Search Results (144)

Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment for hematologic malignancies. However, it is limited by high costs, risk of severe toxicities such as cytokine release syndrome and neurotoxicity, and heterogeneous patient responses. The current therapy monitoring depends largely on subjective symptom assessment, routine laboratory tests, and basic vital signs, without real-time, quantitative evaluation of CAR T-cell expansion or activation in clinical practice. This lack of timely immune monitoring hampers individualized care and contributes to increased treatment costs. To address this need, we present a proof-of-concept, label-free rapid optical imaging (ROI) biosensor with automated machine learning analysis for direct quantification of CAR T-cells from whole blood. This microfluidic platform integrates red blood cell (RBC) removal, CAR T-cell capture, and imaging-based quantification on a single chip, eliminating the need for centrifugation, staining, and operator-dependent interpretation. For validation, 50 μL whole blood samples spiked with Jurkat cells expressing CD19 CARs underwent RBC depletion by agglutination and microfiltration. The remaining blood components were then incubated on a sensor chip functionalized with recombinant CD19 protein. Captured CAR T-cells were imaged by brightfield microscopy and automatically enumerated using a machine learning algorithm trained on fluorescence-validated cells. The CD-19 cells’ capture performance was validated by flow cytometry and fluorescence imaging. The trained machine learning model validated at 88% sensitivity and 96% specificity. Buffer and whole blood calibration curves were established across clinically relevant concentrations (1–1000 cells/µL) with triple replicates. The results showed high correlation (0.975 and 0.990 R²) between the spiked concentration and the detected CAR T-cells, with a 95% certainty limit of detection (LOD) and quantification (LOQ) of 0.6 and 1.1 cells/µL for spiked buffer, and 14 and 67 cells/µL for spiked whole-blood, respectively. Full article

The integration of whole-cell biosensors in miniaturized measuring devices to exploit synergetic effects as small, rapid, cost-effective, sensitive, and highly specific platforms with point-of-care applicability was often discussed in recent years and many different setups have been presented to date. In many cases these setups were envisaged as powerful systems in their respective fields; however, the anticipated success often failed to materialize, and the systems remained a proof-of-concept. We elaborate on the hurdles and possible challenges that have to be overcome for the successful development and application of such systems. Further, we critically discuss and rank the impact of different challenges during system development, application, and commercialization. Finally, we point out possible future applications and conclude future perspectives for whole-cell biosensors integrated into microfluidic platforms. Full article

A high-throughput assay system is developed for measuring communication in microbial biofilms in a 96-well microtiter plate format. In this assay, bioluminescent microbial whole cell biosensor systems (MWCBs) for quorum-sensing molecules (QSMs) are embedded into biofilms, and their response to chemical cues relevant for bacterial communication is assessed. For measuring the response to stress, a sigma factor 54 (σ⁵⁴, RpoN)-dependent MWCB was developed. Biofilms generated in this platform were exposed to gradients of communication signals (QSMs such as N-acetyl-homoserine lactones (AHLs), 3,5- dimethylpyrazin-2-ol (DPO), or phytochemicals that can act as natural quorum-sensing inhibitors (QSIs) such as curcumin or 3,3′-diindolylmethane (DIM)), and the response pattern was monitored. Further, the regulatory role of stressors such as oxidants (H₂O₂) or antibiotics (ciprofloxacin, trimethoprim/sulfamethoxazole) on the communication response is assessed. QSMs induced the MWCBs at 1 h and 4 h in biofilms, but high concentrations inhibited them at 24 h. Curcumin and DIM at higher concentrations lead to inhibition of quorum sensing in biofilms after 4 h and 24 h, but this is not followed by biofilm disintegration. H₂O₂ above 0.002% efficiently inhibited the MWCB activities and led to biofilm disintegration. At lower concentrations of H₂O₂, we observed induction of MWCBs. The antibiotics inhibited the MWCB activity at concentrations above their minimal inhibitory concentration (MIC), but this did not necessarily lead to disintegration of the biofilm. Like low concentrations of H₂O₂, the antibiotics activated the MWCBs at concentrations close to their MIC, possibly as a result of H₂O₂ generated during their bactericidal action. Interestingly, the induction of communication in response to antibiotics can be quenched by iron chelators, suggesting involvement of H₂O₂ and free radicals generated by the Fenton reaction. We hypothesize that the observed response to these stressors reflects increased communication in the biofilm, possibly enhancing tolerance and increasing survival. Full article

