Whole-cell Biosensors Research Articles

Designing a whole-cell biosensor applicable for S-adenosyl-l-methionine-dependent methyltransferases

Designing a whole-cell biosensor applicable for S-adenosyl-l-methionine-dependent methyltransferases

Rapid, Sensitive, and Specific Microbial Whole-Cell Biosensor for the Detection of Histamine: A Potential Food Toxin.

Histamine is a biogenic amine; its level indicates food quality, as elevated levels cause food poisoning. Therefore, monitoring food at each step during processing until it reaches the consumer is crucial, but current techniques are complicated and time-consuming. Here, we designed a Pseudomonas putida whole-cell biosensor using a histamine-responsive genetic element expressing a fluorescent protein in the presence of the cognate target. We improved the performance of the proposed biosensor by optimizing the chassis, genetic regulatory element, and reporter gene. A sensitive and rapid biosensor variant was obtained with a limit of detection (LOD) of 0.39 ppm, manifesting a linear response (R2 = 0.98) from 0.28 to 18 ppm in 90 min. The biosensor showed minimal cross-reactivity with other biogenic amines and amino acids prevalent in food, making it highly specific. The biosensor effectively quantified histamine in spiked fish, prawn, and wine samples with a satisfactory recovery. Additionally, a colorimetric sensor variant PAlacZ was developed enabling histamine quantification in seafood via a smartphone application, with an LODgray of 0.23 ppm, exhibiting a linear response from 0 to 2.24 ppm. Overall, this study reports an efficient, specific, and highly sensitive biosensor with strong potential for the on-site detection of histamine, ensuring food safety.

Decoding wheat contamination through self-assembled whole-cell biosensor combined with linear and non-linear machine learning algorithms

The contamination of mycotoxins is a serious problem around the world. It has detrimental effects on human beings and leads to tremendous economic loss. It is essential to develop a rapid and non-destructive method for contamination recognition particularly for early alarm. In this study, the whole-cell biosensor array was constructed and employed for rapid recognition of wheat contamination by combining with machine learning algorithms. Seven key VOCs were explored through univariate coupling to multivariate analysis of orthogonal partial least squares-discrimination analysis (OPLS-DA) models. The promoters of dnaK, katG, oxyR, soxS obtained from the stress-responsive of key VOCs were fused to the bacterial operon and fabricated on the whole-cell biosensor. The constructed whole-cell biosensor array was consisted with four kinds of sensors and 18 sensor unit. The bioluminescent intensity combined with linear machine learning algorithm of partial least squares discriminant analysis (PLS-DA) and non-linear algorithms of back propagating artificial neural network (BP-ANN) and least square support vector machine (LS-SVM) were employed to establish discrimination models for mold contamination especially for early warning. The Monte-Carlo strategy was performed to generate thirty subsets for modeling to give more reliable results. As a result, the whole-cell biosensor combined with non-linear algorithm of LS-SVM was practicable for detecting mold identification for wheat early-warning with the accuracy of 97.24%. Additionally, this study provides practical and effective methods not only for wheat quality guarantee and supervision but also for other foodstuffs.

Visual signal transduction for environmental stewardship: A novel biosensing approach to identify and quantify chlorpyrifos-related residues in aquatic environments

The pervasive presence of organophosphate pesticides (OPs), such as chlorpyrifos (CPF), in aquatic ecosystems underscores the urgent need for sensitive and reliable detection methods to safeguard environmental and public health. This study addressed the critical need for a novel biosensor capable of detecting CPF and its toxic metabolite, 3,5,6-trichloro-2-pyridinol (TCP), with high sensitivity and selectivity, suitable for field applications in environmental monitoring. The study engineered a whole-cell biosensor based on E. coli strains that utilize the ChpR transcriptional regulator and the vioABCE gene cluster, providing a distinct visual and colorimetric response to CPF and TCP. The biosensor's performance was optimized and evaluated across various water matrices, including freshwater, seawater, and soil leachate. The biosensor demonstrated high sensitivity with a broad linear detection range, achieving limits of detection (LODs) at 0.8 μM for CPF and 7.813 nM for TCP. The linear regression concentration ranges were 1.6–12.5 μM for CPF and 15.6–125 nM for TCP, aligning with environmental standard limits and ensuring the biosensor's effectiveness in real-world scenarios. This innovative biosensing approach offers a robust, user-friendly tool for on-site environmental monitoring, significantly mitigating OPs contamination and advancing current detection technologies to meet environmental protection standards.

