ACS Sensors

Inositol 1,4,5-trisphosphate (IP3) is a key second messenger that regulates diverse physiological processes. Visualization of IP3 dynamics in living cells is therefore important for understanding its signaling processes. In this study, we developed genetically encoded green fluorescent IP3 biosensors named Green iPenguins with distinct half-maximal effective concentrations (EC50) for IP3, enabling detection of IP3 signals over a range of concentrations. The biosensors displayed more than a 4-fold increase in fluorescence intensity upon IP3 and showed high specificity for IP3 over structurally related molecules. When expressed in HEK293T cells, the biosensors enabled visualization of IP3 dynamics involved in different signaling pathways. They were also compatible with dual-color imaging, allowing simultaneous monitoring of IP3 together with cAMP or Ca2+ signals. In addition, the hierarchical relationship between IP3 and Ca2+ signaling was visualized, providing insight into the temporal relationship between these two second messengers. The biosensors are expected to facilitate future studies of physiological processes involving IP3 signaling networks.

Aptamers are attractive receptors for small-molecule biomarker detection in complex samples because of their high stability, affinity, and specificity, but aptamer-based sensors generally lack the sensitivity to detect low-abundance analytes. As a solution, we developed the charge-amplified FET (CAFET) aptamer biosensor, which is designed to amplify the net charge variation within the Debye length that occurs as a consequence of aptamer-target binding. Our sensor utilizes a strand-displacement aptamer switch, which releases an initially-hybridized displacement strand (DS) upon target binding and thus induces a measurable net charge variation within the Debye length that is amplified to a large FET current response as signal readout. This signal can be further enhanced by adding a charge label to the DS. As a consequence, our sensor can achieve far greater sensitivity than previously described aptamer-FET sensors, where the binding-induced local charge variation is modest. We demonstrate 3-hydroxykynurenine and progesterone detection with a picomolar limit of detection in undiluted human plasma--four orders of magnitude lower than the dissociation constant (KD) of the aptamer component. The CAFET sensor design is modular and should be adaptable for the detection of a wide range of clinically-informative low-abundance analytes in complex samples.

Understanding skeletal muscle metabolism involves real-time monitoring of key cellular parameters, such as calcium ions (Ca2+), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), and intracellular temperature. Fluorescent protein (FP)-based biosensors are used for live-cell imaging of these signals with high spatiotemporal resolution. Differentiated myotubes are in vitro models used for physiological muscle metabolism research. However, efficient transfection of FP-based biosensors into these cells is challenging. Here, we developed an electroporation-based strategy for delivering recombinant protein biosensors into fully differentiated myotubes. Biosensors for Ca2+, ATP, cAMP, and temperature were recombinantly produced using Escherichia coli and introduced into myotubes using electroporation. Electroporation conditions were optimised to maximise delivery efficiency, preserve cell viability, and minimise cellular damage. We established a robust intracellular delivery system that effectively demonstrated Ca2+, ATP, and temperature dynamics. Furthermore, we achieved the successful co-delivery of two biosensors that enabled dual imaging of Ca2+ and cAMP in response to stimulation.

Infections caused by multi-drug-resistant organisms (MDROs) pose a significant public health threat, responsible for over 2 million hospitalizations and 23,000 deaths annually in the United States. Microbiome dysbiosis (imbalance) is considered one of the main causes for MDRO colonization and the resulting infections. Rapid detection and intervention of MDRO outbreaks are crucial to alleviating strain on patients and healthcare facilities. Current diagnostic methods for MDRO detection are too slow and costly to provide the rapid MDRO detection necessary for patient care facilities. Here we present a rapid, accurate and cost-effective electrochemical sensor capable of MDRO detection down to [~]104 colony forming units (CFU)/g in mice and human stool samples. Our novel sensor utilizes probe-modified Screen-Printed Electrodes (SPEs) capable of hybridizing target gene sequences associated with MDROs. The resulting probe/target complex generates a unique and highly sensitive signal detectable down to 10 atto molar or 10 CFU/mL of target TEM-1 gene. The use of these pre-functionalized SPEs reduces individual sample analysis time to less than an hour. Several target sequences from two chromosomal target genes (AmpC and AcrB found in E. coli) have been identified and successfully detected in clinical stool samples with results comparable to the standard quantitative PCR method. Additional target genes associated with antibiotic resistance (TEM-1, VanA, KPC and SHV) have also been successfully detected in vitro and are ready for clinical evaluation. Future development includes multiplexing the sensor to simultaneously detect up to three MDROs target genes, including {beta}-lactamases that hydrolyze {beta}-lactams, the most commonly used antibiotics in clinical settings. This novel sensor platform will be a rapid, economical, point-of-care device with little requirement of reagent handling or technical training.

