Analytica Chimica Acta 819 (2014) 102–107
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Amorphous carbon nanoparticle used as novel resonance energy transfer acceptor for chemiluminescent immunoassay of transferrin Hongfei Gao, Wenwen Wang, Zhenxing Wang, Jing Han, Zhifeng Fu ∗ Key Laboratory of Luminescence and Real-Time Analysis (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
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g r a p h i c a l
• Amorphous
carbon nanoparticles (ACNPs) were prepared with a very simple protocol. • The ACNP was employed as a novel energy acceptor for CRET immunosensing platform. • The ACNPs showed higher energy transfer efficiency than graphene oxide. • The proposed platform showed low cost, simple manipulation and high specificity.
Amorphous carbon nanoparticle was synthesized and used as a novel energy acceptor to develop a CRETbased immunoassay platform for biomolecule (TRF as the model) detection.
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a b s t r a c t
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Article history: Received 7 July 2013 Received in revised form 31 January 2014 Accepted 12 February 2014 Available online 15 February 2014 Keywords: Amorphous carbon nanoparticles Immunoassay Chemiluminescence resonance energy transfer Transferrin
Amorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of chemiluminescence (CL) were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a chemiluminescence resonance energy transfer (CRET)-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP–anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP–TRF for HRP–anti-TRF, thus led to the decreased occurrence of CRET. A linear range of 20–400 ng mL−1 and a limit of detection of 20 ng mL−1 were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Chemiluminescence (CL) resonance energy transfer (CRET) involving a nonradiative dipole–dipole transfer of energy from a donor to an acceptor has attracted increasing attention since its
∗ Corresponding author. Tel.: +86 23 6825 0184; fax: +86 23 6825 1048. E-mail address: [email protected] (Z. Fu). http://dx.doi.org/10.1016/j.aca.2014.02.018 0003-2670/© 2014 Elsevier Ltd. All rights reserved.
a b s t r a c t
concept was proposed by Ren’s group in 2006 [1]. Compared with conventional fluorescence resonance energy transfer (FRET), CRET occurs by the oxidation of a luminescent substrate without an excitation source, which results in a lower background signal and an improved sensitivity for bioassay [2]. Consequently, CRET opens up broad prospects for bioassay applications. Up to now, two main kinds of energy acceptors have been applied to CRET for the purpose of achieving efficient resonance energy transfer. The first kind of energy acceptor is fluorophore
H. Gao et al. / Analytica Chimica Acta 819 (2014) 102–107
material. Some fluorophores such as quantum dot (QD) [3–5] and fluorescein [6–8] can be excited by the CL donors via energy transfer and then eradiate their fluorescence (FL) signals. Monitoring the optical signal at a given wavelength enables quantitative detection of the analyte. Recently, Zhao et al. [9,10] employed CdTe QD as energy acceptor in luminol–NaBrO–QD system. Zhang et al. developed a sensitive CRET-based aptasensor involving donor luminol and acceptor fluorescein [11]. However, these CRET approaches using fluorophores require a sophisticated and expensive apparatus to simultaneously detect the multiple emissions from the donor and acceptor at different wavelength, and sometimes suffer from overlapping of the emission spectra of CL donor and FL acceptor. The second kind of energy acceptor for CRET is non-FL quencher that can highly efficient quench the CL from the donor. Generally, this kind of acceptor has strong light absorption and readily absorbs the energy from the CL donor but does not emit luminescence. This mode only requires a simple CL detector to collect the decreased CL signal for quantification purpose. For instance, owing to their high molar absorptivity and wide range of absorption wavelength that overlap CL spectra of certain CL compounds such as luminol, gold nanoparticles (AuNPs) have been used as luminescence quenchers in CRET [12–15]. More recently, some carbon materials such as graphene [16,17], graphene oxide (GO) [18–21] and carbon nanotubes [22,23] have been proved to be a super-quencher of a CL donor in CRET, because of the nonradiative transfer of electrons from the CL excited states to the system of these materials. For example, Lee et al. [16] used graphene as a super-quencher to construct CRET system for homogeneous immunoassay of C-reactive protein. Bi et al. [18] utilized the excellent quenching efficiency of GO with the long-range nano-scaled energy transfer property to construct a CL sensing platform for biorecognition event detection. An amplified CL turn-on sensing platform using single-walled carbon nanotubes as highly efficient quenching acceptors for ultrasensitive DNA detection was also developed by Huang et al. [22]. From these examples, it has been demonstrated that a simple and inexpensive instrument can be used for highly sensitive detection just by monitoring the change of emission intensity from the CL donor when this kind of quencher was employed as the energy acceptor. Whereas, so far there are merely several above-mentioned nanomaterials applied as efficient energy quenchers. Therefore, further efforts are required to seeking more novel CRET acceptors with highly efficient energy quenching to expand the potential applications of this mode. In this investigation, we synthesized novel amorphous carbon nanoparticles (ACNPs) that are expected to be employed as the new energy quenching acceptor for CRET. The prepared ACNPs show some attractive properties such as ease of synthesis, simplicity of conjugation chemistry, good biocompatibility and excellent water solubility. Moreover, the ACNPs show higher energy transfer efficiency than other nano-scaled acceptor for CRET assay. A competitive immunoassay based on CRET by using ACNPs was constructed to assay transferrin (TRF), an important iron-binding protein involved in anemia and inflammation diseases.
