ACS Sensors

Detection of molecules with cell-based sensors allows for conversion of binding events into gene expression outputs. Here, we present a cell-based sensor that can detect extracellular double-stranded DNA. This sensor is based on an engineered receptor which we call Luminescent Ultrasensitive Nucleic Acid Reporter, or LUNAR. LUNAR is based on a recently developed Programmable Antigen-gated G-protein-coupled Engineered Receptor (PAGER). PAGERs are a genetic fusion of an auto-inhibitory peptide, a protein-binding domain, and a modified kappa opioid receptor. PAGERs are gated by two binding events. First, a protein ligand displaces an intramolecular inhibitor, Arodyn, then a second ligand activates the receptor. By replacing the protein-binding domain with a DNA binding zinc finger protein (ZFP) we could detect extracellular DNA in a dose-dependent fashion. Here, we show that first-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells. Future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.

Current technologies to monitor membrane potential are either highly invasive and perturb the integrity of the membrane, use labels that compromise biological behavior, or are limited in sensitivity and do not enable simultaneous monitoring of multiple cells and cell populations. Here, we present Mid-IR Assessment of Conformation in Lipids by Ensemble Sensing (MIRACLES) that, by detection of molecular vibration of lipid acyl chains under cell-membranes electric field dynamics, achieves highly sensitive label-free imaging of membrane potential dynamics in living cells. MIRACLES leverages lipid conformational changes within the plasma membrane as intrinsic indicator for cell membrane depolarization and hyperpolarization. As proof-of-concept, we apply MIRACLES to monitor membrane depolarization during glucose stimulated insulin secretion in {beta}-cells at single-cell level and achieve assessment of {beta}-cell functionality in real time. These results highlight the potential of mid-IR optoacoustic as a powerful tool for indirect, label-free potential assessment of cellular metabolic activities.

Monitoring pH and extracellular acidification rate (ECA) in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins,OEP and OEPK, embedded in a plasticized PVC matrix containing a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime-based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of the protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.

Solid-state nanopores offer a versatile platform for single-molecule sensing owing to their mechanical robustness, tuneable geometry, and compatibility with scalable fabrication. Here, we present a pyramidal silicon nanopore with a 40 nm sensing aperture for label-free characterisation of protein molecules by resistive pulse sensing. The nanopore operates stably over transmembrane voltages ranging from -2 to +2 V and across a broad range of electrolyte concentrations, enabling analysis under diverse experimental conditions. As molecules traverse the confined sensing region, transient ionic current modulations are generated that reflect their excluded volume and molecular geometry. Using this approach, we characterise unlabelled Tau species spanning monomeric proteins, intermediate aggregates, and mature fibrillar assemblies. Analysis of the resulting current signatures, together with simplified geometrical models, enables reconstruction of molecular dimensions and discrimination of distinct Tau populations based on their electrical fingerprints. These results demonstrate that pyramidal silicon nanopores provide a sensitive and scalable platform for label-free monitoring of structurally heterogeneous protein aggregation and establish a framework for investigating protein aggregation using solid-state nanopore sensing.

Digital bioassays enable precise single-molecule quantification but are difficult to adapt to point-of-care testing (POCT) because conventional protocols include off-chip complex processes for sample treatment and sealing, requiring hardware and workflow complexity. We present OASSIS (Open Aqueous two-phase Separation System for Integrated Single- molecule digital bioassay platform), an oil-free and open ATPS platform that localizes both targets and signals in femtoliter-scale dextran (DEX) droplets beneath a polyethylene glycol (PEG) phase. We integrated a CRISPR-Cas13a assay system with a novel, branched fluorescent reporter conjugated to a dextran-binding domain (DBD), which ensures signal retention within the DEX droplets after cleavage. Fluorescence recovery after photobleaching experiments confirmed this robust signal confinement. OASSIS not only performs amplification-free digital RNA detection but also enables serial sample introductions through its open-format architecture that progressively improve sensitivity: the limit of detection (LOD) improved from 1.08 fM (first introduction) to 0.34 fM (third introduction). Furthermore, OASSIS demonstrated specific detection and [~]10-fold enrichment from a complex, denaturant-treated nasopharyngeal swab matrix. Together, these results demonstrate that the open-format architecture of OASSIS provides a practical route toward sensitive, low-complexity POCT and clinical diagnostic applications.

