Near-Infrared Probes for Biothiols (Cysteine, Homocysteine, and Glutathione): A Comprehensive Review

Rahul Kaushik; Nidhi Nehra; Veronika Novakova; Petr Zimcik

doi:10.1021/acsomega.2c06218

. 2022 Dec 28;8(1):98–126. doi: 10.1021/acsomega.2c06218

Near-Infrared Probes for Biothiols (Cysteine, Homocysteine, and Glutathione): A Comprehensive Review

Rahul Kaushik ^†,^‡,^*, Nidhi Nehra ^§, Veronika Novakova ^‡, Petr Zimcik ^‡

PMCID: PMC9835641 PMID: 36643462

Abstract

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Biothiols (cysteine, homocysteine, and glutathione) are an important class of compounds with a free thiol group. These biothiols plays an important role in several metabolic processes in living bodies when present in optimum concentration. Researchers have developed several probes for the detection and quantification of biothiols that can absorb in UV, visible, and near-infrared (NIR) regions of the electromagnetic spectrum. Among them, NIR organic probes have attracted significant attention due to their application in in vivo and in vitro imaging. In this review, we have summarized probes for these biothiols, which could work in the NIR region, and discussed their sensing mechanism and potential applications. Along with focusing on the pros and cons of the reported probes we have classified them according to the fluorophore used and summarized their photophysical and sensing properties (emission, response time, limit of detection).

Introduction

Near-infrared (NIR) probes have been given significant attention in the last few decades due to their applications in the biomedical field.¹⁻⁴ NIR probes absorb low-energy light and emit emissions in the NIR range ca. 650–1700 nm. The NIR range is generally divided into two major windows known as the NIR-I window (650–900 nm) and NIR-II window (900–1700 nm).^5,6 NIR emissive probes allow deep tissue penetration of light and can avoid light scattering, reabsorption, and interference with the fluorescence of endogenous chromophores, typical drawbacks occurring in UV/visible probes.⁷⁻⁹ Due to these advantages, NIR probes have been highly efficient and extensively used for in vivo optical imaging.^10,11

Thiols or mercaptans are the compounds that contain the -SH group and have been extensively used by biologists,^12,13 chemists, and physicists in the development of chemical reactions,¹⁴ hybrid materials,¹⁵ electronic devices,¹⁶ and many more.^17,18 The most common but extremely important biological thiols are cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). As shown in Figure 1, Cys and Hcy have the same active functional groups, i.e., −SH, −NH₂, and −COOH, whereas GSH is usually present in two forms: reduced and oxidized.¹⁹ Reduced GSH consists of a free -SH group, whereas in oxidized GSH (GSSH), free -SH forms a disulfide bond joining two identical reduced GSH molecules. GSH is considered an important tripeptide and bioreducing agent.²⁰ Besides that, the -SH group compounds have played a significant and important role in living beings. Biothiols also play a vital role in physiological processes such as metal binding and help in the removal of xenobiotics, redox biocatalysis, etc.^14,21−24

Chemical structures of Cys, Hcy, and GSH (reduced) and oxidized GSH (GSSH) forms.

Cys, Hcy, and GSH are considered the most abundant nonprotein biothiols, and they are found in living beings in the concentration ranges of 240–360 μM (Cys), 12–15 μM (Hcy), and 1–10 mM (GSH).²⁵ A slight disturbance in the optimum concentration could cause serious health issues such as liver damage, Alzheimer’s disease, neural defects, cardiovascular disease, slowed growth due to muscle and fat loss, skin diseases, etc.^{12−14,19,22,23,26} Currently, several methods have been used for the detection of biothiols such as mass spectrometry, electrochemical assay, capillary electrophoresis, high-pressure liquid chromatography (HPLC), and optical methods. Compared to other methods, optical methods take an upper hand due to their quickness, sensitivity, and ease of sample preparation.^14,22,23

In the last two decades, several probes for biothiols focused on simple organic compounds or metal complexes.^14,21,27,28 Most of the simple organic probes have the disadvantage of absorption in the UV and visible range, which could be harmful for cells due to photodamage risks, which limits their applications in biological systems. On the other hand, nanomaterial and metal complexes probes have the disadvantage of metal ion accumulation after completion of a sensing process and could cause biotoxicity. However, NIR probes could eliminate these serious issues and therefore represent a suitable alternative in sensing systems.²⁹⁻³¹ Among the reported organic probes, NIR absorbing probes were always found to be superior compared to the probes that showed a signal change in the UV and visible spectral ranges. NIR organic probes mentioned in this review have used various mechanisms; the most common are shown in Scheme 1.

Scheme 1 — (a) Disulfide cleavage reaction, (b) Michael addition of thiol at unsaturated conjugated bond, and (c) thiol-assisted ether cleavage reaction. (d) Aromatic nucleophilic substitution-rearrangement reaction. F = fluorophore, A = electron-acceptor, D = electron-donor, Q = quencher, Ar = Aromatic unit.

Several excellent reviews that are focused on organic probes for biothiols have been reported in the past few years.^{14,23,28,32,33} Fan et al.³⁴ have also reported NIR-emitting probes for biothiol detection; however, they have only included the probes from 2017 to 2021. Recently, Jose et al.²⁹ and Yin et al.³⁵ have thoroughly discussed NIR probes for H₂S and their applications in bioimaging, and therefore in the present review, we have not included NIR probes for H₂S. In the present review, we have thoroughly discussed and compared the NIR probes (with λ_em > 650 nm) reported in terms of solvent systems, limit of detection (LOD), reaction kinetics, selectivity, and their potential for application in bioimaging.

Squaraine-Based NIR Probes

Squaraine dyes are one of the most common organic dyes and have been widely used in various applications such as optoelectronic devices, sensing, photodynamic therapy, etc.³⁶⁻³⁹ They typically absorb in the far red and NIR region (640–850 nm) and have shown high stability toward photobleaching.⁴⁰ The core skeleton consists of a resonance-stabilized four-membered ring with electron deficiency, which is prone to nucleophilic attack.

Ajayaghosh et al.⁴¹ have reported π-extended squaraine dye NIR-1, which exhibits both UV–vis and turn off emission change in the presence of Cys and Hcy. NIR-1 showed absorbance peaks at 363 and 750 nm (ϵ = 1.8 × 10⁵ M^–1 cm^–1), and when excited at 730 nm, an emission band appeared at 800 nm in acetonitrile (ACN)/H₂O (1:1, N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, pH 9.6). The presence of Cys and Hcy disturbed the π-conjugation resulting in a decrease in the NIR absorption band at 750 nm with a blue shift of 310 nm, with the new band emerging at 440 nm along with a green to light yellow color change. In emission spectra, the peak at 800 nm decreased (λ_ex = 730 nm), and a new emission band at 592 nm (λ_ex = 410 nm) was observed. These ratiometric changes were observed due to the nucleophilic addition to a cyclobutene ring that altered the π-conjugation as shown in Scheme 2. NIR-1 showed selectivity toward Cys, Hcy, and GSH, among other amino acids, and applications of NIR-1 were explored in reduced human blood plasma (collected before breakfast, after breakfast, and after smoking a cigarette) using microtest assay experiments and by monitoring enhancement in the emission peak at 592 nm, which indicated the presence of free amino thiols. Further, the quantitative value of free amino-thiol content in a plasma sample collected after smoking was calculated as 545 μg L^–1.

Scheme 2 — Images of vials represent the color of **NIR-1** before and after the addition of Cys. Reprinted with permission from ref (41). Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Using the same nucleophilic addition mechanism on the π-extended conjugated system as observed in NIR-1, Fu et al.⁴² have reported squaraine-based colorimetric probe NIR-2 for thiol (GSH, Cys, and Hcy) at pH 7.5 (1 mM cetrimonium bromide (CTAB)). In a GSH environment, the conjugated system of NIR-2 gets disturbed (as shown in Scheme 3), and the absorbance band at 794 nm was decreased with a green to yellow color change. The effect of surfactants (in a buffer) was also explored, and positively charged surfactant CTAB was found better than sodium dodecyl sulfate (SDS) (anionic) and Triton-X (neutral) surfactants. NIR-2 worked well over a wide pH range; the maximum change was recorded in a basic range (pH 7–11), and it could detect GSH with an LOD of 10 nM. Interestingly, NIR-2 showed a reversible phenomenon with Cys when incubated for 9 min, whereas Hcy and GSH showed an irreversible response, which was confirmed by an electrospray ionization mass spectrometry (ESI-MS) analysis. The feasibility of NIR-2 was explored using a spiked assay in synthetic samples containing amino acids with good recovery values.

Scheme 3 — Images of vials represent the color of **NIR-2** before and after the addition of GSH. Reprinted with permission from ref (42). Copyright 2013 Elsevier.

Keeping the squaraine as an NIR-conjugated skeleton but with a different sensing mechanism than that utilized in NIR-1 and NIR-2, Xu et al.⁴³ have designed and synthesized Förster resonance energy transfer (FRET)-based turn on fluorescent probe NIR-3 for GSH in ACN/PBS (2:8, v/v; PBS = phosphate-buffered saline) medium. As shown in Scheme 4, two identical squaraine units were linked via a disulfide bond, which facilitates the self-quenching by FRET and results in weak fluorescence properties. However, GSH cleaves the disulfide bond of NIR-3 and eliminates the FRET process with 2.7-fold fluorescence increase at 665 nm (λ_ex = 610 nm). In absorption spectra, NIR-3 showed a decrease in the absorbance intensity at 596 nm. The limit of detection was calculated as 0.15 μM with the response time of 120 min. NIR-3 showed significant intensity enhancement with GSH over the pH range of 3.9–7 with high selectivity among other thiols including Cys. NIR-3 was characterized by low cytotoxicity and localized in lysosomal compartments. It allowed endogenous and exogenous GSH detection through confocal imaging. Confocal imaging, low toxicity, and high selectivity for GSH makes it advantageous in comparison to the above-mentioned NIR-1 and NIR-2.

Taking advantage of a designed push–pull system in squaraine dyes, Lu et al.⁴⁴ have reported Cys and Hcy sensing probe NIR-4 with a 2,4-dinitrobenzenesulfonyl unit as a thiol reactive site. NIR-4 showed an absorbance band at 634 nm, which decreases in the presence of Cys and Hcy and exhibits a change in color from dark blue to light blue. NIR-4 showed a dull emission peak at 656 nm (Φ_F = 0.006) that increased with Cys (33-fold, Φ_F = 0.162) and Hcy (22-fold, Φ_F = 0.145). The increase in fluorescence was explained by the two-step reaction as shown in Scheme 5. The first step is an irreversible removal of quencher (2,4-dinitrobenznesulfonyl group) from the squaraine fluorophore to produce fluorescence enhancement, whereas in the second step, which is reversible, the thiol group of Cys and Hcy undergoes a nucleophilic addition reaction with the conjugated system of a four-membered squaraine ring to induce an absorbance decrease at 635 nm; however, the fluorescence spectra remained stable. NIR-4 worked well in a physiological pH range and could detect thiol concentration as low as 2 μM with reaction completion recorded in about 20 min, which is quicker than for NIR-3. Confocal cell imaging was done to detect the intracellular biothiols as shown in Figure 2; after an incubation of KB cells in NIR-4 and then treated with Cys, red fluorescence was observed, which confirmed the potential biological application. Importantly, NIR-4 also behaves as a colorimetric sensor for Cys (blue to light blue) and Hcy (blue to light yellow) in ACN/PBS buffer (1:9, v/v, pH = 7), which could be better for on-field Cys detection due to a naked-eye noticeable color change.

Confocal fluorescence microscopic images of **NIR-4** (10 μM) for detection of Cys using living KB cells. (a, d, g) Bright-field images. (b, e, h) Fluorescence images. (c, f, i) Merged images. (a–c) Samples were only incubated with **NIR-4** for 20 min. (d–f) Samples were preincubated with NEM. (g–i) Samples were pretreated with Cys (300 μM). The images were captured with emission at 650–750 nm upon excitation at 633 nm. Reprinted with permission from ref (44). Copyright 2013 The Royal Society of Chemistry.

