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. 2024 Jan 3;2(6):422–431. doi: 10.1021/cbmi.3c00110

Near-Infrared Fluorescent Probe with pH- and Viscosity-Switchable Performance for the Detection of Thrombi in Live Animals and Organs

Jinlan Luo 1, Changyong Song 1, Yunling Chen 1, Keyin Liu 1,*
PMCID: PMC11504539  PMID: 39474516

Abstract

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Blood viscosity changes and blood clots are high-impact diseases, but the pathogenic mechanisms and detection methods are still limited. Due to the complexity of the cellular microenvironment, viscosity is a key factor in regulating the behavior of mitochondria and lysosomes in cells. Conventional fluorescence probes are highly restrictive for complex viscosity detection in live animals. Therefore, we developed two near-infrared fluorescence probes, QL1 and QL2, with dual responses to the pH and viscosity. Notably, QL2 has two maximum fluorescence emissions at 680 and 750 nm, when excitation by 580 and 700 nm, respectively. QL2 exhibited both a pH and viscosity switchable fluorescence response. The two emission peaks exhibited a reverse change trend: the fluorescence at 680 nm decreased by 90%, and the fluorescence at 750 nm increased by about 5-fold with pH from 2 to 10. Meanwhile, both emission peaks show remarkable fluorescence enhancement toward viscosity change, with 185 and 32 times enhancement, respectively. The sensing mechanism and spectral changes are confirmed by DFT calculations. QL2 was further used for viscosity imaging in live cells, zebrafish, and live animals. Most importantly, QL2 is able to successfully track changes in blood clots in live mice and organs, thus enabling the study of blood clots in cerebral strokes and the underlying pathological mechanisms.

Keywords: Fluorescent detection, viscosity, near-infrared emission, mouse organs, live animal

1. Introduction

Viscosity is one of the most important factors impacting the body.1,2 Changes in viscosity also have a significant effect on individual organelles in cells.3 For example, changes in mitochondrial viscosity can lead to disturbances in mitochondrial function, thus further leading to changes in the mitochondrial matrix components. In turn, this can lead to diseases and cellular dysfunctions such as diabetes, Alzheimer’s disease, atherosclerosis, and cellular malignancies.4−8 When blood viscosity increases, it can slow blood flow. Red blood cell aggregation, which is common during aging and atherosclerosis, leads to increased blood viscosity and an insufficient blood supply to the heart and cerebral vessels. When the blood is excessively viscous and in a hypercoagulable state, blood clot formation, ischemic strokes, and ischemic emergencies occur more easily.9,10 The viscosity is different in each area of the cell and the body, and thus, it is difficult to monitor viscosity changes in complicated systems. Thus, developing novel detection tools is significant for investigating viscosity changes and blood clots in live animals.

However, traditional viscometers, such as capillary viscometers and rotational viscometers, can measure only the viscosity of liquids. They are still limited in evaluating viscosity changes at the microscopic level.11−13 Fluorescent probes can interact with analytes to cause changes in the fluorescence properties. Fluorescence imaging is commonly used in biological species detection because of its simple operation, high selectivity, and good temporal and spatial resolution.14−20 Sun et al. designed and synthesized a two-photon fluorescent molecular probe for ratiometric imaging of viscosity and mitochondrial endogenous ONOO–. The probe can target mitochondria.21 Peng et al. reported the first probe for dual-mode fluorescence imaging of intracellular viscosity. The fluorescence lifetime is prolonged in viscous media, which provides not only a basis for ratio measurement but also a condition for fluorescence lifetime imaging.22

However, viscosity may also be influenced by other factors, such as the solvents, ions, and pH of the environment. Intracellular pH plays a critical role in the physiological parameter. It plays an important regulatory role in various cellular events including ion transport, enzyme activity, cell growth, calcium regulation, endocytosis, cell adhesion, etc.23,24 In addition, the pH of each subcellular organelle is different due to its specific function. For example, the pH of the lysosomes is 4–6;25,26 the pH of the cytoplasm is ∼7; and the pH of the mitochondrial and nuclear regions is 8.0.27−29 Thus, our goal here was to measure the viscosity and pH changes simultaneously.