Chemical monitoring of pollutants and hazardous materials in water supply systems traditionally depends on centralized laboratories, advanced instrumentation, and trained personnel, limiting accessibility and preventing real-time, on-site analysis. This work presents an alternative cost-effective, field-deployable approach that uses genetically engineered bioluminescent bioreporters, encapsulated in self-sufficient alginate capsules and integrated with an optoelectronic detection circuit, to detect and quantify target materials in water. We have developed a scalable single-channel prototype featuring four sensing tracks—two for sample measurement, one for clean water, and one for a standard reference solution. The latter employs the standard ratio (SR) method to ensure robust quantification, compensating for batch variability and environmental effects. System characterization showed high uniformity across tracks. Validation with nalidixic acid (NA) demonstrated reliable quantitative performance, with a blind test estimation of 5.6 mg/L for a true concentration of 5 mg/L, well within the calibration error range. Additional sensitivity testing confirmed detection of mitomycin C (MMC) at concentrations as low as 50 µg/L. Overall, the results highlight the potential of bacterial chemical sensing as a practical and scalable tool for real-time, in situ water quality monitoring networks. Full article

Tumor Mutational Burden (TMB) is a critical biomarker used to determine patient eligibility for immunotherapy with immune checkpoint inhibitors. However, its gold-standard assessment via whole exome sequencing is limited by high costs, technical complexity, and lengthy processing times. To address these challenges, we investigated whether Ultra-High-Frequency (UHF) electromagnetic wave sensing could serve as an alternative method for evaluating TMB. We analyzed the dielectrophoresis crossover frequency spectrum and corresponding electromagnetic signature (EMS) of cancer cells using a lab-on-a-chip biosensor that integrates microfluidics with dielectrophoresis-based electro-manipulation. Across seven solid tumor cell lines exhibiting diverse TMB levels, EMS exhibited an upward shift correlated with higher TMB, suggesting a relationship between mutational load and electromagnetic behavior. To further explore this connection, we artificially increased the somatic variant burden by exposing cells to the mutagen N-ethyl-N-nitrosourea (ENU). EMS measurements reliably detected the induced increase in variant load in ENU-treated cells. Overall, these findings demonstrate that EMS can detect both intrinsic TMB differences and experimentally induced increases in mutational burden, enabling refined categorization of cancer cells. Although further validation is required, this work lays the foundation for developing complementary, rapid, and accessible tools to support cancer cell stratification and guide immunotherapy decision-making. Full article

Background: The current rise in multidrug-resistant pathogens highlights the urgent need for the discovery of novel antibacterial agents with potential clinical applications. A considerable proportion of these developed resistances may be attributable to the intrinsic response of bacteria to antibiotic-induced stress conditions in the environment. Consequently, the identification and characterization of genetic alterations in physiological processes in response to antibiotics represent promising strategies for the discovery and characterization of naturally produced novel antibacterial agents. This study investigated the antimicrobial activity of an antimicrobial active isolate Actinomadura coerulea derived from a meerkat fecal sample. Methods: The production of secondary metabolites that potentially compromise bacterial cell wall integrity was confirmed by the induction of promoter activity in whole-cell biosensors in which an antibiotic-inducible promoter was fused to the luciferase cassette. During plate-based biosensor assays, we identified naturally resistant Bacillus subtilis colonies growing in the zone of inhibition around A. coerulea colonies. After these successive rounds of selection, highly resistant spontaneous B. subtilis mutants had evolved that were subjected to whole-genome sequencing. Results: Non-silent mutations were identified in pssA, which encodes a phosphatidylserine synthase; mdtR, as a gene for the repressor of multidrug resistance proteins, and yhbD, whose function is still unknown. A new cinnamycin-like molecule, coerumycin, was discovered based on the physiological role of PssA and comprehensive genomic analysis of A. coerulea. Additional experiments with cell extracts containing coerumycin as well as the cinnamycin-like compound duramycin confirmed that the interaction between coerumycin and the bacterial cell envelope is inhibited by a loss-of-function mutation in pssA. Conclusion: Our approach demonstrates that combining the exploration of niche habitats for actinomycetes with whole-cell biosensor screening and characterization of natural resistance development provides a promising strategy for identifying novel antibiotics. Full article