Development of CadR-based cadmium whole cell biosensor for visual detection of environmental Cd2+

BackgroundAs a threat to human health and public health, cadmium (Cd) pollution has received widespread social concern. Our previously constructed CadR-based bacterial whole cell biosensor (WCB) epCadR5 showed high sensitivity and specificity in cadmium detection. However, the application of the sensor is still hindered by the need for laboratory equipment to read the fluorescence signal output. In this study, we aimed to optimizing the sensor to make it available for visual detection of environmental cadmium and simplify the detection process to advance practical application of the sensor. ResultsBy replacing the constitutive promoter with J110, the fluorescence signal output of the sensor was significantly increased and the fluorescence leakage was decreased. In addition, the fluorescence signal output of green fluorescence protein (GFP) was enhanced by the addition of a 5′ untranslated region (5′-UTR) mlcR10. The fluorescence signal output of the WCB is sufficiently robust to be visible and distinguishable to the naked eye, which is of paramount importance for visual detection. The sensor readout can be conveniently recorded by mobile phone camera and quantified. For ease of on-site application, the WCB's visual detection procedures and conditions were further optimized and simplified. The WCB demonstrated good linearity and detection limit (1.81 μg/L) for visual detection of Cd2+ without the assistance of bulky laboratory equipment. For the detection of real environmental samples, the WCB visual detection results were close to those of WCB-flow cytometry (FACS) and graphite furnace atomic absorption spectroscopy (GFAAS). SignificanceIn this work, we developed an easy-to-use, on-site and visual detection biosensor for monitoring environmental Cd2+. It will advance the utilization of cadmium WCBs in practical settings. The optimization and simplification strategy in the study also provide new insights into the visualization of other bacterial biosensors, and will advance the practical application of WCBs.

A new yeast-based bioreporter for simple, sensitive, and cost-effective detection of dioxin-like compounds

Dioxin-like compounds (DLCs) are environmental xenobiotics that can activate the aryl hydrocarbon receptors (AhR), thereby imposing a significant threat to human health through biomagnifications processes. In this study, a dioxin-activated nano-luminescent Saccharomyces cerevisiae bioreporter, called DnaSc, was developed for simple and rapid detection of DLCs and AhR agonists. The bioreporter used nano-luciferase (NLuc) as a signal generator to emit bioluminescent signals in response to DLCs without cell lysis. Through optimizing AhR nuclear translocator (ARNT) expression and engineering the AhR, the yeast-based bioassay exhibited a detection limit of 10 fM for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within 6 h, making it the most sensitive whole-cell biosensor reported to date. Furthermore, the detection capacity of the dioxin-activated nano-luminescent S. cerevisiae bioreporter (DnaSc) bioassay for DLCs and AhR agonists was characterized. In summary, the yeast-based bioreporter developed in this study provided a simple, sensitive, and cost-effective method for DLCs detection.

Mechanistic insights into the orthogonal functionality of an AHL-mediated quorum-sensing circuit in Yersinia pseudotuberculosis

YpsR, a pivotal regulatory protein in the quorum-sensing (QS) of Yersinia pseudotuberculosis(Y. pstb), is essential for molecular signaling, yet its molecular mechanisms remain poorly understood. Herein, this study systematically investigates the interactions between YpsR and acyl-homoserine lactones (AHLs), shedding light on the selective mechanism of YpsR to various AHL molecules. Using molecular docking and surface plasmon resonance (SPR) analysis, we confirmed YpsR’s binding affinities, with the strongest observed for 3OC6-HSL, which notably inhibited Y. pstb growth. Additionally, we engineered a whole-cell biosensor based on YpsR-AHL interaction, which exhibited sensitivity to the signal molecule 3OC6-HSL produced by Y. pstb. Furthermore, key YpsR residues (S32, Y50, W54, D67) involved in AHL binding were identified and validated. Overall, this research elucidates the mechanisms of QS signal recognition in Y. pstb, providing valuable insights that support the development of diagnostic tools for detecting Y. pstb infections.

