Chemiluminescence of lucigenin-tetracycline and its application for sensitive determination of procyanidin

Abstract

Tetracycline was reported to trigger intense lucigenin chemiluminescence (CL). Based on its significant quenching to lucigenin-tetracycline CL, procyanidin was detected. The mechanism of the lucigenin-tetracycline CL and its quenching by procyanidin were proposed. Under the optimum conditions, the calibration curve is linear in the range of 10–1000 nM for procyanidin with a detection limit of 4.4 nM. The RSD for nine replicate determinations of procyanidin was less than 1.7%. Moreover, this method was satisfactorily applied to the determination of procyanidin in human urine samples and health food capsule of grape seed.

1. Introduction

Chemiluminescence (CL) has been considered as a popular and powerful analytical technique and universally used in chemical and biological sensing fields owing to its simple and relatively cheap instrumentation, quick response, high sensitivity, wide linear range and low background noise [1,2]. Lucigenin (N, N′-dimethyl biacridinium dinitrate), one of the most extensively used chemiluminescent materials, has gained enormous concern due to its excellent CL properties and broad range of analytical applications, including drug analysis, metal ion detection, clinical diagnosis [3–5]. Hydrogen peroxide as a classical co-reactant generates strong CL signals with lucigenin in alkaline medium. However, the poor specificity and instability of the traditional lucigenin-H₂O₂ CL system in the presence of common interferents including metal ions and their complexes result in limitations on its development of sensing analysis [6]. In recent years, metal nanoparticles or quantum dots (carbon dots and black phosphorus quantum dots) have been utilized as catalysts in lucigenin CL to stimulate the emission reaction [3,7–9]. Therefore, it is of great aspiration to explore new lucigenin CL systems to broaden the scope of CL applications.

Tetracyclines belongs to a category of pharmaceutical compounds and are the first antimicrobial agents possessing a broad-spectrum activity against wide range of pathogenic gram-positive and gram-negative bacteria by inhibiting the formation of proteins within bacteria. Such compounds, including tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC), are widely used to prevent different diseases and cure various bacterial infections in humans and animals. In addition, they have also been employed in applications such as feed additives, pest control and preservation of agricultural products [10]. Broad and strong antibacterial efficiency and relatively low cost of TCs make obvious contributions to its wide-ranging clinical benefits [11]. Based on previous reports, reactive oxygen species (ROS) can be generated by TCs involved in photochemical oxidation in aqueous solution and autoxidation [12,13]. In the presence of some metal ions, TCs are involved in the formation of oxygen free radicals [14]. It means that TCs may react with lucigenin to generate CL. However, there are few reports for the CL systems of lucigenin with TCs [15,16].

Procyanidin (PC), widely existing component in plants, is an important polyphenolic compound that plays a significant role in the physiological activities of human body. PC has potential biological activities, such as antioxidative, anti-tumor, anti-aging, and anti-inflammatory effects. Furthermore, PC has a promising anticarcinogenic agent, since it is capable of inhibiting the proliferation of tumor cells of various tumors such as colorectal, gastric and prostatic cancers via the upregulation and down-regulation of mRNA expressions [17–20]. PC resists the fibril formation of human islet amyloid polypeptide and may have potential to treat Alzheimer Disease. PC is also widely used in health food, pharmaceutical industries and functional cosmetics, and is one of the available mature products in the plant extract market. Owing to its considerable clinical anticancer effect, it is absolutely necessary to monitor its biological levels in human beings. Moreover, the detection of PC in the different matrices is highly required to ensure the quality, safety and effectiveness of the manufactured product at the quality control test. In this context, several methods have been reported for the successful determination of PC, including high performance liquid chromatography (HPLC) [21], spectrophotometry [22], capillary electrophoresis [23] and spectrally sensitive pulse photometry [24]. However, these methods still have shown various limitations. For example, chromatographic methods require large amount of capital investment in testing instruments and the complicated procedures [17].

