Langmuir

Coacervate droplets and lipid vesicles are two classes of self-assembled compartments that have been proposed as protocell models. Hybrid protocells, in which a coacervate core is surrounded by a lipid membrane, can integrate the advantages of both protocell systems while overcoming their limitations. Although hybrid protocell membranes have been produced with a variety of diacyl phospholipids related to modern biology and some single-chain amphiphiles inspired by prebiotic scenarios, little is known about how mixtures of single-chain amphiphiles impact hybrid protocell membrane formation and properties. Given the plausible diversity of amphiphiles in the prebiotic milieu, the resulting membranes would have inherently incorporated multiple lipids of different types, potentially altering the properties and viability of hybrid protocells in their environment. Here, we systematically increased the compositional heterogeneity of hybrid protocell membranes by using different prebiotically relevant single-chain amphiphiles of varying head groups and alkyl chain lengths. These membranes were assembled around model coacervate droplets generated from polyallylamine hydrochloride and adenosine diphosphate, and the effect of heterogeneity on membrane properties and stability was evaluated. Compared to protocells with homogeneous membranes, those with heterogeneous amphiphile membranes exhibited higher yields, smaller sizes, and greater sub-compartment formation. Also, they showed increased membrane order, retained similar lateral lipid diffusion, and showed population-level variability in permeability to small anionic molecules. Notably, heterogeneous membranes showed enhanced structural stability under acidic conditions, retaining key properties like size and sub-compartment heterogeneity, thereby broadening the pH range over which hybrid protocells remain intact. These findings suggest that amphiphile diversity not only would have influenced the structural properties of hybrid protocells but also created diversity within the protocell population and enhanced their robustness, thereby playing a crucial role in protocell evolution on early Earth.

Lipid nanoparticles (LNPs) are the most successful drug delivery carrier to date, but optimizing lipid formulations to improve membrane fusion capabilities for effective drug release has been challenging due to lack of a quantitative measure for fusogenicity. Here we introduce a new framework based on small angle X-ray scattering to experimentally measure [Formula] for lipids used in LNP formulations such as glycerol monooleate (GMO) and ionizable lipids (SM-102 and ALC-0315). Q intrinsically captures spontaneous curvature (J0), which is traditionally used to assess fusogenicity. The change of cubic lattice parameters with temperature was measured for GMO-containing lipid mixtures, and the Q extracted quantitatively correlated with LNP fusogenicity power validated by fluorescence-based fusion assays and cryogenic electron microscopy. Fusogenicity of SM-102 and ALC-0315 was quantified by adding them to host membranes and assessing change in Q. This framework provides researchers with the ability to optimize the fusogenicity of LNP formulations for potent drug release and enhances understanding of parameters governing fusion in all biomembranes.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

The apparent pKa of ionizable lipids in lipid nanoparticles (LNPs) is a key determinant of RNA encapsulation during formulation and endosomal release after cellular uptake. However, it is difficult to predict the effective pKa of a given ionizable lipid solely from its solution pKa, because it is sensitive to the membranes composition, as well as solution conditions such as the salt concentration. We developed a simple continuum electrostatics model, based on Gouy-Chapman theory, to predict the shift in effective pKa for ionizable lipids in lipid bilayers as a function of salt concentration and membrane composition. We derive equations for the surface potential and fraction of lipids charged, which are solved self-consistently as a function of solution pH to extract the titration curve and effective pKa. The model shows that the shift in effective pKa is largest when the concentration of titratable lipid is high, and the effect is diminished by increasing salt concentration. We provide a python implementation of the model and an interactive notebook that will allow users to further easily explore the predicted pKa shifts as a function of formulation variables.

