7.2. Fluorescent Protein-Based Redox (FPBR) Analysis

Fluorescent protein-based redox (FPBR) probes are formulated by combining fluorescent and prokaryote redox sensitive proteins. The recombinant proteins are administered to the cells via adenovirus or plasmid and targeted on subcellular organelles, to determine the redox state of certain regions [210]. Recomb-proteins redox dependent fluorescence change is attained by change in structure of disulfide bonds and main chain in oxidized conditions. The FPBR probe affords real time and dynamic detection of change in redox potential of reaction (that involve radicals) without special need for permeation in target cells. The fluorescent proteins (FP) reside in cells and allow long-term detection of cellular radicals. The combination of FP with targeting signal peptide or retention sequence allows FP to target various organelles, and therefore reveals redox status. For redox detection, the FPRB analysis involves numerous colors of redox-sensitive targeting proteins.

Formulation of redox-sensitive green fluorescent protein (roGFP) involves addition of redox reactive cysteine in GFP beta-strand 7 and 10 at site Q204 and S147. Under a reduced environment, it causes formation of disulfide linkage between two domains, which reacts to redox changes in intra/extra-cellular systems, leads to reversible ratiometric change in the intensity of fluorescence [211]. Based on roGFP, numerous probes are developed.

roGFP 1 and 2 are the first two analogues of roGFP that are differentiated based on the amino acid (T65S) unit. These may indicate conversion of dithiol/disulfide that is stimulated through ROS accumulation. These are used to examine variation in thiol/disulfide equilibrium [149]. The cysteine pair of roGFP 1 and 2 is protonated under physiological pH. Rather than direct ROS measurement, roGFP1 and 2 determine dynamic redox change. As roGFP1 and 2 undergo complete oxidation by oxidating organelles (lysosomes and endosomes), they can be appropriate for reduced environments (mitochondria, cytoplasm, peroxisomes, and nucleus) [212]. The sensitivity of roGFP also depends upon pH and speed of reaction. As intensity of roGFP fluorescence does not change quickly with change in redox condition, this indicates suitability of roGFP in monitoring the long-term redox shifts [210]. Moreover, in comparison to roGFP1, roGFP2 is easily influenced by variation in pH (ranged between 6 to 8), hence detection condition is an important aspect to consider.

Depending upon roGFP1 usage in oxidizing environment, the roGFP1-iX was formulated. The formulation of roGFP1-iX involves incorporation of the amino acids next to cys147 and mutation of H148S in roGFP1 beta-strand 7. The roGFP1-iX offers fast reaction speed and low pH sensitivity (ranged from 6 to 8) in comparison to roGFP1. This results in roGFP1-iX suitability in monitoring redox in oxidating organelles such as endoplasmic reticulum (ER) [213,214].

roGFP1 probe was modified to roGFP1-iL for sensing the redox of ER. The roGFP1-iL disulfide bonds are partly oxidized, which generates high reduction potential to examine the variation in redox condition [215]. roGFP1-iL and Grx1 in combination exhibit high sensitivity towards 2GSH or GSSG. In comparison to roGFP1, roGFP1-RX exhibits high reaction speed and dynamic range through addition of three amino acids (carrying positive charge) next to cysteine [216].

Just like roGFP1, derivatives were also created for roGFP2. The broad midpoint potential of Grx1-roGFP2-iL makes it suitable for determination of redox in ER and cytosol. In comparison to roGFP2, the derivative Grx1-roGFP2-iL exhibits higher specificity in measurement of redox potential of GSH [192]. The derivative probe roGFP2-Orp1 is H₂O₂-specific. roGFP2-Orp1 was created by combining roGFP2 with yeast peroxidase Orp1, and redox relay equivalent between the two allows the roGFP2-Orp1 probe to efficiently indicate the H₂O₂ level [217]. The pH stability of this derivative probe makes it applicable for sensing alteration in H₂O₂ levels in mitochondria and cytosol in micromole [218]. The roGFP2-Orp1 probe exhibits superior selectivity towards H₂O₂. It is important to note that none of the studies reported roGFP probes-based analysis to determine the concentration or net formation of H₂O_2. In fact, roGFP probes can only sense the variation in H₂O₂ or redox level instigated by external influences.

These are created using yellow shifted GFP-derived protein (YFP), wherein the cysteine pair is incorporated next to chromophoric domain, thereby forming a reversible disulfide bond. The oxidative environment leads to a change in spatial conformation of YFP that causes decrease in fluorescence intensity (near 527 nm) which allows visualization of cellular redox in vivo [219]. Attributed to suitable midpoint redox potential, the rxYFP can be used to sense the dynamic change in GSH, GSSG, thiol, and disulfide in various reducible cellular regions (such as cytosol, mitochondria, and nucleus) [220]. Generally, rxYFP targets to examine the whole redox change rather than sensing of particular redox couple.

This is the modified probe of rxYFP, which is engineered by combining glutaredoxin-1 of yeast (Grx1p) and rxYFP. This probe is suitable for sensing the redox potential of intracellular GSH [221]. This is more specific to GSH in comparison to H₂O₂, hydroxyethyl disulfide, and cysteine.

