1. Introduction
The specific spectroscopic, electrochemical and magnetic properties of Ln(III) ions make them perfect candidates for use in many chemical, biological and environmental systems. Ln(III) ions in their complexes exhibit unique luminescence properties because of sharp and characteristic emission bands whose positions are not influenced by the physico-chemical properties of the ligand [
1,
2,
3,
4,
5]. In addition, the luminescence spectra of Ln(III) complexes can be recorded in time-resolved mode due to relatively long lifetimes (in the μs-ms scale). This enables the filtering of their signal from the in vivo background originating from organic molecules with luminescent decay on the ns time scale [
1,
2,
3,
4,
5,
6,
7,
8]. The excited states of Ln(III) ions in their complexes are not quenched by O
2 molecules but are influenced by water molecules coordinated to the Ln(III) ion [
1,
2,
3,
4,
5,
6,
7,
8]. Therefore, their luminescence intensity is higher in complexes than in aqua ions. The introduction of a chromophoric group into the ligand significantly increases the luminescence of Ln(III) complexes as consequence of the so-called antenna effect, through which energy strongly absorbed by the chromophoric ligand is efficiently transferred to the Ln(III) ion. This is followed by irradiation of luminescence, with spectra that are characteristic for each Ln(III) ion (see
Figure 1a) [
1,
2,
3,
4,
5,
6,
7,
8,
9,
10]. This phenomenon can be utilized for the structural design of Ln(III) complexes with applications in biology and medicine—clinical diagnostics [
7,
8,
9,
10,
11,
12,
13], mostly as luminescent sensors and probes [
12,
14,
15,
16].
The Ln(III) complexes employed in vivo applications should have high thermodynamic stability as well as kinetic inertness to ensure that toxic Ln(III) ions are not released from their complexes; these ions are capable of substituting Ca(II) ion(s) bound in biomolecules [
5]. The most suitable ligands for the tight binding of Ln(III) ions are highly rigid macrocyclic compounds which fulfil the above-mentioned properties due to the macrocyclic effect [
17,
18,
19]. When the coordination number of the Ln(III) ion (usually about 8–9) is equal to denticity of macrocyclic ligand, complexes with 1:1 stoichiometry are formed [
5,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24]. For these reasons, Ln(III) complexes with macrocyclic ligands (mainly DOTA derivatives) are commonly utilized in medicinal chemistry as radiopharmaceuticals (
90Y,
153Sm,
166Ho,
177Lu) [
18,
25] and contrast agents for MRI (Gd) [
5,
18,
26,
27]. When the denticity of the macrocyclic ligand is lower than that of the Ln(III) ion, there is a higher tendency to form ternary complexes. This kinetically inert binary Ln(III) complex is capable of binding another ligand bearing a functional group with specific physico-chemical properties (e.g., chromophore, fluorophore, electroactive group, etc.) and thus this Ln(III) ternary complex can be tailored according to the desired applications [
10,
17,
28,
29,
30]. The highly luminescent ternary [Eu(DO3A)L] and [Tb(DO3A)L] complexes (see
Figure 1b, L = picolinic/3-isoquinolic acid) can be given as an example [
28,
29,
30]. In these complexes, the sensitization of Ln(III) luminescence by a fluorophore is achieved via efficient energy transfer from the ligand to the Ln(III) ion. The luminescent ternary Eu(III) complex is also electroactive due to possibility of the reduction pathway Eu(III) → Eu(II) occurring alongside the redox processes taking place on the fluorophoric group [
29].