Cyanine Dye-Based NIR Probes

Cyanine dyes have been widely used for fluorescence labeling and sensing in biological applications due to their NIR absorbing and emission properties.⁴⁵⁻⁴⁷ Cyanine dyes belong to the polymethine dye family, as they have a sharp fluorescence band, high molar extinction coefficient, good stability, and high sensitivity.^48,49 Cyanine dyes are also known to behave differently in keto and enol structural isomerism, which results in distinct spectroscopic properties.⁵⁰ This structural behavior has been used by researchers for the sensing of various cations and anions using different dyes including cyanine dyes.⁵¹

As an example, Lin et al.⁵² have synthesized NIR-5 for turn-on detection of Cys in dimethyl sulfoxide (DMSO)/PBS (3:7, pH = 7.4). NIR-5 showed NIR absorbance maximum at 900 nm which shifted to 892 nm in GSH environment. In emission, fluorescence enhancement at 928 nm was observed with addition of GSH and incubation time of 60 min. The LOD for Cys was calculated as 85 nM. NIR-5 was highly selective for Cys and no noticeable change was recorded with other anions including Hcy and GSH. The spectral changes were attributed to a disruption of the photoinduced electron transfer (PET) effect from 3,5-bis(trifluoromethyl)-benzenethiol and heptamethine units as shown in Scheme 6. NIR-5 was successfully explored for in vivo imaging in a tumor mouse model for real-time visualization of GSH.

Yoon et al.⁵³ have also developed cyanine dye-based ratiometric probe NIR-6, which undergoes intramolecular cyclization and consequently keto–enol tautomerism, when allowed to react with Cys. The cyanine skeleton was modified with an acrylate group, which acted as a reactive site for Cys in EtOH/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (1:9, v/v, 10 mM, pH 7.4) medium. NIR-6 showed a ratiometric change in absorption spectra with a decrease at 775 nm and increase at 515 nm upon the successive additions of Cys. The color change from light blue to red was recorded with isosbestic point at 605 nm and a blue shift of 240 nm within 30 min. The emission spectral response of NIR-6 with Cys was also recorded at 780 nm (λ_ex = 720 nm, for only NIR-6) and 570 nm (λ_ex = 520 nm, for only NIR-6 + Cys). Cys addition to NIR-6 decreased the emission peak at 780 nm, whereas the emission peak at 570 nm significantly increased. The spectral change was explained by a two-step pathway, which includes the attack of a thiol group on an allylic double bond followed by the removal of the allyl group due to the formation of a cyclic byproduct and cyanine core unit in keto form as shown in Scheme 7. NIR-6 showed high selectivity for Cys over other amino acids including Hcy and GSH. The pseudo-first-order rate constant was calculated as 0.23, 0.047, and 0.029 min^–1 for Cys, GSH, and Hcy, which also proved the high selectivity toward Cys. The NIR-6 could be effective over the wide pH range of 4–10, and biological applications were confirmed by fluorescence cell imaging using MCF-7 breast cancer cells.

Qian et al.⁵⁴ synthesized NIR-7 for Cys, Hcy, and GSH in DMSO/PBS solution (V/V = 5/5, pH = 7.4) medium. NIR-7 was composed of two fluorophores, i.e., cyanine and 1,8-naphthaleneimide linked by a thioether bond. In the case of Cys, both fluorophores were substituted by the nucleophilic attack of Cys resulting in Cys-cyanine and Cys-naphthaleneimide as products. The emission enhancement observed at 733 and 435 nm was due to Cys-cyanine and Cys-naphthaleneimide, respectively, whereas Hcy and GSH showed only cyanine-based substituted products with emission intensity at 733 nm (for Hcy) and 815 nm (for GSH). Further, a peak at 467 nm was also observed with both Hcy and GSH, which was due to a naphthalimide-based byproduct as shown in Scheme 8. The emission enhancement at different wavelengths proved to be highly advantageous for the discrimination of Cys, Hcy, and GSH. The probe NIR-7 also produced singlet oxygen when irradiated at 660 nm, which was proved by photocytotoxicity experiments in cancer cells. Due to the presence of a cyanine unit, NIR-7 exhibited good mitochondrial targeting properties, which were confirmed by fluorescence imaging in cells and zebra fish.

Chen et al.⁵⁵ have designed and synthesized probes NIR-8a and NIR-8b for the turn-on fluorescent probes of GSH in PBS (pH = 7.4, 15 mM). Like NIR-6 and NIR-7, cyanine dye was used as a fluorophore, whereas electron-withdrawing moieties 2-nitrophenylselane (NIR-8a) and 3-(trifluoromethyl)phenylselane (NIR-8b) were attached to modulate photoinduced electron transfer. Both NIR-8a and NIR-8b showed an absorption band at about 609 nm and an emission maximum at 750 nm (λ_ex = 635 nm, Φ_F = 0.003). In the presence of GSH, both NIR-8a and NIR-8b showed an enhancement in the absorbance and emission intensity at 609 and 750 nm (Φ_F = 0.13), respectively. The emission increment was explained based on a donor-excited photoinduced electron transfer (d-PET) process as shown in Scheme 9. In both NIR-8a and NIR-8b, the d-PET mechanism undergoes from cyanine to the electron-withdrawing moieties 2-nitrophenylselane (NIR-8a) and 3-(trifluoromethyl)phenylselane (NIR-8b), whereas in the presence of thiols, the electron-withdrawing units detached from the cyanine fluorophore. The cleavage eliminates any d-PET process and consequently turns on the fluorescence. Kinetic studies of NIR-8a and NIR-8b with GSH recorded emission enhancement saturation within 3 min. Both NIR-8a and NIR-8b were effective over pH 4.0–8.6 but were not selective among other biothiols. The application in biological samples was explored by detecting GSH through fluorescence cell imaging using RAW 264.7 cells.

Using the similar PET mechanism, which was observed in NIR-8 dyes, Yang et al.⁵⁶ have synthesized NIR-9 for the fluorescent detection of Cys in DMSO-HEPES (1:4, V/V, pH 7.4). Without Cys, NIR-9 exhibited a weak fluorescence maximum at 830 nm (λ_ex = 650 nm), which increased selectively with Cys at 750 nm. The detection limit for Cys was determined as 1.26 μM. Interestingly, GSH induced emission enhancement at 830 nm, whereas dual peak enhancement at 830 and 745 nm was recorded with Hcy, which is better for overall discrimination between Cys, GSH, and Hcy. As shown in Scheme 10, the sensing mechanism follows a biothiol attack on the conjugated carbon and eliminates the PET mechanism from the cyanine moiety to the 4-nitrothiophenol unit. NIR-9 showed good selectivity even with potentially interfering amino acids incubated for 60 min. Low toxicity and fluorescence cell imaging proved the potential for application of NIR-9 in biological samples. Overall, NIR-9 could be better than most of the reported probes, which are incapable of discriminating Cys, GSH, and Hcy.

Using a similar cyanine skeleton, Govindaraju et al.⁵⁷ have synthesized NIR-10 for the detection of GSH, Cys, and Hcy. NIR-10 was composed of cyanine dye as a signaling moiety and 2,4-dinitrobenzenesulfonate as a reactive moiety for thiols and acceptor in PET. NIR-10 showed an absorption band at 390 nm and a weak emission peak at 700 nm (λ_ex = 600 nm). With GSH, the absorption peak at 390 nm was decreased, and two new bands appeared at 476 and 581 nm with a naked-eye detectable light green to blue color change. Emission spectra exhibited more than 25-fold intensity enhancement at 700 nm, and a large Stokes shift of 119 nm was observed. As shown in Scheme 11, the mechanism involving GSH induced the removal of the 2,4-dinitrobenzenesulfonate group, and the formation of the heptamethine-cyanine structure was responsible for absorption changes and turn-on emission. Similar spectral responses were observed with Cys and Hcy. The response time of 2 min was observed with saturation after more than 20 min. NIR-10 showed an emission change over the pH range of 6–10. The biological applications of NIR-10 were confirmed by thiol content quantification in fetal bovine serum and by detection of GSH produced through an enzymatic assay using GSSH with glutathione reductase.

For the thiol’s detection, an aromatic nucleophilic substitution mechanism is also exploited because of the presence of N and S atoms in the biothiols. This mechanism involves two steps; in the first, removal of a halide to form thiolates occurs, and in the second step the thiolates undergo a rearrangement reaction to form an amino-substituted product. Importantly, this mechanism can be employed for the discrimination of GSH from Cys/Hcy.^58,59 Yuan et al.⁶⁰ have reported visible and NIR dual fluorescent probe NIR-11 for the selective detection of biothiols in PBS/ACN (9:1, v/v, pH 7.4). Importantly, NIR-11 has shown distinguishable fluorescence changes with GSH and Cys/Hcy. NIR-11 initially exhibits a nonfluorescent nature due to the PET process from cyanine to the 7-nitro-1,2,3-benzoxadiazole (NBD) unit. The absorption band for NIR-11 was observed at 680 nm and blue-shifted to 460 nm in the presence of Cys/Hcy with emission enhancement at 550 nm (λ_ex = 450 nm). In the case of GSH, emission at 750 nm was red-shifted for about 100–850 nm, and a 45-fold enhancement was observed (λ_ex = 720 nm). The substitution-rearrangement mechanism served us well with Cys/Hcy, whereas a substitution-only mechanism with GSH was responsible for the spectral changes as shown in Scheme 12. Reaction kinetics with thiols showed the plateau that was reached in 60 min. The detection limit was calculated as 75 nM (GSH), 94 nM (Cys), and 160 nM (Hcy). Potential biological applications of NIR-11 were explored in serum samples, live cell imaging using U87 cells, and in a tumor-bearing mouse, which could be advantageous for biomedical applications. As shown in Figure 3, after treatment with N-ethylmaleinimide (NEM) followed by GSH, fluorescence is exclusively observed in a red channel (760–830 nm), while fluorescence was only observed in a green channel for Cys and Hcy (505–550 nm).

Fluorescence confocal cell imaging for the detection of biothiols using **NIR-11** incubated with U87 cells. Fluorescence microscopy images of cells incubated with (a) **NIR-11**, (b) **NIR-11+**NEM, (c) **NIR-11** + NEM + GSH, (d) **NIR-11** + NEM + Hcy, and (e) **NIR-11** + NEM + Cys for 1 h. Reprinted with permission from reference (60). Copyright 2020 The Royal Society of Chemistry.

Yoon et al.⁶¹ have reported and synthesized two cyanine-based probes for GSH sensing, namely, NIR-12a and NIR-12b. Structurally, NIR-12a consists of two cyanine dyes connected with para-phenylene sulfide bonds and is nonfluorescent due to the self-quenching of two neighboring cyanine units, whereas NIR-12b consists of a 3,5-bis(trifluoromethyl)benzenethiol unit and cyanine that is weakly fluorescent due to the PET effect (Scheme 13). As both probes have a similar dye skeleton, an absorption maximum at 660 nm was observed in HEPES/DMSO (99:1, v/v). In a GSH environment, a new absorption band at 780 nm emerged with an emission enhancement at 805 nm (λ_ex = 780 nm). With Cys, the absorption at 660 nm decreased without any significant emission change at 805 nm. A similar decrease in absorption at 660 nm was observed with Hcy along with a slight enhancement of emission at 805 nm. The kinetic studies with Hcy showed a fluorescence intensity increase and then decrease, before the fluorescence attained a plateau. As shown in Scheme 13, the spectral change with GSH was due to the formation of sulfur-substituted products, whereas amino-substituted products with Cys and Hcy were similar to NIR-11. The reaction completion time for the GSH with NIR-12a and NIR-12b was recorded as 10 min, and the detection limit was calculated as 0.63 μM for NIR-12a and 0.33 μM for NIR-12b. Further, biological applications were explored for monitoring GSH in living cells and in vivo fluorescence imaging of SCC7 tumor-bearing mouse model.