Continuous monitoring of proteins in complex biological fluids is essential for advancing personalized medicine, yet existing biosensors are often limited by instability, single-use designs, and insufficient sensitivity. Here, we describe a detailed protocol for the fabrication and operation of molecular pendulum (MP) electrochemical sensors integrated with an active-reset mechanism to enable real-time, reversible, and ultrasensitive protein detection. The protocol is described in two parts. First, we describe the microfabrication of gold microelectrodes and their nanostructured modification, followed by assembly of DNA-based pendulum probes with redox reporters and affinity receptors that translate binding into kinetic electron-transfer shifts. Second, we detail the sensing and active-reset approach, which detects the target analyte and applies tunable oscillatory potentials to accelerate its dissociation, regenerate sensor surfaces, and extend operational lifetime. The protocol includes detailed guidance on device fabrication, surface functionalization, sensing and reset cycles, and data analysis. When implemented, MP sensors with active-reset achieve pg/ml sensitivity, rapid equilibration, and robust performance across biofluids and in situ models, enabling continuous protein monitoring over extended periods. This combined technology represents a biosensing platform with significant potential, opening new avenues for wearable and implantable molecular monitoring, early disease detection, and personalized therapeutic guidance.

BackgroundContinuous glucose monitoring (CGM) systems are used not only for retrospective assessment of the glycemic profile but also for real-time decision-making, including automated insulin delivery. Accordingly, CGM performance characterization must capture not only the agreement of individual paired values but also the systems ability to reproduce the direction, rate, amplitude, and shape of glucose concentration change. Summary metrics, most notably MARD, cannot establish whether an observed deviation reflects an error in the formation of the test profile itself, a constant sensor offset, amplitude compression, a change in response rate, temporal misalignment, or hysteresis. ObjectiveTo adapt a programmable flow-based in vitro platform for the separate assessment of the experimentally delivered glucose profile and the dynamic response of CGM systems, and to propose a set of metrics that decomposes dynamic error into its components. MethodsGLU profiles were generated by programmable mixing of solutions at a constant total flow rate of 2 mL/min. Actual GLU concentration was independently measured with a SUPER GL2 glucose analyzer. Four static levels, three repeats of a 5.5[->]12.0[->]5.5 mmol/L profile, three repeats of a 6.0[->]3.0[->]6.0 mmol/L hypoglycemic profile, three 5.0[->]15.0[->]5.0 mmol/L profiles at different rates, one complex 4[->]18[->]3[->]12[->]5.5 mmol/L profile, and two proof-of-concept sensor experiments at 100- and 200-min transitions were investigated. Dynamic response was characterized by bias, MAE, RMSE, MARD, amplitude transfer coefficient K_A, rate transfer coefficients K_up and K_down, normalized shape RMSE, residual shift, and hysteresis loop area. ResultsAt the static levels, measured GLU exceeded the programmed value by 0.234-0.780 mmol/L. In the repeated 5.5[->]12.0[->]5.5 profiles, the ratio of actual to programmed rate was 0.978-1.083 on the rising phase and 0.987-1.157 on the falling phase, while the amplitude transfer coefficient was 0.967-1.066. In the hypoglycemic profile, minimum GLU was 2.55- 2.96 mmol/L, and time below 3.0 mmol/L was 15.2-72.6 min. The measured rates of 0.0519, 0.1045, and 0.2027 mmol/L/min preserved the intended ratio of approximately 1:2:4. In the complex profile, the programmed plateau of 18 mmol/L was not reached: mean measured GLU was 16.20 mmol/L. For CGM-A, K_A was 0.682 and 0.650, and K_up/K_down were 0.666/0.730 and 0.634/0.626; the corresponding values for CGM-B were 1.228 and 1.128, and 1.564/1.328 and 1.276/1.145. Hysteresis loop area differed 5- to 10-fold between the two sensor responses, exceeding an order of magnitude at the 100-min transition. ConclusionThe programmed concentration should be treated as a control setpoint, rather than as a reference measurement. The "programmed trajectory -- measured glucose -- CGM output" cascade first allows quantitative assessment of the agreement between the programmed and actually realized profile and only then separate characterization of sensor response. Decomposition of dynamic error into amplitude, rate, shape, and hysteresis components reveals differences that a single MARD value or correlation coefficient cannot capture.