Saturated fatty acids such as stearic acid (SA) can exhibit both antimicrobial and growth-promoting effects on bacteria, depending on their concentration and chemical structure. However, the physical properties of the bacterial cell envelope in response to such molecules remain under-explored compared to their biochemical pathways. In this study, a comprehensive investigation is presented on the interaction of SA with the Gram-positive bacterium, Staphylococcus epider-midis (S. epi). SA alters bacterial growth, reflected in a higher maximum specific growth rate, a shorter lag phase, and an extended exponential phase, consistent with a prebiotic effect. Using fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy, we show that SA incorporation leads to significant fluidization of the lipid membrane, characterized by enhanced lateral diffusion and reduced membrane viscosity. Coarse-grained molecular dynamics (CG-MD) simulations demonstrate spontaneous insertion of SA into the membrane and a significant increase in mean-square displacement after insertion, supporting our experimental observations. Importantly, atomic force microscopy measurements show an increase in cell-envelope stiffness, reflected by a higher Youngs modulus which can be attributed to modulations in the glycan-peptide linkage density based on earlier studies that correlate stiffness changes to peptidoglycan (PG) crosslinking in Gram-positive strains [1]. These results provide direct evidence linking membrane fluidization induced by SA and increased cell wall stiffness due to transport modifications in the membrane mediated PG synthesis pathways to enhance bacterial cell viability.

Efficient integration of proteins into amphiphilic polymer membranes offers new opportunities in synthetic biology and nanotechnology. Long-term protein reconstitution into artificial membranes remains challenging due to a lack of stabilising protein-membrane interactions found in native lipid bilayers. Here, we redesigned the transmembrane region of a CytK-4D {beta}-barrel nanopore for stable insertion into 3.5-6.6 nm thick PBD-PEO (poly(1,2-butadiene)-b-poly(ethylene oxide)) bilayers. PBD-PEO membranes offer high mechanical and chemical stability and low electrical noise, but the thick membrane hinders anchoring of biological nanopores. By systematically investigating the elongation of the {beta}-barrel, we engineered nanopore constructs suitable for PBD11PEO8 and PBD22PEO14 membranes. Efficient insertions were observed by adding amino acids that stabilised the transmembrane {beta}-barrel structure and enhanced anchoring of the nanopore into the membrane. Molecular dynamics simulations and single-molecule assays revealed that nanopores folded naturally into PBD-PEO bilayers, enabling successful detection of cyclodextrins and translocation of polypeptides and full-length proteins. Our study offers important lessons for the reconstitution of membrane proteins into artificial membranes. Moreover, these highly robust nanopore-membrane interfaces can be readily integrated into biosensing devices, enabling peptide and protein analysis directly from complex solutions.

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/711661v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@abba74org.highwire.dtl.DTLVardef@5e2b8eorg.highwire.dtl.DTLVardef@7db144org.highwire.dtl.DTLVardef@1034e_HPS_FORMAT_FIGEXP M_FIG C_FIG

The cellular cytosol is a crowded environment. Biomolecular Forster resonance energy transfer (FRET) sensors have been developed to measure crowding in cytosol mimics comprised of synthetic polymers such as polyethylene glycol (PEG) and Ficoll that impart an excluded volume effect. In the current study, we explore the unsolicited role of PEG in driving the phase separation of a protein crowding sensor, AcGFP1/mCherry-FRET crowding helix 2 (CrH2), into fluorescent puncta. In contrast, a DNA-based crowding sensor (CrD), with an Alexa488/Cy5 FRET pair, does not form puncta under the same crowding conditions. Using fluorescence recovery after photobleaching imaging, we uncover the liquid-like physical properties of the PEG-induced puncta. Two-color fluorescence microscopy imaging reveals crowder-induced inhomogeneity, concentration variations, and partition coefficient across the dilute and dense phases of the liquid puncta, which remain largely underexplored in bulk fluorometry measurements. Thus, the average crowding sensor response may originate from an aqueous biphasic system, reporting an erroneous average response instead of distinct levels of crowdedness. A comparison of excluded volume effects conferred by Ficoll and PEGs of various molecular weight ranges shows the influence of size, concentration, excluded volume, and chemical composition on the CrH2 sensor response. We demonstrate that PEGs enable phase separation and alter sensor response through a mechanism that may be driven by polymer interactions with the flexible hinge region of CrH2. Overall, we determine the biophysical mechanisms underlying PEG-induced condensation of CrH2 and demonstrate a CrD sensor as an alternative that does not undergo phase separation.