The same research group⁶² has developed the synthesis of NIR-13 by combining 1,8-napthalimide and cyanine moieties through piperidine as a linker for the colorimetric and fluorescence-based sensing of GSH in HEPES/DMSO (9:1, v/v). NIR-13 showed a light blue to prominent blue color with GSH and showed dual emission peaks at 495 and 795 nm when excited at 370 and 700 nm, respectively. The limit of detection was calculated as 0.153 μM (λ_ex = 495 nm) and 0.171 μM (λ_ex = 795 nm). However, the response time is about 180 min, which is less advantageous than that of NIR-12. As shown in Scheme 14, 1,8-napthalimide dye was detached due to the -SH attack from the GSH molecule, which was responsible for the green channel emission, whereas the cyanine dye linked with piperidine was responsible for the red channel fluorescence. The solvent effect studies were also performed in dimethylformamide (DMF), DMSO, methanol, ethanol, ACN, etc., which showed that NIR-13 aggregates more in an aqueous buffer as compared to organic solvents. Mitochondrial GSH detection was explored by fluorescence cell imaging, which confirmed the potential of NIR-13 in biological applications as shown in Figure 4.

Cell image experiments of **NIR-13** for the detection of GSH in the green and red channels. Images (A, E, and I) correspond to the HeLa cells pretreated with NMM and **NIR-13**, (B, F, and J) correspond to images after cells were incubated with NMM + Cys + **NIR-13**, (C, G, and K) correspond to images after cells were incubated with NMM + Hcy + **NIR-13**, (D, H, and L) correspond to images after cells were incubated with NMM + GSH + **NIR-13** for 30 min in the green channel (D), in the red channel (H), and in the merged image (L). Green channel: λ_ex = 405 nm, λ_em = 460–515 nm; red channel: λ_ex = 635 nm, λ_em = 700–800 nm. Reprinted with permission from ref (62). Copyright 2018 Elsevier.

Kim et al.⁶³ have synthesized GSH sensing probe NIR-14, which worked in pure HEPES buffer with fluorescence turn on at 810 nm (λ_ex = 600 nm). The azo dye with a nitro group acts as a fluorescence quencher in the less-fluorescent NIR-14 (Φ_F = 0.0011). As shown in Scheme 15, addition of GSH to NIR-14 detached the azo dye (quencher unit) from the cyanine dye (fluorophore) thereby exhibiting a fluorescence increase (about 460-fold, Φ_F = 0.187). Reaction with Cys and Hcy led to low and blue-shifted fluorescence at 747 nm (15- and 10-fold increases, respectively). The reason for low fluorescence for these two biothiols was explained on the basis of thiol attack followed by an intramolecular amination reaction. The reaction kinetics recorded about 150 min as the response time, and the limit of detection for GSH, calculated as 26 nM, was better than that of most of the probes discussed above. The solvent effect showed that the rate of reaction in a polar protic solvent (MeOH) was more than 10 times that in a polar aprotic solvent (DMSO). For biological applications, laser confocal scanning microscopy was done using HeLa cells, and the imaging results showed that NIR-14 can detect the mitochondrial GSH by reflecting high fluorescence and also proved to be better than the commercially available mitochondrial tracker.

Zhang et al.⁶⁴ have designed and synthesized NIR-15a as a GSH sensor in an HEPES/DMSO (9:1, v/v) medium. NIR-15a showed an absorption band at 780 nm and weak fluorescence at 818 nm (λ_ex = 710 nm). In the presence of GSH, NIR-15a selectively exhibited fluorescence enhancement without any significant interference from Cys and Hcy, as shown in spectra. As shown in Scheme 16a, GSH induced the removal of the 4-aminothiophenol unit and triggered the emission enhancement. The response time for reaction completion was recorded as 40 min. The high selectivity of NIR-15a could be advantageous for biological applications, which was confirmed by live cell Laser Confocal Fluorescence Microscopic (LCFM) images with HepG2 cells to detect intracellular GSH.

With a similar cyanine dye skeleton and thiol reactive unit as for NIR-15a, He et al.⁶⁵ have synthesized NIR-15b for sensing of Cys and Hcy. NIR-15b showed absorption enhancement at 695 nm and decrease at 840 nm with Hcy and Cys in DMSO/PBS (1:1, v/v) medium. NIR-15b was selective for Cys/Hcy, and no obvious change was observed with GSH/Na₂S. Importantly, color changes from pale yellow to blue (with Cys) and to turquoise (with Hcy) were observed. In fluorescence spectra, enhancement at 776 nm (λ_ex = 650 nm) was observed with Cys (Φ_F = 0.0215) and Hcy (Φ_F = 0.0134). NIR-15b was effective over a wide pH range, and the limit of detection was calculated using emission changes as 0.66 and 1.22 μM for Cys and Hcy, respectively, and using absorbance changes as 0.47 and 0.23 μM for Cys and Hcy, respectively. As shown in Scheme 16b, Cys/Hcy induced the removal of the 4-aminothiophenol unit and the addition of the Cys/Hcy molecule that induced the emission enhancement. Importantly, NIR-15b was utilized for photoacoustic imaging and could be advantageous for biological applications.

With similar solvent conditions (HEPES/DMSO 9:1, v/v) and fluorophore unit as used in NIR-15a, Yoon et al.⁶⁶ have developed two cyanine-based probes NIR-16a and NIR-16b with piperazine as a linker between fluorophore and quencher moieties (Scheme 17). NIR-16a and NIR-16b showed a UV–vis change at 730 nm with emission maxima at 736 nm (λ_ex = 730 nm). NIR-16a showed a change in spectral properties with GSH, Cys, and Hcy, whereas NIR-16b was highly selective toward GSH with a response time of 15 min, and no obvious change was observed with Hcy and Cys. The high selectivity in NIR-16b was rationalized by the flexibility of GSH as compared to Cys and Hcy and the presence of two amide groups in GSH having thus more possibilities for intermolecular interactions than NIR-16a. For example, GSH carboxylate anion formed a H-bond with a piperazine unit as well as an electrostatic interaction with an indolium cation. These interactions made reactive sites (-SH and sulfonamide groups) come closer ensuring high reactivity and selectivity for GSH, whereas this kind of chemistry could not be possible with Cys/Hcy. For a biological application, NIR-16a was explored for biothiol detection in HeLa cells via confocal cell imaging (Figure 5), whereas applications of NIR-16b were explored in GSH detection in the human serum as well as in confocal imaging. NIR-16b was also employed for the detection of oxidant-governed redox status of GSH in the H₂O₂ environment. The application of NIR-16b was extended to in vivo studies; when a probe was intravenously injected into mice, strong red fluorescence was detected, whereas no such fluorescence was observed when mice were pretreated with only N-methylmaleinimide (NMM) or NMM with NIR-16b. These results confirmed the potential application of NIR-16b to detect the GSH in vivo.

Confocal fluorescence microscope imaging experiments of **NIR-16b** in HeLa cells. Fluorescence cell images were captured after (a) incubation of normal cells, (b) cells were incubated with **NIR-16b** only, (c) cells were incubated with NEM followed by **NIR-16b**, and (d) cells were incubated with NEM followed by GSH and finally with **NIR-16b** for 20 min. Images were captured using an excitation wavelength of 635 nm and a band path (655–755 nm) emission filter. Reprinted with permission from ref (66). Copyright 2014 American Chemical Society.

BODIPY Dye-Based NIR Probes

Boron-dipyrromethene (BODIPY) dyes are undoubtedly one of the most studied dyes due to excellent spectroscopic properties. Along with applications in material chemistry, BODIPYs have been extensively used in the field of photodynamic therapy.⁶⁷⁻⁶⁹ Importantly, BODIPYs can cover a large spectrum ranging from the visible to NIR regions by derivatization through extended conjugation. BODIPYs showed high photostability and high quantum yields, which makes them an excellent fluorescent skeleton for sensing and bioimaging applications.^70,71

With the same quencher and Cys reactive moiety as were used in NIR-16b, Jiang et al.⁷² have synthesized turn-on fluorescence probe NIR-17 for Cys (Scheme 18). Cys triggered the cleavage of a sulfonate bond leading to the separation of the 2,4-dinitrobenzenesulfonyl group (quencher) from the aza-BODIPY unit (fluorophore). NIR-17 showed an absorption band at 717 nm (ε = 48 000 M^–1 cm^–1) and weak emission at 734 nm (λ_ex = 670 nm, Φ_F = 0.03) due to an efficient PET from BODIPY to quencher moiety in CH₃CN–H₂O–DMSO (79:20:1, v/v/v; pH = 7.5). In the presence of Cys, the red shift was observed in absorbance (735 nm) and emission (755 nm) bands with increased fluorescence intensity (Φ_F = 0.14). The stability of NIR-17 was confirmed over a wide pH range (5–9). The limit of detection was calculated as 0.7 μM with 50 min of response time, which is less advantageous compared to NIR-16b. Further, spectral change with different thiophenols and 1-octylthiol showed the less-selective nature of NIR-17.

Aldehydes are prone to nucleophilic attack due to a formal positive charge on the carbonyl carbon, and this could be seen in the reaction with thiols. Zhao et al.⁷³ synthesized and reported BODIPY dye with a free meso-aldehyde group as turn-on fluorescent probe NIR-18 for the detection of Hcy. NIR-18 showed an absorbance maximum at 760 nm and nonemissive fluorescence behavior (Φ_F = 0.06) due to an n-π* transition. Upon addition of Hcy to NIR-18, absorption spectra recorded a nearly 100 nm blue shift to 661 nm. In emission, 30 times enhanced intensity (Φ_F = 0.92) was observed with Hcy at 678 nm (λ_ex = 620 nm) in ACN/H₂O (8:2, v/v, pH = 7.2). Cys induced a ninefold enhancement (Φ_F = 0.39), which proved the good selectivity of NIR-18 for Hcy over other amino acids. The proposed mechanism is depicted in Scheme 19, which shows the nucleophilic attack of the Cys/Hcy thiol group on the carbonyl carbon of aldehyde group, thereby forming a cyclized product. However, the fact that NIR-18 has, with Hcy, a reaction completion time of 60 min, the fact that a high percentage of the medium is an organic solvent, and the fact that the compound was not tested for any biological application could be disadvantageous for real-time applications.

Using the acrylate group as a Cys reactive site and an aldehyde group at the ortho position to assist cyclization and to facilitate an excited-state intramolecular proton transfer (ESIPT) mechanism, Zhao et al.⁷⁴ reported BODIPY dye-based probe NIR-19 for a highly selective turn-on sensor for Cys. Solvent-effect experiments confirmed a solvent-dependent property, and further spectroscopic studies showed the absorption band red-shifted from 495 nm (pure ACN) to 530 nm (10% ACN/90% buffer). On the other hand, emission spectra showed almost no wavelength shift with increase in ACN content, but strong enhancement was observed with high ACN content. The absorption and emission maxima were recorded at 527 and 650 nm, respectively, and colorimetric change from orange to rose red was observed in ACN/PBS (1:1, v/v, pH = 7.2). With a response time of 30 min and limit of detection of 0.233 μM, NIR-19 selectively detects Cys over Hcy and GSH. NIR-19 is also effective at physiological pH 6–8, which could be advantageous for exploring biological applications. As shown in Scheme 20, Cys attacks the acrylate group and removes it from NIR-19 by forming a cyclized byproduct. The free aldehyde group is also reactive toward Cys to form a cyclic product. The final products obtained have -NH- (from cyclized Cys) and −OH (obtained after acrylate group removal) in proximity, undergo intramolecular proton transfer, and facilitate the ESIPT mechanism. Biological applications were explored by fluorescent confocal cell imaging using MKN-45 cells. When the cells were incubated with NIR-19, red fluorescence was observed due to the reaction with biothiol present in cells, whereas cells pretreated with NMM showed no red fluorescence, and the fact that only green fluorescence was observed indicated the presence of unreacted NIR-19. A large Stokes shift of 123 nm and high selectivity toward Cys could be advantageous for using NIR-19 in real samples where GSH and Hcy normally coexist.

