Cell-based Biosensors Research Articles

New Insight into Plasmid-Driven T7 RNA Polymerase in Escherichia coli and Use as a Genetic Amplifier for a Biosensor.

T7 RNA polymerase (T7RNAP) and T7 promoter are powerful genetic components, thus a plasmid-driven T7 (PDT7) genetic circuit could be broadly applied for synthetic biology. However, the limited knowledge of the toxicity and instability of such a system still restricts its application. Herein, we constructed 16 constitutive genetic circuts of PDT7 and investigated the orthogonal effects in toxicity and instability. The T7 toxicity was elucidated from the construction processes and cell growth characterization, showing the importance of optimal orthogonality for PDT7. Besides, a protein analysis was performed to validate how the T7 system affected cell metabolism and led to the instability. The application of constitutive PDT7 in functional protein expressions, including carbonic anhydrase, lysine decarboxylase, and 5-ALA synthetase was demonstrated. Furthermore, PDT7 working as a genetic amplifier had been designed for E. coli cell-based biosensors, which illustrated the opportunities in the future of PDT7 used in synthetic biology.

Metallic Nanoparticle-Based Optical Cell Chip for Nondestructive Monitoring of Intra/Extracellular Signals.

The biosensing platform is noteworthy for high sensitivity and precise detection of target analytes, which are related to the status of cells or specific diseases. The modification of the transducers with metallic nanoparticles (MNPs) has attracted attention owing to excellent features such as improved sensitivity and selectivity. Moreover, the incorporation of MNPs into biosensing systems may increase the speed and the capability of the biosensors. In this review, we introduce the current progress of the developed cell-based biosensors, cell chip, based on the unique physiochemical features of MNPs. Mainly, we focus on optical intra/extracellular biosensing methods, including fluorescence, localized surface plasmon resonance (LSPR), and surface-enhanced Raman spectroscopy (SERS) based on the coupling of MNPs. We believe that the topics discussed here are useful and able to provide a guideline in the development of new MNP-based cell chip platforms for pharmaceutical applications such as drug screening and toxicological tests in the near future.

A Rapid Assay to Assess Nitrification Inhibition Using a Panel of Bacterial Strains and Partial Least Squares Modeling.

As a proof of concept, a rapid assay consisting of a cell-based biosensor (CBB) panel of pure bacterial strains, a fluorescent dye, and partial least squares (PLS) modeling was developed to assess the nitrification inhibition potential of industrial wastewater (WW) samples. The current standard method used to assess the nitrification inhibition potential is the specific nitrification rate (SNR) batch test, which requires approximately 4 h to complete under the watch of an experienced operator. In this study, we exposed the CBB panel of seven bacterial strains (nitrifying and non-nitrifying) to 28 different industrial WW samples and then probed both the membrane integrity and cellular activity using a commercially available "live/dead" fluorescent dye. The CBB panel response acts as a surrogate measurement for the performance of nitrification. Of the seven strains, four (Nitrospira, Escherichia coli, Bacillus subtilis, Bacillus cereus) were identified via the modeling technique to be the most significant contributors for predicting the nitrification inhibition potential. The key outcome from this work is that the CBB panel fluorescence data (collected in approximately 10 min) can accurately predict the outcome of an SNR batch test (that takes 4 h) when performed with the same WW samples and has a strong potential to approximate the chemical composition of these WW samples using PLS modeling. Overall, this is a powerful technique that can be used for point-of-use detection of nitrification inhibition.

Development of odor discrimination method with fluorescent image of a cell-based biosensor

Development of odor discrimination method with fluorescent image of a cell-based biosensor

Modulation of Cellular Reactivity for Enhanced Cell-Based Biosensing.