This protein includes addition of three more positive charged cysteine residues; that is, 200R/204R/227R in rxYFP results in formation of rxYFP3R, that exhibits 13 times higher reactivity for GSH when compared with rxYFP [222]. It is important to note that rxYFP modified probes exhibit enhanced specificity for 2GSH and GSSG, and improved stability at physiological pH. rxYFP exhibits pH sensitivity and their chromophores pKa changes as per the pH of environment. In neutral environments, quenching of fluorescence occurs on the rxYFP protonated form, which the reduces fluorescence signal by 2.2 times with nonsignificant excitation wavelength shift [223]. However, this probe is unsuitable for ratiometric quantification of redox.

This is H₂O₂ sensing cpYFP-OxyR recombinant protein. HyPer is engineered by incorporation of circularly permuted yellow fluorescent protein (cpYFP) into regulating domain (RD) of prokaryote H₂O₂ sensing OxyR protein. This creates disulfide linkage between reactive Cys208 and Cys199 of RD on reaction with H₂O₂; which alters conformation of OxyR, that further changes fluorescence intensity and conformation of whole protein and thus correlates alteration in level of H₂O₂ with intensity of fluorescence. HyPer is applicable to various cellular systems and living organisms (caenorhabditis, arabidopsis, yeast, elegans, and mouse) to study intracellular dynamic change and real time analysis of H₂O₂ [224,225]. In comparison to roGFP2-Orp1, the HyPer exhibits high reaction speed and sensitivity towards H₂O₂, attributed to position of redox sensitive cysteine pair in HyPer [226]. To effectively detect the change in H₂O₂ level, the oxidized HyPer must reduce in time, which is mediated through intracellular GSH [227]. Therefore, HyPer can be used to determine equilibrium between GSH and H₂O₂.

These are the HyPer derivative probes that are created via mutation of A406V (for HyPer-2) followed by mutation of H34Y (for HyPer-3) in HyPer [228,229]. HyPer-2 exhibits higher stability than HyPer-3 amongst monomers in OxyR dimers, whereas HyPer-3 exhibits faster response time and reaction speed towards H₂O₂. Both HyPer-2 and 3 exhibits broad dynamic range as compare to HyPer; and used for fluorescence lifetime in vivo imaging of H₂O₂. The change of pH environment is a major challenge for HyPer probes. As in cells the H₂O₂ concentration depends upon disproportion of O₂˙⁻, and an increase in pH may decrease dismutation rate of O₂˙⁻, while using such sensors one must focus systemic or local pH.

The probe of cpYFP (formerly pericam) was initially used to detect calcium, but exertion of ratiometric fluorescence flash by cpYFP/Pericam due to rise in O₂˙⁻ level proved cpYFP as an effective tool for imaging of O₂˙⁻ [230]. Binding of cpYFP to mitochondrium targeting sequence, permits cpYFP to sense subcellular level ROS [231]. One study [232] claims cpYFP responses to variation in pH, which suggests cpYFP specificity towards O₂˙⁻ as point for further discussion. It is important to notice that as cpYFP fluorescence intensity is influenced by mitochondrial matrix alkalinity, hence precaution must be taken while imaging mito-O₂˙⁻.

This gene encoded red fluorescent probe assists in detection of H₂O₂. This HyPerRed probe is engineered by substitution of sensing domain of calcium probe R-GECO1 with OxyR. The disulfide bond formation between Cys208 and Cys199 develops HyPerRed specificity for H₂O₂ identification [233]. The sensitivity and kinetics of HyPerRed for H₂O₂ is just like HyPer probe. HyPerRed has the ability to detect H₂O₂ in low concentration, and its high sensitivity to change in pH necessitates for adjacent analysis, such as HyPer-C199S or HyPerRed-C199S, which are used standard probe for variation in pH [233]. Apart from that, the non-ratiometric reaction among H₂O₂ and HyPerRed (which limits HyPerRed application for quantification) necessitates other GFP sensors as controls for quantification assays.

Based on redox-RFP, several other fluorescent biosensing probes have been constructed. In order to monitor the redox dynamics in case of thioredoxin and thiol/disulfide transformation, two probes, namely TrxRFP1 and cpRFP, were engineered; they assist in the analysis of variation in local or general redox-state in mammalian cells [234,235]. The constructed RFP probes assist in analysis of various redox states and free radicals, but RFP probes also offer some limitations. As construction of RFP probes is a complicated process, during analysis the potential factors must be monitored. For example, the nature of target recipient cells may affect the RFP expression level that in turn impacts the probe capability. The roGFP undergoes reversible oxidation on exposure to oxygen and is less sensitive to oxidants in vivo in comparison to in vitro, attributed to AO presence [236]. The rate of reaction between these RFP proteins and redox substances are slow, which causes less immediacy in intra-cellular radical determination. Just like roGFP2-Orp1 and HyPer, the selectivity of these RFP probes against H₂O₂ is based on a reversible oxidation reduction that is facilitated by H₂O₂ and 2GSH/GSSG couple. However, their reaction capacity difference may cause inaccurate data analysis. As protein-based biosensors offer difficulty in quantitative analysis of oxygen free radicals, it necessitates calibration and verification during analysis. The fluorescence analytical tool also imposes high impact over protein-based biosensors data. Use of microscopy with fluorescence can select various types of cells, but it is unsuitable for large quantity sample imaging. Flow cytometer is commonly used in mammalian cell analysis, whereas laser scanning plate readers are applicable for tissues with high throughput screening (HTS) [237]. Although fluorescent microplate readers offer fluorescence data, they cannot generate information at the cellular or sub-cellular level.