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.

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.

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.

Synthesis of SERS-active core-satellite nanoparticles using heterobifunctional PEG linkers.

Surface-enhanced Raman scattering (SERS) is a sensitive analytical technique capable of magnifying the vibrational intensity of molecules adsorbed onto the surface of metallic nanostructures. Various solution-based SERS-active metallic nanostructures have been designed to generate substantial SERS signal enhancements. However, most of these SERS substrates rely on the chemical aggregation of metallic nanostructures to create strong signals. While this can induce high SERS intensities through plasmonic coupling, most chemically aggregated assemblies suffer from poor signal reproducibility and reduced long-term stability. To overcome these issues, here we report for the first time the synthesis of gold core-satellite nanoparticles (CSNPs) for robust SERS signal generation. The novel CSNP assemblies consist of a 30 nm spherical gold core linked to 18 nm satellite particles via linear heterobifunctional thiol-amine terminated PEG chains. We explore the effects that the varying chain lengths have on SERS hot-spot generation, signal reproducibility and long-term activity. The chain length was varied by using PEGs with different molecular weights (1000 Da, 2000 Da, and 3500 Da). The CSNPs were characterized via UV-Vis spectrophotometry, transmission electron microscopy (TEM), ζ-potential measurements, and lastly SERS measurements. The versatility of the synthesized SERS-active CSNPs was revealed through characterization of optical stability and SERS enhancement at 0, 1, 3, 5, 7 and 14 days.