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9 pages, 2507 KiB

Open AccessArticle

Graphene Oxide-Based Nanostructured DNA Sensor

by Aditya Balaji, Songlin Yang, Jeslyn Wang and Jin Zhang

Biosensors 2019, 9(2), 74; https://doi.org/10.3390/bios9020074 - 30 May 2019

Cited by 30 | Viewed by 7249

Quick detection of DNA sequence is vital for many fields, especially, early-stage diagnosis. Here, we develop a graphene oxide-based fluorescence quenching sensor to quickly and accurately detect small amounts of a single strand of DNA. In this paper, fluorescent magnetic nanoparticles (FMNPs) modified [...] Read more.

Quick detection of DNA sequence is vital for many fields, especially, early-stage diagnosis. Here, we develop a graphene oxide-based fluorescence quenching sensor to quickly and accurately detect small amounts of a single strand of DNA. In this paper, fluorescent magnetic nanoparticles (FMNPs) modified with target DNA sequence (DNA-t) were bound onto the modified graphene oxide acting as the fluorescence quenching element. FMNPs are made of iron oxide (Fe₃O₄) core and fluorescent silica (SiO₂) shell. The average particle size of FMNPs was 74 ± 6 nm and the average thickness of the silica shell, estimated from TEM results, was 30 ± 4 nm. The photoluminescence and magnetic properties of FMNPs have been investigated. Target oligonucleotide (DNA-t) was conjugated onto FMNPs through glutaraldehyde crosslinking. Meanwhile, graphene oxide (GO) nanosheets were produced by a modified Hummers method. A complementary oligonucleotide (DNA-c) was designed to interact with GO. In the presence of GO-modified with DNA-c, the fluorescence intensity of FMNPs modified with DNA-t was quenched through a FRET quenching mechanism. Our study indicates that FMNPs can not only act as a FRET donor, but also enhance the sensor accuracy by magnetically separating the sensing system from free DNA and non-hybridized GO. Results indicate that this sensing system is ideal to detect small amounts of DNA-t with limitation detection at 0.12 µM. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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12 pages, 3444 KiB

Open AccessArticle

In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics

by Kira Erismann-Ebner, Anne Marowsky and Michael Arand

Biosensors 2019, 9(1), 33; https://doi.org/10.3390/bios9010033 - 28 Feb 2019

Cited by 3 | Viewed by 5928

Abstract

In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength [...] Read more.

In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength with highly variable cellular autofluorescence significantly compromised the sensitivity of our analyses. Here, we propose mCerulean3 and mRuby3 as a new FRET pair to potentially overcome these problems. Fusion of the two partners with a trypsin-cleavable linker allowed the direct comparison of the FRET signal characteristics of the associated partners with those of the completely dissociated partners. We compared our new FRET pair with the canonical CFP_YFP and the more recent mClover3_mRuby3 pairs and found that, despite a lower total FRET signal intensity, the novel pair had a significantly better signal to noise ratio due to lower donor emission bleed-through. This and the fact that the mRuby3 emission spectrum did not overlap with that of common cellular autofluorescence renders the mCerulean3_mRuby3 FRET pair a promising alternative to the common CFP_YFP FRET pair for the interaction analysis of membrane proteins in living cells. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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17 pages, 5252 KiB

Open AccessArticle

Energy Transfer between Tm-Doped Upconverting Nanoparticles and a Small Organic Dye with Large Stokes Shift

by Anna López de Guereñu, Philipp Bastian, Pablo Wessig, Leonard John and Michael U. Kumke

Biosensors 2019, 9(1), 9; https://doi.org/10.3390/bios9010009 - 08 Jan 2019

Cited by 18 | Viewed by 5279

Abstract

Lanthanide-doped upconverting nanoparticles (UCNP) are being extensively studied for bioapplications due to their unique photoluminescence properties and low toxicity. Interest in RET applications involving UCNP is also increasing, but due to factors such as large sizes, ion emission distributions within the particles, and [...] Read more.

Lanthanide-doped upconverting nanoparticles (UCNP) are being extensively studied for bioapplications due to their unique photoluminescence properties and low toxicity. Interest in RET applications involving UCNP is also increasing, but due to factors such as large sizes, ion emission distributions within the particles, and complicated energy transfer processes within the UCNP, there are still many questions to be answered. In this study, four types of core and core-shell NaYF₄-based UCNP co-doped with Yb³⁺ and Tm³⁺ as sensitizer and activator, respectively, were investigated as donors for the Methyl 5-(8-decanoylbenzo[1,2-d:4,5-d′]bis([1,3]dioxole)-4-yl)-5-oxopentanoate (DBD-6) dye. The possibility of resonance energy transfer (RET) between UCNP and the DBD-6 attached to their surface was demonstrated based on the comparison of luminescence intensities, band ratios, and decay kinetics. The architecture of UCNP influenced both the luminescence properties and the energy transfer to the dye: UCNP with an inert shell were the brightest, but their RET efficiency was the lowest (17%). Nanoparticles with Tm³⁺ only in the shell have revealed the highest RET efficiencies (up to 51%) despite the compromised luminescence due to surface quenching. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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13 pages, 1081 KiB

