Journal articles: 'Fluorescent biosensor'

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Ma, Xing Meng, Ji Mei Zhang, Zhao Dai, Xiao Yu Chen, Xiao Qing Wang, and Qing Yin Zhang. "Fluorescent DNA Biosensors on Silica Microspheres." Advanced Materials Research 301-303 (July 2011): 195–200. http://dx.doi.org/10.4028/www.scientific.net/amr.301-303.195.

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A novel DNA biosensor system on silica microspheres as solid carriers which based on the fluorescence resonance energy transfer (FRET) was presented in this work when CdTe quantum dots (QDs) were as energy donors and Au nanoparticles (AuNPs) were as energy accepters. Compared with the fluorescent intensity of CdTe QDs, the fluorescent intensity of DNA biosensors decreased extremely, which indicated that the FRET occurred between CdTe QDs and AuNPs. The biosensor system would have a certain degree recovery of fluorescence when the complementary single stranded DNA was introduced into this system. The DNA detection results indicated that this novel fluorescent DNA probe system could recognize the existence of complementary target DNA or not.

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Liu, Xuemei, Kieran J. Germaine, David Ryan, and David N. Dowling. "DEVELOPMENT OF A GFP‐BASED BIOSENSOR FOR DETECTING THE BIOAVAILABILITY AND BIODEGRADATION OF POLYCHLORINATED BIPHENYLS (PCBS)." JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT 15, no. 4 (December 31, 2007): 261–68. http://dx.doi.org/10.3846/16486897.2007.9636939.

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Two whole-cell biosensors were constructed to detect the in situ biodegradation of polychlorinated biphenyl by chromosomal insertion of a mini‐Tn5‐Kmr‐Pm::gfp[mut3]‐T0‐T1 construct into P. fluorescens. In vitro tests showed that the expression of the Pm promoter depended on the growth phase of the biosensors and the concentration of chemical inducers; chlorinated benzoic acid derivatives. A linear relationship between the fluorescent intensity and the log10 concentration of the inducer was observed. One biosensor (F113L::1180gfp) had the ability to degrade PCBs to relevant chlorobenzoic acid derivatives and to induce expression of Gfp. The second biosensor (F113gfp), which cannot degrade PCBs, shows fluorescence after induction by chloro‐benzoic acid derivatives. By using these two biosensors, PCB degradation could be detected in vitro and in soil.

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Tang, Longteng, Shuce Zhang, Yufeng Zhao, Nikita D. Rozanov, Liangdong Zhu, Jiahui Wu, Robert E. Campbell, and Chong Fang. "Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor." International Journal of Molecular Sciences 22, no. 1 (January 5, 2021): 445. http://dx.doi.org/10.3390/ijms22010445.

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Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.

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Lee, Woonwoo, Hyojin Kim, Yerin Kang, Youngshim Lee, and Youngdae Yoon. "A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein." Sensors 19, no. 8 (April 18, 2019): 1846. http://dx.doi.org/10.3390/s19081846.

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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.

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Tenner, Brian, Jason Z. Zhang, Yonghoon Kwon, Veronica Pessino, Siyu Feng, Bo Huang, Sohum Mehta, and Jin Zhang. "FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains." Science Advances 7, no. 21 (May 2021): eabe4091. http://dx.doi.org/10.1126/sciadv.abe4091.

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Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3′,5′-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein–coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

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Manonmani, V., A. Vimala Juliet, J. Ponnidevi, and P. Arumugam. "A Novel Magneto-Fluorescent Biosensor for the Detection of Pathogens in Food." Advanced Materials Research 984-985 (July 2014): 1074–79. http://dx.doi.org/10.4028/www.scientific.net/amr.984-985.1074.

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Food safety has emerged as an important global issue. Illness from the pathogenic bacteria is a significant health concern. Pathogens are potentially harmful and it contaminate food and causes foodborne illness . It is very important for early detection of food borne pathogens. Conventional methods of detecting these pathogens eventhough sensitive, but still it is time consuming,complex and laborious. Biosensors have great potential for the detection of pathogenic bacteria in food. Biosensors are developed to rapidly detect the pathogens. In this paper, we use nanosized magnetite nanoparticles coated with chitosan, a polymer to capture and identify bacteria from contaminated food sample and eliminates the purpose of specific antibody coating. Amine group with magnetite nanoparticles (MNPs) specifically binds with the bacterial cell wall, on their surface of most pathogenic bacteria present in food. Rhodamine as a dye/marker which emits fluorescence in exposure to light used to detect the bacterial concentration in terms of light or fluorescent intensity and analog voltage using Magneto-Fluorescent biosensor. The developed biosensor could be able to detect bacteria in the limit of 10 CFU/ml and the time taken for measurement of a sample using biosensor would be less than 5 minutes.Hence the developed biosensor are non-specific and highly sensitive.

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Stiner, Lawrence, and Larry J. Halverson. "Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds." Applied and Environmental Microbiology 68, no. 4 (April 2002): 1962–71. http://dx.doi.org/10.1128/aem.68.4.1962-1971.2002.

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ABSTRACT A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K app values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

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Goh, Yan Y., Bow Ho, and Jeak L. Ding. "A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria." Applied and Environmental Microbiology 68, no. 12 (December 2002): 6343–52. http://dx.doi.org/10.1128/aem.68.12.6343-6352.2002.

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ABSTRACT Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFPi proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFPi G10 and G12 to lipid A were established. The KD values of 7.16 × 10−7 M for G10 and 8.15 × 10−8 M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFPi biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFPi specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFPi in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

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Seitz, Kati, and Patrick J. Krysan. "Expanding the Toolkit of Fluorescent Biosensors for Studying Mitogen Activated Protein Kinases in Plants." International Journal of Molecular Sciences 21, no. 15 (July 28, 2020): 5350. http://dx.doi.org/10.3390/ijms21155350.