Rapid, selective detection of bacterial pathogens remains a central challenge. Here we report a label-free electrochemical biosensing approach that leverages protein-protein interaction (PPI)-derived peptides as recognition elements for rapid detection of Listeria monocytogenes (LM). The sensor design is inspired by the interaction between the LM virulence factor Internalin A (InlA) and the human host receptor E-cadherin (E-Cad1). Peptides derived from the InlA-binding domain of E-Cad1 were engineered as molecular recognition elements, with the E-Cad1(15-24) peptide displaying micromolar affinity and selective binding towards LM. Immobilization of these peptides on gold electrodes enabled bacterial detection by electrochemical impedance spectroscopy within 10 minutes, without labels or external signal amplification. A low peptide surface density was associated with enhanced binding-site accessibility and may facilitate multivalent interactions between the bacterial surface and the immobilized peptides. The platform produced a detectable response at experimentally tested concentrations as low as 1 CFU mL {superscript 1} and exhibited excellent selectivity under the conditions examined. This work introduces a chemically programmable, PPI-inspired biosensing paradigm that uses a reductionist approach and could potentially be extended to other pathogen targets.

Split aptamer biosensors offer exceptionally low background by assembling only in the presence of a target analyte; however, their performance is frequently limited by the lack of robust design rules for selecting effective split sites. Existing approaches largely rely on heuristic, structure-based assumptions that are poorly validated and often yield suboptimal signal. Herein, we introduce a systematic, data-driven strategy for identifying high-performance split sites within fluorogenic DNA aptamers. Using our massively-parallel aptamer performance analyzer (MAPA) platform, we performed comprehensive single- and double-mutant analysis of the DFAME-binding region of the fluorogenic DNA aptamer Lettuce, informed by its three-dimensional structure. Dimensionality reduction and clustering of the resulting sequence-function landscape revealed mutation-tolerant elements within the binding domain that are suitable for splitting while preserving fluorophore activation. Sensors constructed using these non-intuitive split sites, which are unconventional by standard design principles, exhibited a nearly four-fold improvement in fluorescence signal-to-background ratio for SARS-CoV-2 RNA detection compared to a prior split-Lettuce design. The same split architecture also enabled robust detection of high-pathogenicity H5Nx avian influenza RNA. These results demonstrate that large-scale, data-driven interrogation of aptamer sequence-function relationships can identify non-intuitive split sites and provide a proof-of-concept framework for developing measurement-based design principles for split-aptamer biosensors.

Differentiating overlapping dementia pathologies, such as frontotemporal dementia and Alzheimers disease, calls for the simultaneous measurement of several blood biomarkers, yet electrochemical sensors remain predominantly single-target and dependent on labels and sample preparation. Here we report SMART-NeuroDx, a reagent-free electrochemical array that quantifies pTau217, GFAP, pTau181, and NfL directly from unprocessed plasma and serum in under 35 minutes. Four surface-confined redox-active molecularly imprinted polymer and aptamer-MIP recognition matrices are electropolymerized onto a four-working-electrode porous-gold printed-circuit array, using potential-assisted electrostatic gating keyed to each targets isoelectric point to prevent cross-channel template contamination during synthesis. Label-free Faradaic responses from the redox-active polymer backbone are acquired on a custom battery-powered STM32G4 handheld potentiostat and converted to concentration from five voltammetric features using cross-validated Random Forest and XGBoost regressors. The handheld unit reproduced the baseline fidelity of a commercial benchtop workstation and resolved sub-picogram pTau217 (limit of detection 0.087 pg mL-{superscript 1}) across marker-appropriate dynamic ranges, with inter-chip relative standard deviation at or below 5.34% and coefficients of determination of 0.94 to 0.99 against reference concentrations. Each channel retained selectivity in plasma and serum against competing neurological and inflammatory proteins, with non-specific signal deviation held below 10% by the hydrated PyPEG interfacial shell. The platform establishes reagent-free, simultaneous four-analyte neurodegeneration sensing on a single point-of-care device.

