BACKGROUND: The aggregation of isotropic particles through interparticle reactions poses a challenge in control due to the ability of all surfaces to bind to each other, rendering the quantitative detection of such interparticle reactions based on particle size difficult. Here, we proposed a novel detection scheme for DNA utilizing an assembly of Janus particles (JPs) employing dynamic light scattering (DLS). DNA molecules are tethered on one hemisphere of the JP, while the other hemisphere retains its hydrophobic properties.
RESULTS: Aggregation of JPs was induced by the sandwich hybridization of target DNA between them. The assembly of JPs was effectively monitored by the changes in hydrodynamic diameter detected by DLS, revealing that aggregation peaks at 2-3 particles and further reaction was hindered due to the inability of one hemisphere of the JP to interact with another JP. The target DNA demonstrated detectability at concentrations as low as several tens of pM to several nM using a digital sensing method. The two types of target DNA, such as simple (14 base pairs) and HIV-2 specific sequences (20 base pairs) were detectable at nM and pM levels, respectively. Moreover, we substantiated the robustness of our detection scheme through stoichiometric calculations based on an equilibrium model. The present detection mechanism was well explained based on the binding affinity of DNA hybridization.
CONCLUSIONS: This detection method harnesses the anisotropic nature of JPs and represents the first detection approach based on aggregation. By altering the modification molecules on JPs to match target molecules, such as proteins and organic compounds, a wide range of versatile molecules can be detected using this scheme with high sensitivity. This underscores the broad applicability of the present method.

We introduce aqueous ionic liquid (IL) mixtures, specifically mixtures of 1-butyl-3-imidazoliumtetrafluoroborate (BMImBF4), with water as a minimal model of lipid bilayer membranes. Imidazolium-based ILs are known to form clustered nanoscale structures in which local inhomogeneities, micellar or lamellar structures, are formed to shield hydrophobic parts of the cation from the polar cosolvent (water). To investigate these nanostructures, dynamic light scattering (DLS) on samples with different mixing ratios of water and BMImBF4 was performed. At mixing ratios of 50% and 45% (v/v), small and homogeneous nanostructures can indeed be detected. To test whether, in particular, these stable nanostructures in aqueous mixtures may mimic the effects of phospholipid bilayer membranes, we further investigated their interaction with myelin basic protein (MBP), a peripheral, intrinsically disordered membrane protein of the myelin sheath. Using dynamic light scattering (DLS), continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and small-angle X-ray scattering (SAXS) on recombinantly produced, \"healthy\" charge variants rmC1WT and double cysteine variant C1S17CH85C, we find that the size and the shape of the determined nanostructures in an optimum mixture offer model membranes in which the protein exhibits native behavior. SAXS measurements illuminate the size and shape of the nanostructures and indicate IL-rich \"beads\" clipped together by functional MBP, one of the in vivo roles of the protein in the myelin sheath. All the gathered data combined indicate that the 50% and 45% aqueous IL mixtures can be described as offering minimal models of a lipid mono- or bilayer that allow native processing and potential study of at least peripheral membrane proteins like MBP.

Dynamic light scattering (DLS) enables the characterization of sizes and electrokinetic properties of colloids, polymers, and macromolecules. DNA is a charged semiflexible polyelectrolyte that is condensed or compacted by counterions, proteins, and other condensing agents in processes such as chromosome compaction and gene therapeutic applications. DNA condensation is closely related to charge screening since packaging requires effective neutralization of its surface negative charges. In this chapter, we describe in detail the protocol for DLS DNA-ligand complexes. As an example, we describe data for the condensation of DNA by chitosan and the measurement of size, zeta potential, and electrophoretic mobility of the DNA-ligand complex by DLS.

