Dual emission and its λ-ratiometric detection in analytical fluorimetry Pt. II. Exploration in sensing and imaging

The high-quality spectroscopic instrumentation is needed only on the steps of fluorescence assay development. The choice and testing of fluorescence reporters by their spectroscopic parameters is the necessary procedure. As we will see below, the spectroscopic changes on sensor events are frequently presented in original publications. Then, the simplified version of the technology is described that involves collecting the emission at selected wavelengths or even visual detection.

The λ-ratiometric signal is easily detectable in all instrumental formats (fluorimeters, microscopes, flow cytometers, plate readers, etc) by selecting the filters on fluorescence channels. Simple low-cost ratiometric fluorimeters are known for many years [7]. Diode lasers and digital color cameras [8] introduced compact design and substantial improvement of performance. Modern industry producing optical instruments is oriented on manufacturing of compact handheld and specializes devices [9]. Development of various lab-on-a-chip techniques [10] allowed achieving miniaturization, integration and systematization of various sensor types together with their multi-analyte monitoring in parallel.

A variety of friendly for personal use pocket-size instruments has appeared [11, 12], and the colored smartphone matrices were found ideal for providing the λ-ratiometric output. Recording the ratio of intensities between spectrally resolved emission bands becomes the most precise procedure.

Paper-based sensors are simple to manufacture, inexpensive, portable and disposable. They are quite applicable for diagnostics, human activity monitoring, food safety and environmental detection [13]. The filter paper or microporous membrane can be used as support for printing the fluorescent probes. By immobilizing or dripping λ-ratiometric fluorescent probes onto the surface of solid substrates or incorporating into their porous volume, it is easy to operate with them for realizing the real-time visualized color detection. The novel fluorescent materials, particularly nanomaterials, allowed achieving the brightness sufficiently high for detection together with their wavelength-shifting capability.

However, an important weakness of visual detection is apparent. It can be imprecise due to color mixing [14], since two primary colors can produce an intermediate color, making hard to estimate the contributions of primary colors. It is even harder to operate visually with three-color response [15, 16]. That is why it is important to reduce the effect of color mixing by proper choice of reporters with optimal spectral resolution and optimal spectral location of 'pure colors'. Though a progress has been achieved in visual analysis with paper-based sensors, it is difficult to reach satisfactory sensitivity and accuracy just because our naked eyes are unable to distinguish a slight color change.

Still, the proper choice of analyte-responding and reference fluorophores and the broadest spectral difference of their fluorescence colors may allow achieving good results in visual color-changing analyte detection [17]. Thus, a profuse color-evolution-based fluorescent test paper sensor for rapid and visual monitoring of Cu²⁺ ions in human urine was suggested [15]. It uses the tricolor probe system, selected for optimal visual perception (figure 1). Upon the addition of different amounts of Cu²⁺, the ratiometric fluorescence intensity of this probe varies continuously, leading to color changes from shallow pink to blue with a detection limit of 1.3 nM. The test is simple, rapid and inexpensive. A portable UV lamp is only needed for the detection of target analyte by the naked eye.

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Smartphones have become a part of everyday life. In addition to the ability to make voice calls, they allow using high-resolution cameras, providing data processing, presentation and exploring internet connectivity. Their transformation into multi-channel fluorescence detectors can be easily done with incorporation of diffraction gratings or filters [18]. Using smartphones or their CMOS matrices as detector units, the very inexpensive spectrofluorimeters [19], fluorescence imagers [20] and microscopes [21] were developed. They change the performance of paper strip-based, microfluidic and immunoanalytical techniques and provide easy analysis of microarrays.

The digital imaging features of smartphones make them the outstanding analytical platforms for realizing ratiometric fluorescent strategies to develop the handheld and point-of-care testing sensors. They offer an effective way to establish warning-early mechanisms against environmental and food safety risks, safeguarding public health [22]. Typically for λ-ratiometric detection, two emitters are involved, in one of which the fluorescence is quenched/enhanced in sensing process with a strong change of detected emission color [23, 24]. For quantifying the target concentration with a smartphone, the intensity variations of three primary colors (red-green-blue, RGB) can be transformed into intensity ratio of different colors, such as R/G.