In this work, lucigenin-TC CL was investigated for the first time. TC was able to generate obvious CL signals upon its unique interaction with alkaline solution of lucigenin. Subsequently, the effect of PC on the developed lucigenin-TC system was firstly investigated. It turns out that PC can remarkably inhibit the CL of lucigenin-TC system. By utilizing the quenching effect, a new flow-injection analysis (FIA) for PC sensitive detection was designed, as shown in Scheme S1. Also, the possible mechanism of the lucigenin-TC CL was discussed. This CL approach for PC detection is distinguished by simplicity, fastness, and straightforwardness and shows excellent sensitivity and selectivity in real sample testing.

2. Experimental section

2.1. Materials and reagents

Serine, proline, alanine, glycine, tyrosine, tryptophan, ascorbic acid and phenol were purchased from Beijing Chemical Reagent Company (Beijing, China). Cysteine was acquired from Shanghai Yuanju Biotechnology Co., Ltd. (Shanghai, China). Resorcinol was purchased from Shantou Xilong Chemical Plant Co., Ltd. (Shantou, China). Phloroglucin and 3-aminophenol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Lucigenin, dopamine hydrochloride and glutathione (GSH) were purchased from Shanghai Aladdin Biochemical technology Co., Ltd. (Shanghai, China). Tetracycline hydrochloride and PC were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Lucigenin stock solution (5.0 mM) was prepared by dissolving 0.1021 g of lucigenin in 40 mL water. TC stock solution (5.0 mM) was prepared by dissolving 0.1111 g of TC in 50 mL NaOH (0.1 M) solution. Stock solution of PC (250 μM) was prepared by dissolving 0.0059 g solid PC in 40 mL water. All the chemicals were analytical-reagent grade and used without further purification. Deionized water was used during all the experiments.

2.2. Instruments

The CL signals were measured by utilizing a FIA based CL system which consists of Biophysics CL (BPCL) ultra-weak luminescence analyzer (the Institute of Biophysics, Chinese Academic of Sciences), an intelligent flow injection sampler (IFIS–C mode) (ReMax Inc., Xi’an, China) and a home-made flow cell. The flow cell was put in a light-tight box of the luminescent analyzer. The loop injector was equipped with an injection loop of 50 μL. Unless specifically mentioned, the photomultiplier tube (PMT) voltage was set at 1100 V. Electron paramagnetic resonance (EPR) spectra were measured using a Bruker EMX Plus EPR Spectrometer.

2.3. CL measurement of lucigenin-TC system

Scheme S2 shows the schematic diagram of the FIA system for the CL measurement. In brief, channel 1 and 2 were respectively used to deliver an aqueous solution of 0.5 mM TC and 0.1 M NaOH solution into the flow cell at a flow rate of 2.0 mL/min. Then 200 μL of 0.1 mM lucigenin solution was injected through the loop injector. Finally, the CL signals were measured.

2.4. Procedure for PC detection

0.5 mM TC in water and 0.1 M NaOH solution were pumped into the flow cell through channel 1 and 2 at a flow rate of 2.0 mL/min, respectively. Different concentrations of PC were mixed with 0.1 mM lucigenin first, and then 200 μL of mixture solution was injected through the loop injector, and the CL signals were measured.

2.5. Procedure for PC detection in real samples

Lucigenin-TC system was further applied for the assay of PC in human urine samples and commercially available grape seed extract capsule with as PC main component (GloryFeel GmbH, Germany). The human urine samples were collected from healthy individual. The human urine samples and grape seed extract samples were diluted with double distilled water to minimize the interference of other components and to bring PC concentrations into the linear calibration range. 0.5 mM TC in water and 0.1 M NaOH solution were pumped into the flow cell through channel 1 and 2 at a flow rate of 2.0 mL/min, respectively. Diluted samples were mixed with 0.1 mM lucigenin first, and then 200 μL of mixture solution was injected through the loop injector, and the CL signals were measured. Standard addition method was used for evaluate the percentage recoveries.

3. Results and discussion

3.1. CL of Lucigenin-TC system

It is well known that lucigenin solution generates weak CL signal owing to its reaction with dissolved oxygen. The addition of TC to lucigenin solution leads to a great CL enhancement as illustrated in Fig. S1. This obvious CL signal obtained after the addition of TC to lucigenin solution indicates that TC is a nice coreactant for lucigenin CL. As shown in Fig. 1, the CL spectrum was recorded by measuring CL intensity versus different wavelength filters ranging from 425 to 575 nm. The maximum CL emission wavelength is obtained at 490 nm, which is in accordance with the characteristic wavelength of emission for the excited state of N-methylacridone [25]. Therefore, it also confirms that lucigenin acts as a luminophor in this CL reaction, while TC is the main coreactant.