Deamidation frequently is invoked as an important driver of crystallin aggregation and cataract formation. Here, we characterized the structural and biophysical consequences of cumulative Asn to Asp changes in γD-crystallin. Using NMR spectroscopy, we demonstrate that N- or C-terminal domain-confined or fully Asn to Asp changed γD-crystallin exhibits essentially the same 1H-15N HSQC spectrum as the wild-type protein, implying that the overall structure is retained. Only a very small thermodynamic destabilization for the overall Asn to Asp γD-crystallin variants was noted by chaotropic unfolding, and assessment of the colloidal stability, by measuring diffusion interaction parameters, yielded no substantive differences in association propensities. Furthermore, using molecular dynamics simulations, no significant changes in dynamics for proteins with Asn to Asp or iso-Asp changes were detected. Our combined results demonstrate that substitution of all Asn by Asp residues, reflecting an extreme case of deamidation, did not affect the structure and biophysical properties of γD-crystallin. This suggests that these changes alone cannot be the major determinant in driving cataract formation.

Six derivatives of poly-N-vinylcaprolactam (PNVCL) P1-P6 were synthesized via surfactant-free precipitation polymerization (SFPP) at 70 °C, with potassium persulfate (KPS) as the initiator. P5 and P6 were synthesized using the cross-linker N,N\'-Methylenebisacrylamide (MBA). The conductivity was measured to monitor the polymerization process. The hydrodynamic diameters (HDs) and polydispersity indexes (PDIs) of aqueous dispersions of P1-P6 were determined using dynamic light scattering (DLS) and zeta potential (ZP) using electrophoretic mobilities. At 18 °C for P1-P6, the HDs (nm) were 428.32 ± 81.30 and PDI 0.31 ± 0.19, 528.60 ± 84.70 (PDI 0.42 ± 0,04), 425.96 ± 115.42 (PDI 0.56 ± 0.08), 440.34 ± 106.40 (PDI 0.52 ± 0.09), 198.39 ± 225.35 (PDI 0.40 ± 0.19), and 1201.52 ± 1318.05 (PDI 0.71 ± 0.30), the and ZPs were (mV) 0.90 ± 3.23, -4.46 ± 1.22, -6.44 ± 1.82, 0.22 ± 0.48, 0.18 ± 0.79, and -0.02 ± 0.39 for P1-P6, respectively. The lower critical solution temperature ranged from 27 to 29 °C. The polymers were characterized using the ATR-FTIR method. The study concluded that the physicochemical properties of the product were significantly affected by the initial reaction parameters. Polymers P1-P4 and P6 have potential for use as drug carriers for skin applications.

Biomolecular condensates (BMCs) exhibit physiological and pathological relevance in biological systems. Both liquid and solid condensates play significant roles in the spatiotemporal regulation and organization of macromolecules and their biological activities. Some pathological solid condensates, such as Lewy Bodies and other fibrillar aggregates, have been hypothesized to originate from liquid condensates. With the prevalence of BMCs having functional and dysfunctional roles, it is imperative to understand the mechanism of biomolecular condensate formation and initiation. Using the low-complexity domain (LCD) of heterogenous ribonuclear protein A1 (hnRNPA1) as our model, we monitored initial assembly events using dynamic light scattering (DLS) while modulating pH and salt conditions to perturb macromolecule and condensate properties. We observed the formation of nanometer-sized BMCs (nano-condensates) distinct from protein monomers and micron-sized condensates. We also observed that conditions that solubilize micron-sized protein condensates do not solubilize nano-condensates, indicating that the balance of forces that stabilize nano-condensates and micron-sized condensates are distinct. These findings provide insight into the forces that drive protein phase separation and potential nucleation structures of macromolecular condensation.