In fact, any mobile device can be easily turned into a portable fluorescence light detector by developing simple accessories that create a mini-darkbox around the photocamera, preventing ambient light from interfering with the test's light signal [25]. At present, most reported smartphone sensing processes rely on the dark environmental conditions. Very recently, 3D-printing technology was introduced to integrate with smartphone all necessary optical elements [26].

The smartphone-based sensing platform combined with a paper strip was suggested for the detection of pesticides in fruits in real-time/on-site conditions [27]. It was integrated with a UV lamp and dark cavity by 3D-printing technology and allows visual quantitative detection of pesticide with a dual-emissive ratiometric paper strip (figure 2). Red-emitting CdTe quantum dots (rQDs) were embedded into the silica nanoparticles (SiO₂ NPs) as an internal reference, while blue-emitting carbon dots (bCDs) as a signal report unit were covalently linked to the outer surface of SiO₂ NPs. The blue fluorescence is quenched by gold nanoparticles (Au NPs). Then it is recovered when they dissociate under the interaction with pesticide. The red, green and blue channel values of the generated images were determined by a color recognizer application (APP) installed in the smartphone, and the red/blue values could be used for pesticide quantification with a sensitive detection limit (LOD) of 59 nM.

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It is commonly recognized that enzyme-linked immunosorbent assays (ELISAs) or related fluorescence sandwich immunoassays that are presently applied to a broad range of clinically relevant analytes do not fit for point-of-care applications since they do not satisfy the requirements for speed, low cost, and ease of use [28]. Possessing the advantages of high specificity and sensitivity, they require multiple steps performed by skillful personnel: washing, incubation, external calibration, reagent addition and optimization of assay conditions. Ideally, a diagnostic test should be such that it could be performed anywhere, regardless of facilities [29].

Lateral flow immunoassays (LFIAs) were introduced as cheap and relatively easy-to-use point-of-care tests that are configured to mimic a heterogeneous ELISA-like workflow by use of capillary action to separate bound from unbound material. It allows to capture the analyte-specific detection signal avoiding the sample-to-lab transition and the need for multiple wash steps [30]. However, the LFIAs suffer from limited sensitivity, and their primary use is only as qualitative tests [31]. Most attempts for their improvement were made to increase the brightness of reporting signal for making it visible by naked eye or recorded by simple instrumentation [30].

Several recent publications demonstrated strong improvement of LFIA test sensitivity when, instead of single-color detection, a λ-ratiometric approach was realized. The scheme for such test that allows the naked eye semi-quantitative readout of aflatoxin M₁ concentration is presented in figure 3 [32]. Utility of such approach was also demonstrated on detection of immunoglobulins (IgG) [33]. The red-fluorescent CdSe@ZnS quantum dots (655-QDs) were modified with target-specific antibodies, and the target-independent blue fluorescent polystyrene nanobeads (450-NBs) were immobilized on the test line together with these antibodies. The presence of the target and the subsequent immune-sandwich formation induced the accumulation of the 655-QDs. It was possible to generate a wide color palette (blue, purple, pink, red) on the test line informing on analyte concentration.

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There were many attempts to develop homogeneous immunoassays, in which the reversible interaction (molecular recognition) based on affinity between antigen and antibody could provide direct response rapidly and in one step [29, 34]. The problem is that the signal generation should be directly coupled with molecular recognition and demonstrate sufficiently bright change in self-referenced parameter of fluorescence emission.