The absorption and emission spectra of classical rhodamine derivatives are below 600 nm, which hinder their application in live animal imaging.30−32 As known, the emission wavelength of chromophores can be red-shifted by extending the conjugated π-bonds. The original molecular structure of rhodamine is stable enough with a high fluorescence quantum yield. It can be used as core to construct novel near-infrared rhodamine analogue with larger conjugated π-bonds and excellent near-infrared fluorescence performance. Herein, we focused on the construction of near-infrared emission rhodamine fluorescent probes for viscosity and pH measurements (Scheme 1). The emission and sensing properties of chromophores was improved by enlarging the conjugated π system.

Scheme 1. Design and Response Mechanism of QL1 and QL2 to Viscosity and pH.

Scheme 1

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2. Experimental Section

2.1. Experimental Materials, Reagents, and Equipment

The experimental reagents, solvents, equipment, and procedures are shown in the Supporting Information.

2.2. Synthesis of QL1 and QL2

The methods of preparation of 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydro-xanthylium (compound 1), QL1, and QL2 are shown in the Supporting Information (Scheme S1). In Scheme 2, both probes were synthesized from 4-hydroxyisophthalaldehyde under different conditions, where the 1:1 reaction produced QL1 in 90% yield.33 The 2:1 reaction produced QL2 in 70% yield (Supporting Information). All of the raw materials are inexpensive and readily available, and the reaction conditions are mild with excellent yields.

Scheme 2. Synthesis of Near-Infrared Fluorescent Probes QL1 and QL2.

Scheme 2

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3. Results and Discussion

3.1. UV Absorption Spectra of Probes QL1 and QL2

The photophysical properties of QL1 and QL2 in different pH buffers and solutions of different viscosities were studied by UV absorption and fluorescence spectroscopy. Here, the viscosity was modulated by water with different concentrations of glycerol. The UV absorption spectra show that both QL1 and QL2 had a maximum peak at around 590 nm. With an increasing viscosity, the maximum absorption peak at 590 nm increased significantly (Figure 1a, b). QL2 also showed an extra small absorption peak at 720 nm (Figure 1b), which also increased with the increase in viscosity. However, the maximum absorption peaks of QL1 and QL2 exhibited remarkably different behavior when the pH is changed. With the increase in solution alkalinity, the absorption peak of QL1 decreased, and red shifts from 550 to 600 nm (Figure 1c). As for QL2, the two absorption peaks of QL2 had an inverse trend of change when the pH changed (Figure 1d). The absorption peak at 600 nm decreased with increasing pH, while the maximum absorption peak at 720 nm increased significant.

Figure 1.

Figure 1

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UV–vis absorption spectra of QL1 (a) and QL2 (b) in solutions of different viscosities. UV–vis absorption spectra of QL1 (c) and QL2 (d) in different pH solutions.

3.2. Fluorescence Response Spectra of QL1 and QL2 to pH

The fluorescence spectral characteristics of QL1 and QL2 in different pH solutions were studied. Similar to the absorption spectra change, the maximum emission peak of QL1 was red-shifted from 600 to 740 nm with increasing pH values (Figure S1), which was due to the enhancement of the electron push–pull system. Both emissions at 600 and 740 nm weakened as the pH increased.

QL2 gave a totally different fluorescence change compared with QL1 when the pH changed. QL2 has a major emission peak near 700 nm (Figure S2a), and the fluorescence intensity decreases by over 90% and blue-shifted with increasing pH from pH 2 to pH 10. Interestingly, the fluorescence intensity at 750 nm increased approximately 5-fold over this change from pH 2 to 10, although the fluorescence intensity is weak in the region. Considering the significant signal changes of the two fluorescence emissions, the ratiometric measurement of pH change in solution (F700/F750) can also be obtained. The probe showed good linear fluorescence response and a significant ratiometric fluorescence change (Figure S2b, c).

3.3. Fluorescence Response Spectra of QL1 and QL2 to Viscosity

We evaluated the fluorescence spectra of probes QL1 and QL2 with an increasing solution viscosity. The color of the solution changed significantly with the increase in viscosity. Figure 2a and b shows that QL1 has only weak fluorescence in PBS solution. With increasing viscosity, the fluorescence at 590 and 740 nm increases approximately 5 and 9 times (excitation by 490 and 620 nm, respectively), respectively. By plotting the fluorescence intensity (log I) and solution viscosity (log η) (Figure 2e, f), we see that QL1 had a good linear relationship (R2 = 0.99). Similarly, QL2 has good viscosity response characteristics. At 680 and 750 nm, the fluorescence increases significantly with increasing solution viscosity (Figure 2c, d), and then increases nearly 185 and 32 times (excitation by 580 and 700 nm, respectively), respectively. QL2 has a good linear relationship (R2 = 0.99) from 0.903 to 1410 cP (Figure 2g, h). QL2’s fluorescence changes can also be observed with the naked eye (Figure 2i). These results indicate that both probes can be used as sensitive fluorescent probes to quantitatively detect solution viscosity. Most importantly, QL2 is much more sensitive than QL1. The good fluorescence response of QL2 is due to its low background fluorescence in PBS solution.34

Figure 2.