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Mitogen-activated protein kinases (MAPKs) are key regulators of numerous biological processes in plants. To better understand the mechanisms by which these kinases function, high resolution measurement of MAPK activation kinetics in different biological contexts would be beneficial. One method to measure MAPK activation in plants is via fluorescence-based genetically-encoded biosensors, which can provide real-time readouts of the temporal and spatial dynamics of kinase activation in living tissue. Although fluorescent biosensors have been widely used to study MAPK dynamics in animal cells, there is currently only one MAPK biosensor that has been described for use in plants. To facilitate creation of additional plant-specific MAPK fluorescent biosensors, we report the development of two new tools: an in vitro assay for efficiently characterizing MAPK docking domains and a translocation-based kinase biosensor for use in plants. The implementation of these two methods has allowed us to expand the available pool of plant MAPK biosensors, while also providing a means to generate more specific and selective MAPK biosensors in the future. Biosensors developed using these methods have the potential to enhance our understanding of the roles MAPKs play in diverse plant signaling networks affecting growth, development, and stress response.

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Hansen, Lars Hestbjerg, Belinda Ferrari, Anders Hay Sørensen, Duncan Veal, and Søren Johannes Sørensen. "Detection of Oxytetracycline Production by Streptomyces rimosus in Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry." Applied and Environmental Microbiology 67, no. 1 (January 1, 2001): 239–44. http://dx.doi.org/10.1128/aem.67.1.239-244.2001.

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ABSTRACT Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production byStreptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between thetetR-regulated P tet promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosusintroduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

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Trigo-Mourino, Pablo, Thomas Thestrup, Oliver Griesbeck, Christian Griesinger, and Stefan Becker. "Dynamic tuning of FRET in a green fluorescent protein biosensor." Science Advances 5, no. 8 (August 2019): eaaw4988. http://dx.doi.org/10.1126/sciadv.aaw4988.

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Förster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with B02-dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.1 GHz. These structural data provide the unprecedented opportunity to calculate FRET from the x-ray structure and to compare it to experimental data in solution. We find that interdomain dynamics limits the FRET effect and show that a rigidification of the sensor further enhances FRET.

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Morgan, Mark T., Gi Young Kim, Daniel Ess, Aparna Kothapalli, Byoung Kwon Hahm, and Arun Bhunia. "Binding Inhibition Assay Using Fiber-Optic Based Biosensor for the Detection of Foodborne Pathogens." Key Engineering Materials 321-323 (October 2006): 1145–50. http://dx.doi.org/10.4028/www.scientific.net/kem.321-323.1145.

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Frequent outbreaks of foodborne illness have been increasing the need for simple, rapid and sensitive methods to detect foodborne pathogens. Conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Some immunological rapid assays are developed, but these assays still require prolonged enrichment steps. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Among the biosensors, fiber-optic methods have much potential because they can be very sensitive and simple to operate. Fiber-optic biosensors typically use a light transmittable, tapered fiber to send excitation laser light to the detection surface and receive emitted fluorescent light. The fluorescent light excited by an evanescent wave generated by the laser is quantitatively related to fluorophor-labeled biomolecules immobilized on the fiber surface. A portable and automated fiber-optic biosensor, RAPTOR (Research International, Monroe, WA), was used to detect Salmonella enteritidis in food samples. A binding inhibition assay based on the biosensor was developed to detect the bacteria in hot dog samples. The biosensor and the binding inhibition assay could detect 104 cfu/ml of bacteria in less than 10 min of assay time.

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Schaaf, Tory M., Evan Kleinboehl, Samantha L. Yuen, Lauren N. Roelike, Bengt Svensson, Andrew R. Thompson, Razvan L. Cornea, and David D. Thomas. "Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules That Enhance Ca2+ Transport." Cells 9, no. 5 (May 8, 2020): 1170. http://dx.doi.org/10.3390/cells9051170.

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We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.

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Shokhina, AG, VV Belousov, and DS Bilan. "A genetically encoded biosensor roKate for monitoring the redox state of the glutathione pool." Laboratory diagnostics, no. 1 (March 14, 2019): 86–92. http://dx.doi.org/10.24075/brsmu.2019.013.

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Genetically encoded fluorescent sensors are exploited to study a variety of biological processes in living organisms in real time. In recent years, a whole family of biosensors has been developed, serving to visualize changes in the glutathione redox state. The aim of our experiment was to design a biosensor based on the red fluorescent protein mKate2 for measuring the 2GSH/GSSG ratio. A pair of cysteine amino acid residues were introduced into the structure of the fluorescent protein using site-directed mutagenesis. These residues form a disulfide bridge when the surrounding glutathione pool is oxidized, affecting the spectral characteristics of the protein. Our biosensor, which we called roKate, was tested in vitro on an isolated protein. Specifically, we examined the spectral characteristics, pH and the redox potential of the sensor. Additionally, the performance of roKate was evaluated using the culture of living mammalian cells. The fluorescent signal emitted by the sensor was very bright and remarkably stable under pH conditions varying in the physiological range. Irreversibly oxidized in mammalian cells, roKate stands out from other members of this biosensor family. This biosensor should be preferred in the experiments when the time between the manipulations with the biological object and the subsequent analysis of the induced effect is substantial, as is the case with long sample preparation.