Open AccessArticle

Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B

by Zhaohui Wang and Pak Kin Wong

Biosensors 2018, 8(4), 126; https://doi.org/10.3390/bios8040126 - 10 Dec 2018

Viewed by 4328

Abstract

Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated [...] Read more.

Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA–protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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12 pages, 3840 KiB

Open AccessArticle

A DNA-Based Assay for Digoxin Detection

by Michael V. Kjelstrup, Line D. F. Nielsen, Malthe Hansen-Bruhn and Kurt V. Gothelf

Biosensors 2018, 8(1), 19; https://doi.org/10.3390/bios8010019 - 06 Mar 2018

Cited by 7 | Viewed by 7638

Abstract

The most common method for quantifying small-molecule drugs in blood samples is by liquid chromatography in combination with mass spectrometry. Few immuno-based assays are available for the detection of small-molecule drugs in blood. Here we report on a homogeneous assay that enables detection [...] Read more.

The most common method for quantifying small-molecule drugs in blood samples is by liquid chromatography in combination with mass spectrometry. Few immuno-based assays are available for the detection of small-molecule drugs in blood. Here we report on a homogeneous assay that enables detection of the concentration of digoxin spiked into in a plasma sample. The assay is based on a shift in the equilibrium of a DNA strand displacement competition reaction, and can be performed in 30 min for concentrations above 10 nM. The equilibrium shift occurs upon binding of anti-digoxigenin antibody. As a model, the assay provides a potential alternative to current small-molecule detection methods used for therapeutic drug monitoring. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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Review

Jump to: Research

17 pages, 1866 KiB

Open AccessReview

FRET Microscopy in Yeast

by Michal Skruzny, Emma Pohl and Marc Abella

Biosensors 2019, 9(4), 122; https://doi.org/10.3390/bios9040122 - 11 Oct 2019

Cited by 15 | Viewed by 11592

Abstract

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize [...] Read more.

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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35 pages, 21221 KiB

Open AccessReview

In Vivo Biosensing Using Resonance Energy Transfer

by Shashi Bhuckory, Joshua C. Kays and Allison M. Dennis

Biosensors 2019, 9(2), 76; https://doi.org/10.3390/bios9020076 - 03 Jun 2019

Cited by 31 | Viewed by 11104

Abstract

Solution-phase and intracellular biosensing has substantially enhanced our understanding of molecular processes foundational to biology and pathology. Optical methods are favored because of the low cost of probes and instrumentation. While chromatographic methods are helpful, fluorescent biosensing further increases sensitivity and can be [...] Read more.

Solution-phase and intracellular biosensing has substantially enhanced our understanding of molecular processes foundational to biology and pathology. Optical methods are favored because of the low cost of probes and instrumentation. While chromatographic methods are helpful, fluorescent biosensing further increases sensitivity and can be more effective in complex media. Resonance energy transfer (RET)-based sensors have been developed to use fluorescence, bioluminescence, or chemiluminescence (FRET, BRET, or CRET, respectively) as an energy donor, yielding changes in emission spectra, lifetime, or intensity in response to a molecular or environmental change. These methods hold great promise for expanding our understanding of molecular processes not just in solution and in vitro studies, but also in vivo, generating information about complex activities in a natural, organismal setting. In this review, we focus on dyes, fluorescent proteins, and nanoparticles used as energy transfer-based optical transducers in vivo in mice; there are examples of optical sensing using FRET, BRET, and in this mammalian model system. After a description of the energy transfer mechanisms and their contribution to in vivo imaging, we give a short perspective of RET-based in vivo sensors and the importance of imaging in the infrared for reduced tissue autofluorescence and improved sensitivity. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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14 pages, 853 KiB

Open AccessFeature PaperReview

Homotransfer FRET Reporters for Live Cell Imaging

by Nicole E. Snell, Vishnu P. Rao, Kendra M. Seckinger, Junyi Liang, Jenna Leser, Allison E. Mancini and M. A. Rizzo

Biosensors 2018, 8(4), 89; https://doi.org/10.3390/bios8040089 - 11 Oct 2018

Cited by 13 | Viewed by 5936

Abstract

Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the [...] Read more.

Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors. Full article

(This article belongs to the Special Issue FRET-Based Biosensors)

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