Regarding highly needed detection of antibodies, different suggestions were made on the generation of ratiometric signal in response to binding of analyte antibody with molecular constructions that contained the antigenic site (epitope). In a flexible peptide that incorporates the epitope of 3–4 amino acids in length and terminates from both sides with pyrene groups, these groups may form excimers that become disrupted on interaction with antibody (figure 4). Thus, the λ-ratiometric signal comes from switching between excimer and monomer emissions [35]. Disruption of close interaction between FRET donor and acceptor (that can be presented by two fluorescent proteins) may occur if the extended link between them allows binding to two sites of Y-shaped bivalent antibody [36]. The FRET system can also be formed on binding to the same antibody of two peptides, one containing the donor, and the other the acceptor [37].

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A prototype of λ-ratiometric sensor based on peptide labeled with λ-ratiometric 3-hydroxychromone dye was developed [38]. Using a model of high-affinity interaction between an 18-aminoacid antigenic peptide derived from tobacco mosaic virus coat protein and a recombinant antibody fragment, Fab 57P, a dramatic change of fluorescence spectrum was observed. Due to ESIPT reaction, the ratio of intensities of two bands (I_N*/I_T*) in the dye fluorescence spectrum changed dramatically (from 0.92–0.94 to 0.60) and remained unchanged in the presence of nonspecific fragments (figure 5).

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The spotted microarrays are the assemblies of a great number (hundreds and thousands) of sensor instruments, in which every instrument is represented by a spot on a surface formed by the deposition of one type of sensor molecules or particles. The read-out of reporter signal is made using the specialized fluorescence microscope instruments—the microplate readers. The analyzed sample is exposed to whole microarray, so the binding of different targets to correspondent sensor instruments occurs in essentially the same conditions. Sensor arrays can detect many analytes simultaneously, adapt to several analyte concentration ranges, provide multi-analyte comparison between the samples, realizing the 'chemical nose/tongue' approach [12, 39].

Implementing the two-color readout is important for improving the performance of array-based technique [40], which was shown in high-throughput analysis of proteins [41], disease markers [42] and pathogenic microbes [43]. The attempts to provide multicolor fluorescence reporting were based on 'sensing-with-the-reference' concept, combining two OFF-ON and ON-OFF responses [44], arranging the FRET pairs [45] and ESIPT reactions [43].

For tracing dopaminergic agents that are important in diagnosis of Parkinson's disease in human urine, a fluorescence multicolor electronic tongue was developed [42], figure 6(a). A smartphone camera was used to take photos from the solutions in the wells. Distinct changes in the spectral profiles along with vivid and concentration-dependent color variations led to visual discrimination of dopaminergic agents in a broad concentration range.

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Sensor arrays based on small molecules offer a chemically stable and cost-effective alternative identification of pathogenic microorganisms. The outstanding properties of 3HC dyes to provide an ESIPT-based dual-channel ratiometric response, allowed by using discriminant analysis of the sensor array responses, effective distinguishing between eight bacterial species and recognition of their Gram status [43], see figure 6(b).

Thus, a rainbow of switchable colors of easily generated and recorded fluorescence can be transformed into informative analytical signals with the present-date instrumentation adapted for particular assay. Miniaturization of all optical elements and their fabrication from inexpensive materials and in the form of integrated units are the present tendencies in instrument design. Even in the simple versions of the λ-ratiometry that sometimes look primitive, this concept competes successfully with lifetime and anisotropy techniques that also allow instrument-independent output but use a more complicated type of measurement. New designs and new improvements continue.

The appearance of new bands in absorption and fluorescence spectra as a function of proton dissociation/association in organic dyes is basic in versatile measurements of pH [46]. In the collection of many organic dyes, one may find those changing the emission color in neutral and weakly alkaline pH range [47] or at strongly acidic pH [48]. The fluorescence spectra of one of the dyes titrating in low pH region are presented in figure 7 [49].