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Tewson, Paul H., Scott Martinka, Nathan C. Shaner, Thomas E. Hughes, and Anne Marie Quinn. "New DAG and cAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories." Journal of Biomolecular Screening 21, no. 3 (December 11, 2015): 298–305. http://dx.doi.org/10.1177/1087057115618608.

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Protein-based, fluorescent biosensors power basic research on cell signaling in health and disease, but their use in automated laboratories is limited. We have now created two live-cell assays, one for diacyl glycerol and another for cAMP, that are robust (Z′ > 0.7) and easily deployed on standard fluorescence plate readers. We describe the development of these assays, focusing on the parameters that were critical for optimization, in the hopes that the lessons learned can be generalized to the development of new biosensor-based assays.

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Schaaf, Tory, Ang Li, Benjamin Grant, Kurt Peterson, Samantha Yuen, Prachi Bawaskar, Evan Kleinboehl, Ji Li, David Thomas, and Gregory Gillispie. "Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening." Biosensors 8, no. 4 (October 24, 2018): 99. http://dx.doi.org/10.3390/bios8040099.

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We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

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Houser, Mei CQ, Steven S. Hou, Florian Perrin, Yuliia Turchyna, Brian J. Bacskai, Oksana Berezovska, and Masato Maesako. "A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells." Sensors 20, no. 21 (October 22, 2020): 5980. http://dx.doi.org/10.3390/s20215980.

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Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.

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Wang, Honghong, Hui Wang, Mai Zhang, Yuting Jia, and Zhengping Li. "A label-free aptamer-based biosensor for microRNA detection by the RNA-regulated fluorescence of malachite green." RSC Advances 9, no. 56 (2019): 32906–10. http://dx.doi.org/10.1039/c9ra07552f.

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Almadidy, Amer, James Watterson, Paul AE Piunno, Inge V. Foulds, Paul A. Horgen, and Ulrich Krull. "A fibre-optic biosensor for detection of microbial contamination." Canadian Journal of Chemistry 81, no. 5 (May 1, 2003): 339–49. http://dx.doi.org/10.1139/v03-070.

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A fibre-optic biosensor is described for detection of genomic target sequences from Escherichia coli. A small portion of the LacZ DNA sequence is the basis for selection of DNA probe molecules that are produced by automated nucleic acid synthesis on the surface of optical fibres. Fluorescent intercalating agents are used to report the presence of hybridization events with target strands. This work reviews the fundamental design criteria for development of nucleic acid biosensors and reports a preliminary exploration of the use of the biosensor for detection of sequences that mark the presence of E. coli. The research work includes consideration of the length of the strands and non-selective binding interactions that can potentially block the selective chemistry or create background signals. The biosensors were able to detect genomic targets from E. coli at a picomole level in a time of a few minutes, and dozens of cycles of use have been demonstrated. In a step towards the preparation of a completely self-contained sensor technology, a new intercalating dye known as SYBR 101 (Molecular Probes, Inc.) has been end-labelled to the LacZ nucleic acid probe, to examine whether dye tethered onto an oligonucleotide terminus could fluorimetrically transduce the formation of hybrids. The results obtained from experiments in solution indicate that the use of tethered dye provides fluorescence signals that are due to hybridization, and that this process is functional even in the presence of a high concentration of non-selective background DNA obtained from sonicated salmon sperm. Key words: biosensor, DNA, fibre optic, hybridization, fluorescence, pathogen, E. coli.

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Wu, Jikui, Yunfei Lu, Ningna Ren, Min Jia, Ruinan Wang, and Junling Zhang. "DNAzyme-Functionalized R-Phycoerythrin as a Cost-Effective and Environment-Friendly Fluorescent Biosensor for Aqueous Pb2+ Detection." Sensors 19, no. 12 (June 18, 2019): 2732. http://dx.doi.org/10.3390/s19122732.

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The sensitive detection of Pb2+ is of significant importance for food safety, environmental monitoring, and human health care. To this end, a novel fluorescent biosensor, DNAzyme-functionalized R-phycoerythrin (DNAzyme-R-PE), was presented for Pb2+ analysis. The biosensor was prepared via the immobilization of Iowa Black® FQ-modified DNAzyme–substrate complex onto the surface of SPDP-functionalized R-PE. The biosensor produced a minimal fluorescence signal in the absence of Pb2+. However, Pb2+ recognition can induce the cleavage of substrate, resulting in a fluorescence restoration of R-PE. The fluorescence changes were used to measure sensitively Pb2+ and the limit of detection was 0.16 nM with a linear range from 0.5–75 nM. Furthermore, the proposed biosensor showed excellent selectivity towards Pb2+ even in the presence of other metal ions interferences and was demonstrated to successfully determine Pb2+ in spiked lake water samples.

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Arnold, Maren E., Wolfgang R. Dostmann, Jody Martin, Michael J. Previs, Bradley Palmer, Martin LeWinter, and Markus Meyer. "SERCA2a-phospholamban interaction monitored by an interposed circularly permutated green fluorescent protein." American Journal of Physiology-Heart and Circulatory Physiology 320, no. 6 (June 1, 2021): H2188—H2200. http://dx.doi.org/10.1152/ajpheart.00858.2020.

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This study describes the design and characterization of a novel biosensor that can visualize the interaction of SERCA2a and phospholamban (PLB). The biosensor combines SERCA2a, a circularly permutated green fluorescent protein, and PLB into one recombinant protein (SGP). Proteinkinase A activation results in phosphorylation of the PLB domain and is associated with a marked increase in the fluorescence yield to allow for real-time monitoring of the SERCA2a and PLB interaction in cells.