With better sensitivity but lower selectivity than NIR-19, Zhao et al.⁷⁵ developed NIR-20 for biothiols detection in PBS/DMSO (9:1, v/v, pH = 7.4). NIR-20 showed fluorescence enhancement with Cys and Hcy at two different wavelengths: 540 nm (30-fold, λ_ex = 470 nm) and 730 nm (15-fold, λ_ex = 670 nm). Interestingly, GSH induced a 28-fold enhancement in NIR-20 emission at 730 nm only, which could be helpful for discrimination of GSH from Hcy and Cys. With a better response time than NIR-19, it took only 10 min for NIR-20 to reach the full fluorescence with a limit of detection of 0.008 μM (Cys), 0.17 μM (Hcy), and 0.05 μM for GSH. The emission turn on at 730 nm was reported for the pH 6–10 range for GSH, whereas enhancement at 540 nm with Cys and Hcy was observed between pH 5 and 9. As depicted in Scheme 21, the initial PET process from the BODIPY unit to the nitrobenzoxadiazole (NBD) unit was in the “on” state. When GSH/Cys/Hcy were added, the two dyes get separated due to thiol-assisted ether bond cleavage and disturbed the PET mechanism, and thereby the BODIPY unit exhibited red fluorescence. However, the NBD-GSH product is nonfluorescent due to the thioether bond, whereas the Cys/Hcy-NBD dye is linked through the amino group and therefore shows emission in a green channel (540 nm). Confocal cell imaging of all three thiols (Cys, Hcy, and GSH) with NIR-20 was carried out. The GSH gave fluorescence in cells in the red channel only, whereas Cys and Hcy gave fluorescence changes in both green and red channels. This observation could be advantageous for discriminating Cys, Hcy, and GSH in different channels using NIR-20.

Qian et al.⁷⁶ have synthesized and reported NIR-21 as a probe for the detection of GSH in THF/PBS (1:1, v/v). BODIPY was used as a fluorescent reporter, and the 2,4-dinitrobenzenesulfonyl unit was used as a reactive unit for GSH. In a GSH environment, NIR-21 (Φ_F = 0.03) showed fluorescence intensity enhancement at 658 nm (λ_ex = 646 nm) with a response time of 60 min and limit of detection as low as 131 nM. However, NIR-21 is less selective due to induced enhancement with Cys (Φ_F = 0.24) and Hcy (Φ_F = 0.12) also but less as compared to GSH (Φ_F = 0.48). The GSH sensing mechanism involved the attack of sulfonate by a GSH thiol and detachment of the 2,4-dinitrosulfonyl group (quencher) from the fluorophore BODIPY unit leading to turn-on emission as shown in Scheme 22. Fluorescence cell imaging was performed to explore the practical utility of NIR-21 for detection of the intracellular GSH.

Dicyanomethylene-4H-1-benzopyran Dye-Based NIR Probes

Dicyanomethylene-4H-1-benzopyran can be regarded as a donor−π–acceptor type of structure and can have several significant photophysical properties such as a broad absorption band, NIR emission, high quantum yields, etc. Dicyanomethylene-4H-1-benzopyran has been one of the preferred dyes for researchers of interdisciplinary research.⁷⁷

With the same quencher and sensing mechanism as for NIR-21 and NIR-17, James et al.⁷⁸ have developed dicyanomethylene-4H-1-benzopyran-based GSH sensor NIR-22 with colorimetric response from slight yellow to pink. GSH induced a decrease in the absorbance band at 414 nm, and a new band emerged at 560 nm with an isosbestic point at 446 nm. In emission spectra, NIR-22 showed a fluorescence increase at 690 nm (λ_ex = 560 nm) with an almost 130 nm Stokes shift in DMSO/PBS buffer (1:1, v/v, pH = 7.4). As shown in Scheme 23, a GSH-specific O–S bond cleavage detached the quencher DNBS moiety, and the fluorescently active dicyanomethylene-4H-1-benzopyran unit showed a fluorescence increase. The reaction completion time was observed at about 5 min, which is better than that of NIR-21, and the LOD was calculated as low as 1.8 × 10^–8 M. NIR-21 also showed fluorescence enhancement with dithiothreitol (DTT), Cys, and Hcy and was stable between pH 5.7 and 8.0. The biological applicability of NIR-22 was confirmed by the detection of GSH in HeLa cells using confocal microscopy.

Feng et al.⁷⁹ have developed colorimetric and fluorescent sensor NIR-23 for the detection of biothiols in DMSO/PBS buffer (1:1, v/v, pH 7.4). NIR-23 is composed of conjugated dicyanomethylene-4H-1-benzopyran as a signaling unit and an acrylate group as an electron-withdrawing as well as thiol-responsive unit with the PET “on” process. In the absorption spectra, NIR-23 showed an absorption band at 395 nm, which showed a red shift (∼160 nm) to 555 nm with a light yellow to pink color change (response time ≈ 30 min) upon addition of Cys. With an 81 nM detection limit, emission enhancement was recorded at 706 nm (λ_ex = 560 nm), with response time of 15 min. As shown in Scheme 24, a Cys sensing mechanism involved thiol-assisted cleavage of the acrylate unit from NIR-23 and thereby showed turn “on” fluorescence in the NIR region by blocking the PET process that was effective over the pH 6–10 range. A similar but smaller increment of fluorescence intensity was observed with Hcy and GSH compared to Cys in absorption spectra. For biological applications of NIR-23, live cell imaging with HeLa cells was carried out, and it was found that, when NIR-23 was incubated with HeLa cells without pretreatment with NEM, this sensor can detect the biothiol present in living cells by inducing red fluorescence, whereas no such fluorescence emission was observed when cells were pretreated with NEM (thiol-blocking agent).

Wu et al.⁸⁰ have reported NIR-24 as a Cys sensor that could work in PBS/DMSO (1:1, v/v) medium. NIR-24 showed an absorbance maximum at 500 nm, which red-shifted to 600 nm in a Cys environment. In emission spectra, NIR-24 showed about 40-fold turn on emission in Cys at 760 nm (λ_ex = 600 nm) and a large Stokes shift of 160 nm. The reaction completion time of NIR-24 with Cys was reported as 5 min with an LOD of 48 nM, which showed better sensitivity than NIR-23. NIR-24 is selective for Cys, and only a slight enhancement was observed with Hcy and GSH. As with NIR-23, thiol-assisted cleavage of the acrylate group from NIR-24 gave a cyclized byproduct and dye with a free OH group that was reasoned to stay behind the spectral change with Cys as depicted in Scheme 25. Low toxicity of NIR-24 was observed, and in vitro cell imaging and in vivo mice imaging for Cys detection confirmed its potential biological application.

Disulfide bonds are prone to react with thiol groups, and using this chemistry, Zhu et al.⁸¹ have reported NIR-25 for the sensing of GSH in DMSO/PBS (1:1, v/v). The absorbance maximum of NIR-25 was observed at 435 and 448 nm, whereas weak fluorescence emission was recorded at 568 and 566 nm (λ_ex = 450 nm). Addition of GSH induced a 57 nm red shift in absorption spectra with a color change from yellow to red. Similarly, emission spectra also showed a red shift, and the new band was observed at 665 nm with an isosbestic point at 594 nm. NIR-25 needed 20 min to react completely with GSH, and the limit of detection was calculated as 24 μM. NIR-25 exhibited stability and was effective over the pH range of 6–9 for GSH sensing. However, similar enhancement with Cys, DTT, and Hcy could be a disadvantage in selectivity. The sensing mechanism consists of a two-step process, where the first step is the cleavage of disulfide bond and the second step involves intramolecular cyclization as shown in Scheme 26. Confocal cell imaging experiments support the potential application of NIR-25 for detecting GSH in biological systems.

Hemicyanine Dye-Based NIR Probes

Hemicyanine dye has been extensively used by researchers for the development of colorimetric and fluorescence probes.⁵¹ The hemicyanine skeleton is important due to its ability to modulate the electron-donating properties of attached donors to the acceptor molecules attached to other side in the same molecule by affecting the intramolecular charge transfer (ICT). Generally, a blue shift in the UV–vis or fluorescence spectra is observed when hemicyanine reduces the donating ability of a donor molecule, and a red shift is observed when the donating ability is enhanced. Hemicyanine dyes are also used to extend the conjugation and to generate a signal in long-wavelength regions.⁸²⁻⁸⁵

Using the same disulfide bond-cleavage mechanism used in NIR-25 but with a different signaling unit, Tang et al.⁸⁶ have synthesized GSH sensing probe NIR-26, which works in pure HEPES buffer. NIR-26 showed a turn-on fluorescence change of fivefold at 702 nm (λ_ex = 660 nm) with GSH. High selectivity was recorded among amino acids but not for thiol-group species such as DTT, Cys, and Hcy, which showed similar enhancement. The mechanism involves the attack of GSH to cleave the disulfide bond with subsequent intramolecular cyclization and, thereby, separation of the fluorescent reporting dye and anticancer drug (CPT = camptothecin) as shown in Scheme 27. The probe was effective in the pH range between 6.7 and 8.0, and a photostability experiment showed that NIR-26 is stable for at least 500 s, which is an advantage for applications in cells. The photostability was further supported by fluorescence imaging with three cell lines, namely, Hep G2, HL-7702, and MCF-7, and intense red fluorescence was recorded when the preincubated cells with NIR-26 were treated with GSH. Similarly, high fluorescence in the GSH environment was also recorded through in vivo imaging in an H22 tumor-bearing mouse.

A hemicyanine unit was also employed by Zhang et al.⁸⁷ in NIR-27a for the colorimetric and fluorescence detections of Cys in HEPES/DMSO (19:1, v/v) with excitation and emission wavelengths of 670 and 697 nm (Φ_F = 0.016), respectively. The addition of Cys to NIR-27a induced a purple to cyan color change within 5 min, and the limit of detection was calculated as 0.16 μM (emission) and 0.13 μM (UV–vis). The turn-on emission was also observed at 697 nm with a fluorescence quantum yield of 0.051. The reaction mechanism involved the reaction of Cys with the acrylate moiety of NIR-27a, consequently forming a cyclized byproduct as depicted in Scheme 28. The reaction kinetics was also studied, and a pseudo-first-order rate constant was calculated as 299 and 1.29 M^–1 s^–1 for Cys and Hcy, respectively. The pH reliability for Cys sensing was recorded between 6 and 10, and applications to quantify the thiol group bearing compounds in human serum and confocal imaging were explored using NIR-27a.

Using thianthon-indole salt as a fluorophore and an acrylate group as the recognition unit for Cys, Zeng et al.⁸⁸ have synthesized NIR-27b, which works in EtOH/PBS: v/v = 1/4, pH = 7.4. Intramolecular cyclization followed by elimination of an ester group was responsible for a spectral change with Cys as shown in Scheme 28. The absorbance maximum was observed at 610 nm, which was shifted to 728 nm, and emission at 770 nm increased in the Cys environment. The LOD was calculated as 16 nM and showed good selectivity toward Cys. With low cytotoxicity and lysosomal targeting ability, NIR-27b was used for detection of exogenous and endogenous Cys in HeLa cells.

Chen et al.⁸⁹ have synthesized and reported NIR-28a for turn-on detection of Cys in DMSO/PBS (1:99, v/v) using hemicyanine as a reporter and the dinitrophenyl group as a reactive unit. The detachment of the 2,4-dinitrophenyl group (as depicted in Scheme 29) due to a thiol group attack was responsible for the emission increase at 710 nm when excited at 600 nm. For signal saturation with Cys, NIR-28a consumed about 150 min, which is poorer than that for NIR-27a, whereas the limit of detection for Cys using NIR-28a was calculated as 82 nM, which is better than that for NIR-27a. Except the thiol-bearing and structurally similar Hcy and GSH, NIR-28a showed no change with various amino acids. Further, confocal cell imaging was investigated using three different cell lines: L02, A549, and MCF-7. The cell lines incubated with NIR-28a and pretreated with thiol blocking agent NEM showed no fluorescence. However, in the presence of any of Cys, Hcy, and GSH, a significant increase in red fluorescence was observed in the cells. In in vivo imaging of NIR-28a in an H22 tumor-bearing mouse, high fluorescence was recorded due to the higher concentration of biothiols in tumor cells. These experiments confirmed the application of NIR-28a in a biological field.