Nicotinamide adenine dinucleotide consists of two nucleotides (adenine, nicotinamide) joined by a common phosphate group. Its phosphorylated analogue in the 2′ position on the ribose ring is now recognized as a universal energy carrier performing reversible two-electron transfers in a variety of essential metabolic reactions [
31,
32]. Thus, NAD(P)H/NAD(P)
+ are considered compounds of paramount biological importance because they serve as cofactors in many metabolic pathways. Both are involved in redox reactions because of their ability to carry electrons from one reaction to another (NAD(P)H and NAD(P)
+ are reducing/oxidizing agents able to donate/accept electrons) and therefore they are used as substrate-coenzymes of redox enzymes in chemical reversible reactions [
31,
32]. NAD can be converted into the NAD-phosphate coenzyme, which has usually analogous redox chemistry and serves as a cofactor in anabolic metabolism, e.g., reductive synthetic reactions—synthesis of fatty acids and steroids as well as in oxidant production for antioxidant protection [
31,
32]. On the other hand, the NAD redox pair is generally involved in catabolic processes [
31], e.g., Krebs citric acid cycle, glycolysis, β-oxidation of fatty acids, etc. They also participate in the addition/elimination of chemical groups to or from proteins, e.g., in post-translational modifications; therefore, enzymes participating in NAD metabolism are very often considered targets for the development and testing of new drugs [
32]. The concentration of NAD species can be estimated to tenths-units of mM scale [
32,
33,
34]. The NAD(P)
+/NAD(P)H ratio is an important cell parameter as it reflects both metabolic activity and the health of cells. The NAD
+/NADH ratio (usually > 1) is a complex parameter because of the overall contribution of several key enzymes. Conversely, the NADP
+/NADPH ratio is much lower than one, meaning that NADPH is the dominating species of this coenzyme. This ratio for the same concentration of both species can also be represented as a conditional standard redox potential (pH = 7) for the following redox reaction:
whose value does not differ dramatically (−320 mV for NAD
+, −324 mV for NADP
+ [
32]). Due to different spectral properties of both species, this ratio can be used to monitor enzyme activity by molecular spectroscopy. The direct spectroscopic determination of NAD(P)H is based on the fact that the absorption/excitation band(s) of NAD(P)H are between 280–360 nm, with the maximum occurring at 340 ± 30 nm and the emission band at 460 ± 50 nm. Any absorption/emission band of NAD(P)
+ could not be detected [
34,
35].
A typical example with NAD
+ cofactor is alcohol dehydrogenase (ADH, EC 1.1.1.1), which can catalyze the reversible oxidation of alcohol to the corresponding aldehydes and ketones with the reduction of the nicotine adenine dinucleotide [
35,
36]. The oxidative reaction of ethanol to acetaldehyde coupled with the reduction of NAD
+ to NADH is catalyzed by the ADH enzyme:
This leads to a change in the NAD
+/NADH ratio, which helps follow the metabolic effects of ethanol in the human body [
36]. In addition, it can be also used for the determination of ethanol in food, alcoholic drinks, etc. Some examples are given elsewhere [
35,
37,
38,
39,
40,
41].
In this paper, we first studied the antenna effect of Ln(III) ternary complexes which emit characteristic luminescence spectra in the visible and NIR wavelength ranges. The NAD(P)H quenching phenomena on Ln(III) complex luminescence were investigated. In the last part, the results related to the quenching effects were verified for possible applications in bioanalysis using enzymes with the NAD(P)H/NAD(P) redox couple.
3. Materials and Methods
The macrocyclic ligands (H2DO2A, H3DO3A) were purchased from CHEMATECH (Dijon, France). The Ln(III) chloride salts (Nd(III), Sm(III), Eu(III), Tb(III), Dy(III), Yb(III)) of analytical grade purity were purchased from Alfa-Aesar (Darmstadt, Germany). NADPH and NADH compounds and enzyme alcohol-dehydrogenase (ADH) from Saccharomyces cerevisiae (321 U·mg−1) of biochemical grade purity were purchased from Sigma-Aldrich (St. Louis, MO, USA) and their solutions were prepared fresh daily.