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Jeong, Hee-Jin, Tomoki Kojima, Jinhua Dong, Hiroyuki Ohashi, and Hiroshi Ueda. "One-pot construction of Quenchbodies using antibody-binding proteins." Analytical Methods 8, no. 43 (2016): 7774–79. http://dx.doi.org/10.1039/c6ay02108e.

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Ooi, Lia, Lee Yook Heng, and Asmat Ahmad. "Toxicity Biosensor for Sodium Dodecyl Sulfate Using Immobilized Green Fluorescent Protein ExpressingEscherichia coli." Journal of Sensors 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/809065.

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Green fluorescent protein (GFP) is suitable as a toxicity sensor due to its ability to work alone without cofactors or substrates. Its reaction with toxicants can be determined with fluorometric approaches. GFP mutant gene (C48S/S147C/Q204C/S65T/Q80R) is used because it has higher sensitivity compared to others GFP variants. A novel sodium dodecyl sulfate (SDS) toxicity detection biosensor was built by immobilizing GFP expressingEscherichia coliink-Carrageenan matrix. Cytotoxicity effect took place in the toxicity biosensor which leads to the decrease in the fluorescence intensity. The fabricatedE. coliGFP toxicity biosensor has a wide dynamic range of 4–100 ppm, with LOD of 1.7 ppm. Besides, it possesses short response time (<1 min), high reproducibility (0.76% RSD) and repeatability (0.72% RSD,R2>0.98), and long-term stability (46 days).E. coliGFP toxicity biosensor has been applied to detect toxicity induced by SDS in tap water, river water, and drinking water. High recovery levels of SDS indicated the applicability ofE. coliGFP toxicity biosensor in real water samples toxicity evaluation.

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De Lorimier, Robert M., J. Jeff Smith, Mary A. Dwyer, Loren L. Looger, Kevin M. Sali, Chad D. Paavola, Shahir S. Rizk, et al. "Construction of a fluorescent biosensor family." Protein Science 11, no. 11 (April 13, 2009): 2655–75. http://dx.doi.org/10.1110/ps.021860.

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Andresen, Max, Kwok-Yin Wong, Yun-Chung Leung, Wai-Ting Wong, Pak-Ho Chan, Max Andresen-Vasquez, Leyla Alegria, et al. "Method Based on theβ-Lactamase PenPC Fluorescent Labeled forβ-Lactam Antibiotic Quantification in Human Plasma." BioMed Research International 2016 (2016): 1–6. http://dx.doi.org/10.1155/2016/4307987.

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Recently, Wong et al. have successfully developed a fluorescent biosensor based on the PenPCβ-lactamase which changes its intrinsic fluorescence in presence ofβ-lactam antibiotics (BLAs). Here, we studied systematically this correlation among the fluorescence change of the biosensor and the concentration of different BLAs aimed at developing a novel method for estimating the concentration of a wide range of BLAs. This method showed high precision and specificity and very low interference from clinically relevant samples. We were able to monitor the pharmacokinetics of meropenem in healthy volunteers as well as in an ill animal model too, indicating that the implemented method could be suitable for clinical practice.

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Montecinos-Franjola, Felipe, John Y. Lin, and Erik A. Rodriguez. "Fluorescent proteins for in vivo imaging, where's the biliverdin?" Biochemical Society Transactions 48, no. 6 (November 16, 2020): 2657–67. http://dx.doi.org/10.1042/bst20200444.

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Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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Bai, Yunlong, Tong Shu, Lei Su, and Xueji Zhang. "Fluorescent Gold Nanoclusters for Biosensor and Bioimaging Application." Crystals 10, no. 5 (April 30, 2020): 357. http://dx.doi.org/10.3390/cryst10050357.

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With the rapid development of materials technology, fluorescent gold nanoclusters (AuNCs) are emerging as novel functional materials for diagnostic applications including the detection of biomarkers and bioimaging due to the advantages of their ultra-small size, tunable emissions, size-dependent fluorescence and excellent biocompatibility. In this review, we introduced the synthetic methods, and physical and chemical properties of AuNCs. Subsequently, we described the AuNCs-based design strategies for the detection of biomarkers including small molecules, DNA and proteins. The applications of AuNCs for tumor imaging in vitro and in vivo were also presented. Finally, we discussed the challenges and potential solutions of AuNCs-based nanosensors.

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Gallo, Eugenio, Sophia Wienbar, Avin C. Snyder, Kalin V. Vasilev, Bruce A. Armitage, and Jonathan W. Jarvik. "A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families." Protein & Peptide Letters 21, no. 12 (November 5, 2014): 1289–94. http://dx.doi.org/10.2174/0929866521666140616121800.

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Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.

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Hantani, Rie, Saya Hanawa, Shohei Oie, Kayo Umetani, Toshihiro Sato, and Yoshiji Hantani. "Identification of a New Inhibitor That Stabilizes Interleukin-2-Inducible T-Cell Kinase in Its Inactive Conformation." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 8 (June 27, 2019): 854–62. http://dx.doi.org/10.1177/2472555219857542.

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Interleukin-2-inducible T-cell kinase (ITK) plays an important role in T-cell signaling and is considered a promising drug target. As the ATP binding sites of protein kinases are highly conserved, the design of selective kinase inhibitors remains a challenge. Targeting inactive kinase conformations can address the issue of kinase inhibitor selectivity. It is important for selectivity considerations to identify compounds that stabilize inactive conformations from the primary screen hits. Here we screened a library of 390,000 compounds with an ADP-Glo assay using dephosphorylated ITK. After a surface plasmon resonance (SPR) assay was used to filter out promiscuous inhibitors, 105 hits were confirmed. Next, we used a fluorescent biosensor to enable the detection of conformational changes to identify inactive conformation inhibitors. A single-cysteine-substituted ITK mutant was labeled with acrylodan, and fluorescence emission was monitored. Using a fluorescent biosensor assay, we identified 34 inactive conformation inhibitors from SPR hits. Among them, one compound was bound to a site other than the ATP pocket and exhibited excellent selectivity against a kinase panel. Overall, (1) biochemical screening using dephosphorylated kinase, (2) hit confirmation by SPR assay, and (3) fluorescent biosensor assay that can distinguish inactive compounds provide a useful platform and offer opportunities to identify selective kinase inhibitors.