Cell-based sensing platforms provide functional information on cellular effects of bioactive or toxic compounds in a sample. Current challenges concern the rather extended length of the assays as well as their limited reproducibility and sensitivity. We present a biosensing method capable of appraising, on a short time scale and with exquisite sensitivity, the occurrence and the magnitude of cellular alterations induced by low levels of a bioactive/toxic compound. Our method is based on integrating optogenetic control of non-electrogenic human cells, modified to express light sensitive protein channels, into a non-invasive electro-optical analytical platform enabling quantitative assessment of the stimulus dependent, dynamical cellular response. Our system exploits the interplay between optogenetic stimulation and time lapse fast impedance assays in boosting the platform sensitivity when exposing cells to a model exogenous stimulus, under both static and flow conditions. The proposed optogenetically modulated cell-based sensing platform is suitable for in field applications and provides a new paradigm for impedance-based sensing.

Encapsulation of enzyme by metal-organic framework for single-enzymatic biofuel cell-based self-powered biosensor

Self-powered biosensor, as a new sensing platform, utilizes biofuel cells as synchronous power source and biosensor system, with the advantages of no external power source, simple instrumentation and easy miniaturization. Meanwhile, inspired by biomimics for the encapsulation of soft tissue within protective exteriors in nature, encapsulation of biological macromolecules by metal organic-frameworks (MOFs) have been proved to be an effective strategy for enhancing the biocatalytic activity in maintaining biological functions. Herein, we successfully encapsulated laccase (LAC) in zeolitic imidazolate framework-8 (ZIF-8) and combined it with bacterial cellulose (BC)/carboxylated multi-walled carbon nanotubes (c-MWCNTs) skeleton to prepare highly flexible BC/c-MWCNTs/ZIF-8@LAC electrode. Subsequently, we ingeniously designed an highly flexible self-powered sensing platform based on biofuel-cell driven by single-enzyme, harvesting bisphenol A (BPA) as a model analyte. The maximum power density (Pmax) of single-enzymatic biofuel cell (EBFC) exhibited an excellent linear dynamic range from 0.01 to 0.4 mM with a lower detection limit of 1.95 × 10−3 mM for BPA concentrations. Therefore, the designed single-EBFC-based self-powered sensor is feasible in BPA detection and has a broad application prospect in the field of water pollutants detection.

Assessment of Cypermethrin Residues in Tobacco by a Bioelectric Recognition Assay (BERA) Neuroblastoma Cell-Based Biosensor

This study presents a bioelectric cell-based biosensor for the monitoring of the pyrethroid pesticide cypermethrin, a voltage-gated sodium channel blocker, in tobacco samples. For this purpose, neuroblastoma cells were used as biorecognition elements. The potential interference by the tobacco major alkaloid nicotine on the detection of cypermethrin was also studied. In addition, fluorescence microscopy revealed a specific pattern of neuroblastoma cell calcium efflux (Ca2+) after treatment with nicotine or cypermethrin. Finally, actual field-derived tobacco extracts were used for assessing matrix effects on the biosensor’s performance. The biosensor could detect cypermethrin in concentrations up to 1.5 μg mL−1 without being influenced by the presence of nicotine and possibly other tobacco alkaloids. Though not selective for cypermethrin, the neuroblastoma-based biosensor system appears to be a promising alternative to laborious analysis methodologies for rapid, high throughput and cost-efficient screening of this pyrethroid in tobacco samples in the near future.

New application of microbial fuel cell-based biosensor for monitoring the quality of actual potato chips’ processing wastewater

In this study, a dual-chamber microbial fuel cell (MFC) fed with actual potato chips’ processing wastewater (PCPW) was tested as a biosensor. The performance of MFC-based biosensor was evaluated in terms of the current measurement range, toxicity detection and sensitivity, and the operational stability. The results revealed that the MFC can simply be converted to an online biosensor unit to detect the harmful effect of suspended solids and acidic content in the actual PCPW on the anodic attached biofilm and the values of the generated current as well. A notable decrease in the current values was observed indicating the adverse effects of the harmful matters in the PCPW fed to the biosensor unit. The results proposed a competition between the harmful components and the favorable substrate in binding to the redox complex. An excellent fitting was obtained between the experimental and predicted results by $$ I_{{K_{m} }} $$ model with determination coefficient (R2) and mean-square-error values of 0.927 and 0.363, respectively. Additionally, a new approach was developed based on direct measurement of actual field data to replace the conventional statistical methods.

