A perylenetetracarboxylic dianhydride and aniline-assembled supramolecular nanomaterial with multi-color electrochemiluminescence for a highly sensitive label-free immunoassay

Wei Zhanga, Yue Songa, Yunyun Wanga, Shuijian Heb, Lei Shanga, Rongna Maa, Liping Jiaa and Huaisheng Wang*a
aChemistry of Department, Liaocheng University, Liaocheng, Shandong 252059, China. E-mail: hswang@lcu.edu.cn
bCollege of Materials Science and Engineering, Nanjing Forestry University, Nanjing, 210037, China

Received 26th October 2019 , Accepted 1st February 2020

First published on 5th February 2020


Most electrochemiluminescence (ECL) studies focus on the single emission of luminophores, which severely limits the development of the multi-color ECL fundamental theory and applications. Herein, we prepared a multi-color ECL supramolecular nanomaterial self-assembled by 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and aniline (An) through hydrogen bonding. This PTCDA–An supramolecular nanomaterial simultaneously produced multi-color emissions peaking at 486, 692 and 760 nm with K2S2O8 as a coreactant. These multi-color emissions were assigned to the excited PTCDA monomer (486 nm), H-dimer (692 nm) and J-dimer (760 nm). The simultaneously increased dual-color ECL intensity significantly enhanced the total ECL intensity of PTCDA–An. Furthermore, this highly efficient ECL nanomaterial was used as an ECL platform to construct a label-free immunosensor for tumor marker carcinoembryonic antigen (CEA) detection. The total ECL intensity of the immunosensor sensitively decreased due to the simultaneously decreased ECL of multiple emissions. Also, this immunosensor exhibited a wide linear range from 1 pg mL−1 to 10 μg mL−1 with a low detection limit of 0.23 pg mL−1. The multi-color ECL from the same luminophore PTCDA in this work also provides a new perspective for multi-color ECL biomaterial design.


Introduction

Electrochemiluminescence (ECL) is generated by the excited state of a luminophore on the electrode surface through electron transfer; it does not need an external light source and possesses low background interference, high sensitivity and rapid response. Thus, the ECL technique has been widely applied in many fields such as optoelectronics,1,2 light-emitting devices, biosensing,3–8 and especially for tumor marker detection in clinical diagnostics.9–12 In these ECL studies, the reported luminophores mostly produced single emission and a few multi-color ECL luminophores in organic systems, such as metal complexes13,14 and BODIPY dye-capped PbS nanocrystals,15 were also reported. However, this multi-color ECL in organic systems was not suitable for the biological field. Thus, the design of multi-color ECL nanomaterials in an aqueous system is necessary for biological applications such as the design of more sensitive biosensors or the development of more accurate detection methods. Hence, more efforts have been devoted to prepare multi-color ECL nanomaterials in an aqueous system. Recently, Cui's group prepared an aqueous multi-color ECL nanoluminophore N-(4-aminobutyl)-N-ethylisoluminol/tetra(4-carboxyphenyl)porphyrin/TiO2, which produced potential-resolved multi-color ECL16 and greatly promoted the development of multi-color ECL.

Here, we reported a new multi-color ECL nanomaterial in an aqueous solution, which produced multi-color emissions at the same potential range. This multi-color ECL nanomaterial was prepared by assembling the single-emission luminophore 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) with a coreactant promoter aniline (An). This PTCDA–An nanomaterial produced a stronger emission at 486 nm than the single emission of PTCDA at 483 nm and simultaneously produced two new emission bands at 692 and 760 nm with K2S2O8 as a coreactant (Scheme 1A). The simultaneously produced multi-color emission significantly enhanced the total ECL intensity of PTCDA–An. A possible multi-emission scheme is discussed in this work.


image file: c9tb02368b-s1.tif
Scheme 1 (A) The structure of PTCDA–An (left), multi-color ECL spectrum of PTCDA–An (middle) and the significantly enhanced total ECL of PTCDA–An compared with that of PTCDA (right); (B) the preparation procedure of label-free immunosensor and the detection mechanism.

