Search Results (129)

Sediments are key players in the optimum functioning of ecosystems; however, they also represent the largest known repository of harmful contaminants. The vast variety of these sediment-associated contaminants may exert harmful effects on marine communities and can impair ecosystem functioning. Whole-cell biosensors are a rapid and biologically relevant tool for assessing environmental toxicity. Therefore, in this study, we developed a bioassay-based toxicity measurement system using genetically modified bacteria to create a whole-cell optical biosensor. Briefly, reporter bacteria were integrated and immobilized using a calcium alginate matrix on fiber-optic tips connected to a photon counter placed inside a light-proof, portable case. The calcium alginate matrix acts as a semi-permeable membrane that protects the reporter-encapsulated optical fiber tips and allows the inward passage of toxicant(s) to induce a dose-dependent response in the bioreporter. The samples were tested by directly submerging the fiber tip with immobilized bacteria into vials containing either water or suspended sediment samples, and the subsequent bioluminescent responses were acquired. In addition to bioavailable sediment toxicity assessments, conventional chemical methods, such as liquid chromatography–mass spectroscopy (LC-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES), were used for comprehensive evaluation. The results demonstrated the efficacy of the biosensor in detecting various toxicity levels corresponding to identified contaminants, highlighting its potential integration into environmental monitoring frameworks for enhanced sediment and water quality assessments. Despite its utility, this study notes the system’s operational challenges in field conditions, recommending future enhancements for improved portability and usability in remote locations. Full article

Various high-throughput screening methods have been developed to explore plant phenotypes, primarily at the organ and whole plant levels. There is a need to develop phenomics methods at the cellular level to narrow down the genotype to phenotype gap. This study used double-resonator piezoelectric cytometry biosensors to capture the dynamic changes in mechanical phenotypes of living cells of two rice species, drought-resistant Lvhan No. 1 and drought-sensitive 6527, under PEG6000 drought stress. In rice cells of Lvhan No. 1 and 6527, mechanomics parameters, including cell-generated surface stress (ΔS) and viscoelastic parameters (G′, G″, G″/G′), were measured and compared under 5–25% PEG6000. Lvhan No. 1 showed larger viscoelastic but smaller surface stress changes with the same concentration of PEG6000. Moreover, Lvhan No. 1 cells showed better wall–plasma membrane–cytoskeleton continuum structure maintaining ability under drought stress, as proven by transient tension stress (ΔS > 0) and linear G′~ΔS, G″~ΔS relations at higher 15–25% PEG6000, but not for 6527 cells. Additionally, two distinct defense and drought resistance mechanisms were identified through dynamic G″/G′ responses: (i) transient hardening followed by softening recovery under weak drought, and (ii) transient softening followed by hardening recovery under strong drought. The abilities of Lvhan No. 1 cells to both recover from transient hardening to softening and to recover from transient softening to hardening are better than those of 6527 cells. Overall, the dynamic mechanomics phenotypic patterns (ΔS, G′, G″, G″/G′, G′~ΔS, G″~ΔS) verified that Lvhan No. 1 has better drought resistance than that of 6527, which is consistent with the field data. Full article

This study presents a proof-of-concept evaluation of optimized whole-cell biosensors designed for the real-time detection of crop infections. Genetically engineered luminescent bacterial strains were used to detect volatile organic compounds (VOCs) emitted by crops during spoilage. Key factors investigated include bacterial uniformity, nutrient supply, and temperature effects. The results demonstrated that lower temperatures (+4 °C) yielded higher sensor sensitivity and prolonged bacterial viability. A proof-of-concept evaluation was conducted in storage-like conditions, showing effective infection detection in potatoes. These findings underscore the potential of whole-cell-based biosensors for monitoring postharvest production in cold storage environments. Full article