A LAPS-Based Differential Sensor for Parallelized Metabolism Monitoring of Various Bacteria.

Monitoring the cellular metabolism of bacteria in (bio)fermentation processes is crucial to control and steer them, and to prevent undesired disturbances linked to metabolically inactive microorganisms. In this context, cell-based biosensors can play an important role to improve the quality and increase the yield of such processes. This work describes the simultaneous analysis of the metabolic behavior of three different types of bacteria by means of a differential light-addressable potentiometric sensor (LAPS) set-up. The study includes Lactobacillus brevis, Corynebacterium glutamicum, and Escherichia coli, which are often applied in fermentation processes in bioreactors. Differential measurements were carried out to compensate undesirable influences such as sensor signal drift, and pH value variation during the measurements. Furthermore, calibration curves of the cellular metabolism were established as a function of the glucose concentration or cell number variation with all three model microorganisms. In this context, simultaneous (bio)sensing with the multi-organism LAPS-based set-up can open new possibilities for a cost-effective, rapid detection of the extracellular acidification of bacteria on a single sensor chip. It can be applied to evaluate the metabolic response of bacteria populations in a (bio)fermentation process, for instance, in the biogas fermentation process.

Synthetic Biology Enables Programmable Cell-Based Biosensors.

Cell‐based biosensors offer cheap, portable and simple methods of detecting molecules of interest but have yet to be truly adopted commercially. Issues with their performance and specificity initially slowed the development of cell‐based biosensors. With the development of rational approaches to tune response curves, the performance of biosensors has rapidly improved and there are now many biosensors capable of sensing with the required performance. This has stimulated an increased interest in biosensors and their commercial potential. However the reliability, long term stability and biosecurity of these sensors are still barriers to commercial application and public acceptance. Research into overcoming these issues remains active. Here we present the state‐of‐the‐art tools offered by synthetic biology to allow construction of cell‐based biosensors with customisable performance to meet the real world requirements in terms of sensitivity and dynamic range and discuss the research progress to overcome the challenges in terms of the sensor stability and biosecurity fears.

Detection of Superoxide Alterations Induced by 5-Fluorouracil on HeLa Cells with a Cell-Based Biosensor.

Background: In vitro cell culture monitoring can be used as an indicator of cellular oxidative stress for the assessment of different chemotherapy agents. Methods: A cell-based bioelectric biosensor was used to detect alterations in superoxide levels in the culture medium of HeLa cervical cancer cells after treatment with the chemotherapeutic agent 5-fluorouracil (5-FU). The cytotoxic effects of 5-fluorouracil on HeLa cells were assessed by the MTT proliferation assay, whereas oxidative damage and induction of apoptosis were measured fluorometrically by the mitochondria-targeted MitoSOX™ Red and caspase-3 activation assays, respectively. Results: The results of this study indicate that 5-FU differentially affects superoxide production and caspase-3 activation when applied in cytotoxic concentrations against HeLa cells, while superoxide accumulation is in accordance with mitochondrial superoxide levels. Our findings suggest that changes in superoxide concentration could be detected with the biosensor in a non-invasive and rapid manner, thus allowing a reliable estimation of oxidative damage due to cell apoptosis. Conclusions: These findings may be useful for facilitating future high throughput screening of different chemotherapeutic drugs with a cytotoxic principle based on free radical production.

Real-Time Monitoring of Cellular Cultures with Electrolyte-Gated Carbon Nanotube Transistors.