This highly efficient ECL nanomaterial PTCDA–An was further used to construct a label-free immunosensor for tumor marker detection.17–19 Au nanoparticles (AuNPs) were electrodeposited on the PTCDA–An surface to immobilize the primary antibody (Ab1) for capturing the targets. The carcinoembryonic antigen (CEA) detection was used to provide a proof of concept.20 When CEA was captured on the PTCDA–An surface, the total ECL intensity largely decreased due to steric hindrance, which also decreased the multi-color emissions simultaneously, as shown in Scheme 1B. The results demonstrate that the novel multi-color nanomaterial PTCDA–An is highly sensitive for CEA detection.

Results and discussion

Aniline was used as a coreactant promoter to assemble with PTCDA through hydrogen bonding.21 To demonstrate the formation of PTCDA–An, the UV-vis spectra of aniline, PTCDA and PTCDA–An were recorded. The UV-vis spectrum of An shows an obvious E2 band and B band of the benzene ring at 236 nm and 287 nm, respectively, and both the bands are attributed to the π–π* transition (Fig. 1A, black line). The UV-vis spectrum of PTCDA in an aqueous solution does not display the three well-defined vibronic bands corresponding to the characteristic S1 ← S0 electronic transition of the perylene backbone (Fig. 1A, red line) and its absorption bands obviously red-shift (482, 527 and 588) due to the aggregation of PTCDA.22 In the UV-vis absorption spectrum of PTCDA–An (Fig. 1A, green line), an obvious E2 band and B band of aniline at 230 nm and 278 nm, respectively, and three absorption bands of the perylene units at 482, 527 and 586 nm can be observed, indicating the formation of PTCDA–An. The perylene derivatives can self-organize into supramolecular architectures through π–π stacking (forming sandwich-type H-aggregates)23 and hydrogen bonding (forming slipped J-aggregates).21 Here, PTCDA self-assembled into a nanorod structure, as shown in the SEM image (Fig. S1a, ESI). After PTCDA combines with An, no obvious structural changes can be observed in the SEM image of PTCDA–An (Fig. 1B). Furthermore, the XPS spectrum of PTCDA–An was recorded. The peak of N1s at 400.1 eV in the XPS spectrum further demonstrates the formation of PTCDA–An (Fig. 1C, inset). Thereafter, the ECL experiments of PTCDA and PTCDA–An were performed. After aniline assembled with PTCDA, the ECL intensity significantly increased (Fig. 1D).
image file: c9tb02368b-f1.tif
Fig. 1 (A) The UV-vis spectra of An, PTCDA and PTCDA–An; (B) the SEM image of PTCDA–An; (C) the XPS spectrum of PTCDA–An, inset: the XPS of N1s in PTCDA–An; (D) the ECL intensity of PTCDA-modified GCE and PTCDA–An-modified GCE between −0.1 and −1.9 V.

Spooling ECL spectroscopy involves the real-time monitoring of ECL during a potential scanning process, which allows the visualization of the rise and fall of the ECL intensity in the potentiodynamic process. Fig. S2b (ESI) and Fig. 2A show the spooling spectra of PTCDA-modified GCE and PTCDA–An-modified GCE in the presence of 0.1 M K2S2O8 as the potential is scanned between −1 and −2.0 V for a single cycle with the same scanning speed at 25 mV s−1. All the displayed spooling ECL spectra of PTCDA and PTCDA–An are recorded at the intervals of 4 s. A single ECL emission for PTCDA and three ECL emissions for PTCDA–An at the same potential range were observed.


image file: c9tb02368b-f2.tif
Fig. 2 (A) The spooling ECL spectrum of PTCDA–An between −1 and −2 V; (B) the stacked ECL spectrum of PTCDA–An between −1.6 and −2 V. (C) The CV of bare GCE and GCE/PTCDA/An in PBS solution and GCE/PTCDA/An in PBS solution with 0.1 M K2S2O8; (D) the PL spectrum of PTCDA solution and PTCDA powder.