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Multiple titrating groups and more complicated reaction schemes can be introduced for extending the pH range [50], up to extreme acidic and alkaline values [51]. The need of operation in different detection formats and increased requirements towards chemical stability and photostability of the probes stimulated development of pH-sensing nanostructures [46]. Incorporation into polymer nanoparticles of dyes with different pH range of response allowed extending the dynamic range of pH measurements [52, 53]. Extending the pH range and improvement of performance was also achieved with dual-emitting functionalized gold nanoclusters [53] and metal-organic frameworks [54]. The sensitivity sufficient for visual detection of change in emission color was achieved for some of these structures [55].

Intracellular pH, especially cytoplasmic pH (~7.2) plays a crucial role in cell functions and metabolism, and a number of λ-ratiometric probes were designed for these studies [47, 56, 57]. The pH values in different organelles differ from cytoplasmic pH and their study is of special importance but needs the probes with high affinity to these organelles. There are the λ-ratiometric pH sensors for mitochondria that operate in neutral-alkaline pH range [58]. In contrast, pH in lysosomes is variable in acidic range, and a number of probes were developed for detecting and analyzing these variations [59, 60]. A bicolor fluorescence imaging becomes efficient tool in cellular research [61].

Metal cations are very important for biology and medicine. These include sodium (Na⁺) potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), copper (Cu⁺ and Cu²⁺) and zinc (Zn²⁺) ions. Health and environmental problems may be caused by toxic metal ions, such as lead (Pb²⁺), cadmium (Cd²⁺) and Mercury (Hg²⁺). Perturbation by their charge of π-electronic structure of organic dyes resulting in the appearance of intramolecular charge transfer (ICT) bands is the general principle of generation their λ-ratiometric response [62, 63]. The sensors based on formation of intramolecular and intermolecular excimer complexes or excited-state energy transfer between molecular fragments have also been developed.

The reader is strongly advised to study original paper of Roger Tsien [64] on the development of the first λ-ratiometric Ca²⁺ probes FURA-2 and INDO-1 (figure 8). In these stilbene-like dyes, the bound Ca²⁺ ions interact with the electron-donor nitrogen atom incorporated into the fluorophore. Strong electrostatic interaction with the ion generates a new ground-state form with its light absorption and excitation band shifted to shorter wavelengths. The two forms with bound and unbound analyte behave as different species, and the Ca²⁺ binding-release results in interplay of two excitation spectra. The BAPTA sites serve as the ion chelating groups. The binding of Ca²⁺ to these sites in water, shifts the excitation spectra of FURA-2 and INDO-1 to blue, allowing the excitation-based ratiometry.

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With the emission spectra, the situation is more interesting. Only one band of Ca²⁺ -free form is seen for FURA-2 because the ion dissociates in the excited state. This is due to the reduction on excitation of the electron density of the nitrogen atom conjugated with the electron-withdrawing group of the fluorophore. This causes disruption of the interaction between this nitrogen and bound cation. Consequently, fluorescence emission closely resembles that of the free probe. That is why, the ratio F(λ^ex ₁)/F(λ^ex ₂) of intensities at two band in excitation spectra is used here. In contrast to FURA-2, in INDO-1 the excited-state charge transfer is insufficient to cause the nitrogen–Ca²⁺ bond breaking and, therefore, we observe the λ-ratiometric response in fluorescence. Detailed analysis of this mechanism can be found in [65].

In line with Ca²⁺ ion, the Zn²⁺ ions are very important in biology. Presented example of Zn²⁺ probe, P-Zn (figure 9), allows observing the distribution of these ions in living cells and tissues by comparing the colors of fluorescence emission [66]. Its operation is based on ICT mechanism. The initial emission peak of the present probe at 465 nm decreased with a new peak increased at 550 nm, leading to the ratiometric determination of Zn²⁺ with high accuracy. The two-photon microscopy allowed obtaining the zinc ion distribution in living tissues.

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Regarding sensing the anions, the λ-ratiometric approach is highly popular, but most of developed techniques explore the concept of chemodosimetry (see section 2 of Part I [1]). The reason is that a strong hydration or large size of some anions does not allow strong electrostatic effects and easy generation of reporter signal based on electron, charge or proton transfer. Those are the sensors for F⁻ [67] and CN^- ions [68]. Providing the change in the dye structure, they induce the appearance of new fluorescence bands.

