A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells

Abstract

Nitrate (NO3 –) is a critical source of nitrogen (N) available to microorganisms and plants. Nitrate sensing activates signaling pathways in the plant system that impinges upon, developmental, molecular, metabolic, and physiological responses locally, and globally. To sustain, the high crop productivity and high nutritional value along with the sustainable environment, the study of rate-controlling steps of a metabolic network of N assimilation through fluxomics becomes an attractive strategy. To monitor the flux of nitrate, we developed a non-invasive genetically encoded fluorescence resonance energy transfer (FRET)-based tool named “FLIP-NT” that monitors the real-time uptake of nitrate in the living cells. The developed nanosensor is suitable for real-time monitoring of nitrate flux in living cells at subcellular compartments with high spatio-temporal resolution. The developed FLIP-NT nanosensor was not affected by the pH change and have specificity for nitrate with an affinity constant (K d) of ∼5 μM. A series of affinity mutants have also been generated to expand the physiological detection range of the sensor protein with varying K d values. It has been found that this sensor successfully detects the dynamics of nitrate fluctuations in bacteria and yeast, without the disruption of cellular organization. This FLIP-NT nanosensor could be a very important tool that will help us to advance the understanding of nitrate signaling.

A March to Shape Optical Artificial Olfactory System toward Ultrasensitive Detection of Improvised Explosives

“…From the research on optical detection of IEs from 2010 to 2021 ( Figure ), it can be found that the detection limits of hypochlorite, [ 41–57 ] perchlorate, [ 58–60 ] peroxide, [ 61–77 ] nitrate, [ 78–85 ] and nitrite [ 86–99 ] based on solution reaction are mostly between 10 −5 and 10 −9 M , and it is difficult to achieve higher sensitivity detection by merely optical probes designing. By combining the sensing probes with paper‐based [ 19,100–103 ] and hydrogel‐based [ 28,104–106 ] substrate and optical microscope device, the detection sensitivity has been greatly improved in 2020 and 2021 reaching a level of 10 −15 M. To better improve the detecting sensitivity, optical artificial olfactory system takes chemical sensor array as the detection modules of different IEs, as well as considering the automatic analyte collection with airflow and the ultrasensitive ecognition of optical signals has been explored from the design of different components.…”

“…Researches on optical detection of hypochlorite, [ 41–57 ] perchlorate, [ 58–60 ] peroxide, [ 61–77 ] nitrate, [ 78–85 ] and nitrite [ 86–99 ] IEs from 2010 to 2021 and sensors with paper‐based [ 19,100–103 ] and hydrogel‐based [ 28,104–106 ] substrate and optical microscope device. The first Figure from left on bottom reproduced with permission.…”

“…For the realization of detecting more IEs, our group explored and integrated a series of colorimetric principles for the detection of potassium permanganate, S, urea, sugar, and other components of IEs. [28] From the research on optical detection of IEs from 2010 to 2021 (Figure 3), it can be found that the detection limits of hypochlorite, [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] perchlorate, [58][59][60] peroxide, [61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77] nitrate, [78][79][80][81][82][83][84][85] and nitrite [86]…”

“…[112] Shoronia et al used 4-dimethyl-aminocinnamaldehyde to react with nitrourea in neutral environment to form a red protonated Schiff base structure, which is the condensation reaction product between amino group and carbonyl group, and thus realized the detection of residue on fingers (Figure 4b). [113] The reaction Researches on optical detection of hypochlorite, [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] perchlorate, [58][59][60] peroxide, [61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77] nitrate, [78][79][80][81][82][83][84][85] and nitrite…”

“…Figure3. Researches on optical detection of hypochlorite,[41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] perchlorate,[58][59][60] peroxide,[61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77] nitrate,[78][79][80][81][82][83][84][85] and nitrite[86][87][88][89][90][91][92]…”