scholarly journals A FRET-ICT Dual-Modulated Ratiometric Fluorescence Sensor for Monitoring and Bio-Imaging of Cellular Selenocysteine

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 4999
Author(s):  
Zongcheng Wang ◽  
Chenhong Hao ◽  
Xiaofang Luo ◽  
Qiyao Wu ◽  
Chengliang Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Fluorescence Sensor ◽  
Quantitative Detection ◽  
Fluorescence Signal ◽  
Ratiometric Fluorescence ◽  
Sensitivity And Selectivity

Since the fluctuation of cellular selenocysteine (Sec) concentration plays an all-important role in the development of numerous human disorders, the real-time fluorescence detection of Sec in living systems has attracted plenty of interest during the past decade. In order to obtain a faster and more sensitive small organic molecule fluorescence sensor for the Sec detection, a new ratiometric fluorescence sensor Q7 was designed based on the fluorescence resonance energy transfer (FRET) strategy with coumarin fluorophore as energy donor and 4-hydroxy naphthalimide fluorophore (with 2,4-dinitrobenzene sulfonate as fluorescence signal quencher and Sec-selective recognition site) as an energy acceptor. The sensor Q7 exhibited only a blue fluorescence signal, and displayed two well distinguished emission bands (blue and green) in the presence of Sec with ∆λ of 68 nm. Moreover, concentrations ranging of quantitative detection of Sec of Q7 was from 0 to 45 μM (limit of detection = 6.9 nM), with rapid ratiometric response, high sensitivity and selectivity capability. Impressively, the results of the living cell imaging test demonstrated Q7 has the potentiality of being an ideal sensor for real-time Sec detection in biosystems.

scholarly journals Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252887
Author(s):  
Renate Schneider ◽  
Aline Lamien-Meda ◽  
Herbert Auer ◽  
Ursula Wiedermann-Schmidt ◽  
Peter L. Chiodini ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Melting Curve ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Significant Rise ◽  
Quantitative Detection ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

scholarly journals An Aptamer-Array-Based Sample-to-Answer Biosensor for Ochratoxin A Detection via Fluorescence Resonance Energy Transfer

Chemosensors ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 309
Author(s):  
Yongning Li ◽  
Zhenfei Peng ◽  
Yaxi Li ◽  
Min Xiao ◽  
Gongjun Tan ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Ochratoxin A ◽  
Fluorescence Resonance Energy Transfer ◽  
Limit Of Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Detection Methods ◽  
Fluorescence Signal ◽  
Fluorescence Resonance

Food toxins are a hidden threat that can cause cancer and tremendously impact human health. Therefore, the detection of food toxins in a timely manner with high sensitivity is of paramount importance for public health and food safety. However, the current detection methods are relatively time-consuming and not practical for field tests. In the present work, we developed a novel aptamer-chip-based sample-to-answer biosensor (ACSB) for ochratoxin A (OTA) detection via fluorescence resonance energy transfer (FRET). In this system, a cyanine 3 (Cy3)-labeled OTA-specific biotinylated aptamer was immobilized on an epoxy-coated chip via streptavidin-biotin binding. A complementary DNA strand to OTA aptamer at the 3′-end was labeled with a black hole quencher 2 (BHQ2) to quench Cy3 fluorescence when in proximity. In the presence of OTA, the Cy3-labeled OTA aptamer bound specifically to OTA and led to the physical separation of Cy3 and BHQ2, which resulted in an increase of fluorescence signal. The limit of detection (LOD) of this ACSB for OTA was 0.005 ng/mL with a linearity range of 0.01–10 ng/mL. The cross-reactivity of ACSB against other mycotoxins, ochratoxin B (OTB), aflatoxin B1 (AFB1), zearalenone (ZEA), or deoxynilvalenol (DON), was less than 0.01%. In addition, this system could accurately detect OTA in rice samples spiked with OTA, and the mean recovery rate of the spiked-in OTA reached 91%, with a coefficient of variation (CV) of 8.57–9.89%. Collectively, the ACSB may represent a rapid, accurate, and easy-to-use platform for OTA detection with high sensitivity and specificity.

