FRET-Based Genetically Encoded Sensor to Monitor Silver Ions

Abstract

Silver is commonly used in wound dressing, photography, health care products, laboratories, pharmacy, biomedical devices, and several industrial purposes. Silver (Ag+) ions are more toxic pollutants widely scattered in the open environment by natural processes and dispersed in soil, air, and water bodies. Ag+ binds with metallothionein, macroglobulins, and albumins, which may lead to the alteration of various enzymatic metabolic pathways. To analyze the uptake and metabolism of silver ions in vitro as well as in cells, a range of high-affinity fluorescence-based nanosensors has been constructed using a periplasmic protein CusF, a part of the CusCFBA efflux complex, which is involved in providing resistance against copper and silver ions in Escherichia coli. This nanosensor was constructed by combining of two fluorescent proteins (donor and acceptor) at the N- and C-terminus of the silver-binding protein (CusF), respectively. SenSil (WT) with a binding constant (K d) of 5.171 μM was more efficient than its mutant variants (H36D and F71W). This nanosensor allows monitoring the level of silver ions in real time in prokaryotes and eukaryotes without any disruption of cells or tissues.

Application Effect of Silver-Containing Dressings in the Repair of Chronic Refractory Wounds

“…Silver-containing dressings effectively eliminated susceptible bacteria, avoided the occurrence of clinical complications, and further ensured the cure rate of the disease. e reason why, silver sulfadiazine in silver dressings has a broad antibacterial spectrum and is active against both positive and negative bacteria, including molds and yeasts, especially Staphylococcus aureus and Pseudomonas aeruginosa (Pseudomonas aeruginosa) [23][24][25]. At the same time, the effective action time of the dressing is long, and after the silver ion-containing dressing loses its activity in the bacteria, the silver ion is freed from the bacteria and the bactericidal activity is repeated, with a longer antibacterial effect [26,27].…”

Fluorescent proteins and genetically encoded biosensors

“…In other FRET-based sensors, the Cu + -sensing domains from metallothionines like Atm1 and Ace1 have been inserted between the FRET pair, 273,274 or inserted into the EGFP sequence as BiFC (activation of the FRET-pair needs 4 Cu + ), 275 or into RFP as single-FP based sensors. 276 A FRET-based silver sensor has also been reported using the Cu + -transporting bacterial efflux pump CusF 277 (part of CusCFBA 278 ), with ECFP and Venus (a YFP variant). A single-FP based sensor for Cu 2+ normally uses the fluorescent quenching effect through the Cu 2+ -binding histidine residue on the interior of EGFP 279 or HcRed 280 (a red fluorescent protein from Heteractis crispa).…”

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors

“…For a nanosensor construct pRSET-B_ECFP_SeBP_Venus, inserting the amplified SeBP gene between ECFP and Venus in the pRSET-B vector and fidelity of the construct was confirmed by full-length sequencing (Figure S4). The pYES-DEST52 vector was used to carry the ECFP_SeBP_Venus sequences by a gateway cloning method utilizing the LR-Clonase-II enzyme (Invitrogen, USA) following the manufacturer's instructions 34 for expression of the sensor protein in yeast. The S. cerevisiae/URA3 strain BY4742 was used for transforming the pYDEST-ECF-P_SeBP_Venus plasmid as a eukaryotic expression system.…”