The emission and excitation spectra of the Ln(III) ternary complexes without and in presence of the NADPH compound were recorded on Horiba FluoroMax-4P (Eu—input/output slit 5/0.5 nm, Tb, Sm, Dy—input/output slit 5/2 nm, all integration time 100 ms) and Horiba JY Fluorolog (Yb, Nd—input/output slit 14.7/14.7 nm, integration time 300 ms) spectrofluorometers (both Kyoto, Japan) containing sensitive NIR photomultiplier detectors using air- and water-cooling. The steady-state and time-resolved luminescence studies of Eu(III) complexes in presence of the NADH ligand were carried out on the PTI spectrometer QM 300 Plus (Horiba), operating with a flash 150 W Xe-lamp (frequency 300 Hz) in the wavelength range of 200–900 nm. All emissions were corrected by the wavelength sensitivity (correction function) of the spectrometer using 395 nm filter (for the visible region) and 600 nm (for the NIR region). All measurements were performed at laboratory temperature (~298.2 K).
The HRMS spectra of aqueous solutions of [Ln(DO3A)(IQCA)] complexes (pH ~ 6.0) were recorded on a 6224 Accurate-Mass TOF LC-MS system (Agilent, Santa Clara, California, USA) in negative mode using electrospray ionization (ESI) under following conditions: nitrogen flow 5 L/min, gas temperature 325 °C, nebulizer 45 psig and capillary voltage 2.5 kV.
4. Conclusions
In this paper, new ternary Ln(III) complexes have been investigated from a photophysical point of view. Eu(III) and Tb(III) ternary complexes of DO3A and IQCA ligands exhibit emission spectra with several characteristic peaks and high values of quantum yield, while Sm(III) and Dy(III) complexes have lower values. On the contrary, the Nd(III) and Yb(III) complexes with the lowest quantum yield values demonstrate the highest Stokes shift—550–650 nm—of all the studied Ln(III) complexes.
The “quenching” effect of NAD(P)H on the luminescence spectra of Ln(III) complexes was also investigated and it was proposed that this phenomenon is caused by the shielding of light needed for excitation of Ln(III) complexes by NAD(P)H. Due to the common excitation wavelength for both NAD(P)H and Ln(III) complexes, it is possible to measure both fluorescence signals (NAD(P)H in region 400–750 nm, Ln(III) depends on the type of Ln(III) ion). In some cases, such as Tb(III) or Sm(III), both emission spectra are overlapping with NAD(P)H; therefore, the time-gating mode should be applied to filter both signals leading to a decrease of the Ln(III) complex signal. Therefore, there seem to be more useful applications of Ln(III) complexes emitting in the NIR region, where the Stokes shift is higher, than for Ln(III) complexes emitting in the visible region. Another advantage of using Ln(III) complexes for detection is the higher sensitivity and broader linear concentration range of NAD(P)H than in case of direct fluorescence signal measurement at 460 nm.
The benefits mentioned above can be utilized for monitoring enzymatic reactions where the NAD(P)H/NAD(P)+ redox pair is an essential co-factor. As an example, the enzymatic transformation of ethanol into acetaldehyde catalyzed by alcohol-dehydrogenase (ADH) was chosen and the course of this reaction was monitored by the fluorescence measurement of the indirect signal of the Eu(III) ternary complex and direct signal of NAD(P)H. It was also shown that the Eu(III) complex does not have any inhibiting effect on the enzymatically catalyzed reaction, which exhibits behavior according to the mathematical model postulated by Michaelis and Menten. The calibration plot for ethanol is linear in the 0.1–30.0 mM range. The initial-rate method also enables the estimation of ADH enzyme activity. This new method was verified by the analysis of a real sample of fruit brandy and the results agree with values obtained by GC.
Thus, the results presented in this paper show that Ln(III) complexes are suitable for indirect determination of NAD(P)H compounds and can be used for detection in many biological systems where the NAD(P)H/NAD(P)+ redox pair plays important role, mainly in enzymatic reactions. While the Eu(III) complex is suitable for the detection of the course and end of enzymatic reactions, the Yb(III) and Nd(III) complexes should be employed in steady-state modes at the end of the reaction because of technical difficulties of fluorescent measurements in NIR region. The utilization of these Ln(III) complexes with desired photo-physical properties opens doors for future applications in biochemical and clinical analysis.