HCV RNA concentrations as low as 1.706 fM. This sensitivity is crucial, given the extremely low viral loads present during early infection stages. Our research demonstrates the promise of enzyme-free molecular biosensors for HCV, with the potential to provide cost-efficient, rapid, point-of-care testing, although further sensitivity enhancements are needed to address the challenges of early-stage detection.

Efficient production of uniform gold nanoparticles via a streamlined low-cost, semi-automated, open-source platform.

In the quest to discover dependable and repeatable methods for producing noble metal nanospheres, both commercial and academic scientists have shown great interest. The challenge of precisely controlling the size of these nanospheres is critical, as variations can alter their optical characteristics, leading to complications in subsequent applications. In this context, we present the design and validation of an affordable, semi-automated device that synthesizes gold nanoparticles using the Turkevich method. This device, named 'NanoSynth Mini' and powered by Raspberry Pi, demonstrates the capability to generate gold nanoparticles with diameters ranging from 15 to 60 nanometers with minimal variability. Its design allows for seamless integration into lab processes, providing consistent support for extensive research initiatives.

Freeze-Driven Synthesis of DNA Hairpin-Conjugated Gold Nanoparticle Biosensors for Dual-Mode Detection.

Freeze-based immobilization of deoxyribonucleic acid (DNA) oligonucleotides on gold nanoparticles (AuNPs) is highly efficient for single-stranded oligonucleotides but typically does not accommodate structures such as snap-cooled DNA hairpins (Sc-HPs) and snap-cooled molecular beacons (Sc-MBs) frequently used for biorecognition applications. Recognizing this limitation, we have developed a modified, freeze-based technique specifically designed to enable the adsorption of such hairpin oligonucleotides onto AuNP surfaces while ensuring that they retain their biosensing capabilities. Successful hairpin oligonucleotide conjugation of varying lengths to a wide range of AuNP diameters was corroborated by dynamic light scattering, ζ-potential, and UV-vis spectrophotometry. Moreover, we conducted a thorough evaluation of this modified method, confirming the retention of the sensing functions of Sc-HPs and Sc-MBs. This advancement not only offers a more efficient route for DNA hairpin conjugation but also elucidates the underlying biorecognition functions, with implications for broader applications in molecular diagnostics.

Effect of microchannel geometry and linker molecules on surface immobilization efficiency of proteins in microfluidic devices.

One of the key metrics in the design of biosensors is the presence of an effective capture layer. Surface-immobilized proteins (especially as a part of antibody-antigen combinations) are the most commonly used capture ligands in biosensors. The surface coverage of these proteins in flow-based biosensors are affected by both the linker chemistry used to attach them as well as the microchannel geometry. We used streptavidin as a model protein to compare glutaraldehyde, EDC-NHS, sulfo-SMCC and sulfo-NHS-biotin as linkers inside straight, serpentine and square-wave microchannel geometries. We found that straight microchannels achieve the highest degree of protein immobilization compared to serpentine and square-wave microchannels, irrespective of the linker chemistry used. We also showed that for a given microchannel geometry, sulfo-NHS-biotin leads to the highest immobilization of streptavidin among all the linkers.

3D Printed SERS-Active Thin-Film Substrates Used to Quantify Levels of the Genotoxic Isothiazolinone.

Several reports present methods to fabricate thin-film substrates capable of surface-enhanced Raman scattering (SERS). Substrates synthesized by displacing silver onto copper using facile synthesis methods such as galvanic displacement can generate high levels of SERS enhancement rivaling commercially available substrates manufactured by lithographic methods. Here, we describe the optimization of a novel set of SERS-active thin-film substrates synthesized via the electroless displacement of Ag onto the surface of three-dimensional (3D) printed disks composed of the copper/polymer (PLA) composite filament. The effect of AgNO3 concentration on the deposition, morphology, and overall SERS activity of the substrates has been carefully studied. Two commonly used Raman reporters, 4-mercaptobenzoic acid (MBA) and malachite green isothiocyanate (MGITC), were used to measure the SERS output of the substrates. Good SERS signal reproducibility (RSD ∼16.8%) was measured across the surface of replicate substrates and high-sensitivity detection of MBA was achieved (10-12 M). To test the real-world application of our substrates, we opted to detect 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), which is a genotoxic, biocide common in many household products, known to leach into water supplies. Our newly developed SERS-active substrates could detect CMIT down to 10 ppm when spiked in simulated lake water samples, which is well within current agency standards.

High-mobility group box-1 translocation and release after hypoxic ischemic brain injury in neonatal rats.

Inflammation contributes to neonatal brain injury. Pro-inflammatory cytokines represent key inflammatory meditators in neonatal hypoxic-ischemic (HI) brain injury. The high mobility group box-1 (HMGB1) protein is a nuclear protein with pro-inflammatory cytokine properties when it is translocated from the nucleus and released extracellularly after stroke in adult rodents. We have previously shown that HMGB1 is translocated from the nucleus to cytosolic compartment after ischemic brain injury in fetal sheep. In the current study, we utilized the Rice-Vannucci model to investigate the time course of HMGB1 translocation and release after HI injury in neonatal rats. HMGB1 was located in cellular nuclei of brains from sham control rats. Nuclear to cytoplasmic translocation of HMGB1 was detected in the ipsilateral-HI hemisphere as early as zero h after HI, and released extracellularly as early as 6 h after HI. Immunohistochemical double staining detected HMGB1 translocation mainly in neurons along with release from apoptotic cells after HI. Serum HMGB1 increased at 3 h and decreased by 24 h after HI. In addition, rat brains exposed to hypoxic injury alone also exhibited time dependent HMGB1 translocation at 3, 12 and 48 h after hypoxia. Consequently, HMGB1 responds similarly after HI injury in the brains of neonatal and adult subjects. We conclude that HMGB1 is sensitive early indicator of neonatal HI and hypoxic brain injury.