A Single Molecular Multianalyte Fluorescent Probe and Its Application to Imaging Intracellular Multianalyte Dynamics

Single molecular multianalyte sensors are molecular sensors that can determine multiple analytes by recognition of the various analytes based on the difference of the spectral changes (Figure 1).1,2 To date, many molecular sensors and probes have been developed that focus on a single analyte. The Ca2+ fluorescent probe, fura-2,3 is one of the most well-known examples of such a probe. The authors have also developed highly selective magnesium fluorescent probes, the KMGs.4–6 Intracellular imaging using these cation fluorescent probes has been widely researched. The intracellular signals are based on the correlation and cross-talk of various messengers; thus, multianalyte imaging has become increasingly significant.

Figure 1 - Single molecular multianalyte sensors/probes.

This paper proposes single molecular multianalyte fluorescent probes for intracellular multianalyte imaging. By using single molecular multianalyte fluorescent probes, there is a minimal invasive effect and quantitative analysis without localization, metabolism, or photobleaching. Quantitative analysis can be done with ratiometric calculations.

Figure 2 - Schematic diagram of multi-Ca2+/Mg2+ fluorescent probe, KCM-1, and its intracellular imaging.

Ca2+ is a well-known signal transmitter, and Mg2+ acts as a cofactor in many situations. The authors focused on the correlations of the intracellular Ca2+ and Mg2+ with simultaneous imaging of both cations. The simultaneous imaging of intracellular Ca2+ and Mg2+ using a novel single molecular multianalyte sensor, the multi-Ca2+/Mg2+ fluorescent probe, is shown in Figure 2.

Experimental

Details of the experiment are reported in Ref. 1.

Fluorescence imaging

Figure 3 - Excitation wavelength changeable fluorescent microscope system.

Three images of different excitation wavelengths were evaluated using an ECLIPSE TE300 inverted microscope (Nikon, Tokyo, Japan) equipped with a 40× S Fluor objective lens, 505 dichroic mirror, and 535/55 barrier filter (all from Nikon). A 150-W xenon lamp with a monochromator unit was used for producing three excitation wavelengths (365, 390, and 420 nm), and the fluorescence was measured by a HiSCA charge-coupled device (CCD) camera (Hamamatsu Photonics, Hamamatsu, Japan) (Figure 3).

Cells were incubated with 10 μM KCM-1AM in culture medium for 30 min at 37 °C and were then washed twice with a recording solution, followed by further incubation for 15 min in order to allow complete hydrolysis of the ester form of the KCM-1AM loaded into the cells.

Discussion

Molecular design of multi-Ca2+/Mg2+ fluorescent probe

The concept of a single molecular multianalyte sensor/probe has been proposed. Many molecular sensors and probes have been developed to date, and these molecules are focused with a high selectivity toward a single analyte. The authors focused on the optical discrimination of the analyte, in this case, the molecular design of semiselectivity (insensitivity to analytes that are not of interest) and discrimination of the analytes are required, and the evaluated spectra should be solved by a mathematical solution.

Figure 4 - a) ICT-type mechanism. b) Absorbance spectra of KCM-1 to different Ca2+ and Mg2+ concentrations (measured at pH 7.20, 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 130 mM KCl, 20 mM NaCl).

A multi-Ca2+/Mg2+ fluorescent probe had to be designed to show the different fluorescence (excitation, emission) change from Ca2+ and Mg2+. Other cations should not bind to the probe (semiselectivity). The first multi-Ca2+/Mg2+ fluorescent probe, KCM-1, was designed based on the intramolecular charge transfer (ICT)-type mechanism. This mechanism is explained by the change in the energy gap of the dye molecule, the donor site of the dye having a large coefficient in highest occupied molecular orbital (HOMO), an acceptor site with a large coefficient in lowest unoccupied molecular orbital (LUMO), and a cation interaction stabilizing the energy level according to electronic interactions (Figure 4a).

It was considered that in the case of a donor–acceptor (D–A) type dye, the cation interaction with the donor site of the dye induces a larger HOMO stabilization than LUMO, resulting in a blue shift of the spectra, interaction with the acceptor site that induces a larger LUMO stabilization than HOMO, and a red shift (ICT-type mechanism).

It was assumed that the ICT-type mechanism could be used for the discrimination of multiple analytes; one cation tends to bind to the donor site with a blue shift of the spectra, while the other cation binds to the acceptor site with a red shift. The selective binding sites for Ca2+ and Mg2+ have been known, i.e., bis-(2-aminophenyl)-ethyleneglycol-tetraacetic acid (BAPTA) for the Ca2+ binding site, and the charged beta diketone for the Mg2+ binding site.