Figure 2

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(a, b) Fluorescence spectra of QL1 (10 μM) in PBS and glycerol solution in different ratios (from 0.903 to 1410 cP). (c, d) Fluorescence spectra of QL2 (10 μM) in PBS and glycerol solution. (e, f) Linear responses between logI590, logI740) and log η in PBS-glycerol solution. (g, h) Linear responses between logI680), log I750, and log η in PBS-glycerol solution. (i) QL2 with different viscosity solutions under ultraviolet lamp irradiation (365 nm). Error bar: n = 3.

The good spectroscopic performance of QL2 in different pH buffer and viscosity solutions motivated us to perform further detailed studies of the photophysical properties of QL2 in different pH buffers and glycerol. Interestingly, QL2 showed significantly different photophysical properties under acidic and basic (pH 2.0 and 10.0) conditions during viscosity changes. The maximum absorption peak is mainly at 600 nm when the pH is 2.0 (Figure 3a). As the viscosity increased (Figure 3b, c), the fluorescence intensity of QL2 at 680 nm increased about 9-fold, and the fluorescence change at 750 nm was inconspicuous. When the solution pH was 10.0 (Figure 3d), QL2 gave a shouldered absorption band with maximum peaks at 600 and 720 nm. With the increase in the glycerol volume (Figure 3e, f), the fluorescence intensity of QL2 at 750 nm also increased by 4-fold and 9-fold when excitation by 580 and 700 nm, respectively.

Figure 3.

Figure 3

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Absorption and fluorescence spectra change of QL2 at pH 2.0 (a–c) and pH 10.0 (d–f) solution with different viscosity intensities. (b, e) λex = 580 nm, (c, f) λex = 700 nm.

The photophysical spectral changes indicate that QL2 has a pH-switchable viscosity change. Therefore, it can be used for pH detection, and viscosity detection in both acid and alkaline buffers with distinctive fluorescence change.

3.4. DFT Calculation of QL1 and QL2

To explain the spectral change of QL2, high-density functional theory (DFT) calculations were used to calculate the spectral changes of QL1 and QL2. For both probes, the energy gap become smaller in the deprotonation state, suggesting that a lower excitation energy is needed (Figure 4, Tables S1, S2). The HOMO of QL1-OH and QL2-OH is mainly located in the rhodamine part, and the LUMO is in the 4-hydroxyresorcinol part.

Figure 4.

Figure 4

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Energy state of QL1 and QL2 in acid and basic solutions (QL1-OH and QL2-OH) via DFT calculations.

This indicates that the molecular charge transfer is enhanced after deprotonation.35,36 DFT calculations and fluorescence spectra analysis as a function of pH solution (pH 2–10) showed that the two emission signals of QL2 at 680 and 750 nm are from QL2 and its deprotonation product QL2-OH, respectively. The energy difference between QL2 and QL2-OH further proves that the absorption at 550 nm decreases. The absorption at 650 nm increases with increasing pH values (Figures S7, S8).

3.5. Selectivity of QL2

We next studied the selectivity of QL2 for measuring viscosity. The fluorescence spectra of both probes with other biological species were studied to exclude the interference of other metal ions, amino acids, and oxidants. The results (Figure S3) show that when K+, Ca2+, Zn2+, Na+, Fe3+, Fe2+, NH4+, Cu+, S2O32–, S2–, SO42–, HSO3–, NO2–, Cr2O72–, H2O2, tryptophan, l-cysteine, l-leucine, l-threonine plasma, and amino acids were added, the fluorescence intensity of QL2 did not change significantly. The strong near-infrared radiation of probe QL2 was clearly observed only in highly viscous media.37 The same selectivity result was also obtained for QL1 (Figure S4). To verify the effect of solvents on the probe, we next dissolved QL2 in different solvents to measure the fluorescence. The fluorescence spectra of the probes (Figures S5, S6) indicate that the fluorescence intensities of the probes were weak in both water and other solvents. Thus, QL2 had better performance, and it was selected for the following biological imaging applications.