Microbial biosensors are bioanalytical devices that can measure the toxicity of pollutants or detect specific substances. This is the greatest advantage of microbial biosensors which use whole cells of microorganisms as powerful tools for measuring integral parameters of environmental pollution. This review explores the core principles of microbial biosensors including biofuel devices, emphasizing their capacity to evaluate biochemical oxygen demand (BOD), toxicity, heavy metals, surfactants, phenols, pesticides, inorganic pollutants, and microbiological contamination. However, practical challenges, such as sensitivity to environmental factors like pH, salinity, and the presence of competing substances, continue to hinder their broader application and long-term stability. The performance of these biosensors is closely tied to both technological advancement and the scientific understanding of biological systems, which influence data interpretation and device optimization. The review further examines cutting-edge developments, including the integration of electroactive biofilms with nanomaterials, molecular biology techniques, and artificial intelligence, all of which significantly enhance biosensor functionality and analytical accuracy. Commercial implementations and improvement strategies are also discussed, providing a comprehensive overview of the state-of-the-art in this field. Overall, this work consolidates recent progress and identifies both the potential and limitations of microbial biosensors, offering valuable insights into their future development for environmental monitoring. Full article

Dengue is a neglected disease mainly affecting tropical and subtropical countries. The diagnosis of dengue fever is still a problem since most of it is made from whole or recombinant DENV proteins, which present cross-reactions with other members of the Flavivirus family. Therefore, there is still a huge demand for new diagnostic methods that provide rapid, low-cost, easy-to-use confirmation. Thus, in this study, we developed an affordable electrochemical biosensor for rapidly detecting immunoglobulin G (IgG) serological antibodies in the sera of DENV-infected patients. An identified linear B-cell epitope (DENV/18) specific for DENV 1–4 serotypes recognized by IgG in patient sera was selected as a target molecule after a microarray of peptides using the SPOT-synthesis methodology. After chemical synthesis, the DENV/18-peptide was immobilized on the surface of the working electrode of a commercially available screen-printed gold electrode (SPGE). The capture of DENV-specific IgG allowed for the formation of an immunocomplex that was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a potassium ferrocyanide/ferricyanide ([Fe(CN)₆]^3−/4−) electrochemical probe. An evaluation of the biosensor’s performance showed a detection limit of 100 µg mL⁻¹ for the synthetic peptides (DENV/18) and 1.21 ng mL⁻¹ in CV and 0.43 ng mL⁻¹ in DPV for human serum, with a sensitivity of 7.21 µA in CV and 8.79 µA in DPV. The differentiation of infected and uninfected individuals was possible even at a high dilution factor that reduced the required sample volumes to a few microliters. The final device proved suitable for diagnosing DENV by analyzing real serum samples, and the results showed good agreement with molecular biology diagnostics. The flexibility to conjugate other antigenic peptides to SPEs suggests that this technology could be rapidly adapted to diagnose other pathogens. Full article

This review attempts to summarize my three decades-long involvement in, and contribution to, the design, construction and testing of bioluminescent microbial sensor strains (bioreporters). With the understanding that such a document cannot be completely free of bias, the review focuses on studies from my own lab only, with almost no coverage of the parallel progress made by others in similar fields. This admittedly subjective approach by no way detracts from the achievements of countless excellent researchers who are not mentioned here, and whose contributions to the field are at least as important as that of my own. The review covers basic aspects of microbial sensor design, and then progresses to describe approaches to performance improvement of sensor strains aimed at the detection of either specific chemicals, groups of chemicals sharing similar characteristics, or global effects, such as toxicity and genotoxicity. The need for integration of live sensor cells into a compatible hardware platform is highlighted, as is the importance of long-term maintenance of the cells’ viability and activity. The use of multi-member sensors’ panels is presented as a means for enhancing the detection spectrum and sample “fingerprinting”, along with a list of different purposes to which such sensors have been put to use. Full article