2. Experimental 2.1. Materials and apparatus TRF, HRP labeled mouse monoclonal antibody for TRF, human serum albumin (HSA), human IgA (HIgA), human IgM (HIgM) and human IgG (HIgG) were all purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (China). BSA was obtained from
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Beijing Dingguo Biotechnology Co., Ltd. (China). Sodium chloroacetate (ClCH2 COONa) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Aladdin Chemistry Co., Ltd. (China). N-Hydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) were provided by Sigma–Aldrich Chemical Co., Ltd. (USA). SuperBlock® T20 used as blocking buffer was obtained from Thermo Fisher Scientific Inc. (USA). The ELISA kit for TRF was provided by R&D Systems Inc. (USA). Phosphate buffer saline (PBS) at 0.10 M and pH 7.0 was used as dilution buffer for antigens and antibodies. CL substrate solution for HRP was composed of solution 1 containing 5.0 × 10−5 M luminol (Sigma, USA) and 5.0 × 10−5 M p-iodophenol (PIP) (Aladdin, China) in 0.10 M PBS at pH 8.5, and solution 2 containing 1.0 × 10−2 M H2 O2 (Chengdu Kelong Chemical Reagent Co., Ltd., China). The serum samples were kindly provided by three healthy adult volunteers. All other chemicals were of analytical reagent grade and used without further purification. All solutions were prepared using ultra-pure water (18.2 M) purified by an ELGA PURELAB classic system. The polystyrene 96-well high-affinity microplate was provided by Greiner Bio-One Biochemical Co., Ltd. (Germany). The CL measurements were performed with a MPI-A CL analyzer (Xi’an Remax Electroni Science & Technology Co., Ltd., China) equipped with a photomultiplier operated at −800 V. The ultraviolet visible (UV–vis) absorption spectra were obtained from a UV-2450 UV–vis spectrophotometer (Shimadzu, Japan). The photoluminescence spectrum of luminol was obtained from a F-7000 fluorescence spectrometer (Hitachi, Japan). The FT-IR spectrum was measured by a Bruker IFS66 spectrometer (Bruker, Germany). The morphologies of ACNPs were examined on a F-7500 transmission electron microscope (Hitachi, Japan). X-ray photoelectron spectroscopy (XPS) spectrum was recorded with an ESCALAB-250 X-ray photoelectron spectroscope (Thermo, USA). The X-ray diffraction analysis was conducted by a DMAX-2500 diffractometer using a Cu K␣-1 radiation source (Rigaku, Japan). 2.2. Preparation of ACNPs ACNPs were synthesized by acidolysis of the candle soot. Generally, the crude candle soot was collected by putting a clean glass plate on the top of a smoldering candle. After soaked with acetone overnight and centrifuged to discard the supernatant, the precipitate was dried at 60 ◦ C. Fifty milligrams of the resultant solid was mixed with 15 mL of 5.0 M HNO3 , followed by refluxing at 140 ◦ C for 16 h. After cooled down to room temperature (RT), the solution obtained was centrifuged at 12,000 rpm for 10 min to remove the supernatant. Afterward, black precipitate was washed twice with acetone and dried at 60 ◦ C. The obtained ACNPs were then stored at RT for further use. 2.3. Preparation of ACNP–TRF conjugate For preparation of ACNP–TRF conjugate, firstly, 1.0 mg of ACNPs was dispersed in 1.0 mL of ultra-pure water, followed by addition of 50 mg of NaOH and 50 mg of ClCH2 COONa to transform hydroxyl and carbonyl groups into carboxylic groups. After sonication for 3 h at RT, the resultant mixture was neutralized with 2.0 M HCl aqueous solution and centrifuged at 8000 rpm for 10 min to discard the supernatant. Secondly, TRF was covalently bound to the COOH group of the carboxylated ACNPs to synthesize ACNP–TRF composite. Typically, carboxylated ACNPs were mixed with 0.40 M EDC and 0.10 M NHS in 1.0 mL of MES buffer (pH 5.2) and incubated at RT for 1 h to activate the carboxylate groups. After that, the resulting mixture was washed twice by centrifugation with 0.10 M PBS at pH 7.0 to remove excess EDC and NHS moleculars. The precipitates were then added into 1.0 mL of PBS and sonicated for
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Fig. 1. (A) TEM images of ACNPs. (B) XRD of ACNPs. (C) (a) CL spectrum of luminol and UV–vis absorption spectra of (b) ACNPs, (c) GO and (d) graphene under the same concentration (20 g mL−1 ). (D) FT-IR spectrum of ACNPs.
