Key takeaways from this article:The authors have developed a novel set of dual Nir-IIB ratio fluorescent probes capable of emitting two fluorescence signals of the same wavelength at 1525 nm, with 808 nm excitation fluorescence as the sensing signal and 980 nm excitation fluorescence as the self-calibrated signal, and accurate and reliable detection results were obtained by measuring the Nir-II fluorescence ratio signal (F808EX F980EX). After administration of the probe in arthritic mice, the inflammatory lesion is distinguished from the surrounding normal tissue by responding to the clo- effect at the site of inflammation based on the ratio signal. In addition, the amount of expression clo- in the lesion can be accurately calculated by in vivo images and in vitro detection curves. Considering that the dual NIR-IIB ratio fluorescence signal is not affected by various interferences such as probe concentration and location, it is expected to provide a non-invasive, quantitative, and visual platform for biomedical research and pathological diagnosis.
Figure 1Preparation and characterization of dye@dsnps with dual emission at the same Nir-IIB wavelength.
Of all the dyes, IR-808 was found to be the most effective sensitizer for the prepared DSNPS. Under 808 nm excitation, the IR-808 @dsnps exhibits characteristic sharp fluorescence peaks at 980 and 1525 nm, which can be attributed to the 2F5 2 2F7 2 transition of the Yb3+ ion and the 4i13 2 4i15 2 transition of the ER3+ ion (Figure 1F). As shown in Figure 1h, the absorption spectra show that IR-808 can confer high 808 nm light absorption to IR808@DSNPS, but is almost negligible at 980 nm absorption. IR-808-mediated triplet sensitized DSNPs process the same NIR-IIB emission at 808 or 980 nm excitation for the development of ratiometric fluorescent probes with the same dual emission.
Figure 2IR-808@dsnps@ DSPE-PEG-NH2 nanoprobe with the same NIR-IIB dual emission.
To impart water solubility to IR-808@DSNPs for biological research, encapsulate IR-808@DSNPs with the amphiphilic molecule DSPE-PEG-NH2 to make them dispersible in aqueous solution (Figure 2A). The measured ratio fluorescence signal (F808EX F980EX) is approximately 1 and remains stable in PBS for 7 days without any change, favoring long-term storage of the nanoprobe (Figure 2G). In addition, the obtained NIR-IIB ratio fluorescent probe is essentially non-toxic to 4T1 breast cancer cells and L929 normal cells at a high concentration of 1000 g ml, with a safety profile for biological applications (Figure 2i).
Figure 3Depth-adjustable 1% fat emulsion covered with IR-808 @dsnps@ DSPE-PEG-NH2 at 808 980 nm excitation dual NIR-IIB ratio imaging.
As shown in the NIR-II fluorescence images, the fluorescence intensity of both 808 and 980 nm excitation decreases with increasing penetration depth (Figure 3C,D), but their NIR-II ratio signal remains consistent (Figure 3C,E). These results show that the ratio signal of dual NIR-IIB emission (F980EX F808EX) is independent of the absorption and scattering of 1% fat emulsion. Therefore, IR-808 @dsnps@ DSPE-PEG-NH2 are ideal ratiometric NIR-IIB fluorescent nanoprobes.
Figure 4In vitro dual Nir-IIB ratio fluorescence imaging of IR-808 @dsnps@ DSPE-PEG-NH2 at different CLO concentrations.
It has been reported that IR-808 is able to be oxidized specifically by CLO-, however, the NIR-IIB fluorescence excited at 808 nm is produced by the IR-808-mediated triplet sensitization pathway, so the corresponding emission of ER3+ ions decreases as the amount of CLO- is added (Figure 4A). Conversely, the downshifted fluorescence excited at 980 nm can be retained independently with the addition of CLO-. IR-808 @dsnps@ DSPE-PEG-NH2 can be used for the quantitative detection of CLO- with 808 nm excitation fluorescence as the sensing signal and 980 nm excitation fluorescence as the self-calibration signal (Figure 4B). The ratio signal (F808EX F980EX) was plotted against CL- concentration, and a strong linear correlation was obtained (R2 = 0.).998, Figure 4e). Figure 4f shows that the nanoprobe only reacts to clo-and avoids interference with other similar analytes, providing extremely high sensitivity and selectivity.
Figure 5In situ monitoring of inflammation in arthritis models by dual NIR-IIB ratiometric nanoprobes.
To demonstrate the effectiveness of the NIR-IIB ratio fluorescence signal for accurate in vivo detection, CLO- was visualized using IR-808 @dsnps@ DSPE-PEG-NH2 in a mouse model of arthritis. Use the formula r = 10017-0.0454c (where r represents the ratio of F808EX to F980EX and C represents the concentration of CLO-, Figure 4E) to obtain the amount of Clo- in the inflammatory foot from 095 m raised to 1444 m (Figure 5f). Therefore, the dual NIR-IIB ratio probe is not only able to identify inflammation and normal tissues, but also to quantify the amount of Clo- in vivo in real time.
The authors have developed a novel set of dual Nir-IIB ratiometric fluorescent probes for in vivo detection based on analyte-dependent triplet energy transfer from dye to dsnp. The prepared probe is capable of emitting two fluorescence signals of the same wavelength at 1525 nm, one of which is excited at 808 nm for the sensing channel and the other at 980 nm for the reference channel. Benefit from eliminating interference from light propagation pathways in tissues for accurate and reliable detection by measuring the NIR-II fluorescence proportional signal (F808EX F980EX). After administration of the probe in arthritic mice, the inflammatory lesion can be directly distinguished from the surrounding normal tissue, considering that the proportional signal can respond to the CLO at the site of inflammation. In addition, by comparing in vivo images and in vitro detection curves, the amount of CLO expressed in the lesion can be accurately calculated. Considering that the dual NIR-IIB ratio fluorescence signal is independent of various interferences such as probe concentration and location, it is expected to provide a non-invasive, quantitative, and visualization platform for a wide range of biomedical research and pathological diagnosis.
References
wang, x.; li, m.; zheng, x.; sun, b.; wang, y.; xu, j.; han, t.; ma, s.; zhu, s.; zhang, s., dye-triplet-sensitized downshifting nanoprobes with ratiometric dual-nir-iib emission for accurate in vivo detection. analytical chemistry 2023, 95 (41), 15264-15275.