Proteins in ionic liquids (ILs) and deep eutectic solvents (DESs) have gained significant attention due to their potential applications in various fields, including biocatalysis, bioseparation, biomolecular delivery, and structural biology. Scattering approaches including dynamic light scattering (DLS) and small-angle X-ray and neutron scattering (SAXS and SANS) have been used to understand the solution behavior of proteins at the nanoscale and microscale. This review provides a thorough exploration of the application of these scattering techniques to elucidate protein properties in ILs and DESs. Specifically, the review begins with the theoretical foundations of the relevant scattering approaches and describes the essential solvent properties of ILs and DESs linked to scattering such as refractive index, scattering length density, ion-pairs, liquid nanostructure, solvent aggregation, and specific ion effects. Next, a detailed introduction is provided on protein properties such as type, concentration, size, flexibility and structure as observed through scattering methodologies. This is followed by a review of the literature on the use of scattering for proteins in ILs and DESs. It is highlighted that enhanced data analysis and modeling tools are necessary for assessing protein flexibility and structure, and for understanding protein hydration, aggregation and specific ion effects. It is also noted that complementary approaches are recommended for comprehensively understanding the behavior of proteins in solution due to the complex interplay of factors, including ion-binding, dynamic hydration, intermolecular interactions, and specific ion effects. Finally, the challenges and potential research directions for this field are proposed, including experimental design, data analysis approaches, and supporting methods to obtain fundamental understandings of complex protein behavior and protein systems in solution. We envisage that this review will support further studies of protein interface science, and in particular studies on solvent and ion effects on proteins.

Here, we report a novel kind of protein nanoparticles of 11 nm in size, which have a central protein core surrounded by two layers of lipid. One layer of the lipid was covalently attached to the protein, while the other layer has been physically assembled around the protein core. Particle synthesis is highly modular, while both the size and charge of the protein nanoparticles are controlled in a predictable manner. Circular dichroism studies of the conjugate showed that the protein secondary structure is retained, while biophysical characterizations indicated the particle purity, size, and charge. The conjugate had a high thermal stability to steam sterilization conditions at 121°C (17 psi). After labeling the protein core with few different fluorescent dyes, they were strongly fluorescent with the corresponding colors independent of their size, unlike quantum dots. They are readily digested by proteases, and these water-soluble, non-toxic, highly stable, biocompatible, and biodegradable conjugates are suitable for cell imaging and drug delivery applications.

Porous hybrid microparticles are characterized by their densities and porosities. Consequently, the evaluation for density and porosity of porous hybrid microparticles in liquids is crucial for predicting the transport of particles in the atmosphere, human body, and industrial processes. However, direct measurement of the density and porosity of porous hybrid microparticles in liquids remains a challenge. In this study, we investigated the centrifugal sedimentation of polystyrene-silica hybrid microparticles with and without gas-containing closed pores. A centrifugal liquid sedimentation-dynamic light scattering combined analytical method was employed to determine the apparent densities of hybrid microparticles with and without gas-containing closed pores. The porosity of the hybrid microparticles with gas-containing closed pores was elucidated based on the inner buoyancy, which is a centrifugal force generated by the presence of low-density gas inside numerous closed pores. Further, the inner gas buoyancy was analyzed to estimate the particle porosity in liquids. The results obtained in this study confirmed the feasibility of utilizing the proposed method to determine the apparent density and porosity of porous hybrid microparticles in liquids.

A novel construction strategy is introduced for an ultrasensitive dynamic light scattering (DLS) immunosensor targeting alpha fetoprotein (AFP). This approach relies on a self-assembled heptamer fusion protein (A1-C4bpα), incorporating the dual functions of multivalent recognition and crosslinking aggregation amplification due to the presence of seven AFP-specific A1 nanobodies on the A1-C4bpα heptamer. Leveraging antibody-functionalized magnetic nanoparticles for target AFP capture and DLS signal output, the proposed heptamer-assisted DLS immunosensor offers high sensitivity, strong specificity, and ease of operation. Under the optimized conditions, the designed DLS immunosensor demonstrates excellent linear detection of AFP in the concentration range 0.06 ng mL-1 to 512 ng mL-1, with a detection limit of 15 pg mL-1. The selectivity, accuracy, precision, practicability, and reliability of this newly developed method were further validated through an assay of AFP levels in spiked and actual human serum samples. This work introduces a novel approach for constructing ultrasensitive DLS immunosensors, easily extendable to the sensitive determination of other targets via simply replacing the nanobody sequence, holding great promise in various applications, particularly in disease diagnosis.