For analyzing and visualizing various types of biological processes, it is important to detect gaseous signaling molecules carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H₂S). They are neutral molecules of very small size, and it is hard to realize here multiple weak interactions for applying the molecular recognition principle. Luckily, they are highly reactive species, and their high and specific chemical reactivity can be in the background of their detection. Thus, the principle of chemodosimetry was found to be the most efficient [69, 70]. Based on this principle, numerous fluorescent probes have been constructed for their analysis [71, 72].

For the λ-ratiometric detection of CO, the probe was synthesized based on attachment of a CO-specific carbamate derivative to the amino unit of naphthalimide [73]. In this case, ICT is restricted and the emission wavelength is blue-shifted, located at 472 nm (figure 10). When CO was added, the allyl acetate group was eliminated and amino-naphthalimide was released. The ICT process was restored and the emission wavelength was red-shifted to 545 nm. This probe system shows excellent sensing properties for CO, including rapid response, high selectivity and sensitivity in aqueous solution under mild conditions.

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For imaging of CO in mitochondria, a ratiometric fluorescent probe was developed, in which the mitochondria-targeting unit (triphenylphosphonium moiety) and CO-responsive unit (allyl ether moiety) were covalently linking into the single molecule [74]. Treating with CO resulted in the cleavage of allyl ether element, leading to the new band in emission that allows λ-ratiometric measurements.

An interesting idea of coupling chemodosimetry with ESIPT was realized in ratiometric fluorescence probe for NO [75]. Initially, the probe (figure 11) exhibited a fluorescence emission peak at 470 nm, and interaction with NO initiated the ESIPT process leading to the formation of a new band at longer wavelengths. This spectral transformation can be plotted as a ratiometric change in fluorescence intensity (F₅₆₀/F₄₇₀) as a function of NO concentration.

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Providing fluorescence sensing of H₂S in different cell compartments, one has to account that its pK_a value is 7.05 (at 25 °C), so that the ratio of its natural and proton-dissociated (HS^-) forms may differ. The chemodosimeter approach may operate providing switching between the ground-state forms generating the change in excitation spectra as well as in fluorescence emission spectra [76]. Efficiency of this approach based on modulation of ICT reaction [77] is demonstrated in figure 12. The ratiometric detection in fluorescence can be based on a large red shift, over 62 nm. A dramatic change of color of both absorbed and emitted light can be detected visually.

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There were many other probes for hydrogen sulfide imaging in vivo, and the most efficient of them are based on λ-ratiometric principle. Typically, the ability of H₂S to reduce the probe azide group to an amino group was explored. The electron-withdrawing group becomes converted into an electron donor group, which leads to the spectral shift [78]. The pyrene-containing probes responding by H₂S-induced monomer-excimer transition were also suggested [79]. The λ-ratiometric probes for H₂S detection become available for the near-IR region [80].

Rapid and simple blood glucose monitoring is very important for diabetes patients. Here fluorescence sensing is competing with the self-test glucose measurement systems based on redox-couple-mediated enzymatic oxidation of glucose with electrochemical detection. To win this competition, the methods that allow visual detection of changes in emission color have to be developed.

The glucose binding power of boronic acid has been explored for a long time. The ratiometric signal of excimer formation was obtained on binding to glucose of two boronic-modified pyrene derivatives [81]. There were many other suggestions to use coupling of color-switching dyes with boronic acid [82]. Color change due to a dual-emission response to H₂O₂ generation coupled with enzymatic glucose oxidation was realized in nanostructures [83, 84]. Among recent developments are the dual red-green emission-switching nanocomposites based on quantum dots that allow visualizing glucose content with a naked eye [85].

The skill of researcher in generating new emission bands was demonstrated in sensing phosphate anions. It was shown [86] that the conformational change occurring on PPi binding generates the ESIPT reaction resulting in strongly shifted (by ~100 nm) fluorescence band (figure 13).