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Constantinou, Antony, and Karen M. Polizzi. "Opportunities for bioprocess monitoring using FRET biosensors." Biochemical Society Transactions 41, no. 5 (September 23, 2013): 1146–51. http://dx.doi.org/10.1042/bst20130103.

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Bioprocess monitoring is used to track the progress of a cell culture and ensure that the product quality is maintained. Current schemes for monitoring metabolism rely on offline measurements of samples of the extracellular medium. However, in the era of synthetic biology, it is now possible to design and implement biosensors that consist of biological macromolecules and are able to report on the intracellular environment of cells. The use of fluorescent reporter signals allows non-invasive, non-destructive and online monitoring of the culture, which reduces the delay between measurement and any necessary intervention. The present mini-review focuses on protein-based biosensors that utilize FRET as the signal transduction mechanism. The mechanism of FRET, which utilizes the ratio of emission intensity at two wavelengths, has an inherent advantage of being ratiometric, meaning that small differences in the experimental set-up or biosensor expression level can be normalized away. This allows for more reliable quantitative estimation of the concentration of the target molecule. Existing FRET biosensors that are of potential interest to bioprocess monitoring include those developed for primary metabolites, redox potential, pH and product formation. For target molecules where a biosensor has not yet been developed, some candidate binding domains can be identified from the existing biological databases. However, the remaining challenge is to make the process of developing a FRET biosensor faster and more efficient.

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Ning, Yi, Qiang Gao, Xiaoqing Zhang, Ke Wei, and Lingli Chen. "A Graphene Oxide–Based Sensing Platform for the Determination of Methicillin-Resistant Staphylococcus aureus Based on Strand-Displacement Polymerization Recycling and Synchronous Fluorescent Signal Amplification." Journal of Biomolecular Screening 21, no. 8 (July 10, 2016): 851–57. http://dx.doi.org/10.1177/1087057116653564.

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To develop new technology for detecting methicillin-resistant Staphylococcus aureus (MRSA), a novel fluorescent biosensor based on Klenow fragment (KF)–assisted target recycling amplification and synchronous fluorescence analysis was created. Carboxy-fluorescein (FAM)–labeled single-stranded DNA (ssDNA) containing a capture probe and a signal probe was adsorbed onto the surface of graphene oxide (GO) via π-stacking interactions, resulting in the fluorescence quenching of the dye. When target and primer were introduced, the fluorescence was restored due to P0 being completely released from the surface of the GO. Meanwhile, by using the KF and exploiting the synergistic effect of FAM and the double-stranded DNA (dsDNA)–SYBR Green I duplex structure, the fluorescence in this detection system was considerably amplified and the sensitivity was improved. The proposed strategy for mecA gene analysis showed a good linear range from 1 to 40 nmol/L, with a lower limit of detection of 0.5 nmol/L. In addition, a bacterial sample harboring the mecA gene was also detected, and its lower detection limit was up to 300 colony-forming units (CFU)/mL. Accordingly, this biosensor exhibits high sensitivity and selectivity and has great potential for early clinical diagnosis and treatment.

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Kim, Gi Young, Mark T. Morgan, Daniel Ess, Byoung Kwon Hahm, Aparna Kothapalli, Angela Valadez, and Arun Bhunia. "Detection of Listeria Monocytogenes Using an Automated Fiber-Optic Biosensor: RAPTOR." Key Engineering Materials 321-323 (October 2006): 1168–71. http://dx.doi.org/10.4028/www.scientific.net/kem.321-323.1168.

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Fiber-optic biosensor uses light transmittable tapered fiber to send excitation laser light and receive emitted fluorescent light. The fluorescent light excited by an evanescent wave generated by the laser is quantitatively related to biomolecules immobilized on the fiber surface [1]. An automated fiber-optic biosensor based detection method for Listeria monocytogenes was developed in this research. Detections of Listeria monocytogenes in hotdog sample were performed to evaluate the method. By using the detection method with automated fiber-optic biosensor, 5.4×107 cfu/ml of Listeria monocytogenes was able to detect.

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Tsai, H. C., and R. A. Doong. "Optimization of sol-gel based fibre-optic cholinesterase biosensor for the determination of organophosphorus pesticides." Water Science and Technology 42, no. 7-8 (October 1, 2000): 283–90. http://dx.doi.org/10.2166/wst.2000.0580.

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A sol-gel based fiber-optic biosensor with acetylcholinesterase as the biorecognition element has been developed for the rapid determination of organophosphorus pesticides. Nine fluorescent indicators, acridine, acridine orange, neutral red, DAPI, rhodamine B, fluorescein, umbelliferone, FITC on celite and FITC-dextran, have been examined to optimize the fiber-optic system. Results showed that acridine and FITCs were sensitive to the change of pH value caused by the enzyme-substrate catalysis reaction. However, the sensitivity of acridine was 260 times lower than that of FITCs. Higher toxicity of acridine to acetylcholinesterase than FITC was also observed. Moreover, the high-molecular-weight FITC-dextran showed low leakage rate when immobilizing using sol-gel technology, showing that the FITC-dextran was a suitable pH sensitive fluorescent indicator for the OPPs biosensor. The response of the fiber-optic biosensor to the substrate, acetylcholine, was highly reproducible (RSD=3.5%). A good linearity of acetylcholine in the range from 0.5 to 20 mM was also obtained (R2=0.98). Furthermore, a 30% inhibition can be achieved in 30min when 152 ppb paraoxon was added into the system. The results show the possibility for real-time determination of organophosphorus pesticides by using the biosensor developed in this study.