In the first multi-Ca2+/Mg2+ fluorescent probe, KCM-1, coumarin was used as the D–A type fluorophore. BAPTA was used for the Ca2+ binding site at the donor site of the dye and the charged beta diketone at the acceptor site, assuming a blue shift for Ca2+ and a red shift for Mg2+. The dissociation constants of the BAPTA derivative are typically on the order of 0.1–1 μM for Ca2+ and 1 mM for Mg2+, while those of the charged beta diketone are on the order of 10 mM for Ca2+ and 1–10 mM for Mg2+. A fluorine-substituted BAPTA was chosen as the Ca2+ selective binding site due to its low Mg2+ affinity. KCM-1 was successfully synthesized by an 11-step synthesis.

Properties of multi-Ca2+/Mg2+ fluorescent probe

Figure 4b shows the absorbance spectra of KCM-1 with Ca2+ and/or Mg2+ under biological conditions. Upon complexation to Ca2+, KCM-1 shows a 45-nm blue shift in absorbance and a 5-nm blue shift in the fluorescence spectrum. For Mg2+, a 21-nm red shift in absorbance and a 5-nm red shift in fluorescence occurred.

The binding characteristics of fluorescent probes are often compared based on their dissociation constant, Kd. The dissociation constants of KCM-1 with Ca2+ or Mg2+ were calculated to be 14 μM in the case of Kd (Ca2+) and 26 mM for Kd (Mg2+). While the intracellular Ca2+ concentration is on the order of 0.1 μM to 1 μM and the Mg2+ concentration is 1 mM, the experimentally determined dissociation constants indicate relatively low affinities. However, low-affinity indicators are reportedly useful for the quantification of high concentrations and transient responses. The properties are consistent with the molecular design consideration.

Chemical equilibrium and quantification of multifluorescent probe

Once the difference spectral changes to Ca2+ and Mg2+ have been achieved, how can the multi-Ca2+/Mg2+ fluorescent probe work in the presence of both Ca2+ and Mg2+? The optical properties of KCM-1 were measured for different Ca2+ and Mg2+ concentrations, and the response was compared to the mathematical simulation that assumes two chemical equilibriums:

Based on the combination of the two equations, the fluorescence intensity can be solved as follows:


Where K represents the association constant, I is the indicator (fluorescent probe), ICa2+ and IMg2+ are the indicator–ion complex, and ƒ is a proportionality coefficient for the individual fluorescent compounds.

This assumption fully agreed with the experimental data for concentrations less than 10 mM Ca2+ and 10 mM Mg2+ (R>0.999); in the low concentration of Ca2+ and Mg2+, the two chemical equilibriums Eqs. (1) and (2) were worked out. While there is much free KCM-1, the binding of the two cations can be treated independently.

At high Ca2+ and Mg2+ levels, the binding of both analytes to KCM-1 deviated from Eq. (3). However, the effect was small. The binding of one cation to the multiprobe affected the second binding site by an electron withdrawing effect and reduced its affinity, which can be considered as an allosteric effect in an indicator.

For imaging by fluorescent microscopy, Eq. (3) can be solved using the fluorescent ratio:

Where Rmn represents the observed ratio of the fluorescence intensity Fm/Fn (excited at m, n = 1 for 390 nm, 2 for 365 nm, 3 for 420 nm), rImn is the ratio of the free indicator, rCamn is the ratio for an excess Ca2+ (1 mM), rMgmn is the ratio for an excess Mg2+ (500 mM), α is the ratio of the fluorescent intensity at 1 mM Ca2+/free indicator at 365 nm, and β is the ratio of the fluorescent intensity at 500 mM Mg2+/free indicator at 365 nm. To solve the equation, at least three fluorescent intensities at different excitation/emission wavelengths are required, since there are two unknown parameters in the system, [Ca2+] and [Mg2+], and one wavelength for evaluating the fluorescent ratio (to erase the terms of the concentrations of the probe)

A microscope system with changeable excitation wavelengths was constructed (Figure 3). The excitation light can be quickly changed by an optical chopper at 360/390/420 nm. First, the calibration data and the fluorescent intensity of the KCM-1, with excess Ca2+ and Mg2+, should be measured at the three excitation wavelengths. By measuring the fluorescent images of the PC12 cells imaged at the three wavelengths, the Ca2+ and Mg2+ image can be calculated.