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Recognition and sensing of nucleotides, and of ATP or GTP in particular, is an active area of research. The fluorescence probes are targeted to two recognition sites, the purine bases and triphosphate groups. Here the π-π stacking interactions together with H-bonding and steric effects can be very efficient. In this respect, a λ-ratiometric ATP sensor based on disruption of pyrene excimer is a classical example [87]. The, imidazolium receptors bearing two pyrene groups allow sensing ATP molecules that are sandwiched between two pyrene groups breaking the formation of excimer. Such sensor displays a unique selectivity for ATP over other nucleoside triphosphates. [88] demonstrates another possibility of realizing this idea, by using the cyclophanes.

Intracellular thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play key roles in biological systems. They regulate oxidation/reduction potential in cells and serve as potent antioxidants, but in many respects their function vary; therefore their differential detection is needed [89]. They may form different ground-state species producing different intramolecular charge transfer (ICT) effects and generating interplay between two bands in excitation spectra [90, 91]. Manipulating with the targeted chemical transformation in π-conjugation length of the probe based on fluorescein and coumarin platforms, the dual-mode and λ-ratiometric response to Cys over Hcy/GSH was achieved [92]. In another Cys probe, shrinking such conjugation resulted in dramatic shifts of excitation and emission spectra to shorter wavelengths [90].

Coupling chemodosimetry with the excited-state reaction (ICT, ESIPT, EET) allowed producing in sensing thiols the ratiometric response in fluorescence spectra only. An example of λ-ratiometric fluorescent probe based on ESIPT reaction designed for the detection of Cys is presented in figure 14. The probe BTP-Cys uses the acrylates moiety as a recognition site. This site also serves as the protection unit preventing the ESIPT reaction; this reaction is activated on bond splitting. In the absence of Cys, the emission is blue. After splitting and following ESIPT, a very large (113 nm) shift to green is observed [93]. The change in F₅₂₃/F₄₁₀ ratio of intensities fits nicely the desired range of Cys concentrations.

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In sensing glutathione, the excitation energy transfer (EET) between two fragments of the sensor molecule (rhodamine derivatives) is used [94]. The reaction with analyte destroys the light absorbance of EET acceptor, and the donor starts emitting light itself, providing good possibilities for λ-ratiometric analyte detection.

Fluorescence methods for the detection of cholesterol are generally based on the detection of hydrogen peroxide (H₂O₂) that appears as a result of its transformation in enzyme reaction with cholesterol oxidase (ChOX). A variety of these methods were based on construction of nanocomposites with λ-ratiometric response that include immobilized ChOX with the fluorescence-responsive and reference factors [95, 96]. Non-enzymatic λ-ratiometric determination of cholesterol can be based on its functional role to modify the structure and dynamics of biological membranes. The model system involving the liposomes with incorporated ratiometric fluorescent dye was suggested [97], and as a method for the determination of cholesterol it deserves further development.

Figure 15 demonstrates this possibility to use functional nanostructures together with probing fluorophore to sense the biologically important molecules such as cholesterol. Since the natural function of cholesterol is its participation in formation of biological membranes and providing its dynamic ordering, the modulation of this ordering in a model of small vesicles (liposomes) may provide the direct λ-ratiometric analytical response of the dye demonstrating ESIPT reaction [97]. Other attempts in this direction included the formation of Langmuir-Blodgett sensor surfaces formed of phospholipids [98].

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Determination of hydrogen peroxide H₂O₂ is of its own value as an indicator of oxidative stress, and its role can be extended as an output mediator in the essays of different metabolites being the product of their oxidation by correspondent enzymes. One of these reactions catalyzed by enzyme uricase was used for the determination of uric acid [99, 100]. Selective quenching of fluorescence in designed nanocomposites provides λ-ratiometric signal. The coupling of hydrogen peroxide detection with ESIPT reaction allows making the fluorescence probe for λ-ratiometric imaging in mitochondria [101]. Based on these ideas, many other organelle-targetable probes for hydrogen peroxide have been synthesized. They are based on chemical transformation starting the excited-state process. New fluorescence bands are generated for providing efficient λ-ratiometric detection and color-changing in imaging [102–104].