In order to elucidate various ECL peaks, the stacked ECL spectra of PTCDA-modified GCE and PTCDA–An-modified GCE in PBS solutions with K2S2O8 as a coreactant during a cycle scanning potential between −1.6 and −2.0 V are shown in Fig. S2c (ESI) and Fig. 2B. The PTCDA-modified GCE produced strong emission at 483 nm (ECL-1 peak) between −1.85 and −2.0 V (Fig. S2c, ESI). The PTCDA–An-modified GCE exhibited an ECL-1 peak at 486 nm, ECL-2 peak at 692 nm and an additional ECL-3 peak at 760 nm between −1.85 and −2.0 V (Fig. 2B). All the emission intensities for PTCDA–An were found to be larger than that of PTCDA (Fig. S3, ESI) at the same potential, indicating that the multi-emissions simultaneously increased and the three emissions integrated into strong ECL in the ECL intensity–time curve, as shown in Fig. 1D, red line. To determine the origin of these ECL peaks, the ECL spectra of PTCDA-modified GCE in a PBS solution without K2S2O8 and bare GCE in a PBS solution containing K2S2O8 were recorded (Fig. S4, ESI). For bare GCE in the PBS solution containing K2S2O8, there is a weak ECL signal attributed to the emission of excited oxygen,24 (Fig. S4a, ESI) whose ECL peak has been reported to be located at 575 nm.25 However, its ECL intensity is too small to record the ECL emission spectra in this work (Fig. S5, ESI). No ECL signal was observed for PTCDA in the PBS solution without K2S2O8 (Fig. S4b, ESI). In addition, aniline is not an ECL luminophore and cannot produce ECL.25 Thus, the multi-color ECL at 486 (483), 692 and 760 nm should be due to the coreaction of PTCDA and S2O82−.

To explore the ECL scheme of PTCDA and PTCDA–An, the CV tests of bare GCE, PTCDA-modified GCE and PTCDA–An-modified GCE in PBS solutions in the presence and absence of K2S2O8 were performed (Fig. 2C and Fig. S2a, ESI). There is one additional reduction peak at −1.3 V in the curve of bare GCE in the presence of K2S2O8 (Fig. S2a, red line, ESI), which should be due to the reduction of S2O82− on the GCE surface. The CV curves of PTCDA-modified GCE and PTCDA–An-modified GCE in PBS solutions show two irreversible redox peaks (Fig. 2C, red line and Fig. S2a, green line, ESI), indicating the two one-electron transfer processes of PTCDA (PTCDA/PTCDA and PTCDA/PTCDA2−), which are in accordance with that of other perylene derivatives.26–28 In the presence of K2S2O8, the reduction current of PTCDA largely increased, demonstrating the strong oxygenation between PTCDA and K2S2O8 (Fig. 2C, green line and Fig. S2a, blue line, ESI).

To demonstrate that ECL resulted from PTCDA, the PL spectra of PTCDA and PTCDA–An in aqueous solutions were recorded. There are two vibronic bands at 411 and 437 nm, corresponding to the characteristic S1 ← S0 electronic transition of the perylene backbone in the excitation spectra of the PTCDA solution (Fig. S6, ESI). The monomeric emission bands at 484 and 511 nm and the emission bands of the aggregates at 613 and 660 nm are shown in the PL spectra of PTCDA (Fig. 2D, dark line). In addition, the PL spectra of PTCDA–An are shown in Fig. S7 (ESI), both of which demonstrate similar vibronic bands (412, 434 nm) and monomer emission bands (482, 511 nm) to that of PTCDA. The PL emission of PTCDA and PTCDA–An (Fig. 2D, dark line and Fig. S7, red line, ESI) shows the maximum emission wavelength at 484 (482) nm, which matches well with its ECL at 483 (486) nm, providing further evidence that the luminophore is PTCDA in the S2O82− solution and the excited state of the PTCDA monomers is responsible for the ECL-1 emission of both PTCDA and PTCDA–An.