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Zhang, Song Bai, Meng Tang, Li Ying Zheng, Xia Hu, Guan Gyu Shen, Xue Wen Liu, Jin Lin Lu, Yuan Dao Chen, Li Ping Qiu, and Shi Biao Zhou. "Molecular Beacon-Based Fluorescent Biosensor for Sensitive Detection of Single Nucleotide Polymorphism." Applied Mechanics and Materials 651-653 (September 2014): 256–59. http://dx.doi.org/10.4028/www.scientific.net/amm.651-653.256.

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A sensitive fluorescent biosensing strategy based on structure-switching of molecular beacon for the detection of single nucleotide polymorphism is proposed in this study. In the absence of target DNA, only low background fluorescence can be detected since the formation of hairpin structure induces close proximity of fluorophore and quencher. On the contrary, the fluorescence intensity increases significantly after introduction of target DNA because structure-switching of molecular beacon results in separation of fluorophore and quencher. The change of fluorescence intensity is linear with concentration of target DNA, and a molecular beacon-based sensing system for fluorescent detection of single nucleotide polymorphism is fabricated. The target DNA can be sensitively detected in a linear dynamic range from 17.58nM-1.125μM with a low detection limit of 8 nM. Moreover, good reproducibility and high specificity are achieved.

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Joglekar, S. S., P. V. Pimpliskar, V. V. Sirdeshmukh, P. S. Alegaonkar, and A. A. Kale. "FITC Embedded ZnO/Silica Nanocomposites as probe for detection of L-lactate: Point-of-Care diagnosis." MRS Advances 4, no. 46-47 (2019): 2533–40. http://dx.doi.org/10.1557/adv.2019.158.

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Abstract:A novel Fluorescence Resonance Energy Transfer (FRET) based ‘Turn-ON’ biosensor has been developed using fluorescent ZnO/APTMS-FITC (ZFA) nanoflakes as sensing probe. In this biosensor, Lactate Dehydrogenase (LDH) is used for the detection of L-lactate, a diagnostic marker for abnormal physiological conditions like muscular dystrophy, myocardial infraction, abnormal tissue formation and tissue damage. Lactate Dehydrogeanse (LDH) catalyses the conversion of L-Lactate to L-Pyruvate, in presence of β-NAD reducing to β-NADH. We tried to explore this mechanism with FRET based system for highly sensitive detection of L-Lactate. The fluorescence of these nanoflakes can be reversibly quenched in the presence of β-NAD.

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Tao, Wen, Michael Rubart, Jennifer Ryan, Xiao Xiao, Chunping Qiao, Takashi Hato, Michael W. Davidson, Kenneth W. Dunn, and Richard N. Day. "A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy." American Journal of Physiology-Cell Physiology 309, no. 11 (December 1, 2015): C724—C735. http://dx.doi.org/10.1152/ajpcell.00182.2015.

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The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

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Soto, Dagoberto, Camila Silva, Cristian Ugalde, Kwok-Yin Wong, Yun-Chung Leung, Lok-Yan So, and Max Andresen. "Effect of Lactamase Inhibitors on the Biosensor Penp during the Measurement of Lactam Antibiotics Concentration." Sensors 19, no. 5 (March 12, 2019): 1237. http://dx.doi.org/10.3390/s19051237.

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PenP is a fluorescent biosensor of lactam antibiotics (LA). It is structurally derived from the mutant lactamase TEM-1 comprising the substitution E166C, where fluorescein is covalently linked to cysteine. The presence of LA in the medium produces a change in the intrinsic fluorescence level of the biosensor, and the integral of the fluorescence level over time correlates directly with the LA concentration. Previously, we have successfully used PenP to determine the concentration of lactam antibiotics in clinical samples. The use of lactamase inhibitors (LI) is a common strategy to enhance the effect of LA due to the inhibition of an important resistance mechanism of pathogenic microorganisms. Structurally, LI and LA share the common element of recognition of lactamases (the lactam ring), but they differ in the reversibility of the mechanism of interaction with said enzyme. Because the biological recognition domain of PenP is derived from a lactamase, LI is expected to interfere with the PenP detection capabilities. Surprisingly, this work provides evidence that the effect of LI is marginal in the determination of LA concentration mediated by PenP.

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Krull, Ulrich J., Michael S. Heimlich, Krishna M. R. Kallury, P. A. E. Piunno, John D. Brennan, R. Stephen Brown, and Dimitrios P. Nikolelis. "1994 McBryde Medal Award Lecture Investigations of organized monolayer films for biosensor development." Canadian Journal of Chemistry 73, no. 8 (August 1, 1995): 1239–50. http://dx.doi.org/10.1139/v95-152.

