Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish

Abstract

DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene-4H-pyran, DCM-NH₂) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor-acceptor DCM-NH₂ is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference.

Keywords: DPP IV; NIR probe; bioimaging; ratiometric fluorescent sensor; two-photon excitation.

Scheme 1. Synthesis of DCM-NH-Pro-Gly

Figure 1

(A) Evolution of the absorption spectra of DCM-NH-Pro-Gly (10 μM) with DPP IV (10  μg mL^–1) in PBS/DMSO (7/3, v/v) every 30 min for 8.5  h at 37  °C. (B) Maximum absorbance values at 440 nm (black line) and 500 nm (blue line) vs time. (C) Evolution of the emission spectra of DCM-NH-Pro-Gly (10 μM) with DPP IV (10 μg mL^–1) observed every 30 min for 8.5 h by excitation at 463 nm at 37 °C. (D) Maximum fluorescence intensity at 662 nm (red line) and the intensity decrease at 550 nm (black line) vs time. (E) Ratiometric measurements of fluorescence signals of I₆₆₂/I₅₅₀ of DCM-NH-Pro-Gly (10 μM) with different enzyme concentrations vs time. (F) Initial rates from ratiometric measurements vs enzyme concentrations.

Figure 2

Ratiometric measurements of fluorescence signals of I₆₆₂/I₅₅₀ of DCM-NH-Pro-Gly (10 μM) in PBS/DMSO (7/3, v/v) after 80 min of incubation in the presence of different enzymes at the same concentration (5 μg mL^–1) by excitation at 463 nm at 37 °C.

Figure 3

(A) Intensity values of green (λ_em = 533–557 nm) and red (λ_em = 648–722 nm) channels and ratio maps using one-photon excitation at 488 and two-photon excitation at 800 nm of blood plasma sample. Whiskers represent the standard error (SE). (B) Representative ratio R/G maps of healthy human blood plasma with DCM-NH-Pro-Gly with DPP IV (10 μg mL^–1) at different incubation times (λ_ex = 800 nm). C) Ratio R/G average values of blood plasma with the probe DCM-NH-Pro-Gly images registered at different incubation times (λ_ex = 800 nm). Line represents a visual aid. Whiskers represent the SE.

Figure 4

(A) Images of a representative sample of the live Caco-2 cell line incubated with DCM-NH-Pro-Gly (2.5 μM) recorded in the red (left, λ_ex = 450 nm, λ_em = 648–722 nm) and green (middle, λ_ex = 450 nm, λ_em = 533–557 nm) channels at six different time points. Ratio R/G images (right) obtained at the same times. (B) Representation of the average values of the ratio R/G from five independent experiments. Error bars represent SE. (C) Double-logarithmic representation of the kinetics. Error bars represent SE. The red line is a linear fit with an intercept of 0.964 ± 0.004 and a slope of 0.215 ± 0.003 (R² = 0.995).

Figure 5

(A) R/G ratio maps of the live Caco-2 cell line at different times after adding DCM-NH-Pro-Gly (5 μM) without (top) and with (bottom) the DPP IV inhibitor sitagliptin (50 μM). λ_ex = 450 nm. Ratio image is calculated by dividing red (λ_em = 648–722 nm) and green (λ_em = 533–557 nm) channels. (B) Representation of the R/G ratios from microscopy images without (circles) and with (squares) sitagliptin. Scale bars represent 10 μm.

Figure 6

(A) Images obtained from live Caco-2 cells with two-photon excitation at 800 nm (λ_em = 650–720 nm). Scale bars represent 5 μm. (B) Representative image of the red channel obtained from BxPC-3 tumors using two-photon microscopy with excitation at 800 nm (λ_em = 650–720 nm). Scale bars are 10 μm. (C) Representative images of the intensity red (λ_em = 650–720 nm) and green (λ_em = 502–538 nm) channels and the ratio R/G images of BxPC-3 tumors after adding DCM-NH-Pro-Gly (10 μM) using two-photon microscopy with excitation at 800 nm. Scale bars are 10 μm. (D) Representation of the R/G ratio values from microscopy images. Boxes represent the 25th, 50th, and 75th percentiles. Whiskers represent the SE.

Figure 7

(A) Living zebrafish embryos and larvae incubated with 5 μM DCM-NH-Pro-Gly for 2 h at different dpf; red fluorescent (left), brightfield (center), and merge (right) images are shown measured by the stereo microscope (λ_ex = 458 nm, λ_em = 680 nm). Scale bars: 1 dpf: 250 μm, 3–7 dpf: 500 μm. (B) Detail (head with central nervous system) of a living zebrafish larva at 5 dpf. Scale bars: 200 μm. (C) Living zebrafish larva at 5 dpf preincubated for 3 h with 250 μM sitagliptin and incubated with 5 μM DCM-NH-Pro-Gly for 2 h. (D) Intensity values of NIR emission of zebrafish at different dpf, incubated with 5 μM DCM-NH-Pro-Gly in the presence or absence of the inhibitor sitagliptin. Boxes represent the 25th, 50th, and 75th percentiles. Whiskers represent the SE.