The rapid, sensitive and selective detection of free bilirubin in water and urine was achieved with hybrid ratiometric fluorescence sensor composed of Rhodamine B and polymer nanoscale aggregate composing a FRET pair [105]. A blue (465 nm) to orange (577 nm) fluorescence color change is used in this fluorescence assay.

Rapid, simple, accurate and highly sensitive detection of enzymes is essential for early screening and clinical diagnosis of many diseases. Fluorescence spectroscopy, microscopy and flow cytometry with λ-ratiometric recording satisfies these requirements in optimal way.

For the detection of alkaline phosphatase (ALP), a ratiometric fluorescent probe FCP was designed based on the hydroxyl electron-donating group in fluorescein-coumarin conjugate protected by the phosphate group [106]. The splitting by ALP of phosphate group resulted in the change of emission color from blue to green (figure 16).

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The ALP-produced removal of phosphate protection of the electron-donating group was also used in other assays. In hemicyanine dye, it allowed for the λ-ratiometric determination of ALP in the near-IR region, at 590 and 670 nm [107]. The suppression in FRET reaction from blue-emitting carbon dots to the formed by ALP activity yellow fluorescent energy acceptor 2,3-diaminophenazine (DPA) generated a powerful λ-ratiometric signal [108].

A dual-emission fluorescent nanoscale probe for acetylcholine esterase was reported [109]. The probe showing a fast emission color change (620 to 662 nm) in response to β-galactosidase activity was also developed and applied for fluorescence microscopy and flow cytometry [110]. The drug-metabolizing enzyme cytochrome P450 1A was visualized with a two-photon fluorescent probe based on 1,8-naphthalimide that provided changing the emission color from 452 to 564 nm [111]. The λ-ratiometric probes targeting other enzymes were developed [112] with particular emphasis on fluorescence response in the near-IR region [113].

The appearance of neurodegenerative diseases is related with the formation of abnormal protein aggregates. The λ-ratiometric probes have started to play important role in targeted recognition of these aggregates and in studying the conditions for their appearance. They were suggested for the aggregate labeling, inhibiting their assembly, and studying the possibility to reduce their cytotoxicity [114, 115].

Because the understanding of amyloid fibrillation in molecular detail is essential for the development of strategies to control this process, it becomes natural to search for fluorophores that can be the most efficient in these studies. Those are the dyes of 3-hydroychromone (3HC) family that provide multiparametric spectral response to polarity and hydration of their environment [116, 117], see section 5.3 of Pt. I [1]. The two-band deconvolution of their excitation spectra and three-band deconvolution of emission spectra [118] allowed obtaining simultaneously the information on the changes in polarity and hydration of probes attached to aggregating molecules. The α-synuclein, that exhibits pathogenic folding leading to Parkinson disease, and a number of its mutated forms were in focus of these studies [119], figure 17.

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Characterizing simultaneously the polarity and hydration (proticity) of probe environment by observing the interplay of N*, T* and H-N* contributions to 3HC probe emission, it became possible to distinguish between polymorph forms derived from naturally occurring point mutations in α-synuclein structure ([119]. The sensitivity of such a simple external probe to conformational alterations induced by point mutations is unprecedented and provides new insight into key phenomena in the formed amyloid fibrils: plasticity, polymorphism, propagation of structural features, and structure-function relationships underlying toxicity. Moreover, the results of monitoring the kinetics of aggregation of α-synuclein, allowed providing the information on early and intermediate stages of the overall reaction [120]. The unexpected result was that the reaction intermediates differ significantly not only from monomeric but also from fibrillar amyloid forms of α-synuclein.