Cell-based biosensors constitute a fundamental tool in biotechnology, and their relevance has greatly increased in recent years as a result of a surging demand for reduced animal testing and for high-throughput and cost-effective in vitro screening platforms dedicated to environmental and biomedical diagnostics, drug development, and toxicology. In this context, electrochemical/electronic cell-based biosensors represent a promising class of devices that enable long-term and real-time monitoring of cell physiology in a noninvasive and label-free fashion, with a remarkable potential for process automation and parallelization. Common limitations of this class of devices at large include the need for substrate surface modification strategies to ensure cell adhesion and immobilization, limited compatibility with complementary optical cell-probing techniques, and the need for frequency-dependent measurements, which rely on elaborated equivalent electrical circuit models for data analysis and interpretation. We hereby demonstrate the monitoring of cell adhesion and detachment through the time-dependent variations in the quasi-static characteristic current curves of a highly stable electrolyte-gated transistor, based on an optically transparent network of printable polymer-wrapped semiconducting carbon-nanotubes.

Extracellular electrophysiological based sensor to monitor cancer cells cooperative migration and cell-cell connections

Herein, we describe an electrophysiological based sensor that reproducibly monitors and quantifies in real-time collective migration and the formation of cell-cell junctions by C6 glioma cells seeded on top of electrodes. The signal amplitude and frequency generated by the migrating cells changed over time and these parameters were used to accurately calculate the migration speed. Electrophysiological measurements could also distinguish individual from collective cell migration. The migration of densely packed cells generated strong signals, while dispersed cells showed weak bioelectrical activity. We propose this electrophysiological technique as a cell-based biosensor to gain insight into the mechanisms of cooperative migration of cancer cells. Possible applications include screening for anti-migratory compounds, which may lead to the development of novel strategies for antineoplastic chemotherapy.

Implementation of Combinatorial Genetic and Microenvironmental Engineering to Microbial-Based Field-Deployable Microbead Biosensors for Highly Sensitive and Remote Chemical Detection

Bioreporters, microbial species genetically engineered to provide measurable signals in response to specific chemicals, have been widely investigated as sensors for biomedical and environmental monitoring. More specifically, the bioreporter encapsulated within a biocompatible material, such as a hydrogel that can provide a suitable microenvironment for its prolonged activity as well as efficient scalable production, has been viewed as a more broadly applicable mode of biosensors. In this study, alginate-based microbeads encapsulated with the bacterial bioreporter capable of expressing green fluorescence protein in response to nitro compounds (e.g., trinitrotoluene and dinitrotoluene) are developed as biosensors. To significantly enhance the sensitivity of the microbial-based microbead biosensors, "multifaceted" modification strategies are simultaneously employed: (1) multiple genetic modifications of the bioreporter, (2) tuning the physicomechanical properties of the encapsulating microbeads, (3) controlling the initial cell density within the microbeads, and (4) enrichment of nitro compounds inside microbeads via functional nanomaterials. These microbial and microenvironmental engineering approaches combine to significantly enhance the sensing capability, even allowing highly sensitive remote detection under a low-vapor phase. Thus, the strategy developed herein is expected to contribute to various cell-based biosensors.

A novel fluorescent recombinant cell-based biosensor for screening NLRP3 inflammasome inhibitors

Nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 (NLRP3) inflammasomes trigger a defense mechanism in response to physiological or pathological reactions caused by various injuries, and they have critical roles in the development of many major diseases. The development of plant source anti-inflammatory agents and the assessment of their anti-inflammatory properties still face many challenges, such as high costs and long testing periods. Exogenous anti-inflammatory factors can be evaluated at the protein level using the NLRP3-green fluorescent protein (GFP) THP-1 cell-based biosensor. To achieve this, THP-1 cells were stably transfected with a plasmid encoding the GFP reporter gene that is under the transcriptional regulation of the NLRP3 promoter. Because the NLRP3 promoter controls the green fluorescent signal, the fluorescence is an indicator of NLRP3 activity. Our results show the fluorescence intensity was dependent on both the degree of inflammation and the effect of the anti-inflammatory agent. Here, we successfully used the biosensor to screen an NLRP3 inhibitor in the presence of polyphenols. Our results demonstrate the potential of this cell-based biosensor in monitoring the development of inflammation and in screening inflammatory inhibitors.