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Our interests have focused on the investigation and development of biosensors that use chemically selective membranes to measure the concentration of specific species in complex media. One fundamental idea is that protein, which can bind selectively to a specific organic or biochemical species, can be incorporated into an ordered lipid or surfactant membrane assembly such that selective binding events lead to changes in the structure of the membrane (transduction) that can be measured quantitatively. The primary advantage of this method of detection is that it is applicable to interactions of enzymes, antibodies, receptors, and lectins, and it may be extended to investigations of DNA/RNA hybridization. This detection method therefore provides a sensitive generic strategy for sensor applications. The central problem to be solved is how the alteration of the structure of a membrane that is caused by binding events of protein or genetic material can give rise to an analytical signal. We have been focusing our efforts in the areas of fluorescence spectroscopy and electrochemical methods. The electrochemical methods rely on detection in changes of the permeability of membranes to ions and provide systems with very low background signal, leading to the possibility of detection of single molecular-binding events. Fluorescent systems operate on the basis that a chemically selective membrane containing a fluorescent indicator can provide an analytical signal caused by the change of the structure of the membrane due to the binding events. Keywords: fluorescence, lipid membrane, electrochemistry, receptor, biosensor.

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Wang, Rongji, Zhihua Wang, Honghong Rao, Xin Xue, Mingyue Luo, Zhonghua Xue, and Xiaoquan Lu. "A two fluorescent signal indicator-based ratio fluorometric alkaline phosphatase assay based on one signal precursor." Chemical Communications 57, no. 36 (2021): 4444–47. http://dx.doi.org/10.1039/d1cc00244a.

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Vásquez-Bochm, L. X., Isabel Velázquez-López, Rachel Mata, Alejandro Sosa-Peinado, Patricia Cano-Sánchez, and Martin González-Andrade. "Application of a Fluorescent Biosensor in Determining the Binding of 5-HT to Calmodulin." Chemosensors 9, no. 9 (September 5, 2021): 250. http://dx.doi.org/10.3390/chemosensors9090250.

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Here, we show the utility of the fluorescent biosensor hCaM-M124C-mBBr in detecting and determining the affinity of serotonin (5-HT). We obtained a Kd of 5-HT (0.71 μm) for the first time, the same order of magnitude as most anti-CaM drugs. This data can contribute to understanding the direct and indirect modulation of CaM on its binding proteins when the 5-HT concentration varies in different tissues or explain some of the side effects of anti-CaM drugs. On the other hand, molecular modeling tools help the rational design of biosensors and adequately complement the experimental results. For example, the docking study indicates that 5-HT binds at the same site as chlorpromazine (site 1) with a theoretical Ki of 2.84 μM; while the molecular dynamics simulations indicate a stability of the CaM–5-HT complex with a theoretical ΔG of −4.85 kcal mol−1, where the enthalpy contribution is greater. Thus, the combination of biotechnology and bioinformatics helps in the design and construction of more robust biosensors.

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Ding, Lu, Zhenwen Qin, Chunlan Xiang, and Gang Zhou. "Novel fluorescent organic nanoparticles as a label-free biosensor for dopamine in serum." Journal of Materials Chemistry B 5, no. 15 (2017): 2750–56. http://dx.doi.org/10.1039/c6tb03077g.

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Shokhina, Arina G., Alexander I. Kostyuk, Yulia G. Ermakova, Anastasiya S. Panova, Dmitry B. Staroverov, Evgeny S. Egorov, Mikhail S. Baranov, et al. "Red fluorescent redox-sensitive biosensor Grx1-roCherry." Redox Biology 21 (February 2019): 101071. http://dx.doi.org/10.1016/j.redox.2018.101071.

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Carter, Robert M., Robert C. Blake, Harmony P. Mayer, Alexander A. Echevarria, Trong D. Nguyen, and Levon A. Bostanian. "A Fluorescent Biosensor for Detection of Zearalenone." Analytical Letters 33, no. 3 (January 2000): 405–12. http://dx.doi.org/10.1080/00032710008543061.

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Morii, Takashi, Kenji Sugimoto, Keisuke Makino, Masami Otsuka, Keiji Imoto, and Yasuo Mori. "A New Fluorescent Biosensor for Inositol Trisphosphate." Journal of the American Chemical Society 124, no. 7 (February 2002): 1138–39. http://dx.doi.org/10.1021/ja016824d.

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Liang, Aoming, Yafang Shen, Yawen He, Jianping Wang, and Yanbin Li. "An Automated Magnetic Separation Device Coupled with a Fluorescent Biosensor for Detection of Antibiotic Residues." Transactions of the ASABE 64, no. 1 (2021): 23–30. http://dx.doi.org/10.13031/trans.14076.

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HighlightsA practical magnetic separation device was designed, fabricated, and evaluated for enrofloxacin detection.Coupled with a fluorescent biosensor, the device could automatically process a sample in 50 min.The device performed incubation and magnetic separation using a pipette method.The device has the advantages of low-cost and feasibility for on-site detection.Abstract. Antibiotic residues have been a continuing concern in food safety, raising a great issue in human health. For rapid detection of antibiotics, an automated device was developed that can capture and separate a target analyte based on immunomagnetic beads. This automated separation device is suitable for separating the magnetic beads in a preprocessing step, with liquid transfer and magnetic enrichment functions. The device was combined with a fluorescent biosensor to simplify the cumbersome pretreatment of enrofloxacin. In our experiments, enrofloxacin in water samples was used as the detection object, and the entire process could be completed in less than 50 min with automated operation. The lower limit of detection reached 54 ng mL-1 (S/N = 3). The fluorescent biosensor has been enhanced with this automated separation device for more sensitive rapid detection of antibiotic residues in the food supply chain and environment. Keywords: Antibiotic detection, Automation, Fluorescent biosensor, Immunomagnetic separation, Sample pretreatment.

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Siqian, L., S. Lei, and L. Ying. "Development of a cellular biosensor system for genotoxicity detection based on Trp53 promoter." Human & Experimental Toxicology 35, no. 10 (July 19, 2016): 1102–7. http://dx.doi.org/10.1177/0960327115621364.