Efficient drug delivery using nanoparticles (NPs) critically depends on their ability to diffuse through biological tissues to reach target cells at therapeutic concentrations. The extracellular matrix (ECM) poses a key barrier to such transport, which directly influences bio-distribution, cellular uptake, and overall therapeutic efficacy. A key regulator of this transport is hyaluronic acid/hyaluronan (HA), a major ECM polysaccharide that forms a hydrated, viscoelastic network. Increased/reduced hyaluronan concentration can elevate/decrease ECM bulk and effective viscosity. Increase in effective viscosity at the nanometer/micrometer length scales can hinder NP mobility through steric obstruction and hydrodynamic drag. There is a large variability in the HA molecular weights and concentrations, especially across age, tissue/organ, and pathological conditions. This work aims to study the diffusion of different NP types in the mixtures of HA polymers with variable molecular weights using the dynamic light scattering technique (DLS). Furthermore, we perform coarse-grained molecular dynamics (CG-MD) simulations for a model system to complement our findings from the dynamic light scattering experiments. We observe NP undergo anomalous diffusion, which is strongly dependent on the ratio of particle size/HA network mesh size, especially for higher molecular weight mixtures. This is strongly influenced by the effective viscosity, which is defined at the local environment experienced by the NPs. Our work highlights developing a simplified predictive framework coupled with simulations for a target-specific extracellular matrix environment.

Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR-Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.

mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further accelerate translation. Here, we present an approach to improve the liquid shelf-life of mRNA-LNPs that does not rely on modifications to the mRNA or LNP chemistry. In particular, we show that bleb formation induced by high ionic strength acidic citrate buffers during LNP formation reduces mRNA degradation and retains in vitro activity during extended liquid storage. We observed an increase in the in vitro activity storage half-life from 2.8 to 18.9 days at 25{degrees}C when prepared using high ionic strength buffers translating into a [~]7-fold improvement in the liquid shelf-life of MC3-LNPs. This enhanced stability of LNPs with large amount of bleb formation was mainly attributed to reduced rates of lipid-mRNA adduct formation and mRNA fragmentation. Furthermore, the acidic buffer dependent stabilization was observed across different ionizable lipids with the extent dependent on the ionizable lipid head group. We envision that the induction of bleb formation via selection of appropriate acidic mixing buffers may represent a universal approach to enhance mRNA-LNPs stability and enable extended long-term refrigerated storage.

Protein cages are versatile platforms capable of encapsulating a wide range of nanoparticle cargo within biocompatible protein shells while providing tunable functionalities. Here, we investigated a self-assembly system that forms vesicle-like protein cages while simultaneously encapsulating nanoparticles at high density, yielding pomegranate-like protein- nanoparticle hybrid materials. Amphiphilic recombinant fusion protein building blocks based on elastin-like polypeptides, leucin zippers, and fluorescent proteins were employed to assemble vesicle-like protein cages via temperature-triggered liquid-liquid phase separation in the presence of fluorescent polystyrene nanoparticles. Analysis of nanoparticle encapsulation density and protein cage size indicates cooperative interactions between protein building blocks and nanoparticles that mediate the formation of protein-nanoparticle coacervate intermediates, which subsequently convert into core-shell hybrid protein cages, as further supported by kinetics studies. We demonstrate the self-assembly hybrid protein cages incorporating a fluorescent calcium sensor protein and titanium oxide nanoparticles, which exhibit a drastic enhancement in their calcium-sensing capability as a result of nanoparticle encapsulation. This platform offers a broadly applicable strategy that integrates protein biofunctionality with diverse nanoparticle properties for development of advanced hybrid materials.