Lin et al.⁹⁰ have also developed hemicyanine-based fluorescent probe NIR-28b with an emission maximum at 716 nm (λ_ex = 690 nm) that detects biothiols in a PBS/ACN (7:3,v/v, pH = 7.4) medium. Structurally, NIR-28b is composed of hemicyanine as a reporter unit and the 2,4-dinitrobenzenesulfonyl group as a reactive unit, facilitating a nucleophilic attack of biothiols. For Cys, reaction completion was observed within 60 s. The quick response, NIR absorption, low toxicity, and cell membrane permeability facilitate the use of NIR-28b in biological applications, and this is well-supported by biothiol detection in new-born calf serum solution and NIR imaging of thiols using fluorescence imaging of thiols in HeLa cells.

Chen et al.⁹¹ have synthesized turn-on probe NIR-29 for the detection of Cys in PBS/EtOH (99:1, v/v) with emission wavelength at 710 nm (λ_ex = 630 nm, Φ_F = 0.15). In a Cys environment, the emission peak at 710 nm decreased, and a new peak at 626 nm emerged with emission enhancement at this wavelength and overall higher fluorescence (Φ_F = 0.48). In absorption spectra, a blue shift of about 120 nm was observed from 660 to 540 nm. NIR-29 consumed about 8 min for reaction completion, and the limit of detection was calculated as 0.15 μM for Cys. High selectivity including Hcy and GSH makes NIR-29 better than the aforementioned hemicyanine-based probes. As shown in Scheme 30, removal of thiophenol and attachment of a Cys molecule by its amino group were responsible for the spectral change. The pH range of 4.5–10 is effective for Cys sensing, and fluorescence imaging along with an in vivo application using zebra fish as a testing model makes NIR-29 a potential candidate for Cys sensing.

Wang et al.⁹² have reported NIR-30a as fluorescent sensor for Cys in pure PBS medium. With a 12-fold enhancement of emission at 680 nm (λ_ex = 540 nm), NIR-30a possessed high selectivity among important competitive anions including Hcy and GSH. The limit of detection for Cys sensing was calculated as 91 nM, and signal saturation was observed in about 10 min after the addition of Cys. As per the mechanism shown in Scheme 31, NIR-30a has two reactive sites; one is the acrylate, which is more reactive, and other is the −Cl group, which is less reactive. At a low concentration, Cys (0–5 μM) reacted with the acrylate group site, which triggered an increase in fluorescence at 680 nm, whereas, at a high concentration, Cys (5–200 μM) reaction takes place at both sites and, therefore, exhibits different concentration-dependent spectral changes. Confocal microscopy and in vivo imaging in living mice also supported the potential biological applications of NIR-30a. In cell imaging, cells were incubated with NIR-30a and then treated with a low concentration (5 μM) and high concentration (500 μM) of Cys. The low concentration of Cys exhibited red fluorescence, whereas the high concentration of Cys exhibited bright orange fluorescence under a confocal microscope. Importantly, pure aqueous conditions and the high selectivity of NIR-30a are comparable to those of NIR-29, but a low detection limit makes it better than NIR-29.

A thiol-assisted quencher-removal mechanism to revive the fluorescence was also used by Lin et al.⁹³ in NIR-30b for the detection of Cys in PBS/EtOH (9:1, v/v, pH = 7.4). Initially, NIR-30b showed weak fluorescence at 680 nm (λ_ex = 550 nm) due to an attached 2,4-dinitrobenzenesulfonyl group and showed concentration-dependent spectral changes. At a low concentration of Cys (0–50 μM), the emission band at 680 nm increases, whereas at higher concentration of Cys (50–500 μM), the emission band at 625 nm increases. The selectivity was also observed to be concentration-dependent; at a low concentration, selectivity was lower, and an emission change was observed with Hcy, GSH, and Na₂S, whereas at a higher concentration improved selectivity was observed, as only Hcy and Cys showed change, due to structural similarities. NIR-30b has followed the same mechanism as discussed with NIR-30a. NIR-30b was further used to detect the Cys concentration in living cells, and in vivo sensing studies were carried out in mice with low and high concentrations of Cys, in red and orange channels, respectively. The concentration-dependent properties of NIR-30b make it a rare kind of probe, which could detect Cys in both high and low concentrations in different channels.

Xanthene-Based NIR Probes

Xanthene-based dyes are considered one of the excellent fluorescent dyes due to the conversion of a carboxyl group into spirolactam or a spirolactone ring. Besides fluorescence properties, a high absorption coefficient and good water solubility have widened their scope for bioimaging applications. Spirolactam ring formation in the core structure of a xanthene dye leads to fluorescence quenching, whereas the open form with a free carboxyl group introduces a high fluorescence emission in the probes.⁹⁴

Using a rhodamine-hemicyanine dye as a fluorescence reporter, Qian et al.⁹⁵ have synthesized GSH sensor NIR-31, which works in EtOH/PBS (1:1, v/v) medium. NIR-31 worked as both a colorimetric (yellow to green color change) and turn-on fluorescent sensor for GSH at the emission wavelength of 739 nm (λ_ex = 600 nm). A quick response time of 5 s for a naked-eye color change and about 50 s for emission change saturation was recorded for NIR-31. A limit of detection as low as 0.01 μM and a high selectivity for GSH with 90-fold emission enhancement (Φ_F = 0.26) were observed. The color and fluorescence change were attributed to the GSH attack on the terminal unsaturated double bond followed by intramolecular cyclization to release a free −COOH group in the dye as shown in Scheme 32. NIR-31 works well and efficiently over the wide pH range of 5–12, and endogenous and exogeneous fluorescence imagings for GSH detection were carried out in MCF-7 cells, which supports biological applications of NIR-31.

A similar structural motif in a reporter dye was used by Li et al.,⁹⁶ who have designed and synthesized fluorescence turn-on and colorimetric probe NIR-32 for GSH in H₂O/DMSO (9:1, v/v). NIR-32 showed an absorbance change at 698 nm and exhibited a yellow to green color change in coexistence with GSH. In the emission spectra, NIR-32 showed weak emission (Φ_F > 0.01) due to spirolactam ring formation, which opens in the GSH environment with 75-fold turn on emission at 735 nm (λ_ex = 698 nm, Φ_F = 0.43). NIR-32 consumed 3 min for reaction completion; the limit of detection was calculated as 0.15 μM, and high selectivity among competitive amino acids, including Cys and Hcy, was observed. As shown in Scheme 33, a sensing mechanism involved the attack of GSH on the aldehyde group followed by the formation of a cyclized byproduct and leaving free −COOH group containing a rhodamine unit similarly as in NIR-31. Density functional theory (DFT) calculations were performed to understand the optical behavior and mechanism of NIR-32, and a biological application was explored using fluorescence imaging in live HeLa cells. When NIR-32 was incubated with HeLa cells, bright fluorescence was observed, and cells pretreated with NEM showed no fluorescence enhancement, which led to the conclusion that NIR-32 could be used for detecting endogenous GSH. Further, confocal imaging was also done using NIR-32 in rat live tissues up to 40–120 μm in depth. The tissue without any treatment showed red fluorescence due to GSH concentration, whereas no fluorescence was observed when a tissue slice was pretreated with thiol-blocking agent NEM.

Huo et al.⁹⁷ have designed and synthesized NIR-33 for the detection of Cys in DMSO/PBS (1:1, v/v) with emission recorded at 688 nm (λ_ex = 620 nm). Initially, NIR-33 showed no absorbance at 620 nm, but the addition of Cys, GSH, and Hcy induced an absorbance increase. Similarly, in emission spectra, from almost no emission at 688 nm, Cys, GSH, and Hcy have induced enhancement in fluorescence intensity. Reaction between NIR-33 and Cys proceeded with the detachment of the 2,4-dinnitrosulfonyl group from the dibenzoxanthene fluorophore and, thereby, revived the fluorescence, as shown in Scheme 34. The reaction was completed in 20 min, and the limit of detection was calculated as 2.93 μM (Cys), 1.29 μM (Hcy), and 59 nM (GSH). Being effective over the 5–11 pH range, NIR-33 selectively detects intracellular thiols, which was confirmed by live cell imaging using A549 cells.

Using the same fluorophore unit as in NIR-33 and exploiting a Cys-assisted acrylate group substitution mechanism, Feng et al.⁹⁸ reported NIR-34 as a colorimetric and fluorescent probe for Cys that works in DMSO/PBS (4:6, v/v). NIR-34 showed a colorimetric response from colorless to blue color change with Cys with the absorbance intensity rise at 620 nm. With the emission wavelength of 690 nm (λ_ex = 612 nm), a 560-fold emission enhancement was observed with Cys due to the removal of both acrylate groups as shown in Scheme 35. The reaction was completed in 10 min with a detection limit of 0.18 μM for Cys. The sensing of Cys with NIR-34 was observed over pH range of 2–9 with high selectivity including GSH and Hcy. Further, biological applications of NIR-34 were explored through cell imaging and recorded that NIR-34 could detect Cys at concentrations as as low as 20 μM.

Using the same mechanism as was discussed for NIR-35 and as shown in Scheme 36, Tan et al.⁹⁹ have developed NIR-35, which could work as both fluorescent and colorimetric probes for Cys in DMSO/PBS (2:8, v/v). The absorption spectra of NIR-35 showed a red shift from 589 to 692 nm in a Cys environment with the color change from blue to green. This sensor behaved as a ratiometric probe since a decrease at 660 nm and an increase at 716 nm (λ_ex = 610 nm) were detected. The limit of detection of 0.083 μM was calculated for Cys with high selectivity and effectivity over the pH range of 5–7. Further, NIR-35 was tested to detect endogenous Cys in HeLa cells by showing turn-on red fluorescence in the incubated cells observed by using confocal microscopy.

Lin et al.¹⁰⁰ have synthesized NIR-36 with a 2,4-dinitrobenzenesulfonyl unit as a quencher for the sensing of Cys in buffer/DMSO (8:2, v/v, pH 7.2). The absorption band of NIR-36 was recorded at 550 nm with emission at 665 nm (λ_ex = 580 nm) and with a Stokes shift of 110 nm. In a Cys environment, a sixfold fluorescence enhancement was observed, and the limit of detection was calculated as 43 μM with a response time of 10 min. The detachment of the 2,4-dinitrosulfonyl group from the fluorophore was responsible for the emission enhancement and spectral changes as shown in Scheme 37. However, a similar emission enhancement with Hcy and GSH could be a matter of concern for the selective detection of Cys using NIR-36. Furthermore, NIR-36 was used for the detection of Cys in live cells and in vivo experiments on mice.

Connecting BODIPY (donor unit) and rhodamine (acceptor unit) with a thiol-reactive disulfide bond, Zhao et al.¹⁰¹ have developed probe NIR-37, which utilizes the FRET process for biothiol detection in EtOH/PBS (1:2, v/v, pH = 7.4). NIR-37 showed absorbance band maxima at 501 and 625 nm corresponding to BODIPY (donor unit) and rhodamine (acceptor unit), respectively. Initially, the FRET on state showed NIR emission of rhodamine (λ_em = 656 nm) when BODIPY was excited at λ_ex = 480 nm, whereas, in the GSH environment, the two units were separated due to cleavage of the disulfide bond, and consequently FRET was turned off as shown in Scheme 38. The FRET process inhibition induced emission enhancement at 512 nm, when excited at 480 nm. Similar spectral changes were observed with Cys and Hcy. For GSH, the reaction time and limit of detection were calculated as 60 min and 0.26 μM, respectively. Further, confocal imaging was carried out to confirm the biological applications as shown in Figure 6. HeLa cells were incubated in NIR-37; green fluorescence from a BODIPY donor was observed, and almost no red fluorescence from the rhodamine acceptor was observed. When incubated with NEM, a significant increase in red fluorescence and a decrease in green fluorescence were observed due to FRET, whereas, when cells were incubated in GSH, strong BODIPY fluorescence was observed. This confirmed the applicability of NIR-37 for confocal cell imaging.