Disentangling the Regulatory Response of Agrobacterium tumefaciens CHLDO to Glyphosate for Engineering Whole-Cell Phosphonate Biosensors.

Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25-50 μM range in various samples, including soil and water.

Antimony and arsenic detection: review on electrochemical biosensors and their applications

ABSTRACT Arsenic (As) and antimony (Sb) contamination poses significant health risks, manifesting in a spectrum of ailments such as cardiovascular, respiratory, and skin diseases and the development of cancer. When toxic elements enter the body through different exposure routes, they can cause serious health issues. This highlights the critical need to reduce their presence in the environment to protect public health. When detecting As and Sb in different samples, researchers prefer to use whole-cell and optical biosensors rather than electrochemical (EC) biosensors for reliable detection. Utilizing EC biosensors can significantly enhance the detection thresholds and accuracy of monitoring environmental changes. It is particularly well suited for applications requiring high sensitivity, rapid detection, portability, and cost-effectiveness. For fast onsite environmental monitoring, its portability and quick reaction times are essential. The augmentation of EC biosensors via AI integration has the potential to transform pollution detection, healthcare diagnostics, and food safety monitoring, in addition to enhancing their sensitivity and accessibility. This review highlights the potential benefits of EC biosensors and their diverse applications in environmental monitoring. It critically evaluates strategies aimed at enhancing the efficiency of environmental monitoring processes.

Advances in microbial whole-cell sensors for the detection of aromatic compounds

The inherent stability and recalcitrance of benzene ring structures render aromatic compounds a major ecological concern and a substantial risk to human health. Hence, developing a facile and efficacious detection technique for aromatic compounds is essential. As our comprehension of aromatic compound characteristics deepens, microbial cell-based biosensors have emerged as increasingly popular tools in the detection of aromatic compounds. This article introduces the operational principles of microbial whole-cell biosensors and elucidates the construction techniques and applications of electroactive biofilm-based microbial whole-cell sensors, transcription factor-based microbial whole-cell sensors, and degradation gene promoter-dependent microbial whole-cell sensors in the detection of aromatic compounds. In addition, we review the methodologies for improving the performance of microbial whole-cell sensors based on surface display, logic gate construction, genetic circuit modification, and quorum sensing signal amplification.

Utilizing a high-throughput visualization screening technology to develop a genetically encoded biosensor for monitoring 5-aminolevulinic acid production in engineered Escherichia coli

5-Aminolevulinic acid (5-ALA) is a non-protein amino acid widely used in agriculture, animal husbandry and medicine. Currently, microbial cell factories are a promising production pathway, but the lack of high-throughput fermentation strain screening tools often hinders the exploration of engineering strategies to increase cell factory yields. Here, mutant AC103-3H was screened from libraries of saturating mutants after response-specific engineering of the transcription factor AsnC of L-asparagine (Asn). Based on mutant AC103-3H, a whole-cell biosensor EAC103-3H with a specific response to 5-ALA was constructed, which has a linear dynamic detection range of 1–12 mM and a detection limit of 0.094 mM, and can be used for in situ screening of potential high-producing 5-ALA strains. With its support, overexpression of the C5 pathway genes using promoter engineering assistance resulted in a 4.78-fold enhancement of 5-ALA production in the engineered E. coli. This study provides an efficient strain screening tool for exploring approaches to improve the 5-ALA productivity of engineered strains.

An Inducible Whole-Cell Biosensor for Detection of Formate Ions

Ten strains of the yeast Yarrowia lipolytica were constructed, the genomes of which contain the hrGFP gene under regulation of the formate dehydrogenase promoters. The resulting strains can act as whole-cell biosensors for the detection of formate ions in various media. By visual assessment of the biomass fluorescence, we selected the three most promising yeast strains. The main biosensor characteristics (threshold sensitivity, amplitude, and response time) of the selected strains were measured. As a result, in terms of characteristics, the B26 strain was recognized as the most suitable for the detection of formate ions. A carbon source for the nutrient medium that does not reduce the activation of the biosensor was selected. Furthermore, we showed that unlike formate and formaldehyde, methanol practically does not induce the biosensor fluorescence response.