3.6. Viscosity and Blood Clot Imaging Experiment

To simulate blood viscosity changes and blood clot formation in vitro, we used CMC (carboxymethyl cellulose) and gelatin to simulate the liquid-to-solid phase transition. We then compared the different proportions of glycerol via an in vivo imaging instrument. Viscosity change and clot formation were measured via QL2.

With the increase in glycerol viscosity, the fluorescence intensity of channels 1 and 2 increased nearly 10-fold (Figure 5). QL2 can also detect the sol–gel transition process: The fluorescence of both channels gradually increased with an increase in the gelatin content. Bright fluorescence was observed in both channels after gel formation (Figure 5c, d). By comparing the imaging of different contents of glycerol and gelatin, we found that QL2 could respond to both viscosity change and clots formation. It had a better response to viscosity changes.

Figure 5.

Figure 5

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Imaging of different contents of glycerol and gelatin. (a, b) Image of viscosity change simulated by glycerol. (c, d) Blood clots simulated by the carboxymethyl cellulose (5%, wt %) and gelatin (0.01%–0.04%). Histogram of relative strength in different glycerol contents (e) and gelatin (f). Channel 1: λex = 580 nm, collection: 600–700 nm; Channel 2: λex = 700 nm, collection: 710–800 nm. Error bar: n = 3.

3.7. Fluorescence Imaging of Viscosity Change in Cells

The cytotoxicity of the probe is one of the key factors for its application in organisms. MTT is a common biological tool to detect the cytotoxicity of probes;38 thus, we used MTT to verify the cytotoxicity of the probes (Figure S9). QL2 has no obvious cytotoxicity to cells when the probe concentration reaches 20 μM, thus, indicating that the probes have good biocompatibility. Co-localization experiments were performed to explore the intracellular behavior of the probe based on the good biocompatibility. HepG2 cells were incubated with QL2, and then treated with commercial lysosomal (Lyso-Tracker Red) and mitochondrial dyes (MTG), respectively.39−42 The results show that QL2 have a higher overlap rate with commercial MTG (Figure S10) than lysosomal tracker (Figures S11, S12). The Pearson correlation coefficient with MTG was 0.85. Therefore, the probe can be well positioned in the cell mitochondria area.

Next, the ability of QL2 to image the viscosity in a live cell environment was further evaluated. HepG2 cells were incubated with nystatin35,43 and monensin44,45 (30 μM) for 30 min, respectively. They were then incubated with QL2 in a 37 °C incubator for another 30 min followed by fluorescence imaging. Figure 6 shows that both the red and green channels have weak fluorescence when only QL2 is present. However, the fluorescence of the red and green channels was significantly enhanced when nystatin or monensin was added. Figure 6q shows that the fluorescence intensity of the nystatin or monensin group of both channels is significantly enhanced when only the probe is present. Combined with the previous spectral changes with viscosity, these results show that QL2 can detect the endogenous viscosity change in live cells.

Figure 6.

Figure 6

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Images of HepG2 cells induced by monensin and nystatin. (a-d) Blank, (e-h) Cells incubated with QL-2 (10 μM). (i-l) Cells treated with monensin (30 μM) and QL2 (10 μM) for 30 min. (m-p) Cells treated with nystatin (30 μM) and QL2 (10 μM) for 30 min. Green channel: pseudocolor, λex = 580 nm, λem = 600–700 nm; red channel: λex = 680 nm, λem = 700–750 nm. (q) Histogram of fluorescence intensity of green channel and red channel. Error bar: n = 3 Scale: 10 μm.

3.8. Fluorescence Imaging of pH and Viscosity in Zebrafish

Finally, we studied zebrafish to evaluate QL2 under different pH and viscosity conditions. The pH of the medium was adjusted from 4.0 to 10.0 using H+/K+ ionophores.46 Zebrafish were cultured in media with different pH values for 30 min the QL2 (20 μM) was then added for another 30 min. Figure S13 shows that QL2 exhibits a dual channel response property. At pH 4.0, the fluorescence of QL2 is the strongest in the green channel and the weakest in the red channel. At pH 10.0, the fluorescence in the green channel weakened and the red channel became the strongest. Therefore, ratiometric fluorescence imaging was obtained to measure the pH changes in zebrafish. QL2 was then used to image the viscosity in zebrafish. Zebrafish costained with QL2 showed weak fluorescence in the red and green channels (Figure S14). The other two groups prestimulated with monensin or nystatin and then treated with QL2 had enhanced fluorescence intensity in both channels.43,44,47