Heme, a protoporphyrin IX iron complex, functions as an essential prosthetic group in hemoglobin and myoglobin, mediating oxygen storage and transport. Additionally, heme serves as a critical cofactor in various enzymes such as cytochrome c, enabling electron transfer within the mitochondrial respiratory chain. Unlike protein-bound heme, free or labile heme exhibits cytotoxic, pro-oxidant, and pro-inflammatory properties. Elevated levels of free heme are associated with various pathophysiological conditions, including hemolytic disorders such as sickle cell disease, malaria, and sepsis. In this review, we introduce the physiological roles of heme and its involvement in human health and disease. We also examine the mechanisms of heme sensing and regulation in bacterial cells. A variety of analytical methods have been developed to detect and quantify heme, enabling differentiation between protein-bound and free forms. These tools are discussed in the context of their applications in studying cellular heme regulation and their use in monitoring pathological conditions in humans. In particular, we describe examples of biosensors employing bacterial heme sensor proteins as recognition elements. Full article

This article is devoted to the synthesis and investigation of a family of new benzimidazole compounds with a propylsulfonate moiety, synthesized by condensation of salicylic aldehyde or its 5-substituted derivatives with 3-(2,3-dimethylbenzimidazol-1-ium-1-yl)propane-1-sulfonate. The structure of the obtained dyes was confirmed using NMR, FT-IR, and HRMS. Absorption and photoluminescence properties were studied in phosphate buffers over a wide pH range, and changes in the absorption and fluorescence spectra of DMSO solutions upon titration with DIPEA and HCl were also studied. It was found that all the target compounds possess pH-sensitive optical properties and can be used as fluorescent probes, while methoxycarbonyl-substituted derivative 3c demonstrated the most prominent optical and fluorescent response starting from pH ~ 4.5. The toxicity of the compounds was studied using whole-cell bioluminescent bacterial sensors. The effect on the biomass and metabolic activity of strains Staphylococcus aureus ATCC 6538-P FDA 209-P and Escherichia CDC F-50 bacterial biofilms was also investigated. In the final stage of the study, bioimaging experiments were carried out using the selected most promising dye 3c and biofilms. It was demonstrated that the dye can be excited by light with wavelengths of 458 nm or 750 nm in multiphoton mode. Importantly, when biofilms are incubated in the dye solution for 3 h, only the extracellular matrix is stained. However, if the staining time is increased to 24 h, dye penetration into bacterial cells is observed, resulting in a second photoluminescence maximum during sample analysis. It is important to note that when biofilms are incubated in a dye solution for 3 h, only the extracellular matrix is stained, while with longer staining, penetration of the dye into bacterial cells is observed, and a second photoluminescence maximum appears during sample analysis. The results obtained demonstrate a high potential of using benzimidazole-based compounds as pH-sensitive fluorescent probes operating in a biologically relevant pH range, which can be used for imaging of bacterial biofilms. Full article

