scholarly journals Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Ruphi Naz ◽  
Mohammad K. Okla ◽  
Urooj Fatima ◽  
Mohd. Mohsin ◽  
Walid H. Soufan ◽  
...  

N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...


2000 ◽  
Vol 38 (8) ◽  
pp. 2929-2932 ◽  
Author(s):  
J.-M. Costa ◽  
C. Pautas ◽  
P. Ernault ◽  
F. Foulet ◽  
C. Cordonnier ◽  
...  

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify Toxoplasma gondii DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 × 106 to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to ≤0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.


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