Fig. 1.

CL spectrum of lucigenin-TC system. The wavelengths of the filters are 425 nm, 440 nm, 460 nm, 490 nm, 535 nm, 555 nm and 575 nm, respectively. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

It is well-known that ROS usually play a critical role in the CL system of lucigenin [9]. Three main ROS, including singlet oxygen (¹O₂), hydroxide radical (HO^•) and superoxide anion radical ( ${\text{O}}_2^{•-}$), are commonly incorporated in CL triggering from lucigenin species. So the effect of radical scavengers including NaN₃, thiourea, and superoxide dismutase enzyme (SOD) for ¹O₂, HO^•, and ${\text{O}}_2^{•-}$ on the CL intensity were investigated respectively. Fig. S2 shows the effect of radical scavengers on lucigenin-TC CL. NaN₃ and thiourea have little influence on CL intensity; while SOD inhibits CL intensity much. This clearly implies that ${\text{O}}_2^{•-}$ plays pivotal role in lucigenin-TC CL. To further explore the mechanism, the effect of oxygen on this CL system was investigated. Before the measurement, nitrogen gas was bubbled through the reacting solution for 30 min to ensure the removal of dissolved oxygen. As illustrated in Fig. S3, the CL intensity decreased about 43% after the dissolved oxygen was removed from the reactant solutions. Moreover, electron paramagnetic resonance (EPR) spectrum was measured to confirm the formation of ${\text{O}}_2^{•-}$ in the lucigenin-TC system. 5,5′-Dimethyl-1-pyrroline-N-oxide (DMPO) can capture ${\text{O}}_2^{•-}$ to form the spin adduct of $\text{DMPO-}{\text{O}}_2^{•-}$. Fig. S4 shows the characteristic peak of $\text{DMPO-}{\text{O}}_2^{•-}$ spin adduct with the addition of DMPO on this system. These results testify the critical involvement of ${\text{O}}_2^{•-}$ generated from dissolved oxygen in the lucigenin-TC CL.

Taking all these experiments into consideration, the possible reaction mechanism is proposed as shown in Scheme S3. The 4-dimethylamino group of TC is dissociated in alkaline solutions, leading to the generation of TC anionic radical and secondary amine radical (Eq 1 in Scheme S3). These intermediate radicals are converted to quinone and imine products via an oxidation reaction, accompanied by the formation of ${\text{O}}_2^{•-}$ (Eq 2 in Scheme S3) [13,26]. Lucigenin cation radical (Luc^•+) can be produced through one electron reduction reaction in alkaline conditions. And then, Luc^•+ reacts with ${\text{O}}_2^{•-}$ to form highly unstable intermediate lucigenin dioxetane (LucO₂) (Eq 3 in Scheme S3), which subsequently decomposes to the electronically excited N-methylacridone. Finally, the excited N-methylacridone is spontaneously returned to its ground state, resulting in strong CL emission (Eq 4 in Scheme S3) [27,28].

3.2. PC-induced CL quenching of lucigenin-TC system

Figure 2 shows the effect of PC on lucigenin-TC CL. The lucigenin-TC system shows much stronger CL than the lucigenin-PC system. However, the CL intensity of the lucigenin-TC system was notably decreased with the addition of PC. The inhibition on the lucigenin-TC CL in the presence of PC may be caused by the consumption of ${\text{O}}_2^{•-}$ and its effect on reaction process. There are some reports that PC has strong antioxidant activity and is an efficient free radical scavenger even at low concentrations [29]. Therefore, the lucigenin-TC CL system could be used for the determination of PC.

Fig. 2.