Whole-cell biosensors represent one of the tools used for assessing the effects of various agents on living cells. Here we have constructed and tested whole-cell lux-biosensors to detect membrane damage in both Gram-negative and Gram-positive bacteria using the stress-inducible promoter of the pspA gene from Escherichia coli and Bacillus subtilis fused to the lux genes from Photorhabdus luminescens. These biosensors increase their luminescence in response to treatment with a number of known membrane-damaging compounds, such as ethanol, Triton X-100, polymyxin B, dimethylsulfoxide (DMSO) and melittin. E. coli- and B. subtilis-based biosensors demonstrated differences in response to the action of the same membrane-damaging agent. Thus, ethanol and polymyxin B specifically induced the pspA promoter in both lux-biosensors, but the induction amplitude was higher in the E. coli. Triton X-100 and melittin specifically induced the pspA promoter exclusively in B. subtilis cells, while DMSO induced it only in E. coli cells. This indicates a difference in the stress response of the Psp system to membrane-damaging agents in E. coli and B. subtilis cells. Thus, we demonstrated the functionality and efficiency of the constructed lux-biosensors and, using them, showed that some of the tested compounds are able to specifically activate Psp stress response systems in case of membrane damage. Full article

With the aim of developing a new tool to meet the increasing demand for food safety, a whole-cell-based system able to detect the presence of cobalt contamination along the pasta production chain was constructed. This system is based on bacterial cells engineered with a plasmid containing the eGFP gene under the control of a promoter sequence, and is able to elicit a fluorescence signal when activated. The promoters of four stress-responsive genes (DnaK, GroE, UspA, and ZntA) were used to test their responsiveness to cobalt; the promoter of the UspA gene, coding for a universal stress protein, was chosen. The UspA promoter was activated by cobalt, and the system described was highly sensitive, successfully detecting low concentrations of cobalt within complex food matrices derived from durum wheat seeds when exogenous cobalt was added. In food matrices tested alone, a fluorescence signal was present only in bran and fine bran, confirming that these parts of the wheat seed are the ones in which contaminants accumulate. Conversely, in the other matrices derived from the inner part of grains, no signal was detected. The findings reported contribute to the development a new, effective and sensitive tool for monitoring cobalt contamination, offering a valuable approach to enhance food safety control. Full article

Food matrices such as phytic acid, starch, and proteins can chelate heavy metals, acting as stabilizers that significantly hinder accurately detecting heavy metal contamination. This study proposes a biological digestion strategy to overcome such interference. The gene sequences for phytase (appA) from Escherichia coli (E. coli), α-amylase (amyA) from Escherichia coli (E. coli), and protease (AO090120000474) from Aspergillus oryzae were identified via bioinformatics screening. Whole-cell biosensors were then developed to simultaneously detect mercury ions (Hg²⁺) and digest phytate, starch, and proteins. In the presence of 100 μM Hg²⁺, biosensor responses improved by 1.43-, 1.38-, and 1.11-fold, respectively. A “heavy metal pollutant bio-digestion pathway” was constructed by integrating genes for synthesizing phytic acid, starch, and protein with those for Hg²⁺ detection. In the presence of 100 μM Hg²⁺, the detection effect was improved by 1.36-fold. The detection limit of the BαAP whole-cell biosensor was 0.082 μM, while the limit of quantitation was 0.272 μM. The study effectively addresses the limitations of biosensor performance in real sample detection. Full article

Environmental contamination is becoming an increasingly evident risk to human health worldwide. The small, free-living nematode Caenorhabditis elegans (C. elegans) has become a compelling model organism for environmental toxicity studies in recent years, owing to its numerous advantages, including its transparent body, small size, well-characterized biology, genetic tractability, short lifespan, and ease of culture. Several assays have been developed using C. elegans to enable a better understanding of toxicant effects, from whole-animal to single-cell levels. While these methods can be extremely useful, they can be time-consuming and cumbersome to perform on a large scale. Recent advances in microfluidics have adapted many of these assays to enable high-throughput analysis of C. elegans, greatly reducing time and resource consumption while increasing efficiency and scalability. Further integration of these microfluidic platforms with machine learning expands their analytical capabilities and accuracy, revolutionizing what can be achieved with this model organism. This article will review the physiological basis of C. elegans as a model organism for environmental toxicity studies, and recent advances in integrating microfluidics and machine learning which could lead to using C. elegans as a promising living biosensor for environmental sensing. Full article