There is currently a critical need for understanding how the response and activity of whole-cell bacterial reporters positioned in a complex biological or environmental matrix are impacted by the physicochemical properties of their micro-environment. Accordingly, a comprehensive analysis of the bioluminescence response of Cd(II)-inducible PzntA-luxCDABE Escherichia coli biosensors embedded in silica-based hydrogels is reported to decipher how metal bioavailability, cell photoactivity and ensuing light bioproduction are impacted by the hydrogel environment and the associated matrix effects. The analysis includes the account of (i) Cd speciation and accumulation in the host hydrogels, in connection with their reactivity and electrostatic properties, and (ii) the reduced bioavailability of resources for the biosensors confined (deep) inside the hydrogels. The measurements of the bioluminescence response of the Cd(II) inducible-lux biosensors in both hydrogels and free-floating cell suspensions are completed by those of the constitutive rrnB P1-luxCDABE E. coli so as to probe cell metabolic activity in these two situations. The approach contributes to unraveling the connections between the electrostatic hydrogel charge, the nutrient/metal bioavailabilities and the resulting Cd-triggered bioluminescence output. Biosensors are hosted in hydrogels with thickness varying between 0 mm (the free-floating cell situation) and 1.6 mm, and are exposed to total Cd concentrations from 0 to 400 nM. The partitioning of bioavailable metals at the hydrogel/solution interface following intertwined metal speciation, diffusion and Boltzmann electrostatic accumulation is addressed by stripping chronopotentiometry. In turn, we detail how the bioluminescence maxima generated by the Cd-responsive cells under all tested Cd concentration and hydrogel thickness conditions collapse remarkably well on a single plot featuring the dependence of bioluminescence on free Cd concentration at the individual cell level. Overall, the construction of this master curve integrates the contributions of key and often overlooked processes that govern the bioavailability properties of metals in 3D matrices. Accordingly, the work opens perspectives for quantitative and mechanistic monitoring of metals by biosensors in environmental systems like biofilms or sediments. Full article

Multiple antibiotic resistance regulators (MarRs) control the transcription of genes in the mar operon of Escherichia coli in the presence of salicylic acid (SA). The interaction with SA induces conformational changes in the MarR released from the promoter of the mar operon, turning on transcription. We constructed an SA-specific E. coli cell-based biosensor by fusing the promoter of the mar operon (P_marO) and the gene that encodes an enhanced green fluorescent protein (egfp). Because SA and aspirin are structurally similar, a biosensor for monitoring aspirin can be obtained by genetically engineering MarR to be aspirin (ASP)-responsive. To shift the selectivity of MarR toward ASP, we changed the residues around the ligand-binding sites by site-directed mutagenesis. We examined the effects of genetic engineering on MarR by introducing MarRs with P_marO-egfp into E. coli. Among the tested mutants, MarR T72A improved the ASP responses by approximately 3 times compared to the wild-type MarR, while still showing an SA response. Although the MarR T72A biosensor exhibited mutual interference between SA and ASP, it accurately determined the ASP concentration in spiked water and medicine samples with over 90% accuracy. While the ASP biosensors still require improvement, our results provide valuable insights for developing E. coli cell-based biosensors for ASP and transcription factor-based biosensors in general. Full article

We present a generic quantitative chemical sensing methodology for assessing the concentration of a target material (TM) in an aqueous solution by using bioluminescent microbial bioreporters as the core sensing elements. Such bioreporters, genetically engineered to respond to the presence of a TM in their microenvironment by emitting bioluminescence, have previously been mostly designed to report the presence or absence of the TM in the sample. We extend this methodology to also assess the TM concentration, by exploiting the dose-dependency of the TM-induced luminescence. To overcome luminescence intensity variations due to bacterial batch differences and the ambient temperature, simultaneous measurements were carried out on sample solutions containing known concentrations of the TM. A “standard ratio” parameter, defined as the ratio between the two measurements, is shown to be independent of the bacterial batch and the temperature, and hence provides the conceptual basis for a generic quantitative chemical sensing methodology. Assessment of 2,4-dinitrotoluene (DNT) concentration in solutions is demonstrated with an accuracy of 2.5% over a wide dynamic range. Full article