A living plant cell-based biosensor for real-time monitoring invisible damage of plant cells under heavy metal stress

Heavy metals inevitably cause invisible or visible damage to plants, leading to significant economic losses. Therefore, it is necessary to develop a method for timely monitoring the damage of plants under the stress of heavy metals. Here, vitronectin-like proteins (VN) on the surface of plant cells is as an important biomarker for monitoring damage of plants under the stress of heavy metals. A living plant cell-based biosensor is constructed to monitor invisible damage of plant cells induced by cadmium [Cd(II)] or lead [Pb(II)]. To fabricate this sensor, l-cysteine was first modified on the glassy carbon electrode followed by the modification of anti-IgG-Au antibody. Then, the living plant cells, incubated with the anti-VN, were modified onto the electrode. The sensor worked by determining the change in electrochemical impedance. Cd(II) and Pb(II) was detected in the linear dynamic range of 45–210 and 120–360 μmol·L−1, respectively. And the detection limit of Cd(II) and Pb(II) of this biosensor was 18.5 nmol·L−1 [with confidence interval (95%) 18.4–18.6 nmol·L−1] and 25.6 nmol·L−1 [with confidence interval (95%) 25.4–25.8 nmol·L−1], respectively. In both Arabidopsis and soybean, when the content of VN increased by about 20 times under the stress of Cd(II) or Pb(II), which means when the electron-transfer resistance increased by 35%, chlorophyll content showed significant decrease about 17%. Therefore, by establishing a quantitative relationship among the content of biomarker, the electron-transfer resistance and chlorophyll content in plant cells, the invisible damage of plants under the stress of heavy metals was detected. These results can provide a reference method for early-onset warning systems for heavy metal pollution in the environment.

Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation

Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer’s disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation.

Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein.

Microbial cell-based biosensors, which mostly rely on stress-responsive operons, have been widely developed to monitor environmental pollutants. Biosensors are usually more convenient and inexpensive than traditional instrumental analyses of environmental pollutants. However, the targets of biosensors are restricted by the limited number of genetic operon systems available. In this study, we demonstrated a novel strategy to overcome this limitation by engineering an enhanced green fluorescent protein (eGFP). It has been reported that combining two fragments of split-eGFP can form a native structure. Thus, we engineered new biosensors by inserting metal-binding loops (MBLs) between β-strands 9 and 10 of the eGFP, which then undergoes conformational changes upon interaction between the MBLs and targets, thereby emitting fluorescence. The two designed MLBs based on our previous study were employed as linkers between two fragments of eGFP. As a result, an Escherichia coli biosensor exhibited a fluorescent signal only when interacting with cadmium ions, revealing the prospect of a new biosensor for cadmium detection. Although this study is a starting stage for further developing biosensors, we believe that the proposed strategy can serve as basis to develop new biosensors to target various environmental pollutants.

Development of a sediment microbial fuel cell-based biosensor for simultaneous online monitoring of dissolved oxygen concentrations along various depths in lake water

A novel multi-cathode, single-anode system integrating a sediment microbial fuel cell -based biosensor was developed for in-situ, continuous, and online monitoring of dissolved oxygen (DO) concentrations along various depths of lake water. The signal feedback mechanism was evaluated based on a relationship between voltage and DO concentration at corresponding depths. With an external resistance of 1000 Ω, a linear relationship was found (regression coefficient, R2 = 0.9576) between voltage and DO in the range of 0–9 mg L−1. The sensor performance was further optimized under various influence factors. The results of indoor experiments indicated that the optimal anode to single cathode area ratio was 11:1. The sensor signal could also be significantly influenced by organic matter content in sediment; thus, the addition of 5% organic matter could obtain a stable anode potential and a high voltage output. Furthermore, the sensor was operated in-situ for 67 days in a lake environment, which also led to a good correlation between the voltage and DO (R2 = 0.8897). Thus, this integrated system has great potential as an early-warning program to help identify environmental risks in aquatic environments.