3.9. In Vivo Imaging of QL2 in Live Mice and Organs

We next evaluated the ability of QL2 to detect viscosity changes in live animals and organs. Carrageenan was used to induce blood viscosity change and produce blood clots in live mice. The mice had black tails due to blood clots.48−50 The exogenous viscosity change was measured in mice via QL2. After exogenous injection of 50%, 70%, 80%, and 90% glycerol, the fluorescence of the blood clot group in both channels gradually increased, compared to that of the control group (Figure 7).

Figure 7.

Figure 7

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Imaging of viscosity change in live mice. (a1–a5, c1–c5) Fluorescence images of mice with intraperitoneal injection of 20 μM QL2 and different viscosity solutions; excitation was at 580 and 700 nm (b1–b5, d1–d5). Healthy mice were given 20 μM QL2 and the same volume of saline. (e, f) Relative average fluorescence intensity changes were measured in the selected area. (a, b) λex=580 nm, collection: 600–700 nm; (c, d): λex=700 nm and collection: 710–800 nm. Error bar: n = 3.

The fluorescence increased nearly 20-fold and 7-fold, respectively, when exited by 580 and 700 nm. Only weak fluorescence was observed in both channels in the control group (healthy mice injection of saline). Therefore, these studies showed that QL2 can detect viscosity change in live animals.

Finally, the organs from both groups were harvested to investigate the effect of carrageenan during blood clots formation. As shown in Figure 8, the fluorescence is different between the organs. In the blood clots group, all the organs had remarkable fluorescence when incubated with QL2 for 70 min. With the incubation time prolonged, the fluorescence in kidneys is stronger than others. On the contrary, there was almost no obvious fluorescence in the normal group when treated with QL2 under the same condition. Fluorescence was mainly observed in heart and liver after incubation for 70 min (Figures S15, S16). These results indicated that carrageenan incubation had server influence on kidneys; the blood circulation in these organs was blocked due to viscosity change after blood clot formation (Figure 8). The fluorescence signals of spleen, lung, and kidney stimulated with carrageenan were significantly higher than those in the control group, indicating that QL2 could effectively track the viscosity in live organs.

Figure 8.

Figure 8

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Fluorescence imaging of mice organs from different group with QL2. (a, b) Organs from blood clots group and (c, d) organs from normal group were incubated with 10 μM QL2 for 70 min, and images were collected at intervals. Organs sequence: heart, liver, spleen, lung, and kidney. (e–h) The relative average fluorescence intensity change in selected area. λex = 580 nm, collection: 600–700 nm; λex = 700 nm, collection: 710–800 nm. Error bar: n = 3.

4. Conclusion

In summary, blood viscosity changes are a key syndrome closely related to many diseases such as cerebral stroke and thrombus. We prepared two near-infrared fluorescent probes QL1 and QL2 with dual pH and viscosity responses and used them to detect blood clots. The fluorescence behavior of QL2 in solution and the biological environment was thoroughly investigated. QL2 was used to image the viscosity change in mitochondria of HepG2 cells and zebrafish with a sharp fluorescence enhancement. Moreover, the viscosity changes in live mice and organs induced by carrageenan were successfully detected by QL2 with remarkable fluorescence increases in both channels. These superior characteristics of this new probe can monitor the viscosity of mouse organs and living mice for the first time. The present fluorescence detection of viscosity and blood clots in live animals can provide guidance for further understanding the function of viscosity in blood clot formation in live animals.

Acknowledgments

This work was financially supported by the Shandong Province Natural Science Foundation(ZR202306050008, Innovation Team Project of 20 items-university of Jinan (202228073), NSFC (No. 31971605), the Pilot Project for Integrating Science, Education and Industry (2022PYI007), and supported by the Taishan Scholars Program.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.3c00110.

  • Absorption spectral data of the probe; scheme of reaction mechanism; MTT assay, HR-MS, 1H NMR, and 13C NMR spectra of the probe and intermediates (PDF)

Author Contributions

† J.L., C.S., and Y.C. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

im3c00110_si_001.pdf (1.9MB, pdf)

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