Fluorescence imaging of HeLa cells incubated with (a) **NIR-37** only, (b) cells preincubated with NEM followed by **NIR-37**, and (c) cells preincubated with NEM followed by GSH and **NIR-37**. Scale bar: 20 μm. Reprinted with permission from ref (101). Copyright 2020 Elsevier.

Nile Red Dye-Based NIR Probes

Nile red dye is a neutral dye with no formal charge, and it belongs to the benzophenoxazine family. Nile red is used in industries for dyeing fabrics due to its intense color and lipophilic nature. Nile red also exhibits high quantum yields of fluorescence in an aprotic medium and is often used for staining lipids. Although Nile red has poor solubility in water and must be formulated with the addition of organic cosolvents, it and its derivatives have been explored for the different applications.^102,103

Lin et al.¹⁰⁴ have developed and reported NIR-38 for the detection of GSH, Cys, and H₂S in PBS/ACN (7:3, v/v). The excitation wavelength of 510 nm was used, and emission maxima at 559 and 655 nm were recorded, selectively, for GSH, Cys, and H₂S. The response times of NIR-38 were recorded as 17, 25, and 7 min, and limits of detection were 0.05, 0.11, and 0.02 μM for GSH, Cys, and H₂S, respectively. NIR-38 is composed of a Nile red dye skeleton as fluorophore and NBD dye as quencher. An attack by GSH detached the two units, and red fluorescence was observed, whereas in the case of Cys, a similar detachment leads to red fluorescence; interestingly, a byproduct formed with an NBD-Cys reaction showed green fluorescence as shown in Scheme 39. NIR-38 possesses low toxicity, which was confirmed by a cytotoxicity assay, and was further used with confocal cell imaging to detect Cys, GSH, and H₂S. For in vivo studies a zebra fish model and living nude mice were used to detect Cys, GSH, and H₂S, and the results support the biological utility of NIR-38.

Benzothiazole-Based NIR Probes

Benzothiazole-based compounds are known for their multiple biological properties. Along with excellent biological properties, the benzothiazole moiety is also known for its photophysical properties, especially high fluorescence quantum yields and high stability in different solvents. Therefore, benzothiazole-based compounds have been used for applications in sensing, supramolecular chemistry, material chemistry, etc.^105−113

Zhang et al.¹¹⁴ have reported turn-on fluorescent sensor NIR-39 for Cys in PBS/EtOH (1:1, v/v), prepared through a Cys-induced removal of the 2,4-dinitrobenzenesulfonyl quencher unit (Scheme 40), with emission enhancement at 670 nm (λ_ex = 345 nm). NIR-39 showed a detection limit of 0.1 μM for Cys with a reaction completion time of about 30 min. The anionic product formed after the Cys attack showed solvent-dependent properties. Solvent studies confirmed that deprotonation is more feasible in a polar solvent that shifts the absorption/emission spectrum to a higher wavelength as compared to nonpolar solvents. NIR-39 showed good selectivity for thiols over amino acids and cations. NIR-39 was further applied for the detection of Cys in living cells using cell imaging experiments.

Zhang et al.¹¹⁵ have designed and synthesized GSH sensing probe NIR-40a, which worked in PBS/DMSO (1:1, v/v) with excitation wavelength of 350 nm, and emission was initially recorded at 426 nm (Φ_F = 0.138). The emission at 426 nm decreased with GSH addition, and a new peak emerged at 665 nm (Φ_F = 0.245). The absorbance at 516 nm increased readily with addition of GSH, and a colorless to red color change was observed. The response time for NIR-40a was recorded as 120 min, and the limit of detection was calculated as 0.35 μM. The pH range for NIR-40a utility was found out to be 6–8. The application of NIR-40a was confirmed by monitoring the fluorescence change in HeLa cells using confocal microscopic imaging. Under controlled conditions, HeLa cells pretreated with NEM exhibited no fluorescence, whereas strong red fluorescence was observed when the cells were incubated under a GSH environment.

Using the same signaling unit basis as was used with probe NIR-40a, Xu et al.¹¹⁶ have synthesized NIR-40b but with a methacrylate as a Cys-reactive unit. The absorption and emission maxima of NIR-40b were recorded at 343 and 431 nm (λ_ex = 396 nm), respectively, in PBS/DMF (1:1, v/v, pH 7.4). Selectively with Cys, the 343 nm absorption band decreased, and a new peak emerged at 533 nm with an isosbestic point at 396 nm and a change in color from colorless to pink. In emission spectra, a peak at 431 nm gradually decreased, and a new emission was observed in the NIR region at 710 nm. The Cys-assisted removal of the methacrylate unit from NIR-40b, thereby facilitating the ICT process on, was responsible for the spectral change as shown in Scheme 41. The limit of detection was calculated as 0.5 μM, and fluorescence imaging of Cys in live cells was carried out for exploring biological applications of NIR-40b.

Song et al.¹¹⁷ have designed and synthesized NIR-41 for the detection of Cys in DMSO/PBS (1:99, v/v) through the ICT process. In the presence of Cys, the original absorption band of NIR-41 at 540 nm increases, and an additional new band at 584 nm was formed. When excited at 670 nm, only weak emission at 720 nm (Φ_F = 0.01) was observed for the probe, but this was substantially enhanced with Cys (Φ_F = 0.15). Acrylate group removal due to thiol attack was responsible for the spectral change as shown in Scheme 42. With a response time of about 20 min, the limit of detection was calculated as 0.20 μM. In an interference study, only Hcy caused a slight change, but other analytes did not induce any noticeable change. NIR-41 works well between pH 6 and 9, and the mechanism was supported by DFT calculations. Biological applications were explored by quantifying Cys in human serum and with fluorescence cell imaging of endogenous Cys in BEL-7402 cells using confocal laser scanning microscopy.

Coumarin-Based NIR Probes

Coumarin derivatives are often used for the detection of small analytes, and due to their highly fluorescent nature and photostability, their applications were studied in biological samples via cell imaging.^118−120 Feng et al.¹²¹ have synthesized and reported NIR-42, which behaved both as a colorimetric and fluorescent sensor for the detection of Cys in PBS/DMSO (9:1, v/v) through the ICT effect. With Cys, NIR-42 showed a yellow to purple colorimetric change, easily detected by naked eye. In absorption spectra, an increase of the band at 335 nm and a decrease of the band at 423 nm were observed initially. At higher concentrations of Cys, the new band at 335 nm decreased, and a new band emerged at 584 nm. With Cys, the weak emission (Φ_F = 0.002) showed an enhancement of 60-fold (Φ_F = 0.031) at 675 nm, when excited at 574 nm. The mechanism involved the removal of the acrylate group and the formation of a free phenolic −OH, which gives the ICT effect and transfers the charge to the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile (TCF) unit, as depicted in Scheme 43. A spectral change of NIR-42 with Cys was observed at a concentration as low as 0.2 μM (LOD), and the response time was recorded as 10 min with an effective pH range of 6–9. The low toxicity of NIR-42 was observed, and biological application was further extended to fluorescence cell imaging using HeLa cells. In the Cys environment, red fluorescence was recorded in the cells, and therefore NIR-42 could be used for detection of an intracellular level of Cys.

Yin et al.¹²² have reported NIR-43 as the fluorescent probe for Cys with EtOH/PBS (1:1, pH = 7.4) medium as a working solvent mixture (Scheme 44). With Cys, the absorption spectral response showed that the weak band at 345 nm decreased and that a strong absorption peak at 450 nm red-shifted to 500 nm. NIR-43 showed emission intensity increased at 680 nm (λ_ex = 510 nm) in the Cys environment. The limit of detection was calculated as 0.053 μM, and the response time of 5 min was noticed. As shown in Scheme 8, a Cys-assisted removal of the acrylate group followed by cyclization was responsible for emission enhancement and, thereby, an exhibition of high fluorescence. NIR-43 was further utilized for a fluorescence imaging of intracellular Cys in HeLa cell lines due to low toxicity, as 90% of cell survival was observed after 12 h of incubation at a concentration less than 20 μM.

Yin et al.¹²³ have synthesized NIR-44 and used it as a sensor for the simultaneous detection of Cys/Hcy and GSH in PBS/MeOH (8:2, v/v, pH = 7.4) solvent medium. NIR-44 showed a decrease in the absorbance peak at 578 nm and an increase at 417 nm with Cys. This blue shift was attributed to the Michael addition of Cys to the conjugated bond connecting the indole unit and the coumarin unit with a reaction time of 60 min. A similar trend was observed with Hcy but at a slow reaction time (90 min) due to the kinetically less-stable eight-membered transition state. In the case of GSH, a blue shift of 169 nm was observed with a decrease in the initial absorbance at 578 nm and an increase at 409 nm; this was attributed to the nucleophilic addition of the amine group of GSH to the C=C next to the indole unit with a reaction time of 80 min (as shown in Scheme 45). In emission spectra, the peak at 724 nm (λ_ex = 510 or 555 nm) decreased, and a new peak emerged at 564 nm (λ_ex = 510 nm) and 600 nm (λ_ex = 555 nm); the emission change was more with Cys/Hcy than with GSH. The LOD was calculated using the absorbance change at 578 nm for Cys (2.965 μM), Hcy (6.140 μM), and GSH (6.847 μM). Further, cytotoxicity tests confirmed the low toxicity of NIR-44, and in vitro cell imaging using HepG2 cells and in vivo imaging using mice confirmed the biological significance of NIR-44.

Miscellaneous Probes

Gong et al.¹²⁴ have synthesized NIR-45, which is based on a donor-π-acceptor structure for the detection of Cys in PBS/DMF (1:1, v/v). A malononitrile unit was used as an acceptor, whereas a phenolic group (obtained after reaction with Cys) acts as a donor unit (Scheme 46). The phenolic −OH was protected initially with methacrylate and therefore exhibited a fluorescence off state. NIR-45 showed an absorbance change from 480 nm (decrease) to 614 nm (increase) with an orange-red to violet color change in a Cys environment. Initially, weak emission was observed at 655 nm (λ_ex = 614 nm, Φ_F = 0.033), which increased with an addition of Cys (Φ_F = 0.192). Reaction of NIR-45 with Cys was completed within 15 min, and the limit of detection was calculated as 1.06 μM. NIR-45 selectivity is fair, but GSH and Hcy also showed noticeable spectral changes. In confocal imaging, NIR-45 could detect intracellular Cys when incubated in the HeLa cells. Pretreatment with thiol-blocking agent NEM showed no fluorescence in cells, and bright red fluorescence was noticed when the sensor was incubated in Cys.

Wang et al.¹²⁵ have developed NIR-46a for the detection of biothiols in PBS/EtOH (1:1, v/v) medium. NIR-46a was composed of the 2,4-dinitrobenzenesulfonyl group, which acts as a quencher of fluorescence and a reactive unit toward thiols. A colorimetric change from pink to purple color and a fluorescence increase was recorded at 680 nm (λ_ex = 560 nm). The reaction completion times were recorded as 6 min (Cys), 12 min (GSH), and 14 min (Hcy), whereas detection limits of 36.93 nM (Cys), 32.56 nM (GSH), and 65.03 nM (Hcy) were calculated. The spectral change was due to the removal of the 2,4-dinitrobenzenesulfonyl quencher group, thereby blocking the PET from the fluorophore as depicted in Scheme 47a. NIR-46a was explored for biological applications through confocal fluorescence imaging to detect Cys, Hcy, and GSH in HeLa cells and also to detect the exogenous Cys. Along with biological application, NIR-46a was successfully utilized for Cys detection in real water samples with good recovery values.

Peng et al.¹²⁶ have synthesized NIR-46b for the selective detection of Cys in DMSO/PBS (4:1, v/v, pH 7.4). The absorption maximum of NIR-46b was observed at 525 nm and selectively shifted toward the higher wavelength of 550 nm after an addition of Cys with a color change from pink to purple. When excited at 550 nm, the weak emission maximum of NIR-46b was observed at 693 nm, and this was also enhanced selectively with Cys. The detection limit was calculated as 1.29 μM, and reaction completion was observed within 10 min. The spectral change induced by Cys was due to the removal of the acrylate group from the fluorophore as shown in Scheme 47b. The fluorescence intensity was also found to be polarity-dependent and was observed to trend as DMSO > DMS > MeOH > THF > ACN. Live cell imaging experiments were carried out using HeLa cells to detect intracellular and exogenous Cys. Overall, NIR-46b is more selective but less sensitive than NIR-46a.