PTCDA has been reported to form dimers in its crystal structure.26,29 The PL spectra of a solid PTCDA powder shows a wide dimeric emission band (from 600 to 900 nm) with the maximum emission wavelength at 670 nm, which includes the ECL-2 peak (692 nm) and ECL-3 peak (760 nm); this indicated that the excited state of the PTCDA dimers was responsible for the ECL-2 and ECL-3 emissions.30,31 The ECL-3 wavelength at 760 nm has an obvious 68 nm red-shift compared with the ECL-2 wavelength (692 nm). The separation of 68 nm may be explained by the structural differences between the H-aggregated excimers and the J-aggregated excimers.21,22,32 The energy emitted by the J-aggregated excimers is lower than the energy of the H-aggregated excimers.22 Thus, ECL-2 was assigned to the H-dimer33 and ECL-3 was assigned to the J-dimer.34 Furthermore, the ECL-3 intensity was much smaller than the ECL-2 intensity, indicating that the formed excimers were predominantly H-dimers.

Aniline acted as a coreactant promoter to accelerate the production of SO4˙, due to which the ECL-1 intensity at 486 nm largely increased and the other two emissions at 692 and 760 nm were simultaneously produced. The simultaneously increased multi-color ECL significantly enhanced the total ECL. The dual-color ECL mechanism of PTCDA–An28,35,36 should be as follows (Scheme 2).


image file: c9tb02368b-s2.tif
Scheme 2 The multi-color ECL mechanism of PTCDA–An.

From −1.85 to −2.0 V,

 
PTCDA + e → PTCDA (1)
 
PTCDA + e → PTCDA2− (2)
 
S2O82− + e → SO4˙ + SO42− (3)
 
An + S2O82− → An+˙ + SO4˙ + SO42− (4)
 
PTCDA2− + SO4˙ → PTCDA* + SO42− (5)
 
PTCDA* → PTCDA + (486 nm) (6)
 
PTCDA + SO4˙ → PTCDA+˙ + SO42− (7)
 
PTCDA2− + PTCDA → 2PTCDA˙ (8)
 
2PTCDA+˙ + 2PTCDA˙ → (H-dimer)* + (J-dimer)* (9)
 
(H-dimer)* → 2PTCDA + (692 nm) (10)
 
(J-dimer)* → 2PTCDA + (760 nm) (11)

This highly efficient multi-emission nanomaterial PTCDA–An was dropped on the GCE surface, forming the PTCDA–An/GCE interface. AuNPs were electrodeposited on the PTCDA–An surface. Some round dots dispersed on the nanorods, as shown in the SEM image (Fig. S1c, ESI), indicating the successful immobilization of AuNPs. Then, PTCDA–An/AuNPs were used as an ECL platform to prepare a label-free immunosensor and ECL tests were performed during cycle potential scanning from −0.8 V to −1.9 V.

The preparation process of the ECL immunosensor was characterized by electrochemical impedance spectroscopy (EIS) with [Fe(CN)6]3−/4− as the redox probe.8,37,38 The electron transfer resistance (Ret) of PTCDA–An was lower than that of the bare electrode (Fig. S8b, red dot, ESI), which was because the positive charges of PTCDA–An assigned to the protonation of the O and N atoms in PTCDA–An promoted [Fe(CN)6]3−/4− to diffuse from the solution to the electrode surface. After AuNPs were electrodeposited on the PTCDA–An surface, a smaller Ret was observed (Fig. S8b, green dot, ESI). However, when Ab1 and BSA were individually adsorbed onto the AuNP film surface, Ret became increasingly larger due to the poor conductivity of the Ab1 and BSA layers (Fig. S8a, blue dot, cyan dot and magenta dot, ESI).