Multiplexed imaging of biomolecular networks in living cells is limited by the small number of spectral separable fluorophores and the need to monitor dynamic processes in real time. Here, we present logicFRIES, a fluorogenic RNA (FR)-based platform that enables eight-plex live-cell imaging through programmable, logic-gated activation coupled with sequential fluorescence imaging. By integrating small-molecule-binding RNA aptamers into dye-activating fluorogenic RNAs, we engineered trigger-responsive FR reporters. This design implements an AND-gated mechanism in which fluorescence activation requires both a cognate trigger molecule and its corresponding fluorogenic dye, thereby expanding multiplexing capacity without adding new fluorophores. Using three membrane-permeable triggers, tetracycline, ASP2905, and guanine, we generated three distinct trigger-defined activation states for each engineered Broccoli and Pepper FR. Combined with orthogonal Corn/DFHO and DNB/TMR-DN reporter pairs, logicFRIES supports eight-plex imaging through sequential trigger/dye addition, imaging, and wash-based stripping cycles. We demonstrate robust, specific, and reversible fluorescence switching of these multiplexed FR reporters in living HEK293T and SKBR3 cells. Overall, logicFRIES extends live-cell imaging beyond conventional spectral limitations and provides a modular foundation for potentially developing multiplexed sensors targeting endogenous RNAs, proteins, and small molecules in complex cellular systems.

Genetically encoded fluorescent biosensors have gained traction in neuroscience as continuous, reagentless reporters of cellular activity in situ. These sensors, often soluble, can also provide time-resolution in multiple biosensing form factors: benchtop and wearable devices, lyophilized powder tests, and smartphone-based diagnostic tests. These biosensors often take advantage of naturally occurring conformation-switching but require extensive screening to optimize the linkers to a fluorescent reporter. Each new target ligand often requires its own engineering campaign. We asked whether sensors evolved towards a particular target retain useful recognition scope for others. We screened a family of 18 OpuBC-cpGFP sensors evolved toward nicotinic agonists, SSRIs, opioids, and other neural drugs, against 63 structurally diverse compounds. We found that 24 ligands activated at least one biosensor with {Delta}F/F0 > 0.3, sufficient to begin directed evolution, with 8 of those ligands activating at least one biosensor with {Delta}F/F0 > 1.0, the regime of dynamic range usable in end applications. With 124 ligand-biosensor pairs in total, we found multiple leads suitable for directed evolution. Most notably, ligands participating in hits spanned well beyond neural drugs and included DEHP, ergothioneine, ciprofloxacin, thiamine, betahistine, L-carnitine, and L-thyroxine. Across the biosensor family, mutation distance weakly predicted substrate scope. In particular, we observed sequence-function cliffs that could be exploited for future protein engineering campaigns. Thus, broad screening of performant scaffolds offers rapid bootstrapping in biosensor engineering particularly for exogenous molecules.

Fluorescence imaging in the second near-infrared window (NIR-II, 950-1700 nm) offers reduced scattering, lower autofluorescence, and deeper tissue penetration than NIR-I imaging, but its adoption is limited by the need for custom-synthesized fluorophores. Here, we identify commercially available dyes that exhibit usable NIR-II emission. Eleven visible, far-red, and NIR-I fluorophores were screened under twelve acquisition configurations combining 670, 760, and 808 nm excitation with band-pass (950 nm, 1400 nm) or long-pass (1000 nm, 1250 nm) emission filters. Output varied markedly with fluorophore identity and excitation/emission configuration. Among hydrophobic dyes, DiR exhibited strong emission across almost all excitation and emission filters. Among hydrophilic dyes, strong NIR-II fluorescence was observed for IRDye 680RD (excitation at 670 nm), sulfo-cyanine 7 (excitation at 670 nm and 760 nm), and indocyanine green (excitation at 808 nm). DiR showed a linear concentration-response under 760 nm excitation with BP1400 detection. Upon encapsulation in PEGylated liposomes, strong NIR-II fluorescence was retained. In an in vivo study in mice, NIR-II resolved vasculature that NIR-I could not consistently delineate, and enabled pharmacokinetic analysis. Both windows returned similar ex vivo organ distributions. NIR-II imaging is therefore accessible using commercial off-the-shelf fluorophores, provided the dye is matched to the intended excitation/emission configuration.