Nitroaromatic compounds that are extensively used in different industries pose a serious pollution threat. They can be detected applying benzimidazole-based D-π-A fluorophores with λ-ratiometric response [121]. Moreover, these dangerous compounds are explosive, and their discriminative detection in trace amounts has to be provided. This can be done in ratiometric way with blue emissive carbon dots in combination with yellow and red emissive CdTe quantum dots [122]. The sensor array demonstrates a promising capacity to detect structurally similar nitroaromatics in mixtures and complex media of soil and groundwater samples.

Phosgene is a highly toxic gas that is a serious threat to human health and public safety. A novel fluorescent probe for phosgene detection based on ESIPT reaction in quinolone fluorophore was suggested [123]. The amidation reaction between phosgene and quinolone suppresses effectively the ESIPT process in the probe, providing rapid change of emission color (red to green).

Thiophenol belongs to a class of highly reactive and toxic aromatic thiols with widespread applications in the chemical industry for preparing pesticides, polymers, and pharmaceuticals. For its ratiometric detection, a chlorinated BODIPY-based fluorescent probe was reported [124]. The ratio of their fluorescence intensities at 581 and 540 nm could remarkably increase (up to 640-fold) in the presence of excess amount of thiophenol. For its detection, a FRET-ICT-based ratiometric fluorescent and colorimetric probe was also developed [125]. With broad spacing of two emission peaks (133 nm) upon thiophenol addition, the latter probe exhibits drastic (over 2100-fold!) enhancement of the fluorescence intensity ratio.

Hazardous food contaminants require rapid methods for their detection [22]. Quantitative determination of hazardous synthetic colorants in food was achieved with green/red dual emissive carbon dots as the probes [126]. Antibiotics with their great contributions to the improvement of human health become dangerous if they are found in food and water; there are many λ-ratiometric methods developed for their detection [127–129]

Pesticide residues have seriously threatened human health and affected the sustainable development of the environment. Some of them show their own ability to absorb and emit light that can be used in sensing [130]. An interesting example of using the reporting abilities of excimer-monomer transition of pyrene derivatives incorporated into nanocomposites based on cyclodextrin [131] is presented in figure 18.

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The ratiometric fluorescence probes are attractive for the nucleic acids research, particularly for their in situ real-time tracking in living cells and tissue sections and in the studies of their dynamics-function relationships. It was demonstrated that the D–π–A–π–D-type ICT probe was able to respond to dynamic behavior of nucleic acids during different cellular processes in real time, such as the aggregation and separation of chromatin in apoptotic cells [132].

Fluorescent amino acids (such as 3-hydroxychromone derivatives) when incorporated into specific peptides can be used to study the interactions of their zinc-finger sites with nucleic acids [133]. Their dual emission resulting from ESIPT reaction indicates such highly selective binding (figure 19).

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The development of ratiometric emissive nucleoside analogues have stimulated the studies of nucleic acids [134, 135]. They found applications in probing the local structure and dynamics of nucleic acids, typing single-nucleotide polymorphism (SNP), investigations of DNA-proteins interactions, etc Prospective candidates for this role are 3-hydroxychromones due to extreme sensitivity of their ESIPT reaction to local environment and their small size compatible with that of a base pair, ~10 Å [136]. Featuring exceptional environmental sensitivity by switching between two well-resolved fluorescence bands, they can serve as highly responsive biosensors to site-specific probing of the proximal environment of nucleic acid structures [134, 137, 138]. Some of these results are presented in figure 20.

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Many other applications are outside this Review because of its limited space and inability to cover all of them by a single author. Among them are the λ-ratiometric sensors for viscosity [139], temperature [140] and pressure [141] on a molecular scale. Detection of pathogenic bacteria [142], checking their drug resistance [143] and identifying the spores [144] benefit from using ratiometric fluorescence sensing. Viruses, including SARS-CoV-2, can be detected by λ-ratiometric visualization [145] or by two-color immunoassays [146].