The rapid and sensitive detection of bacterial contaminants using low-cost and portable point-of-need (PoN) biosensors has gained significant interest in water quality monitoring. Cell-imprinted polymers (CIPs) are emerging as effective and inexpensive materials for bacterial detection as they provide specific binding sites designed to capture whole bacterial cells, especially when integrated into PoN microfluidic devices. However, improving the sensitivity and detection limits of these sensors remains challenging. In this study, we integrated CIP-functionalized stainless steel microwires (CIP-MWs) into a microfluidic device for the impedimetric detection of E. coli bacteria. The sensor featured two parallel microchannels with three-electrode configurations that allowed simultaneous control and electrochemical impedance spectroscopy (EIS) measurements. A CIP-MW and a non-imprinted polymer (NIP)-MW suspended perpendicular to the microchannels served as the working electrodes in the test and control channels, respectively. Electrochemical spectra were fitted with equivalent electrical circuits, and the charge transfer resistances of both cells were measured before and after incubation with target bacteria. The charge transfer resistance of the CIP-MWs after 30 min of incubation with bacteria was increased. By normalizing the change in charge transfer resistance and analyzing the dose–response curve for bacterial concentrations ranging from 0 to 10⁷ CFU/mL, we determined the limits of detection and quantification as 2 × 10² CFU/mL and 1.4 × 10⁴ CFU/mL, respectively. The sensor demonstrated a dynamic range of 10² to 10⁷ CFU/mL, where bacterial counts were statistically distinguishable. The proposed sensor offers a sensitive, cost-effective, durable, and rapid solution for on-site identification of waterborne pathogens. Full article

Microbioreactors increase information output in biopharmaceutical screening applications because they can be operated in parallel without consuming large quantities of the pharmaceutical formulations being tested. A capillary wave microbioreactor (cwMBR) has recently been reported, allowing cost-efficient parallelization in an array that can be activated for mixing as a whole. Although impedance spectroscopy can directly distinguish between dead and viable cells, the monitoring of cells in suspension within bioreactors is challenging because the signal is influenced by the potentially varying properties of the culture medium. In order to address this challenge, an impedance sensor consisting of two sets of microelectrodes in a cwMBR is presented. Only one set of electrodes was covered by a two-photon cross-linked hydrogel to become insensitive to the influence of cells while remaining sensitive to the culture medium. With this impedance sensor, the biomass of Saccharomyces cerevisiae could be measured in a range from 1 to 20 g L⁻¹. In addition, the sensor can compensate for a change in the conductivity of the suspension of 5 to 15 mS cm⁻¹. Moreover, the two-photon cross-linking of hydroxyethyl starch methacrylate hydrogel, which has been studied in detail, recommends itself for even much broader sensing applications in miniaturized bioreactors and biosensors. Full article

The ability of aquatic mesofauna representatives involved in trophic chains to sorb and accumulate toxicants is important for understanding the functioning of aquatic ecosystems and for fishing industry. This study investigated the capacity of marine amphipod Gammarus oceanicus and freshwater amphipods Eulimnogammarus vittatus and Gammarus lacustris to absorb the DNA-alkylating agent methyl methanesulfonate (MMS). The presence of alkylating agents in the environment and in the tissues of the amphipods was determined using whole-cell lux-biosensor Escherichia coli MG1655 pAlkA-lux, in which the luxCDABE genes from Photorhabdus luminescens, enabling the luminescence of the cell culture, are controlled by the P_alkA promoter of DNA glycosylase. It was shown that within one day of incubation in water containing MMS at a concentration above 10 μM, the amphipods absorbed the toxicant and their tissues produce more alkylation damage to biosensor cells than the surrounding water. Concentrations of MMS above 1 mM in the environment caused the death of the amphipods before the toxicant could be significantly concentrated in their tissues. The sensitivity and the capacity to absorb MMS were found to be approximately the same for the marine amphipod G. oceanicus and the freshwater amphipods E. vittatus and G. lacustris. Full article