Ionizable aminolipids enable lipid nanoparticles (LNPs) to encapsulate nucleic acids at neutral pH and to release their cargo upon endosomal acidification. The discrepancy between this effective, acidic LNP pKa and the basic intrinsic pKa of aminolipids, however, remains poorly understood. Here, we performed microsecond constant-pH molecular dynamics simulations of five widely used aminolipids (DODAP, DLin-MC3-DMA, DLin-KC2-DMA, ALC-0315, and SM-102) embedded in different LNP-relevant ternary DOPC/D-SPC-cholesterol membranes to quantify how aminolipid structure and membrane composition jointly govern aminolipid protonation and the associated pH-dependent membrane remodeling. Across all systems, membrane embedding lowers the apparent aminolipid pKa, yielding physiologically relevant values of 6-7.5 corresponding to shifts by up to 3.5 pKa units or approx. 20 kJ mol-1 with respect to the intrinsic pKa. Strikingly, the magnitude of the pKa shift correlates with pH-driven membrane remodeling upon deprotonation: polyunsaturated aminolipids undergo surface-to-core translocation, branched aminolipids preferentially form laterally segregated surface domains, and DODAP remains interfacially anchored through sustained hydration and hydrogen bonding. Saturated helper lipids (DSPC) systematically enhance segregation and amplify pKa shifts relative to DOPC. Together, these results identify membrane phase behavior as a primary regulator of aminolipid protonation equilibria and establish quantitative design principles for tuning LNP composition toward desired pKa, membrane remodeling, and delivery performance.

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

The acoustoelectric (AE) effect, in which acoustic waves modulate the electrical properties of a conductive medium, holds significant potential for biomedical imaging. While classic models describe the phenomenon through conductivity modulation, a detailed understanding of its microscopic origins, particularly the role of ion behaviours, remains lacking. This study introduces a novel electrokinetic perspective by investigating how ultrasound modulates ion-solvent interactions, thereby bridging macroscopic AE signals with underlying ion dynamics. Through finite element simulations of a dilute NaCl solution, we demonstrate that acoustic pressure waves induce local variations in ion mobility and diffusion by altering ion hydration shells and solvent viscosity. These changes disrupt the balance among Coulombic, diffusive, and frictional forces on individual ions, leading to the local conductivity modulation. Furthermore, simulations reveal that acoustic perturbation of the electrode-electrolyte interface (EEI) significantly enhances AE signal generation, highlighting the EEIs critical role in AE-related applications. By linking acoustic modulation to fundamental ion-solvent interactions, this work not only provides a foundation for more accurate, microscopically grounded models of the AE effect but also connects AE effect modelling to the active research of solvation dynamics in physical chemistry.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Understanding lipid metabolism in peripheral glial cells is crucial for elucidating the molecular mechanisms underlying neurodegeneration, cancerogenesis and therapy resistance. Here, we introduce a spectrolipidomic sensing approach that integrates Raman, FT-IR, and AFM-IR spectroscopy to monitor nanoscale cholesterol remodeling in glial cells exposed to cannabidiol (CBD). Deuterated cholesterol (dChol) was employed as an intrinsic, spectroscopically active molecular probe, enabling selective tracking of cholesterol transformations through characteristic C-D vibrational signatures within the 2300-2000 cm-1 silent spectral region. Multimodal vibrational spectroscopy provided label-free, spatially resolved insight into lipid organization, redistribution, and metabolic reprogramming across micro- and nanoscales. The dChol probe enabled semi-quantitative evaluation of cholesterol uptake, esterification, and membrane integration, revealing that the sequence of CBD exposure, before or after probe addition, triggers distinct lipid metabolic pathways. Raman spectroscopy demonstrated superior sensitivity, with reliable detection of intracellular dChol at concentrations as low as 10 {micro}M, outperforming FT-IR imaging and confirming its suitability for cell lipid sensing. This analytical platform establishes deuterium-labeled lipids as powerful vibrational sensors for probing lipid metabolism and CBD-induced remodeling in situ. The presented spectrolipidomic framework paves the way for next-generation, spectroscopy-based biosensing systems capable of visualizing lipid dynamics, membrane restructuring, and drug- lipid interactions under pharmacological or environmental stress conditions. HighlightsO_LIDeuterated cholesterol (dChol) used as an intrinsic vibrational sensor C_LIO_LILower detection threshold of intracellular dChol for Raman than FT-IR C_LIO_LIAFM-IR reveals phases of lipid droplet formation in nanoscale C_LIO_LICBD alters cholesterol uptake, esterification, and lipid unsaturation profiles C_LI