Detection of Short-Chain Chlorinated Aliphatic Hydrocarbons through an Engineered Biosensor with Tailored Ligand Specificity.

Short-chain chlorinated aliphatic hydrocarbons (SCAHs), commonly used as industrial reagents and solvents, pose a significant threat to ecosystems and human health as they infiltrate aquatic environments due to extensive usage and accidental spills. Whole-cell biosensors have emerged as cost-effective, rapid, and real-time analytical tools for environmental monitoring and remediation. While the broad ligand specificity of transcriptional factors (TFs) often prohibits the application of such biosensors. Herein, we exploited a semirational transition ligand approach in conjunction with a positive/negative fluorescence-activated cell sorting (FACS) strategy to develop a biosensor based on the TF AlkS, which is highly specific for SCAHs. Furthermore, through promoter-directed evolution, the performance of the biosensor was further enhanced. Mutation in the -10 region of constitutive promoter PalkS resulted in reduced AlkS leakage expression, while mutation in the -10 region of inducible promoter PalkB increased its accessibility to the AlkS-SCAHs complex. This led to an 89% reduction in background fluorescence leakage of the optimized biosensor, M2-463, further enhancing its response to SCAHs. The optimized biosensor was highly sensitive and exhibited a broader dynamic response range with a 150-fold increase in fluorescence output after 1 h of induction. The detection limit (LOD) reached 0.03 ppm, and the average recovery rate of SCAHs in actual water samples ranged from 95.87 to 101.20%. The accuracy and precision of the proposed biosensor were validated using gas chromatography-mass spectrometry (GC-MS), demonstrating the promising application for SCAH detection in an actual environment sample.

Metabolic engineering-enabled dual-color biosensor for discriminative and sensitive detection of toxic lead and mercury in environmental waters

The ubiquity of toxic heavy metals, particularly lead (Pb) and mercury (Hg), in environmental waters severely threatens ecosystems and human health. We developed a metabolic engineering-based dual-color bacterial biosensor for the sensitive and discriminative detection of Pb(II) and Hg(II) at nanomolar levels. The biosensor leverages the synthetic biology approach to integrate two pigments, prodeoxyviolacein (PDV) and deoxyviolacein (DV), into a single construct that responds distinctly to Pb(II) and Hg(II), respectively. The engineered E. coli biosensor exhibits a detection range of 0.732–3000 nM for Pb(II) and 0.183–750 nM for Hg(II), with low detection limits of 0.732 nM for Pb(II) and 0.183 nM for Hg(II). The biosensor demonstrated high sensitivity, selectivity, and accuracy with a recovery rate close to 1 in freshwater and seawater matrices. Notably, the biosensor's visual readout allows for rapid, equipment-free assessment. It is a practical tool for real-world monitoring of heavy metal pollution, especially in remote or resource-limited settings. This study underscores the potential of whole-cell biosensors as a sustainable and effective alternative to traditional heavy metal detection methods.

Yeast Surface-Displayed Quenchbody as a Novel Whole-Cell Biosensor for One-Step Detection of Influenza A (H1N1) Virus.

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 μg/mL, with a half-maximal concentration of 2.60 μg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 104 to 1.5 × 107 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

