Light emission enhancement by supramolecular complexation of chemiluminescence probes designed for bioimaging

Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents.


Proposed chemiluminescence enhancement mechanism
To better understand the enhancement mechanism of the TMCD with Schaap's 1,2dioxetane, we have examined the fluorescence enhancement exhibited for the emitter (benzoate) and TMCD. The direct chemiluminescence generated by emission of the corresponding dioxetane probe in water is directly affected from the fluorescence efficiency of the benzoate ester. As presented in Figure S10, the fluorescence of 3hydroxybenozte (3-HB) was significantly higher after incubation with TMCD compared to that of the control experiment. The complexation with TMCD improved the fluorescence intense signal of 3-HB with almost 13-folds. This result supports our proposed CL enhancement mechanism using supramolecular enhancer adducts. The chemiluminescence probe is encapsulated with TMCD to form a stable inclusion, then, activation of the probe led to the generation of the excited benzoate to emit an enhanced chemiluminescent light. Figure S10: Fluorescence spectra of 3-HB with and without TMCD.

Correlation between TMCD and TMCD-FITC Chemiluminescence Enhancement
In order to examine the complexation effect of TMCD-FITC compared to that of TMCD, we have plotted the relative chemiluminescence enhancement of TMCD-FITC as a S17 function of the relative enhancement achieved with TMCD for each probe (1-4). As shown in Figure S11, an excellent linear correlation was obtained with R 2 of 0.975. This result means that the complexation effect and strength of the CD derivatives is similar with probes 1-4. Therefore the only difference is the energy transfer achieved with the TMCD-FITC, which leads to further 30-folds higher light emission. Figure S11: Correlation between the relative chemiluminescence enhancement using TMCD and TMCD-FITC with probes 1-4.
The equilibrium constant calculation:

Ethics Statement
All animal procedures were performed in compliance with Tel Aviv University, Sackler School of Medicine guidelines and protocols approved by the Institutional Animal Care and Use Committee.

Intravital non-invasive chemiluminescence imaging of probe 5 activation in mice
To induce acute inflammation, 1 mL (0.1 mg/mL) of LPS (Lipopolysaccharides from Escherichia coli 055:B5, Sigma) was injected into the peritoneal cavity (i.p.) of Balb/c mice. A second control group of mice was injected i.p. with 1 mL of PBS. Four hours later, both mice groups were additionally injected i.p. with 100 µL of 100 µM of probe 5 or100 µL of 100 µM of probe 5 and 300 μM of TMCD-Cy5. Twenty minutes later, mice were anesthetized using ketamine (100 mg/kg) and xylazine (12 mg/kg), and imaged by S20 BioSpace Lab PhotonIMAGER TM . Activated probe 5 chemiluminescence signal was quantified as total signal of photons/exposure time (sec). Data is expressed as mean ± S.D.

P-NMR titration study
To determine the complexation constant of TMCD with probe 1, titration experiment was carried out directly in an NMR tube. The kinetic of the complexation reaction was studied by 31 P-NMR spectroscopy. Typical procedure: 5 mg of the appropriate probe 1 was dissolved in 1000 µL D 2 O. The latter solution was divided to two 500 µL solutions, A and B. Solution A was transferred to an NMR tube, and to solution B was added 50 mg of TMCD. The spectra were measured after the addition of various amounts of solution B (17, 33, 65, 129, 257, 500 µL) to solution A.

H-NMR crystallization experiment
The crystallization experiment was carried out by typical crystallization protocol. 960 mg of TMCD was dissolved in 950 µL of water and 50 µL of DMF, then 80 mg probe 2 was