AMPLIFIED BIOSENSING STRATEGIES FOR THE DETECTION OF BIOLOGICALLY RELATED MOLECULES WITH SILICA NANOPARTICLES AND CONJUGATED POLYELECTROLYTES

COSMOS ◽  
2010 ◽  
Vol 06 (02) ◽  
pp. 207-219
Author(s):  
LIHUA WANG ◽  
YANYAN WANG ◽  
JIE ZOU ◽  
BIN LIU ◽  
CHUNHAI FAN
Keyword(s):  
Silica Nanoparticles ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Cost Effective ◽  
High Specific Surface Area ◽  
Conjugated Polyelectrolytes ◽  
Signal Amplifier ◽  
Biomedical Diagnostics ◽  
Sensitivity And Selectivity

Development of rapid, field-portable and cost-effective sensors with high sensitivity and selectivity is of great importance for biomedical diagnostics, food safety and environmental monitoring. Silica nanoparticles (SiNPs) have great potential in sensor application due to their biocompatibility, controllable surface modification, excellent chemical stability and high specific surface area. On the other hand, conjugated polyelectrolytes (CPEs) have been widely used in sensor design due to their efficient Förster resonance energy transfer (FRET) to dyes and unique interaction with biomolecules. In this contribution, we briefly summarize the recent development of silica-related NP-based assays that incorporate CPEs as the signal amplifier or reporter. The silica-related NPs are used for probe immobilization, target recognition and separation, while CPEs provide amplified fluorescence signals and high sensitivity. These assays have been proven efficient for the detection of DNA, proteins, and small molecules through specific biorecognition events, such as DNA hybridization, antibody–antigen recognition and target–aptamer binding.

scholarly journals A Ratiometric Fluorescent Aptamer Homogeneous Biosensor Based on Hairpin Structure Aptamer for AFB1 Detection

2022 ◽  
Author(s):  
Beibei Feng ◽  
Fei Zhao ◽  
Min Wei ◽  
Yong Liu ◽  
Xinyu Ren ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Resonance Energy Transfer ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Hairpin Structure ◽  
Fluorescence Signal ◽  
Detection Range ◽  
Quenching Effect ◽  
Double Stranded Dna ◽  
Aflatoxin B

Abstract On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B1 (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescent aptamer sensor demonstrates a weak fluorescence emission of Cy3 and a high fluorescence emission of Cy5 due to the quenching effect of BHQ2. The double-stranded DNA structure will be disintegrated in the presence of AFB1, resulting the removal of Cy3 and the close of Cy5 with BHQ2. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of FCy3 to FCy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, indicating it had good application potential in practical samples.

scholarly journals Mechanism of DNA surface exploration and operator bypassing during target search

2020 ◽  
Author(s):  
E. Marklund ◽  
B. van Oosten ◽  
G. Mao ◽  
E. Amselem ◽  
K. Kipper ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Single Molecule ◽  
Time Scale ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Signal ◽  
Target Search ◽  
Fluorescence Correlation ◽  
Speed Accuracy

SummaryMany proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1–5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 μs. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

Amplified Fluorescence Turn-on Assay for Mercury(II) Based on Conjugated Polyfluorene Derivatives and Nanospheres

2008 ◽  
Vol 1134 ◽  
Author(s):  
Yusong Wang ◽  
Bin Liu
Keyword(s):  
Metal Ion ◽  
Resonance Energy Transfer ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Probe Design ◽  
Turn On ◽  
Sensitivity And Selectivity ◽  
Fluorescence Turn On ◽  
Polyfluorene Derivatives

AbstractDetection of mercury with high sensitivity and selectivity constitutes a significant research concern. Here, we report an amplified fluorescence turn-on assay for mercury(II) with an improved performance. This sensing system takes advantage of optically amplifying fluorescent conjugated polyfluorene derivatives and DNA immobilized silica nanospheres (NSs) in addition to the specific thymine- mercury(II)-thymine(T- Hg2+-T) interaction. The employment of ion-specific T- Hg2+-T coordination increases the melting temperature (Tm) of the double-stranded DNA (dsDNA) on the hybridized NS surface. After thermal washing at 45 °C, the Hg2+ treated sample (dsDNA-NS) was effectively differentiated from that treated with nonspecific ions through monitoring fluorescence emission of fluorescein (Fl) labeled target DNA remained on the NS surface. Finally, a cationic conjugated polyfluorene derivative (CCP) was introduced to electrostatically associate with the DNA molecules on the NS surface, resulting in an amplified Fl signal via fluorescence resonance energy transfer (FRET) from the CCP to the dye molecule. In comparison with the use of Fl alone as a signal reporter, the presence of CCP significantly enhances the detection fluorescence intensity, reduces false-positive signal, and improves the detection selectivity for mercury(II). Further improvement in the probe design could yield more efficient metal ion sensors, which have the potential to be operated at room temperature and for the detection of other metal ions besides mercury(II).

scholarly journals Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5′-Exonuclease, and Hybridization Probe Assays

2003 ◽  
Vol 69 (8) ◽  
pp. 4753-4759 ◽  
Author(s):  
D. T. Newby ◽  
T. L. Hadfield ◽  
F. F. Roberto
Keyword(s):  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sybr Green ◽  
Sybr Green I ◽  
Fluorescence Signal ◽  
Insertion Element ◽  
Hybridization Probe

ABSTRACT Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5′-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

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