The ECL property of the constructed immunosensor was investigated. On increasing the CEA concentration (CCEA), the multi-emission (ECL-1, ECL-2 and ECL-3) intensity simultaneously decreased until CCEA was increased up to 10 μg mL−1 (Fig. 3A). It should be noted that the immobilized CEA on PTCDA–An hindered the coreaction between PTCDA and S2O82− and the reaction between An and S2O82− like an insulator (Scheme 2). The logarithmic value of I2/I1 (log(I2/I1)) linearly depended on the logarithm of the CEA concentration (log[thin space (1/6-em)]CCEA) in the range from 10−3 to 104 ng mL−1 (Fig. 3B). The regression equation was log(I2/I1) = −0.45856 + 0.07108[thin space (1/6-em)]log[thin space (1/6-em)]CCEA/ng mL−1 with the correlation coefficient 0.9861 for this ratiometric CEA immunosensor (Fig. 3B). The total ECL intensity was also recorded, which largely decreased on increasing the CEA concentration (Fig. 3C). The total ECL difference (ΔECL) for the immunosensor in the presence and absence of CEA showed a linear response to log[thin space (1/6-em)]CCEA from 1 pg mL−1 to 10 μg mL−1. The regression equation was ΔECL = 6461.8 + 1135.7[thin space (1/6-em)]log[thin space (1/6-em)]CCEA/ng mL−1 with the correlation coefficient 0.9995. It can be seen that the results using total ΔECL as the detection signal are better than that for the ratiometric CEA immunosensor (Fig. 3D). Thus, the total ΔECL values were taken as the detection signal in this work and its detection limit was 0.23 pg mL−1 with 3σ (according to the criterion of IUPAC recommendation).


image file: c9tb02368b-f3.tif
Fig. 3 (A) The multi-color ECL spectra of the immunosensor at different CCEA from (a) 0 pg mL−1, (b) 1 pg mL−1, (c) 10 pg mL−1, (d) 100 pg mL−1, (e) 1 ng mL−1, (f) 10 ng mL−1, (g) 100 ng mL−1, (h) 1 μg mL−1 and (i) 10 μg mL−1; (B) the relationship between log(I2/I1) and log[thin space (1/6-em)]CCEA; (C) the ECL intensity of the immunosensor at different CCEA from (a) 0 pg mL−1, (b) 1 pg mL−1, (c) 10 pg mL−1, (d) 100 pg mL−1, (e) 1 ng mL−1, (f) 10 ng mL−1, (g) 100 ng mL−1, (h) 1 μg mL−1 and (i) 10 μg mL−1; (D) the relationship between ΔECL and log[thin space (1/6-em)]CCEA. The ECL tests of fabricated immunosensor were performed during cycle potential scanning from −0.8 V to −1.9 V.

The fabricated immunosensor showed a wide detection range, high sensitivity and low detection limit compared with other immunosensors (Table S1, ESI).

We prepared five parallel immunosensors with CCEA at 1 ng mL−1 using five different GCE electrodes to investigate reproducibility. The immunosensors with CCEA at 1 ng ml−1 possessed a consistently high signal difference with the control immunosensors without CEA (0 ng mL−1) (Fig. S9A, ESI), indicating the good reproducibility of the immunosensors. The ECL intensity of the fabricated immunosensor remained almost the same after 10 tests (Fig. S10A, ESI). In addition, we also kept the fabricated immunosensors with CCEA at 1 ng ml−1 in a refrigerator from 0 to 24 days at 4 °C to investigate the stability. We tested their ECL property at an interval of six days, as shown in Fig. S9B (ESI). With increasing the number of days, the ECL intensity gradually decreased and reached 85% on the 24th day, demonstrating good stability of the immunosensor.