Yuan et al.¹²⁷ have synthesized NIR-47 for the sensing of Cys in pure PBS buffer medium with excitation and emission wavelengths as 530 and 674 nm, respectively. The solvent studies confirmed the dependence of optical properties on different solvents, with fluorescence intensity in the following order: DMSO > EtOH > H₂O > PBS, whereas PBS was used for studies due to its biocompatibility. The limit of detection was calculated as 0.96 μM. NIR-47 showed high selectivity toward Cys over other biothiols and amino acids. Advantageously, NIR-47 is one of the probes for Cys, which could work in a pure aqueous medium. Initially, NIR-47 exhibited the ICT “off” mechanism, whereas emission enhancement was attributed to the Cys-assisted removal of the acrylate unit. Consequently, it facilitates the ICT process as depicted in Scheme 48. Finally, confocal cell imaging was successfully carried out for exploring applications of NIR-47. When HeLa cells were incubated with NIR-47, red fluorescence was observed due to the presence of thiol at cellular levels. However, when HeLa cells were pretreated with thiol blocking agent NEM and then incubated with NIR-47, no fluorescence was detected.

Hua et al.¹²⁸ have synthesized NIR-48 for the colorimetric and fluorescent detection of Cys via a carbonyl-assisted cycloaddition process in DMSO/HEPES (1:1, v/v) with emission maximum at 670 nm (λ_ex = 452 nm). In a Cys environment, NIR-48 absorption bands at 378 and 616 nm were decreased, and new bands that emerged at 296 and 452 nm showed a naked-eye detectable color change from greenish-black to orange. The limit of detection using an absorption change was calculated as 0.44 μM, whereas the values of 0.37 and 0.39 μM were calculated using emission changes at 670 and 452 nm, respectively. The mechanism involved the Cys-assisted removal of the protecting succinimidyl group followed by the formation of an intramolecularly cyclized product, as depicted in Scheme 49. The biological application of NIR-48 was confirmed by detecting Cys through fluorescence cell imaging in HeLa cells.

Zhang et al.¹²⁹ have designed and synthesized three different probes NIR-49a–NIR-49c for sensing of GSH in pure PBS buffer medium. The emission wavelength was recorded at 650 nm when excited at 605 nm. All three probes NIR-49a–NIR-49c have shown a change in color from blue to colorless in the presence of GSH and the response time of 5 s only for completion of reaction. The limits of detection were 0.059 μM for NIR-49a, 0.148 μM for NIR-49b, and 0.020 μM for NIR-49c. Comparing all three probes, NIR-49a showed excellent reusability in both color and emission change for GSH. The sensing mechanism involved the Michael addition reaction, which was also facilitated by the presence of a Si atom in the pyronine moiety as shown in Scheme 50. Further, time-resolved fluorescence imaging was carried out for visualizing the GSH fluctuation in HeLa cells and supported the use of NIR-49a in potential applications in biological fields.

Li et al.¹³⁰ designed and synthesized probe NIR-50 for the detection of Cys in DMSO/PBS buffer (3:7, v/v, pH 7.4). The NIR-50 was composed of N,N-dimethylamino units as electron donors, quinoline cations as electron acceptors, and acrylate as the recognition sites of Cys (Scheme 51). In the absorption spectra, the initial peak at 560 nm blue-shifted to 440 nm, when Cys was added. However, fluorescence emission at 700 nm increased 12-fold with high selectivity toward Cys. The biological application was studied indicating high biocompatibility, low-cytotoxicity, and good brain-barrier penetration. The in vitro and in vivo studies confirmed the good impact of NIR-50 in biological applications.

Xu et al.¹³¹ reported 1,8-naphthalimide and tricyanofuran-based water-soluble probe NIR-51 for Cys. Initially, the low fluorescence of NIR-51 was enhanced in the presence of Cys at the emission maximum of 665 nm (λ_ex = 620 nm). The low fluorescence in the OFF state based on ICT quenching was switched ON when Cys reacted with an acrylate group (as shown in Scheme 52). Additionally, a color change from light yellow to purple appeared in the presence of Cys with fast response, high selectivity, and high sensitivity. The limit of detection was calculated as 0.093 μM. The probe NIR-51 was successfully employed for the detection and imaging of Cys in HeLa cells and zebra fish, which confirmed that NIR-51 could have potential applications for intracellular Cys detection.

Sun et al.¹³² have developed NIR-52 for the detection of Cys and GSH in DMSO/PBS (v/v = 1/4, pH = 7.4) medium. In the Cys environment, the emission band at 670 nm (λ_ex = 490 nm) was enhanced fourfold with a large Stokes shift of 180 nm. The sensing mechanism involved conjugated nucleophilic addition by a thiol attack on the acrylate group followed by an intramolecular cyclization reaction as shown in Scheme 53. For biological applications, NIR-52 showed good lysosomal targeting ability due to the presence of a morpholine group in its structure and was used to detect exogenous and endogenous biothiols.

Chen et al.¹³³ synthesized 7,8-pyranocoaumarin-based probe NIR-53 for the detection of Cys, Hcy, and GSH. With the dinitrobenzenesulfonyl group as a recognition unit, a nucleophilic substitution reaction mechanism was responsible for the spectral change as shown in Scheme 54. NIR-53 initially showed absorbance at 566 nm, which was red-shifted to 723 nm upon meeting Cys, Hcy, or GSH in PBS/DMSO medium (v/v: 4/1, pH = 7.4). In emission, the fluorescence intensity in the presence of Cys/Hcy/GSH increased at 751 nm (λ_ex = 680 nm) with reaction completion time less than 5 s. The LOD for Cys, Hcy, and GSH was calculated as 0.08, 0.20, and 0.11 μM, respectively. The solvent studies confirmed that, with an increase in the amount of DMSO, both absorption and fluorescence intensities increase. The capacity for biological application was explored and confirmed by low cytotoxicity, good compatibility, and mitochondrial targeting ability. Further, the NIR-53 probe was explored for in vitro and in vivo studies in a mouse model.

Conclusions and Perspectives

In this review, we mainly focused on the NIR absorbing/emitting organic probes for detection of biothiols. The NIR region has gained immense interest and has been explored for several biological applications both in vitro and in vivo. Mainly, organic dyes such as squaraine, cyanine, BODIPY, hemicyanine, dicyanomethylene-4H-1-benzopyran, xanthene (rhodamine/fluorescein), Nile red, etc. are commonly used for introducing the NIR photophysical properties. The core skeletons of these NIR dyes have several advantages, for example, intense absorption bands with a high extinction coefficient; they also possess strong emission, high biocompatibility, and thermal stability. However, chemical instability, multistep synthesis, and self-aggregation are major issues while working with these dyes, especially squarine, cyanine, BODIPYs, and Nile red core skeletons. Multistep synthesis is not only time-consuming but also enhances the overall cost of the probes. Making the probes water-soluble is also a common issue while working with these core skeletons, which can be easily seen from the probes discussed in the review.

Regarding the sensing principles, nucleophilic attack-assisted removal of the quencher groups such as the 2,4-dinitrobenzenesulfonyl unit or 2,4-dinitrobenzene unit are mostly used. Removal of acrylate from OH with subsequent induction of a strong donor–acceptor system is another important and widely utilized principle in the reported probes. However, these reaction-based sensors often consume more time for complete saturation of signal with no reusability (the reaction is irreversible), which could be disadvantageous for the monitoring of thiols in real time. However, this could be eliminated by a metal displacement approach, which often shows the reusability of the probe due the reversible mechanism with alternate addition of the metal and biothiol (analytes). All the discussed probes exhibit fluorescence changes, which is highly suitable for in vivo, in vitro detection, but it may limit their utilization to instrument-based detection. Therefore, probes with dual response (both colorimetric and fluorescence changes) need to be developed in the future.

For selectivity, most of the probes are selective for Cys, Hcy, and GSH among other amino acids. But, while considering the selectivity among only the biothiols (Cys, Hcy, and GSH), the majority of reported probes fails to discriminate them from one another with few exceptions (Table 1). Most of the probes have a detection limit in the range of μM levels, and therefore more probes with sensitivity in the nM or lower range need to be developed. To improve the selectivity, an aromatic substitution mechanism seems to be comparatively a better approach, as it could provide the emission signals at different wavelengths for Cys, Hcy, and GSH. The analyte-dependent signal allows one to discriminate between thiols, and BODIPYs and cyanine dyes appear to be good candidates for designing such probes due to their wide range of spectrum absorption/emission. The water solubility can be improved by introducing water-solubilizing groups such as sulfonate or quaternary amino groups to the probes. Further, two-photon and photoacoustic-based probes could be an alternate for the detection of thiols in the NIR region.

Table 1. Comparative Table for the Reported NIR Probes for Biothiols.