Fluorescent biosensors have proven valuable for revealing the spatio-temporal dynamics of protein conformation in live cells and animals. The great majority of biosensors are genetically encoded, but genetic encoding is difficult or impossible to apply in many cases, including cells or animals with poorly understood genomes, no DNA, or sensitive to manipulation. Using biosensors without genetic manipulation could greatly simplify studies in animals, expand the range of accessible organisms, and ultimately enable application in humans. Here we explore using a membrane-permeable small molecule as a fluorescent biosensor. The drug trifluoperazine, which binds only to the active conformation of calmodulin, was covalently linked to an environment-sensing merocyanine dye to create CaMero, a biosensor of calmodulin activation. Simple incubation of CaMero in the extracellular medium, or injection in the tail vein of mice, led to sensitive real time reporting of calmodulin activity. The dye underwent a 12-fold change in fluorescence intensity upon binding to activated calmodulin, revealing waves of activation in peristaltic intestine, localization and kinetics of calmodulin activation during serum stimulation in fibroblasts, and localized activation in the single-celled marine protist foraminifera.

Simple, modular platforms for detecting biologically relevant proteins are critical for applications in clinical diagnostics, healthcare, and research. Here, we have combined aptamer-based protein recognition with our conformationally-responsive DNA nanoswitches to enable simple, sensitive and specific protein detection. We demonstrate dual detection of two clinically relevant blood proteins, thrombin and VEGF as initial proof of concept.

Extracellular pH is a key microenvironmental factor shaping cell physiology and disease, creating a need for quantitative biosensors that can capture dynamic changes in pHe at the surface of individual living cells. Here, we develop a genetically encoded, ratiometric extracellular pH biosensor through systematic screening of a modular library of membrane-display designs that combine SEpHluorin with a pH-stable reference fluorophore. Screening identified a cell-surface-localised mKate2-SEpHluorin construct, named SurpHer, that exhibits dynamic ratiometric responses across the pHe range of 6 - 7.8. SurpHer shows robust membrane localisation and extracellular pH responsiveness across diverse human cell types including HEK293T, PANC-1 and MDA-MB231 cells. Following stable integration in MDA-MB-231 cells, SurpHer enabled time-course imaging of pHe gradients in a microfluidic platform for modelling tumour microenvironments. SurpHer enables real-time interrogation of the pericellular pH environment of tumor cells and, more broadly, provides a strategy to probe microenvironmental pH dynamics across diverse biological contexts.

Cancer is a significant contributor to global mortality and places a substantial burden on healthcare systems, underscoring the need for improved strategies for developing and evaluating new therapies. Electrochemical impedance monitoring of in vitro cancer models is a promising technique for evaluating treatment effectiveness, particularly for evaluating how well a drug may kill cancer cells. This approach is advantageous over conventional end-point assays because it is non-destructive, label-free, and can provide temporal information on cell behavior and drug kinetics. However, traditional impedance devices are limited in that they do not support three-dimensional cell culture that has become standard in cancer studies. Typical devices are planar substrates that support monolayer culture, which has been shown to overestimate drug effectiveness. In this work, we propose 3D printed bioelectronic scaffold devices that provide 3D cancer cell culture while functioning as an on-chip readout for monitoring changes in cell characteristics via impedance. We describe device development and demonstrate reproducible fabrication, stable electrochemical properties, cell detection by impedance, and proof-of-concept monitoring of cytotoxicity in response to a chemotherapeutic drug. Overall, this technology offers a promising platform that could be further developed for compound screening as part of drug development or precision medicine.