Comparison diagram of different CL with PC. C(lucigenin), 0.1 mM; C(PC), 1 μM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

3.3. Optimization of experimental conditions

It is reported that alkaline conditions are advantageous to the CL emission with great intensity. Therefore, the CL intensity of the lucigenin-TC system in different concentrations of NaOH solutions was measured. Figure 3A shows the influence of NaOH concentration on the CL inhibition efficiency. (I₀ – I)/I₀ is the CL inhibition efficiency, I₀ is the CL intensity in the absence of PC, and I is the CL intensity in the presence of PC. The inhibition efficiency increased rapidly in the NaOH concentration range 0.05–0.1 M and gradually decreased as the NaOH concentration increased further. With the increase of NaOH concentrations, the background CL intensity of lucigenin in the presence of TC enhanced continually due to the faster deprotonation and decomposition of reactant. While the concentration of NaOH was above 0.1 M, the higher concentration probably caused more side reactions in the more alkaline environment, leading to the decrease of CL inhibition efficiencies. Therefore, 0.1 M NaOH was used for the subsequent experiments. In addition, the effect of TC concentrations on the CL system was studied. As shown in Fig. 3B, the CL intensities keep increasing as TC concentrations increase because of the generation of more reactive species. The CL inhibition efficiency of PC increased with the concentration of TC rising to 0.5 mM and then decreased slightly as its concentration was more than 0.5 mM, resulting from synergistic effect of PC and TC [30]. Consequently, 0.5 mM was selected as the optimal TC concentration in the following experiments.

Fig. 3.

(A) Effects of NaOH concentrations on the CL inhibition efficiencies. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(PC), 1 μM; PMT voltage, 900 V. (B) Effects of TC concentrations on the CL inhibition efficiencies (red line) and CL intensity (black line). C(lucigenin), 0.1 mM; C(NaOH), 0.1 M; C(PC), 1 μM.

3.4. Detection of PC

Figure 4 shows that effect of PC concentrations on the CL intensities of the lucigenin-TC system. It was obvious that the CL intensities decreased with increasing PC concentrations. The CL inhibition efficiency is linear with the concentrations of PC in the range 10–1000 nM. The linear equation is (I₀ – I)/I₀ = 0.097 + 4.27 × 10⁻⁴ C (where C is the PC concentration in nM) (R = 0.996). The limit of detection (LOD) was calculated to be 4.4 nM at a signal-to-noise ratio of 3. The RSD for nine successive detections at the concentration 1 μM is about 1.7% (Fig. S5), indicating the good reproducibility of this detection method. By comparison, our method has the advantages of simple instrument, fast response and high sensitivity, as shown in Table S1.

Fig. 4.

(A) CL emission–time curves in different concentrations of PC from 10 to 1000 nM and (B) PC calibration curve. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

3.5. Selectivity of this method for PC

The effect of the common interfering substances on PC detection was evaluated including dopamine, phenol, resorcinol, phloroglucin, 3-aminophenol, ascorbic acid, cysteine, glutathione, serine, proline, alanine, glycine, tyrosine, tryptophan, Ni²⁺, Ca²⁺, Mg²⁺, Fe³⁺, Zn²⁺, Pb²⁺, and Ag⁺. As shown in Fig. S6, the CL intensity of lucigenin-TC system decreases significantly in the presence of PC. Other substances have much smaller effects on the CL intensities. By comparison, PC has stronger reducing power and more reducing groups than others, resulting in fine selectivity.

3.6. Determination of PC in real samples

In order to study the applicability of the proposed method, we have determined PC in human urine samples and commercially available grape seed extract capsule. To evaluate the recoveries, the human urine samples were diluted 100-fold with double distilled water to minimize interference from potential interferents. The diluted urine samples were respectively spiked with different known concentrations of PC. In addition, 6.0 mg of grape seed extract samples was added in 10 mL deionized water. The solutions were then diluted with deionized water to bring the calculated concentration of PC to 500 nM based on the production information. To evaluate the recoveries, the diluted sample solutions were respectively spiked different known concentrations of PC. As shown in Table S2, the recoveries for PC determination in human urine and grape seed extract capsule were in the range of 94.9–100.2% and 101.6–104.1%, respectively. Moreover, we detected PC in grape seed extract capsule using HPLC-UV. As shown in Table S3, the measured values by our method and HPLC-UV method are similar and are also similar to that (95.1%) stated by the manufacturer. It suggests that this method is promising for the detection of PC in real samples.