about 10 min to harvest a homogeneous dispersion. Subsequently, 100 L of 1.0 mg mL−1 TRF was mixed with the resultant mixture followed by stirring overnight at 4 ◦ C. After the reaction, BSA solution (1.0 mg mL−1 ) was added as a blocking agent, and the reaction continued for another 1 h. Finally, the ACNP–TRF was purified and washed by centrifugation thrice at 7000 rpm for 10 min with PBS buffer. The resulted conjugate was re-dispersed in 1.0 mL of PBS at pH 7.0, and stored at 4 ◦ C until use. 2.4. Assay procedure Typically, a polystyrene 96-well microplate was coated with 100 L of 40 ng mL−1 HRP–anti-TRF dispersed in 0.10 M Tris–HCl buffer at pH 8.0 overnight at 4 ◦ C. Subsequently, the plate was washed thrice with 300 L of washing buffer (PBS containing 0.05% Tween 20) to discard the unbound antibodies. And that, the microplate was blocked with 150 L of blocking buffer per well for 1.5 h at 37 ◦ C, followed by washing thrice with washing buffer. The mixture containing 50 L of ACNP–TRF (50 g mL−1 ) and 50 L of TRF at different concentrations was delivered into the well and incubated for 1.5 h at 37 ◦ C for competitive immunoreactions. After thorough washing, 80 L of CL substrate (solution 1) was pipetted into the well, and the peak height of the total emission was collected at 60 s after injection of solution 2.
(b), indicating that they were probably a suitable couple of donor/acceptor and could be applied for CRET system. Meanwhile, FT-IR spectroscopy (Fig. 1D) of the ACNPs showed the absorption peaks at around 3400 cm−1 ( OH) and 1650 cm−1 (C O), suggesting that ACNPs can be used for bioconjugation by covalently binding carboxylated ACNPs with amine groups in biomolecules. XPS was also used to characterize the original synthesized ACNPs. As seen in Fig. S1 of Supplementary Data, the binding energy of the core electrons for the C 1s line at 284.6 eV was attributed to sp2 C C, while the peaks located at the binding energies of 287.2 and 288.8 eV were assigned to C O, O C O, respectively [24]. Therefore, ACNPs containing a plenty of bonds proned to occurrence of electronic transference. We anticipated that the ACNPs can serve as the energy quenchers in CRET assay. In addition, we also evaluated the number of TRF molecules conjugated to one single ACNP with a protocol reported by Sardesai et al. [25]. The number of TRF in the obtained ACNP–TRF conjugate dispersion was evaluated to be 5.0 × 1016 by the fluorescence emission at 340 nm. The viscosity of the ACNP–TRF dispersion was measured to be 1.015 after it was diluted for 10 times, thus the number of ACNPs in the undiluted dispersion was calculated to be 1.8 × 1015 . Therefore, the TRF/ACNP ratio was estimated to be about 28:1. 3.2. Principle of CRET-based immunoassay
3. Results and discussion 3.1. Characterization of ACNPs The TEM images (Fig. 1A) of the ACNPs showed that they were spheres with an average diameter of 40 nm. Amorphous nature of the particles was demonstrated by the results of powder XRD data (Fig. 1B), in which no peak of crystalline origin was found. UV–vis absorption spectra were recorded as shown in Fig. 1C. It was noted that the CL emission spectrum of luminol (a) was largely overlapped by the absorption spectrum of this material
The principle of the designed CRET-based immunoassay was illustrated in Fig. 2. In this protocol, luminol emitter was chosen as the CRET donor and participated in the HRP-catalyzed luminol/H2 O2 CL reaction. The as-synthesized ACNP was adopted as the acceptor to label the TRF antigen (ACNP–TRF). With the absence of ACNP–TRF, the intensity of luminol emission catalyzed by the HRP-labeled immunocomplex reached a maximum value (control value) since CRET did not occur. When only ACNP–TRF participated into the immunoreaction, the donor and the acceptor were brought to close proximity to enable the occurrence
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Fig. 2. Schematic illustrations of (A) the CRET process from the luminol donor to the ACNP acceptor and (B) the interrupted CRET process in a competitive immunoassay.