Biomarkers in sweat and saliva offer a promising avenue for non-invasive health monitoring. Electrochemical sensors have the potential to measure such biomarkers simultaneously. However, they are limited in discriminating individual biomarkers in mixtures, as redox potentials often overlap, resulting in current signatures that cannot be deconvoluted. This study focuses on differentiating biomarkers using orthogonal sensing materials combined with machine learning. We introduce a flexible electrochemical sensor array comprising carbon flower electrodes modified with poly(vinylidene fluoride) (PVDF) or poly(4-vinylpyridine) (P4VP) for the detection of estradiol (E2), ascorbic acid (AA), serotonin (5-HT), and melatonin (Mel). The two polymers act by altering the redox potential and current response of each biomarker, thereby enhancing signal diversity and enabling peak separation. Using multi-output regression models on 450 single and mixture measurements, the array accurately predicts concentrations (R2 = 0.95) over a wide dynamic range spanning nanomolar to micromolar levels. Polymer-resolved analysis reveals that PVDF-modifications enhance E2 and Mel detection, while P4VP-modifications improve AA and 5-HT quantification, highlighting the benefit of complementary orthogonal sensing electrodes. This finding is further supported by feature attribution analysis, which shows that the machine learning model relies on polymer-specific electrochemical signatures, directly linking improved performance to distinct polymer-analyte interactions. Overall, these results demonstrate that combining polymer-modified orthogonal electrodes with machine learning enables accurate, multiplexed sensing in complex mixtures, advancing selective detection strategies for future sensor platforms.

In todays world, point-of-care nucleic acid detection still remains extensively constrained and limited by the heavy dependence on centralized urban instrumentation facilities and complex assay workflows. Here, we elucidate a glucometer-based analytical platform that enables label-free detection of nucleic acids and the nucleic acid amplification products through a simple redox-mediated mechanism. The approach leverages the potassium ferricyanide (K3[Fe(CN)6])/ potassium ferrocyanide (K4[Fe(CN)6]), redox system, which is intrinsic to commercial glucometers, complementing with interactions between methylene blue (MB) and nucleic acids. These interactions transduce concentration differences in nucleic acids into quantifiable electrochemical signal readouts. Distinct varied signal outputs are observed between single-stranded and double-stranded DNA, enabling the direct detection as well as integration with nucleic acid amplification tests (NAATs), including polymerase chain reaction, rolling circle amplification, and loop-mediated isothermal amplification. Optimization of reaction parameters and conditions leads to enhancement of the overall signal discrimination and sensitivity across various assay formats. This innovation repurposes widely available off-the-shelf glucometers as a low-cost, portable nucleic acid detectors, thus eliminating the need for any specialized instrumentation. Our results enumerate and establish a generalized and scalable strategy for nucleic acid sensing. The platform thus supports sustainable and environmentally responsible point-of-care testing, thereby enabling improved accessibility and public health monitoring at resource-limited and remote settings.

Double-stranded RNA (dsRNA) is a potent immunogenic impurity and its detection is a critical quality attribute in characterizing mRNA therapeutics. Standard analytical methods (e.g., sandwich ELISA) are only able to resolve the bulk presence of dsRNA and cannot characterize the different sub-species that may be present within a mRNA sample.. In this study, we use mass photometry (MP) as a single-molecule analytical platform for the simultaneous detection and characterization of dsRNA impurities in mRNA samples. We demonstrate how ionic strength can interfere with the stability of the mAb/dsRNA complex and measure the binding affinity (1 nM) under a set of parameters for reproducible characterization of the complex. We then leverage the J2 antibody to identify antibody/dsRNA complexes that then resolve dsRNA-positive species within an mRNA sample based on discrete molecular weight profiles. Furthermore, we introduce a novel MP assay that harnesses the repulsive surface chemistry of uncoated glass to exclude the bulk mRNA analyte to enable the use of higher loading concentrations to sensitively profile trace dsRNA impurities as antibody-bound species. This work establishes MP as a valuable next generation mRNA analytical tool for analyzing dsRNA byproducts within mRNA samples.