In vivo detection of endogenous toxic phenolic compounds of intestine

Phenol and p-cresol are two common toxic small molecules related to various diseases. Existing reports confirmed that high L-tyrosine in the daily diet can increase the concentration of phenolic compounds in blood and urine. L-tyrosine is a common component of protein-rich foods. Some anaerobic bacteria in the gut can convert non-toxic l-tyrosine into these two toxic phenolic compounds, phenol and p-cresol. Existing methods have been constructed for measuring the concentration of phenolic compound in feces. However, there is still a lack of direct visual evidence to measure the phenolic compounds in the intestine. In this study, we aimed to construct a whole-cell biosensor for phenolic compounds detection based on the dmpR, the regulator from the phenol metabolism cluster. The commensal bacterium Citrobacter amalonaticus PS01 was selected and used as the chassis. Compared with the biosensor based on ECN1917, the biosensor PS01[dmpR] could better implant into the mouse gut through gavage and showed a higher sensitive to phenolic compound. And the concentration of phenolic compounds in the intestines could be observed with the help of in vivo imaging system using PS01[dmpR]. This paper demonstrated endogenous phenol synthesis in the gut and the strategy of using commensal bacteria to construct whole-cell biosensors for detecting small molecule compounds in the intestines.

Genetically engineered bacterial biofilm materials enhances portable whole cell sensing

In recent years, whole-cell biosensors (WCBs) have emerged as a potent approach for environmental monitoring and on-site analyte detection. These biosensors harness the biological apparatus of microorganisms to identify specific analytes, offering advantages in sensitivity, specificity, and real-time monitoring capabilities. A critical hurdle in biosensor development lies in ensuring the robust attachment of cells to surfaces, a crucial step for practical utility. In this study, we present a comprehensive approach to tackle this challenge via engineering Escherichia coli cells for immobilization on paper through the Curli biofilm pathway. Furthermore, incorporating a cellulose-binding peptide domain to the CsgA biofilm protein enhances cell adhesion to paper surfaces, consequently boosting biosensor efficacy. To demonstrate the versatility of this platform, we developed a WCB for copper, optimized to exhibit a discernible response, even with the naked eye. To confirm its suitability for practical field use, we characterized our copper sensor under various environmental conditions—temperature, salinity, and pH—to mimic real-world scenarios. The biosensor-equipped paper discs can be freeze-dried for deployment in on-site applications, providing a practical method for long-term storage without loss of sensitivity paper discs demonstrate sustained functionality and viability even after months of storage with 5 μM limit of detection for copper with visible-to-naked-eye signal levels. Biofilm-mediated surface attachment and analyte sensing can be independently engineered, allowing for flexible utilization of this platform as required. With the implementation of copper sensing as a proof-of-concept study, we underscore the potential of WCBs as a promising avenue for the on-site detection of a multitude of analytes.

Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules.

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.

Expanding salivary biomarker detection by creating a synthetic neuraminic acid sensor via chimeragenesis.

Accurate and timely diagnosis of oral squamous cell carcinoma (OSCC) is crucial in preventing its progression to advanced stages with a poor prognosis. As such, the construction of sensors capable of detecting previously established disease biomarkers for the early and non-invasive diagnosis of this and many other conditions has enormous therapeutic potential. In this work, we apply synthetic biology techniques for the development of a whole-cell biosensor (WCB) that leverages the physiology of engineered bacteria in vivo to promote the expression of an observable effector upon detection of a soluble molecule. To this end, we have constructed a bacterial strain expressing a novel chimeric transcription factor (Sphnx) for the detection of N-acetylneuraminic acid (Neu5Ac), a salivary biomolecule correlated with the onset of OSCC. This WCB serves as the proof-of-concept of a platform that can eventually be applied to clinical screening panels for a multitude of oral and systemic medical conditions whose biomarkers are present in saliva.

Advances in the Development of Bacterial Bioluminescence Imaging.

Bioluminescence imaging (BLI) is a powerful method for visualizing biological processes and tracking cells. Engineered bioluminescent bacteria that utilize luciferase-catalyzed biochemical reactions to generate luminescence have become useful analytical tools for in vitro and in vivo bacterial imaging. Accordingly, this review initially introduces the development of engineered bioluminescent bacteria that use different luciferase-luciferin pairs as analytical tools and their applications for in vivo BLI, including real-time bacterial tracking of infection, probiotic investigation, tumor-targeted therapy, and drug screening. Applications of engineered bioluminescent bacteria as whole-cell biosensors for sensing biological changes in vitro and in vivo are then discussed. Finally, we review the optimizations and future directions of bioluminescent bacteria for imaging. This review aims to provide fundamental insights into bacterial BLI and highlight the potential development of this technique in the future.