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Objective: To develop a mouse cell biosensor system for the high-throughput genotoxicity detection of chemicals, such as environmental pollutants. Method: We developed a novel reporter vector pGL4-GFP, wherein the firefly luciferase reporter gene in the pGL4.82 vector was replaced by the green fluorescent protein (GFP) gene from the pAcGFP1-N1 vector. To construct the reporter pGL4-p53-GFP (p53 promoter linked to GFP), a fragment containing the p53 gene promoter was generated by amplifying a region from −481 to +180 of mouse genomic DNA isolated from mouse tail tissue. We developed a mouse cell biosensor system for the high-throughput genotoxicity detection of new drugs by stably integrating the reporter plasmid of pGL4-p53-GFP into the mouse embryonic fibroblast cells. Various genotoxic agents were used to treat this biosensor system. The resulting fluorescence was directly observed under a fluorescence microscope, and the GFP protein level was measured through Western blot analysis. Result: The biosensor system was treated with genotoxic agents, such as doxorubicin, cyclophosphamide, and benzo(a)pyrene. The GFP protein expression was significantly increased in cells exposed to genotoxic agents but negatively responded to the non-genotoxic agent dimethyl sulfoxide, thereby proving the specificity and sensitivity of the biosensor system. Conclusion: This novel in vitro biosensor system can be especially useful in genotoxicity detection.

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Albrecht, Simone C., Mirko C. Sobotta, Daniela Bausewein, Isabel Aller, Rüdiger Hell, Tobias P. Dick, and Andreas J. Meyer. "Redesign of Genetically Encoded Biosensors for Monitoring Mitochondrial Redox Status in a Broad Range of Model Eukaryotes." Journal of Biomolecular Screening 19, no. 3 (August 16, 2013): 379–86. http://dx.doi.org/10.1177/1087057113499634.

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The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential ( EGSH). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.

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Hara, Manami, Vytautas Bindokas, James P. Lopez, Kelly Kaihara, Luis R. Landa, Mark Harbeck, and Michael W. Roe. "Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice." American Journal of Physiology-Cell Physiology 287, no. 4 (October 2004): C932—C938. http://dx.doi.org/10.1152/ajpcell.00151.2004.

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The use of biosynthetic fluorescent sensors is an important new approach for imaging Ca2+ in cells. Genetically encoded indicators based on green fluorescent protein, calmodulin, and fluorescence resonance energy transfer (FRET) have been utilized to measure Ca2+ in nonmammalian transgenic organisms and provide information about the organization and regulation of Ca2+ signaling events in vivo. However, expression of biosynthetic FRET-based Ca2+ indicators in transgenic mammals has proven to be problematic. Here, we report transgenic expression of an endoplasmic reticulum (ER) Ca2+ biosensor in mouse pancreas. We targeted expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin I promoter. YC3.3er protein expression was limited to pancreatic β-cells within islets of Langerhans and absent in the exocrine pancreas and other tissues. Animals developed and matured normally; sensor expression was unaffected by age. Glucose tolerance in transgenic mice was also unaffected, indicating the transgenic biosensor did not impair endocrine pancreas function. ER Ca2+ responses after administration of thapsigargin, carbachol, and glucose were measured in individual β-cells of intact islets using confocal microscopy and confirmed the function of the biosensor. We conclude that controlling transgene transcription with a cell-specific promoter permits transgenic expression of FRET-based Ca2+ sensors in mammals and that this approach will facilitate real-time optical imaging of signal transduction events in living tissues.

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Abraham, Sam, James Chin, Huub J. M. Brouwers, Bernadette Turner, Ren Zhang, and Toni A. Chapman. "Green Fluorescent Protein-Based Biosensor To Detect and Quantify Stress Responses Induced by DNA-Degrading Colicins." Applied and Environmental Microbiology 77, no. 18 (July 29, 2011): 6691–93. http://dx.doi.org/10.1128/aem.00534-11.

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ABSTRACTHere we report the development of a whole-cell biosensor to detect and quantify the induction of the SOS response activated by DNA-degrading colicins. This biosensor utilizes the SOS-responsivecdapromoter to regulate the expression of green fluorescent protein. The biosensor assay revealed induction of stress for all DNA-degrading reference colicins (E2, E7, and E8).

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Mie, Masayasu, Rena Hirashima, Yasumasa Mashimo, and Eiry Kobatake. "Construction of an Enzymatically-Conjugated DNA Aptamer–Protein Hybrid Molecule for Use as a BRET-Based Biosensor." Applied Sciences 10, no. 21 (October 29, 2020): 7646. http://dx.doi.org/10.3390/app10217646.

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DNA-protein conjugates are useful molecules for construction of biosensors. Herein, we report the development of an enzymatically-conjugated DNA aptamer–protein hybrid molecule for use as a bioluminescence resonance energy transfer (BRET)-based biosensor. DNA aptamers were enzymatically conjugated to a fusion protein via the catalytic domain of porcine circovirus type 2 replication initiation protein (PCV2 Rep) comprising residues 14–109 (tpRep), which was truncated from the full catalytic domain of PCV2 Rep comprising residues 1–116 by removing the flexible regions at the N- and C-terminals. For development of a BRET-based biosensor, we constructed a fusion protein in which tpRep was positioned between NanoLuc luciferase and a fluorescent protein and conjugated to single-stranded DNA aptamers that specifically bind to either thrombin or lysozyme. We demonstrated that the BRET ratios depended on the concentration of the target molecules.

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Journal articles on the topic 'Fluorescent biosensor'

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