Bioluminescence resonance energy transfer (BRET) systems are widely used for live-cell spectroscopy and biosensor engineering, yet the intrinsic pH sensitivity of commonly used BRET components has not been systematically examined. Here, we show that major BRET luciferase donors, fluorescent acceptors, and donor-acceptor assay pairs exhibit pronounced pH-dependent spectroscopic behavior across physiologically relevant conditions, identifying environmental pH responsiveness as a fundamental property of widely used BRET systems and a potential source of previously underappreciated assay artifacts. Leveraging these principles, we engineered ORION (ratiOmetRIc prOton seNsor), a genetically encoded ratiometric BRET pH sensor based on the NanoLuc-mVenus fusion. ORION exhibited strong brightness, an approximately 9-fold dynamic range, and robust responsiveness across a substantially broader pH range than that of existing genetically encoded sensors. Compared to pHluorin2, ORION maintained substantially improved quantitative performance at acidic pH values below 6.0. To demonstrate its utility in a biological application, we applied ORION across diverse cancer cell models and identified heterogeneous acid imprinting states, suggesting that tumor cells can retain persistent physiological memory of adaptation to acidic microenvironments even after prolonged ex vivo culture. Together, these findings establish pH responsiveness as a fundamental property of BRET systems and position ORION as a best-in-class platform for interrogating and quantifying pH regulation of biology in living systems.

Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven cyanine monomer and dimer dyes (TOTO, BOBO, YOYO, and POPO series, SYTOX Green, SYTOX Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO-3 and SYTOX Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO-3, YOYO-3, BOBO-3, POPO-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms.

Fibrogenesis is essential to wound healing, but aberrant fibrogenesis is a driver of many chronic diseases and cancers. Lysyl oxidases (LOX) play a pivotal role in fibrogenesis by catalyzing the oxidation of lysine residues to reactive aldehydes (allysine) in collagens and elastin, resulting in the crosslinking and excessive deposition of these extracellular matrix components. Currently, rapid and robust histological assays to visualize the spatial distribution of LOX activity are lacking, hindering the precise validation of anti-fibrotic therapies. Here, we present a histological fluorescent staining method to visualize fibrogenesis (active fibrosis) and LOX activity in tissue sections utilizing a bioorthogonal tag and a click reaction with a turn-on fluorophore. Notably, requiring only two commercial reagents, this protocol can be completed in under two hours and is compatible with other imaging modalities, including second-harmonic generation and immunofluorescence staining. We validated this method across various healthy and fibrotic mouse and human tissue specimens.

Recent advances in plasmonic biosensing and imaging have enabled label-free analysis of single biological nanoparticles. We previously developed PlAsmonic NanOapeRture lAbel-free iMAging (PANORAMA) for isolation and purification-free, digital counting and precise localization of small extracellular vesicles (sEVs), with complementary fluorescence interrogation of surface and intravesicular biomarkers for quantitative molecular profiling. The fact that no isolation and purification or isolation is needed represents a crucial advantage because various specificity, efficiency, and time-consumption issues hinder quantitatively reproducible extraction of sEVs from biological fluids. PANORAMA achieves ultrahigh refractive-index sensitivity through arrayed gold nanodisks on invisible substrates (AGNIS) fabricated by nanosphere lithography (NSL). However, despite its simplicity and low cost, NSL is frequently constrained by poor large-area uniformity, which hinders scalable fabrication. Here, we introduce nanosphere settling lithography (NSSL) as an alternative to the gold-standard Langmuir-Blodgett trough (LBT) process, enabling highly uniform, large-area monolayers with reduced process stringency. AGNIS fabricated via NSSL exhibits high refractive-index sensitivity with low spatial variability across 60 mm x 24 mm substrates, sufficient for 60-well in standard 384-well plate format. The platform demonstrates exquisite sensitivity through PANORAMA digital counting and sizing of 25, 50, and 100 nm polystyrene beads, as well as single-vesicle characterization of sEVs derived from H460 lung cancer cells. For the first time, combined PANORAMA and fluorescence imaging enables quantitative analysis of microRNA-21 (miR-21) expression in sEVs to identify "cancer-suspicious" sub-population from liver cancer patient plasma in an unbiased fashion allowing both highly sensitive detection of individual sEVs and simultaneous molecular profiling. Collectively, NSSL enables uniform, high-performance plasmonic biosensing over large areas, providing a scalable and economical pathway for high-throughput, digital single-sEV analysis and translational liquid biopsy applications.