4. Conclusion

In conclusion, TC was employed as an efficient coreactant for lucigenin CL in the absence of specific catalysts or luminescent materials. Notably, PC was found to significantly suppress CL emission of lucigenin-TC system for the first time. This PC-induced CL quenching for lucigenin-TC system was mainly ascribed to its antioxidant effect. A new strategy in combination with flow-injection analysis for sensitive and selective PC determination was established based on this quenching effect on the lucigenin-TC CL. Moreover, the proposed method exhibiting good reproducibility has been successfully applied for the determination of PC in human urine samples and health food capsule of grape seed. With the merits of biocompatibility, simplicity and robustness, our newly developed lucigenin-TC system provides a new way for the biological detection of PC and thus holds great promise in health food, pharmaceutical industries and functional cosmetics.

Appendix

Scheme S1

Schematic of TC induced CL of lucigenin for PC detection.

Scheme S2

Diagram of flow injection analysis for lucigenin-TC CL system.

Scheme S3

Mechanism of lucigenin-TC CL system.

Fig. S1

Comparison diagram of different CL. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

Fig. S2

CL intensity of lucigenin-TC system in the absence (a) and presence of radical scavengers (b) 0.5 mM NaN₃, (c) 1.0 mM thiourea, (d) 1 μg/mL SOD. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

Fig. S3

CL emission-time curves recorded in the presence (red line) and absence of oxygen (black line). C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M; PMT voltage, 700 V.

Fig. S4

The EPR spectra of the adduct DMPO-O₂^•− in the proposed CL system. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

Fig. S5

CL intensity of lucigenin-TC system at the PC concentration of 1 μM under 9 consecutive detections. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

Fig. S6

Interference study of PC detection. The concentrations of PC, dopamine (DA), phenol (Phe), resorcinol (Res), phloroglucin (Phl), 3-aminophenol (3-Amp), ascorbic acid (AA), cysteine (Cys) and glutathione (GSH) are 1 μМ. The concentrations of serine (Ser), proline (Pro), alanine (Ala), glycine (Gly), tryptophan (Try), tyrosine (Tyr), Ni²⁺, Ca²⁺, Mg²⁺, Fe³⁺, Zn²⁺, Pb²⁺, and Ag⁺ are 10 μM. C(lucigenin), 0.1 mM; C(TC), 0.5 mM; C(NaOH), 0.1 M.

Table S1.

Comparison of some previously reported assays for PC detection.

Method	Linear range	LOD	Reference
RP-LC-UV	34.6 – 259.3 μM	5.0 μM	[31]
MIPs-SPE	10.8 – 172.9 μM	5.2 nM	[32]
LC–MS	2.3 – 777.9 μM	103.7 nM	[33]
RP-HPLC-DAD	0.2 – 34.6 μM	43.4 nM	[34]
Nano-LC-TOF-MS	0.2 – 3.6 μM	13.5 nM	[35]
UHPLC–MS/MS	0.2 – 10.7 μM	1.0 nM	[36]
CL	10 – 1000 nM	4.4 nM	This work

Table S2.

Results of the detection of PC in real samples.

Samples	Spiked (nM)	Found (nM)	Recovery (%)	RSD (n = 3, %)
Urine	0.0	–	–	2.4
	200.0	199.1	99.6	2.8
	500.0	474.5	94.9	1.5
	700.0	701.7	100.2	4.8
Grape seed extract capsule	0.0	485.2	–	4.1
	100.0	609.4	104.1	1.7
	200.0	696.3	101.6	2.4
	400.0	907.9	102.6	1.4

Table S3.

Comparison of PC content in grape seed extract capsule detected by HPLC-UV and CL.

Sample	Found by HPLC-UV [37] (%)	Found by CL method (%)
Grape seed extract capsule	96.8	90.2

Funding Statement

This project was kindly supported by the National Natural Science Foundation of China [No. 21804127 and 21874126], and the Chinese Academy of Sciences (CAS)-the Academy of Sciences for the Developing World (TWAS) President’s Fellowship Programme.