Glucose (Gluc), human serum albumin (HSA), thrombin (Thr), bovine serum albumin (BSA), uric acid (UA), ascorbic acid (AA), cholesterol (Chol), alpha fetoprotein (AFP) and prostate-specific antigen (PSA) were taken as the interferences to investigate the selectivity of the immunosensor. The ECL experiments of the immunosensor toward 20 mg mL−1 Gluc, 600 mg mL−1 HSA, 10 μg mL−1 Thr, 10 μg mL−1 BSA, 700 mg mL−1 UA, 700 mg mL−1 AA, 20 mg mL−1 Chol, 10 μg mL−1 AFP, 10 μg mL−1 PSA and 1 μg mL−1 CEA were performed (Fig. S10B, ESI). These ECL values were nearly the same as that for the blank solution. In addition, the ECL experiments of the immunosensor in mixtures including 20 mg mL−1 Gluc, 600 mg mL−1 HSA, 10 μg mL−1 Thr, 10 μg mL−1 BSA, 700 mg mL−1 UA, 700 mg mL−1 AA, 20 mg mL−1 Chol, 10 μg mL−1 AFP, 10 μg mL−1 PSA and 1 μg mL−1 CEA were performed and no obvious changes were observed in comparison to the ECL intensity of the immunosensor in a 1 μg mL−1 CEA solution (Fig. S10B, ESI). These results demonstrated the high selectivity of the fabricated immunosensor.

The feasibility of the fabricated immunosensor in human serum samples was investigated by testing the serum samples of different types of cancers (liver cancer, breast cancer and stomach cancer). The CCEA in these samples tested by the fabricated ECL immunosensor and that tested by ELISA are shown in Table 1. The relative errors were in the range from 5.34% to 7.86% and the RSD values were in the range from 1.58% to 4.32%, indicating an acceptable range. Thus, the fabricated label-free ECL immunosensor showed good feasibility for detecting CEA in real samples.

Table 1 Detection of CEA in human serum samples from various cancers
Samples Proposed method (ng mL−1) ELISA (ng mL−1) Relative error (%) RSD (%)
Liver cancer 1.58 1.49 6.04 4.32
Breast cancer 2.17 2.06 5.34 1.58
Stomach cancer 4.53 4.20 7.86 2.33


Conclusions

In conclusion, we fabricated a novel multi-color ECL nanomaterial PTCDA–An, which produced stronger emission at 486 nm than that of PTCDA and other two new emissions at 692 and 760 nm with K2S2O8 as a coreactant in the same potential range. The multi-emissions were assigned to the excited PTCDA monomer (486 nm), H-dimer (692 nm) and J-dimer (760 nm). The simultaneously increased multi-color ECL intensity significantly enhanced the total ECL intensity of PTCDA–An. Also, this highly efficient ECL nanomaterial was used to fabricate a label-free ECL CEA immunosensor based on the steric hindrance of CEA. The total ECL intensity of the immunosensor sensitively decreased due to the simultaneously decreased ECL of multiple emissions. Moreover, this immunosensor showed high sensitivity and wide linear range with a low detection limit for CEA detection. Additionally, the proposed method to fabricate the ECL immunosensor can be applied in the clinical diagnosis field and the disposable-printed/screen-printed electrode can be used instead of GCE. Thus, we are trying to use the disposable-printed/screen-printed electrode as a working electrode to fabricate an immunosensor in the future. In addition, this work provides a new perspective for designing a multi-color ECL nanomaterial in the precise bio-imaging field.

Conflicts of interest

There are no conflicts to declare. The human serum samples were obtained from 10 cancer volunteers in the Liaocheng People's Hospital (Liaocheng, China). All cancer volunteers gave informed consent. This study was performed in strict accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (WHO/CIOMS, 2002) and was approved by the Liaocheng University Institutional Review Board (IRB).

Acknowledgements

This work was financially supported by the Natural Science Foundation of Shandong Province (ZR2017BB084 and ZR2018BB059) and National Natural Science Foundation of China (21804063, 21505063, 21405070, 21375055 and 21427808).

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Footnote

Electronic supplementary information (ESI) available: The CV, spooling ECL spectroscopy and ECL spectra of PTCDA; ECL spectroscopy of PTCDA and PTCDA–An; the FL excitation spectra of PTCDA; the FL excitation and emission spectra of PTCDA–An in aqueous solution; the EIS of immunosensor fabrication process; comparison of the proposed ECL nanomaterial with other single-emission ECL nanomaterials for CEA detection. See DOI: 10.1039/c9tb02368b

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