Food safety hazards induce diverse cellular damages including DNA damage, oxidative stress, proteotoxic stress, and membrane disruption, ultimately contributing to various human diseases. Conventional toxicity assays, while effective, are often resource-intensive and lack the capacity to distinguish among these different damage types, thereby limiting insight into toxic responses and the development of effective strategies for targeted risk mitigation. Here, we constructed a panel of Escherichia coli whole-cell biosensors capable of distinguishing distinct categories of cellular damage. Specifically, an optimized RecA-LexA-based DNA damage biosensor that precisely controls the exogenous expression of the transcriptional repressor LexA achieved a 35.5% reduction in baseline signal and a 36.6-fold induction of fluorescence. In parallel, systematic promoter screening identified P_fpr, P_katG, P_grpE, and P_fabA as effective modules for constructing oxidative, proteotoxic, and membrane stress biosensors. These biosensors exhibited high specificity and sensitivity, generating dose-dependent responses to model toxicants and enabling discrimination of cellular damage induced by typical hazards such as norfloxacin and ciprofloxacin. Notably, the DNA damage biosensor detected norfloxacin with a limit of detection (LOD) of 1.3 ng/mL in standard solution and 3.0 ng/mL in milk, comparable to that of high-performance liquid chromatography (HPLC). Together, our work not only provides a versatile, cost-effective, and sensitive tool for assessing diverse cellular damages induced by food safety hazards, but also demonstrates potential utility for practical food safety monitoring. Full article

Pseudomonas aeruginosa is a clinically significant pathogen with high antibiotic resistance, necessitating rapid and reliable diagnostic methods. In this study, we developed a whole-cell aptamer selection method for P. aeruginosa using an Eppendorf-tube-based SELEX system, where bacterial cells were directly incubated with an ssDNA library. This configuration enhanced the recovery of bound aptamers and overcame the cell quantity limitations often encountered in microtiter-plate-based SELEX. After 10 selection rounds, six aptamer candidates were obtained and evaluated for affinity. Molecular docking analysis revealed that aptamer T1 possessed the highest target selectivity. To demonstrate diagnostic applicability, aptamer T1 was integrated into a hybrid lateral flow immunoassay (LFIA), replacing the conventional detection antibody. In this format, the AuNP–aptamer complex bound to the target bacteria and was captured by a specific antibody immobilized on the test line. The LFIA achieved a visual detection limit of 2.34 × 10² CFU/mL within 15 min, showing high specificity and suitability for point-of-care applications. This study presents the first demonstration of an aptamer–antibody hybrid LFIA for bacterial detection and highlights the potential of aptamers as low-cost, rapidly synthesized recognition elements adaptable for the detection of other infectious agents. Full article

TtgR, a transcriptional repressor from Pseudomonas putida, plays a key role in regulating multidrug resistance by controlling the expression of genes in response to various ligands. Despite its broad specificity, TtgR represents a promising candidate for the development of transcription factor (TF)-based biosensors. In this study, we utilized TtgR and its native promoter region (P_ttgABC) as genetic components to construct TF-based biosensors in Escherichia coli. By coupling TtgR and P_ttgABC with egfp, we developed a biosensor responsive to diverse flavonoids. To enhance the selectivity and specificity of the biosensor, we genetically engineered a TtgR-binding pocket. Engineered TtgR variants exhibited altered sensing profiles, enabling the development of biosensors with tailored ligand responses. Computational structural analysis and ligand docking provided insights into the interaction mechanisms between TtgR variants and flavonoids. Notably, biosensors based on wild-type TtgR and its N110F mutant were capable of quantifying resveratrol and quercetin at 0.01 mM with >90% accuracy. Although the precise molecular mechanisms involved remain unclear and further optimization is needed, the biosensors developed herein demonstrate strong potential for applications in numerous fields. This study lays the foundation for future research that could extend the utility of TtgR-based biosensors to synthetic biology, metabolic engineering, and beyond. Full article