Flexible and biocompatible piezoelectric materials are crucial for next-generation wearable and bio-integrated electronics. In this work, we report a sustainable bio-composite film by incorporating lysozyme, a naturally abundant protein, into a polyvinyl alcohol matrix to achieve efficient electromechanical conversion. The composite exploits the intrinsic molecular dipoles of lysozyme, which are effectively stabilized and aligned within the polymer network. Under applied bending strain and vertical pressure, the film exhibits a pronounced piezoelectric response, as evidenced by time-dependent electrical measurements under forward and reverse bias conditions. The deformation of -helices and other helical structures within lysozyme induces dipole reorientation and charge separation, generating a measurable electrical output. In contrast, pure polyvinyl alcohol films show no detectable response, confirming the essential role of lysozyme in the observed piezoelectricity. Furthermore, the device enables real-time human motion sensing, highlighting its potential for flexible, eco-friendly, and biocompatible electronic applications.

Nanomaterials have been proposed as drug delivery vehicles to enhance targeting and efficiency of traditional and novel therapeutics and have subsequently been studied for potential ecotoxicity. Previous studies have identified size, surface charge, and volume exclusion as factors that influence nanomaterial diffusion and retention. However, there is little accepted or successful quantification of how these parameters influence nanomaterial penetration relative to biological adaptation and biological response. Part of the challenge is the response of living biological interfaces to many of these nanomaterial delivery vehicles and nanosized drugs. This study aimed to emulate key physicochemical barriers to diffusion found in living biomaterials by developing a tunable, synthetic hydrogel. Through the controlled exposure of 150 kDa and 2 MDa nanodextrans with neutral and negative surface charge, we evaluated the systems ability to emulate three core physicochemical features often implicated in biofilm-associated transport resistance: size exclusion, charge interactions, and volume exclusion. We demonstrated a 30% statistically significant decrease in partition coefficients for 2 MDa nanodextran from 150 kDa nanodextran, confirming the ability of the nanocellulose-based microcaps to mimic the permeability of hydrated biomaterial matrices. These findings reflect patterns observed in, for example, living biofilm studies, where size-based diffusion hinderance is commonly reported, but charge-based interaction and volume exclusion are more context-dependent. This controllable system can be coupled with in silico modeling to understand interfacial transport phenomena for nanomaterial-biomaterial interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/703274v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@13c1a34org.highwire.dtl.DTLVardef@dc6c5borg.highwire.dtl.DTLVardef@14dcbd4org.highwire.dtl.DTLVardef@80f70c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gel electrophoresis has been a cornerstone laboratory technique for decades, yet it is often viewed as cumbersome, costly, and has remained confined to laboratory settings. Recent advances in DNA nanotechnology have repurposed electrophoresis as a primary readout for some biosensing applications such as DNA nanoswitches, where a conformational change in a DNA structure indicates the presence of a target molecule. Conventional gel electrophoresis setups not ideal for such targeted applications, with moderate equipment cost, excessive reagent use, and time-consuming processes. Here, we adopt a reductionist, application-driven approach to redesign gel electrophoresis specifically for DNA nanoswitch-based detection. We present a fully 3D-printable mini gel electrophoresis system that incorporates conductive plastic electrodes, demonstrating performance comparable to conventional systems using platinum electrodes. By optimizing the inter-electrode distance and running parameters, our system resolves the on/off states of DNA nanoswitches in as little as one minute. We further show that the device operates reliably at low voltages, including when powered by a USB power bank, and even enables instrument-free nanoswitch readout using an LED with a cell-phone camera. Our design substantially reduces the cost, voltage requirements, material usage, operational complexity, and experiment time. These improvements make gel-based biosensing more practical outside traditional laboratory environments, paving the way for broader adoption of gel electrophoresis in point-of-care and resource-limited settings.