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that remains a prevalent clinical and environmental challenge. Quorum-sensing (QS) molecules are effective biomarkers in pinpointing the presence of P. aeruginosa. This study aimed to develop a convenient-to-use, whole-cell biosensor using P. aeruginosa reporters individually encapsulated within alginate-poly-L-lysine (alginate-PLL) microbeads to specifically detect the presence of bacterial autoinducers. The PLL-reinforced microbeads were prepared using a two-step method involving ionic cross-linking and subsequent coating with thin layers of PLL. The alginate-PLL beads showed good stability in the presence of a known cation scavenger (sodium citrate), which typically limits the widespread applications of calcium alginate. In media containing synthetic autoinducers—such as N-(3-oxo dodecanoyl) homoserine lactone (3-oxo-C₁₂-HSL) and N-butanoyl-L-homoserine lactone (C₄-HSL), or the cell-free supernatants of planktonic or the flow-cell biofilm effluent of wild P. aeruginosa (PAO1)—the encapsulated bacteria enabled a dose-dependent detection of the presence of these QS molecules. The prepared bioreporter beads remained stable during prolonged storage at 4 and −80 °C and were ready for on-the-spot sensing without the need for recovery. The proof-of-concept, optical fiber-based, and whole-cell biosensor developed here demonstrates the practicality of the encapsulated bioreporter for bacterial detection based on specific QS molecules. Full article

Hepatocellular carcinoma is currently the most common malignancy of the liver. It typically occurs due to a series of oncogenic mutations that lead to aberrant cell replication. Most commonly, hepatocellular carcinoma (HCC) occurs as a result of pre-occurring liver diseases, such as hepatitis and cirrhosis. Given its aggressive nature and poor prognosis, the early screening and diagnosis of HCC are crucial. However, due to its plethora of underlying risk factors and pathophysiologies, patient presentation often varies in the early stages, with many patients presenting with few, if any, specific symptoms in the early stages. Conventionally, screening and diagnosis are performed through radiological examination, with diagnosis confirmed by biopsy. Imaging modalities tend to be limited by their requirement of large, expensive equipment; time-consuming operation; and a lack of accurate diagnosis, whereas a biopsy’s invasive nature makes it unappealing for repetitive use. Recently, biosensors have gained attention for their potential to detect numerous conditions rapidly, cheaply, accurately, and without complex equipment and training. Through their sensing platforms, they aim to detect various biomarkers, such as nucleic acids, proteins, and even whole cells extracted by a liquid biopsy. Numerous biosensors have been developed that may detect HCC in its early stages. We discuss the recent updates in biosensing technology, highlighting its competitive potential compared to conventional methodology and its prospects as a tool for screening and diagnosis. Full article

Iron is a key micronutrient essential for various essential biological processes. As a consequence, alteration in iron concentration in seawater can deeply influence marine biodiversity. In polar marine environments, where environmental conditions are characterized by low temperatures, the role of iron becomes particularly significant. While iron limitation can negatively influence primary production and nutrient cycling, excessive iron concentrations can lead to harmful algal blooms and oxygen depletion. Furthermore, the growth of certain phytoplankton species can be increased in high-iron-content environments, resulting in altered balance in the marine food web and reduced biodiversity. Although many chemical/physical methods are established for inorganic iron quantification, the determination of the bio-available iron in seawater samples is more suitably carried out using marine microorganisms as biosensors. Despite existing challenges, whole-cell biosensors offer other advantages, such as real-time detection, cost-effectiveness, and ease of manipulation, making them promising tools for monitoring environmental iron levels in polar marine ecosystems. In this review, we discuss fundamental biosensor designs and assemblies, arranging host features, transcription factors, reporter proteins, and detection methods. The progress in the genetic manipulation of iron-responsive regulatory and reporter modules is also addressed to the optimization of the biosensor performance, focusing on the improvement of sensitivity and specificity. Full article