Simultaneous intracellular multianalyte (Ca2+, Mg2+) imaging

KCM-1 has five carboxyl acids and is highly soluble in water; therefore, in the case of staining a cell that has a lypophilic cell membrane, derivatization is required. The acetoxymethyl ester derivative multi-Ca2+/Mg2+ fluorescent probe, KCM-1AM, was synthesized. The acetoxymethyl esters can penetrate the cell membrane and can be cleaved by the intracellular esterase; thus KCM-1 can be loaded into a cell.

The cytoplasm of PC12 cells was stained effectively with KCM-1AM and imaged by the fluorescent microscope system. Eqs. (4) and (5) were solved to evaluate the Ca2+ and Mg2+ images of KCM-1-loaded PC12 cells.

Figure 5 - Simultaneous imaging of intracellular Ca2+ and Mg2+ using the multi-Ca2+/Mg2+ fluorescent probe, KCM-1. PC12 cells stained by KCM-1AM, imaged by the fluorescent microscope system. The image is converted. a) Ca2+ image. b) Mg2+ image of PC12 cells after stimulation by FCCP.

Figure 5 shows the converted Ca2+ and Mg2+ image after the p-trifluoromethoxyphenyl carbonyl cyanide phenylhydrazone (FCCP) addition to PC12 cells. By adding mitochondrial uncoupler, FCCP, to the PC12 cells, increases in both Ca2+ and Mg2+ were observed based on the release of mitochondrial Ca2+ and Mg2+.1,4,6 Intracellular Ca2+ and Mg2+ were successfully imaged by a single multi-Ca2+/Mg2+ fluorescent probe.

Conclusion

A multi-Ca2+/Mg2+ fluorescent probe, KCM-1, was developed. With KCM-1, intracellular Ca2+ and Mg2+ were successfully imaged simultaneously. With multianalyte imaging using a multi-Ca2+/Mg2+ fluorescent probe, the correlation of the intracellular Ca2+ and Mg2+ in many biological situations, such as mitochondrial mechanisms, apoptosis, and energy metabolism, could be clarified.

The concept of molecular multianalyte sensors can be extended to other molecular sensors such as chromoionophores, green fluorescent protein (GFP)-based sensors, and contrast agents. Observations of multiple signal transmitter dynamics will clarify the correlation and cross-talk of biological signals in the near future.

References

  1. Komatsu, H.; Miki, T.; Citterio, D.; Kubota, T.; Shindo, Y.; Kitamura, Y.; Oka, K.; Suzuki, K. Single molecular multianalyte (Ca2+, Mg2+) fluorescent probe and applications to bioimaging. J. Am. Chem. Soc.2005, 127(31), 10,798–9.
  2. Komatsu, H.; Citterio, D.; Fujiwara, Y.; Minamihashi, K.; Araki, Y.; Hagiwara, M.; Suzuki, K. Single molecular multianalyte sensor: Jewel Pendant Ligand. Org. Letters2005, 7(14), 2857–9. 
  3. Grynkiewicz, G.; Poenie, M.; Tsien, R.Y. J. Biol. Chem.1985, 260(6), 3440–50.
  4. Komatsu, H.; Iwasawa, N.; Citterio, D.; Suzuki, Y.; Kubota, T.; Tokuno, K.; Kitamura, Y.; Oka, K.; Suzuki, K. Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D-Mg2+ imaging. J. Am. Chem. Soc.2004, 126(50), 16,353–60.
  5. Suzuki, Y.; Komatsu, H.; Ikeda, T.; Saito, N.; Araki, S.; Citterio, D.; Hisamoto, H.; Kitamura, Y.; Kubota, T.; Nakagawa, J.; Oka, K.; Suzuki, K. Design and synthesis of Mg2+- selective fluoroionophores based on a coumarin derivative and application for Mg2+ measurement in a living cell. Anal. Chem. 2002, 74(6), 1423–8.
  6. Kubota, T.; Shindo, Y.; Tokuno, K.; Komatsu, H.; Ogawa, H.; Kudo, S.; Kitamura, Y.; Suzuki, K.; Oka, K. Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells. Biochim. et Biophys. Molec. Cell Res.2005, 1744(1), 19–28.

    Dr. Komatsu is an Instructor, Dept. of Applied Chemistry, and Prof. Oka is a Professor, Dept. of Bioscience and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan; tel.: +81 45 566 1568; fax: +81 45 564 5095; e-mail: komatsu@applc. keio.ac.jp. Prof. Suzuki is a Professor, Dept. of Applied Chemistry, Keio University, and a Research Director, Core Research for Evolutional Science and Technology (CREST), JST Agency, Saitama, Japan.