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1

Leukocyte immunoglobulin-like receptor B4 (LILRB4, ILT3) is an inhibitory immune checkpoint expressed on myeloid cells, where it contributes to immunosuppression within the tumor microenvironment. Secretogranin 2 (SCG2) has recently been identified as a functional ligand of LILRB4, yet small molecule modulators of this interaction remain unexplored. Here, we report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to interrogate the LILRB4 (ILT3)-SCG2 interaction. The assay demonstrated robust performance and was validated using a blocking anti-LILRB4 antibody, consistent with orthogonal ELISA measurements. Pilot screening of chemical libraries identified 23 primary hits, of which two compounds, BMS-813160 and PSB-603, showed reproducible, dose-dependent inhibition with TR-FRET IC50 values of 26.7 {+/-} 1.03 {micro}M and 37.2 {+/-} 2.14 {micro}M, respectively. Activity was confirmed by ELISA, supporting the robustness of the assay. This platform enables high-throughput discovery of first-in-class small molecule modulators of the LILRB4-SCG2 immune checkpoint and provides a foundation for targeting myeloid-driven immunosuppression.

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

Red-emitting carbon quantum dots (HP-CQDs) were synthesised for the first time from aqueous leaf extracts of Hamelia patens through single-step, reagent-free microwave-assisted carbonisation (750 W). The resulting nanoparticles displayed a narrow hydrodynamic size distribution centred at 3.9 nm, consistent with atomic force microscopy measurements showing a maximum height of 2.81 nm. Under 400 nm excitation, the CQDs exhibited a characteristic red emission maximum at 675 nm, representing a rare example of long-wavelength-emitting green CQDs derived from plant biomass. UV-Vis absorption bands at 224 and 256 nm were assigned to {pi}-{pi}* transitions of aromatic carbon domains and n-{pi}* transitions associated with carbonyl-containing surface groups, respectively. X-ray photoelectron spectroscopy (XPS) indicated a carbon-rich composition (C: 67.24%, O: 31.25%, N: 1.52%) with prominent C-O (42.67%) and C-C/C=C (42.64%) contributions. ATR-FTIR further confirmed the retention of hydroxyl, ether, and aliphatic functionalities following carbonisation. The excitation-wavelength-independent emission peak position implicates discrete surface molecular states rather than a heterogeneous distribution of emitters. HP-CQDs exhibit potent DPPH radical scavenging activity (IC50 = 141.8 {micro}g mL-1), comparable to ascorbic acid (IC50 = 114.8 {micro}g mL-1), and maintain >95% cell viability in both HeLa and RPE-1 cells up to 250 {micro}g mL-1. Confocal microscopy demonstrates concentration-dependent cytoplasmic accumulation and selective perinuclear localization at 300 {micro}g mL-1. In vivo biodistribution in zebrafish larvae confirms systemic uptake with statistically significant fluorescence enhancement at 500 {micro}g mL-1 (p < 0.01), establishing HP-CQDs as biocompatible red-fluorescent probes with dual imaging-antioxidant functionality. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/724069v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1dbe864org.highwire.dtl.DTLVardef@763ed0org.highwire.dtl.DTLVardef@115e9b9org.highwire.dtl.DTLVardef@1a3941e_HPS_FORMAT_FIGEXP M_FIG C_FIG