structure	solvent (v/v)	λ_ex/λ_em (nm)	color change	response time (min)	detected thiol	LOD
NIR-1	ACN/CHES buffer (1:1), pH = 9.6	750/800	green to light yellow		GSH, Cys	NA
NIR-2	PB/CTAB, (1 mM) pH = 7.5		green to yellow	9	GSH	10 nM
NIR-3	ACN/PBS (2:8), pH = 7.4	610/665		120	GSH	0.15 μM
NIR-4	ACN/PBS (1:9), pH= 7.0	634/656	blue to light blue	20	Cys	2 μM
NIR-5	DMSO/PBS (3:7), pH = 7.4	808/928		60	Cys	85 nM
NIR-6	EtOH/HEPES (1:9) pH = 7.4	720/780 and 520/570	light blue to red	30	Cys
NIR-7	DMSO/PBS buffer (5:5, v/v, pH = 7.4)	360/435,475	green to red	50 (Cys)	Cys, Hcy	27.9 nM (Cys)
		660/733,815		75 (Hcy)		30.1 nM (Hcy)
NIR-8a	PBS buffer pH = 7.4	635/750		3	GSH
NIR-8b
NIR-9	DMSO/HEPES buffer (1:4), pH 7.4	650/750		60	Cys	1.26 μM
NIR-10	PBS buffer	600/700	light green to blue	2	GSH	5 μM
NIR-11	PBS/ACN (9:1, pH 7.4).	720/750		60	GSH	75 nM (GSH), 94 nM (Cys), and 160 nM (Hcy)
					Cys
					Hcy
NIR-12a	HEPES/DMSO (99:1)	780/805		10	GSH	0.63 μM
NIR-12b	pH = 7.4					0.33 μM
NIR-13	HEPES/DMSO (9:1)	370/495	light blue to blue	180	GSH	0.153 μM (Vis),
	pH = 7.4	700/795				0.171 μM (NIR)
NIR-14	HEPES/buffer	600/810		150	GSH	26 nM
	pH = 7.4
NIR-15a	HEPES/DMSO (9:1), pH = 7.4	710/818		40	GSH
NIR-15b	DMSO/PBS (1:1, v/v)	650/776	pale yellow to blue (with Cys) and to turquoise (with Hcy)		Cys, Hcy	Emission: 0.66 μM (Cys) 1.22 μM (Hcy)
						Absorbance: 0.47 μM (Cys) 0.23 μM (Hcy)
NIR-16a	HEPES/DMSO (9:1)	730/736		15	GSH, Cys, Hcy
NIR-16b	pH = 7.4				GSH
NIR-17	ACN/H₂O/DMSO (79:20:1) pH = 7.5	717/734		50	Cys	0.7 μM
NIR-18	ACN/H₂O	620/678		60	Hcy, Cys
	(8:2)
	pH = 7.2
NIR-19	ACN/PBS buffer (1:1)	527/650	orange to rose red	30	Cys	0.233 μM
	pH = 7.2
NIR-20	PBS/DMSO (9:1)	470/540 and 670/730		10	Cys	0.05 μM (GSH),
	pH = 7.4				GSH	0.08 μM (Cys),
					Hcy	0.17 μM (Hcy)
NIR-21	THF/PBS (1:1)	646/658		60	GSH	131 nM
	pH = 7.4,
NIR-22	DMSO/PBS buffer (1:1, pH = 7.4	560/690	slight yellow to pink	5	GSH	18 nM
NIR-23	DMSO/PBS (1:1)	560/706	light yellow to pink	15	Cys	81 nM
	pH = 7.4
NIR-24	PBS/DMSO (1:1)	600/760		5	Cys	48 nM
	pH = 7.4
NIR-25	DMSO/PBS (1:1) pH = 7.4	450/665	yellow to red	20	GSH	24 μM
NIR-26	HEPES buffer pH = 7.4	660/702			GSH
NIR-27a	HEPES/DMSO (19:1)	670/697	purple to cyan	5	Cys	0.16 μM (emission), 0.13 μM (UV–vis)
NIR-27b	pH = 7.4 ethanol/PBS (1/4, v/v, 10 mM, pH 7.4)	700/770		5	Cys	16 nM
NIR-28a	DMSO/PBS (1:99) pH = 7.4	600/710		5	Cys	82 nM
NIR-28b	PBS/CH₃CN (7:3) pH = 7.4	690/716		60 s	Cys
NIR-29	PBS/EtOH (99:1)	630/710		8	Cys	0.15 μM
	pH = 7.4
NIR-30a	PBS	540/680		10	Cys	91 nM
	pH = 7.4	550/680		10 (680 nm)	Cys
NIR-30b	PBS/EtOH (9:1)			60 (630 nm)
	pH = 7.4
NIR-31	EtOH/PBS (1:1)	600/739	yellow to green	50 s.	GSH	0.01 μM
	pH = 7.4
NIR-32	H₂O/EtOH(9:1)	698/735	yellow to green	3	GSH	0.15 μM
	pH = 7.4
NIR-33	DMSO/PBS (1:1)	620/688		20	Cys	2.93 μM
	pH = 7.4
NIR-34	DMSO/PBS (4:6)	612/690	colorless to blue color	10	Cys	0.18 μM
NIR-35	PBS/DMSO (8:2)	610/660 and 716	blue to green		Cys	0.083 μM
	pH = 7.4
NIR-36	Buffer/DMSO (8:2)	580/665		10	Cys	43 μM
	pH = 7.2
NIR-37	EtOH/PBS (1:2)	480/656		60	GSH	0.26 μM
	pH = 7.4
NIR-38	PBS/ACN(7:3)	510/559, 655		Cys (17), GSH (25)	Cys, GSH,	0.05 μM (Cys),
						0.11 μM (GSH)
	pH = 7.4
NIR-39	PBS/EtOH (1:1)	345/670		30	Cys	0.1 μM
	pH = 7.4
NIR-40a	PBS/DMSO (1:1)	350/665	colorless to red colorless to pink	120	GSH	0.35 μM
NIR-40b	PBS/DMF (1:1)	396/710			Cys	0.5 μM
NIR-41	DMSO:PBS (1:99)	670/720		20	Cys	0.20 μM
	pH = 7.4
NIR-42	PBS/DMSO (9:1), pH = 7.4	574/675	yellow to purple-blue	10	Cys	0.2 μM
NIR-43	EtOH/PBS (1:1, pH = 7.4)	510/680		5	Cys	0.053 μM
NIR-44	PBS/MeOH (8:2, v/v, pH = 7.4)	510 or 555/724		60 (Cys)	Cys, GSH, Hcy	2.965 μM (Cys), 6.847 μM (GSH), 6.140 μM (Hcy)
				80 (GSH)
				90 (Hcy)
NIR-45	PBS/DMF (1:1), pH = 7.4	614/655	light orange to violet	15	Cys	1.06 μM
NIR-46a	PBS/EtOH (1:1)	560/680	pink to purple	6 (Cys)	Cys, GSH, Hcy	36.93 nM (Cys), 32.56 nM (GSH), 65.03 nM (Hcy)
	pH = 7			12 (GSH)
NIR-46b	DMSO/PBS (4:1)	550/693	pink to purple	14 (Hcy)	Cys	1.29 μM (Cys)
	pH = 7.4			10 (Cys)
NIR-47	PBS buffer pH = 7.4	530/674			Cys	0.96 μM
NIR-48	DMSO/HEPES (1:1)	452/670	greenish-black to orange		Cys	0.44 μM (UV–vis),
	pH = 7.4					0.37 μM (λ_em = 670 nm),
NIR-49 (a-c)	PBS buffer pH = 7.4	605/650	blue to colorless	5 s	GSH	0.059 μM (NIR-47a), 0.020 μM (NIR-47b), 0.148 μM (NIR-47c)
NIR-50	DMSO/PBS buffer (3:7, v/v, pH 7.4)	440/700		15 min	Cys	20.8 nM
NIR-51	PBS buffer solution (10 mM, pH = 7.4)	620/665	pale yellow to blue-violet	20 min	Cys	0.093 μM
NIR-52	DMSO/PBS (v/v = 1/4, pH = 7.4)	490/670		5 min	Cys, GSH	35.2 nM (GSH) 34.8 nM (Cys)
NIR-53	PBS/DMSO system (v/v = 4/1 pH = 7.4)	680/751		5 s	Cys, GSH, Hcy	0.08 μM(Cys), 0.20 μM(Hcy) and 0.11 μM(GSH)

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Finally, we believe that there is still a vast scope of applications to explore and develop more advanced NIR sensors for thiols by introducing new NIR fluorophores with properties such as better solubility in water media, reusability, sensitivity, a probe that can discriminate between Cys, Hcy, and GSH and is not limited to sensing but can also be useful for interdisciplinary applications.

Acknowledgments

R.K. is grateful to The Director, CSIR-NIO, India, for his support and encouragement. R.K. would like to acknowledge funding from Project Nos. OLP 2006 and MLP 2019. This manuscript represents the CSIR-NIO MS ref No. 10867 and MS contribution number 7008. N.N. would like to thank IACS for RA-I position. R.K., V.N., and P.Z. would like to thank EFSA-CDN Project No. (Reg. No. CZ.02.1.01/0.0/0.0/16_019/0000841) cofunded by ERDF for financial support. V.N. would also like to acknowledge the financial support from Charles University (PRIMUS/20/SCI/013).

The authors declare no competing financial interest.

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PERMALINK

Near-Infrared Probes for Biothiols (Cysteine, Homocysteine, and Glutathione): A Comprehensive Review

Rahul Kaushik

Nidhi Nehra

Veronika Novakova

Petr Zimcik

Abstract

Introduction

Figure 1.

Scheme 1. General Sensing Mechanisms Used by Reaction-Based Biothiol Probes.

Squaraine-Based NIR Probes

Scheme 2. Proposed Sensing Mechanism of NIR-1 for Thiols.

Scheme 3. Proposed Sensing Mechanism of NIR-2 for Thiols.

Scheme 4. Proposed Sensing Mechanism of NIR-3 for GSH.

Scheme 5. Proposed Sensing Mechanism of NIR-4 for Cys/Hcy.

Figure 2.

Cyanine Dye-Based NIR Probes

Scheme 6. Proposed Sensing Mechanism of NIR-5 for GSH.

Scheme 7. Proposed Sensing Mechanism of NIR-6 for Cys.

Scheme 8. Proposed Sensing Mechanism of NIR-7 for Cys.

Scheme 9. Proposed Sensing Mechanism of NIR-8a and NIR-8b for GSH.

Scheme 10. Proposed Sensing Mechanism of NIR-9 for Thiols.

Scheme 11. Proposed Sensing Mechanism of NIR-10 for GSH.

Scheme 12. Proposed Sensing Mechanism of NIR-11 for GSH, Cys, and Hcy.

Figure 3.

Scheme 13. Proposed Sensing Mechanism of NIR-12a and NIR-12b for GSH, Cys, and Hcy.

Scheme 14. Proposed Sensing Mechanism of NIR-13 for GSH.

Figure 4.

Scheme 15. Proposed Sensing Mechanism of NIR-14 for GSH.

Scheme 16. Proposed Sensing Mechanism of NIR-15a for GSH and NIR-15b for Cys and Hcy.

Scheme 17. Proposed Sensing Mechanism of NIR-16a for GSH and NIR-16b for GSH, Cys, and Hcy.

Figure 5.

BODIPY Dye-Based NIR Probes

Scheme 18. Proposed Sensing Mechanism of NIR-17 for Cys.

Scheme 19. Proposed Sensing Mechanism of NIR-18 for Cys and Hcy.

Scheme 20. Proposed Sensing Mechanism of NIR-19 for Cys.

Scheme 21. Proposed Sensing Mechanism of NIR-20 for GSH, Cys, and Hcy.

Scheme 22. Proposed Sensing Mechanism of NIR-21 for GSH.

Dicyanomethylene-4H-1-benzopyran Dye-Based NIR Probes

Scheme 23. Proposed Sensing Mechanism of NIR-22 for GSH.

Scheme 24. Proposed Sensing Mechanism of NIR-23 for Cys.

Scheme 25. Proposed Sensing Mechanism of NIR-24 for Cys.

Scheme 26. Proposed Sensing Mechanism of NIR-25 for GSH.

Hemicyanine Dye-Based NIR Probes

Scheme 27. Proposed Sensing Mechanism of NIR-26 for GSH, whereas CPT = Camptothecin.

Scheme 28. Proposed Sensing Mechanism of NIR-27a for Cys and Hcy and NIR-27b for Cys.

Scheme 29. Proposed Sensing Mechanism of NIR-28a for Cys and NIR-28b for Biothiols.

Scheme 30. Proposed Sensing Mechanism of NIR-29 for Cys.

Scheme 31. Proposed Sensing Mechanism of NIR-30a and NIR-30b for Cys.

Xanthene-Based NIR Probes

Scheme 32. Proposed Sensing Mechanism of NIR-31 for GSH.

Scheme 33. Proposed Sensing Mechanism of NIR-32 for GSH.

Scheme 34. Proposed Sensing Mechanism of NIR-33 for GSH, Cys, and Hcy.

Scheme 35. Proposed Sensing Mechanism of NIR-34 for Cys.

Scheme 36. Proposed Sensing Mechanism of NIR-35 for Cys.

Scheme 37. Proposed Sensing Mechanism of NIR-36 for GSH, Cys, and Hcy.

Scheme 38. Proposed Sensing Mechanism of NIR-37 for GSH.

Figure 6.

Nile Red Dye-Based NIR Probes

Scheme 39. Proposed Sensing Mechanism of NIR-38 for GSH/Cys.

Benzothiazole-Based NIR Probes

Scheme 40. Proposed Sensing Mechanism of NIR-39 for Cys.

Scheme 41. Proposed Sensing Mechanism of NIR-40a for GSH and NIR-40b for Cys.

Scheme 42. Proposed Sensing Mechanism of NIR-41 for Cys.

Coumarin-Based NIR Probes

Scheme 43. Proposed Sensing Mechanism of NIR-42 for Cys and Hcy.

Scheme 44. Proposed Sensing Mechanism of NIR-43 for Cys.

Scheme 45. Proposed Sensing Mechanism of NIR-44 for Cys/Hcy and GSH.

Miscellaneous Probes

Scheme 46. Proposed Sensing Mechanism of NIR-45 for Cys and Hcy.

Scheme 47. Proposed Sensing Mechanism of NIR-46 for Biothiols.

Scheme 48. Proposed Sensing Mechanism of NIR-47 for Cys.

Scheme 49. Proposed Sensing Mechanism of NIR-48 for Cys.

Scheme 50. Proposed Sensing Mechanism of NIR-49a-c for GSH.

Scheme 51. Proposed Sensing Mechanism of NIR-50 for Cys.

Scheme 52. Proposed Sensing Mechanism of NIR-51 for Cys.

Scheme 53. Proposed Sensing Mechanism of NIR-52 for Cys.

Scheme 54. Proposed Sensing Mechanism of NIR-53 for Biothiols.

Conclusions and Perspectives

Table 1. Comparative Table for the Reported NIR Probes for Biothiols.