Cytoskeletal plasticity is a defining feature of cancer progression, enabling tumor cells to adapt their morphology, mechanics, and migratory behavior during invasion and metastasis. Although actin filaments, microtubules, and intermediate filaments are known to cooperate in these processes, the molecular mechanisms coordinating their dynamics remain incompletely understood, particularly the role of post-translational modifications (PTMs). Here, we developed CytoFRET2, a multiparametric cytometry-based FRET platform that enables real-time and simultaneous monitoring of the dynamics of actin filaments, microtubules, and vimentin in living suspension cells. The system combines fluorescently tagged cytoskeletal reporters with spectral flow cytometry, allowing simultaneous high-content analysis of multiple cytoskeletal networks while overcoming autofluorescence and fluorescence interference from small molecules. Using spectral CytoFRET2, we screened a small library of epigenetic compounds targeting regulators of acetylation and methylation pathways. The screen revealed that inhibition of lysine deacetylases (KDACs) and sirtuins promoted stabilization of both microtubules and vimentin filaments, without impacting actin filament organization. In contrast, inhibition of lysine acetyltransferases (KATs), particularly with garcinol and anacardic acid, induced rapid vimentin disassembly. Mechanistically, the study reveals acetylation as a key post-translational modification regulating the dynamics of microtubules and vimentin filaments, with KAT inhibitors emerging as potent modulators of vimentin organization. Together, the findings establish spectral CytoFRET2 as a versatile platform for systematic investigation of cytoskeletal regulatory networks and therapeutic vulnerabilities in cancer.

Biomarker analysis requires detecting analyte classes that span nucleic acids, proteins, small molecules, and metabolites, yet testing remains fragmented across target-specific assays and instruments. Here we report a molecular information-transduction strategy that converts target recognition across molecular classes into a common, error-tolerant DNA code readable by nanopore sequencing. Target recognition triggers a hybridization chain reaction that generates concatemers containing periodically repeated 10-nucleotide target-specific barcodes. A matched-filter decoder exploits this periodicity and the linear scaling of read length with match count to reject spurious matches by two to three orders of magnitude. Multi-class detection is demonstrated for a small molecule (ATP), two cardiovascular-associated microRNAs and thrombin in singleplex and multiplexed assays. By separating molecular recognition from sequence readout, this architecture provides a modular framework for converting heterogeneous analytes into a shared, redundancy-encoded signal for high-fidelity molecular sensing.

Interleukin-6 (IL-6) is a pleiotropic cytokine whose aberrant elevation drives life-threatening conditions, including sepsis, cytokine storm, and autoimmune disorders, yet existing clinical detection methods demand centralized laboratory infrastructure and multi-hour assay times incompatible with rapid point-of-care decision-making. Here, we report an impedimetric aptasensor built on a programmable tetrahedral DNA nanostructure (TDN) interface anchored to a disposable gold screen-printed electrode (Au-SPE) for the ultrasensitive, label-free detection of IL-6. By systematically varying the number of thiolated base vertices from zero to three, we establish a clear and previously unreported structure-function relationship between multipodal anchoring geometry and charge-transfer resistance modulation: tripodal thiolation yields the most rigid, upright, and electrochemically responsive interface, producing the steepest analytical signal gain upon IL-6 binding at the apex-localised aptamer. Under optimised conditions (pH 7.0, 0.05 uM TDN, MCH passivation), the aptasensor exhibits a linear dynamic range of 0.0001-0.001 pg/mL, a limit of detection of 55 ag/mL, and a sensitivity of 1.55x107 ohm (pg mL-1)-1. Selectivity evaluation against seven physiologically relevant interferents such as TNF-, BSA, glucose, urea, ascorbic acid, glycine, and cysteine confirms negligible cross-reactivity, with relative responses ranging from 0.57% to 14.35% of the IL-6 signal. Spike-recovery experiments in human serum yield recoveries of 74.0-87.6% (%RSD < 4.5%), and the sensor retains functional activity for at least 21 days under refrigerated storage. This work demonstrates that thiolated vertex number is a critical and tunable design parameter for TDN-based biosensors, offering a modular, disposable platform for sub-femtogram cytokine detection with direct applicability to early sepsis diagnosis and inflammatory disease monitoring.

Rapidly detecting infectious diseases such as Covid-19 is essential to control outbreaks and treat patients early. However, no available screening method combines low cost, portability, speed (<20 min, ideally <5 min), and ultrasensitivity (e.g., <1 virus/L): lateral flow assays are fast, portable, and inexpensive but insensitive, whereas ultrasensitive assays require centralized labs with long turnaround times. We recently developed an ultrasensitive immunoassay that captures, separates, and counts saliva biomarker molecules using buoyant microbubbles and magnetic microspheres, but the original assay took 55 minutes and was not readily deployable. Here, we redesigned the assay protocol and reader for emergency medicine and mobile care by streamlining the workflow, collecting saliva with larger swabs, filtering it through a 10 m cap, and using larger microbubbles to accelerate flotation. A paramedic successfully ran the assay on the back of a parked medical van in 3.5 minutes (spit-to-results) while achieving a 1.3 fg/mL analytical detection limit for SARS-CoV-2 nucleocapsid protein (~0.04 virus1/L). The assay remained positive across 9 orders of magnitude. We describe the challenges and opportunities ahead for point-of-care deployment.

Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.

Background: Rapid and accurate measurement of procalcitonin (PCT) is useful for diagnosing bacterial infections and guiding antibiotic therapy, yet current laboratory-based immunoassays require centralised infrastructure, delaying clinical decision-making and limiting access in low-resource settings. We developed a battery-free, smartphone-connected electrochemical lateral flow assay with linked analytics for the detection of PCT (ELLA-PCT) enabling quantitative testing at the point of care without conventional laboratory instrumentation. Methods: We designed a competitive electrochemical lateral flow assay using gold nanoparticles co-functionalised with a PCT-specific DNA aptamer and ferrocene hexanethiol as a redox reporter. A miniaturised near-field communication (NFC) potentiostat embedded within a disposable cassette enabled wireless electroanalytical measurements using a smartphone. Analytical performance was assessed in buffer and serum, including limit of detection (LOD), linearity, specificity, and stability. Clinical evaluation was performed on 27 serum samples from nine adults undergoing antibiotic treatment for suspected bacterial infection, with results compared against the reference Time-Resolved Amplified Cryptate Emission (TRACE) assay. Findings: ELLA-PCT achieved an LOD of 46 pg/mL and a linear detection range of 0.5-100 ng/mL. The ELLA-PCT assays remained stable for three months under ambient storage conditions, and cross-reactivity with calcitonin, C-reactive protein, and interleukin-6 remained below clinically relevant thresholds. Clinical results showed strong correlation with TRACE (R2 = 0.979, p < 0.0001), with a mean bias of 0.05 ng/mL and narrow 95% limits of agreement (-0.69 to 0.89 ng/mL). Importantly, ELLA-PCT delivered results in only 30 minutes without requiring external power or clinical laboratory instrumentation. In comparison to TRACE, the total turnaround time was reduced by at least 50%, which typically requires at least one hour from sample collection to results at a hospital setting. Interpretation: ELLA-PCT is an antibody-free, smartphone-connected test that provides laboratory-grade quantitative PCT measurement using only an NFC-enabled electroanalytical sensor and a mobile phone. The platform has a strong potential to decentralise diagnosis of infectious diseases, support antibiotic stewardship, and enable remote, real-time monitoring in outpatient and resource-limited settings. Larger studies are needed to evaluate the use of whole-blood samples and the integration of the platform into digital clinical workflows.

Reading both physical and chemical properties of a microenvironment from a single fluorophore remains a challenge. Here we demonstrate that two coexisting molecular equilibria within one near-infrared cyanine, CyC4, encode two mechanistically distinct ratiometric reporting channels. A meso-amino group and a pendant carboxylate form a tunable intramolecular hydrogen bond that toggles the dye between closed (700 nm) and open (780 nm) emissive conformers. Time-dependent density functional theory (TD-DFT) calculations show that the hydrogen bond raises the LUMO and blue-shifts the emission, establishing the 700/780 emission ratio as a local reporter of hydrogen bonding and polarity. Independently, the chromophore self-associates under crowding- and cosolvent-rich conditions into an aggregate with a blue-shifted, H-type absorption signature near 530-540 nm and a distinct emission near 610 nm upon 540 nm excitation. The intensity of this aggregate band relative to the monomer emission (Ra) serves as a ratiometric reporter of crowding and self-association. Because the two channels arise from distinct molecular equilibria (intramolecular hydrogen bonding vs. intermolecular self-association) they are largely decoupled: a glycerol titration series confirms that the self-association channel (Ra) can be moved while the hydrogen-bonding channel stays essentially fixed. Applied to protein-PEG biomolecular condensates, the two ratios move oppositely with increasing salt, showing that the interior's chemical (polarity, hydrogen bonding) and physical (packing, self-association) environments co-vary across the salt series; a single CyC4 measurement thereby maps this coupled microenvironment, providing a general strategy for multiparametric, ratiometric sensing of crowded microenvironments.