of CRET. Thus, the CL signal was the weakest (blank value) because of the transfer of the most energy from the donor to the acceptor and thus the strongest quenching of luminol CL. For a competitive immunoassay, TRF at different concentration competed with ACNP–TRF at a fixed amount for HRP–anti-TRF immobilized on the microplate. Therefore the CL intensity increased with the increasing amounts of TRF because of the decreased acceptors coupled with the donors, and thus the decreased occurrence of CRET. The relationship between the quenching of CL intensity and the TRF concentration showed a good linearity, indicating ACNPs can be used to design a CRET-based quantitative immunoassay. To demonstrate the CRET process occurred in the immunoassay, the CL reaction catalyzed by HRP was investigated by adding different amounts of unconjugated ACNPs. As shown in Fig. S6 of Supplementary Data, even though 50 g mL−1 unconjugated ACNPs were added into the CL reaction system, only a very slight decrease of 6.2% was observed. This result demonstrated that the unconjugated ACNPs could not efficiently quench the CL signal when the ACNP was far from the CL emitter. Only after the HRPcatalyzed donor and the acceptor (ACNP) were brought to close proximity by the immunoreaction, the CL signal showed obvious decrease. It was found that the prepared ACNPs absorbed strongly at the CL emission band of HRP-catalyzed luminol system (Fig. 1C). Moreover, the ACNPs show high molar extinction coefficient at 425 nm which was calculated to be 1.5 × 107 L cm−1 M−1 according to Beer’s law. These facts suggest that the quenching of CL signal should be mainly caused by the resonance energy transfer
phenomenon resulting from the immunoreaction [15,16,26]. However, work remains to be done before exact mechanism can be stated. 3.3. The quenching efficiency of ACNPs As shown in Fig. 1C, compared with other carbon nanomaterials such as GO and graphene, which have recently received increasing attention due to its remarkable electron transport and accepting ability [27,28], the ACNPs (b) showed absorption value five and twice times higher than those of GO (c) and graphene (d) at 425 nm, respectively, implying that ACNP may be a more powerful CL quencher. Up to date, GO had been widely reported to act as a super quencher of CL donor and fluorophore in resonance energy transfer, owing to it excellent quenching efficiency [18–21]. The quenching efficiencies of ACNPs and GO were compared in the same CRET protocol. As seen in Fig. 3A, the maximum quenching efficiency was only 33.4% in the GO-based CRET system, while that of ACNPsbased CRET system was 52.8%, demonstrating that ACNP was a more powerful CL quencher. 3.4. Optimization of detection conditions Some condition parameters were compared to obtain optimal performance for CL immunoassay (CLIA). Figs. S2–S5 in Supplementary Data showed the effect of the concentration of HRP–anti-TRF, the volume ratio of probe (ACNP–TRF) to sample (TRF), the blocking
Fig. 3. (A) The relationship between the concentration of TRF and the CL intensity using ACNP–TRF, GO–TRF and BSA-conjugated ACNPs, respectively. (B) Specificity of the proposed CRET-based immunoassay. The concentrations of HSA, HIgA, HIgM, HIgG and TRF were all 100 ng mL−1 .
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Fig. 4. (A) The CL responses of the CRET-based immunoassay for analyzing the target TRF at the concentrations of (a) 0 (blank), (b) 20, (c) 50, (d) 100, (e) 200, (f) 400 ng mL−1 and (g) in the absence of ACNP–TRF and TRF (control). (B) Calibration curve for TRF detection with the concentration of 20, 50, 100, 200 and 400 ng mL−1 . Error bars are standard deviation of five repetitive measurements.
Table 1 Recoveries of TRF spiked in the human serum samples obtained by the proposed CRET-based immunoassay and the ELISA kit. The proposed method (n = 5)a Sample number −1
Initial (ng mL ) Added (ng mL−1 ) Found (ng mL−1 ) RSD (%) Recovery (%) a
1
2 a
302.2 20.0 320.2 6.0 90.0
a
50.0 350.6 7.8 96.8
ELISA (n = 5) 3
258.7 20.0 276.3 4.6 88.0
1 a
50.0 303.5 6.5 89.6
271.3 20.0 293.4 5.1 110.5
2
303.5 × 10 20.0 × 104 322.4 × 104 3.8 94.5 4
50.0 324.1 3.4 105.6
3
261.6 × 10 20.0 × 104 283.6 × 104 7.7 110.0 4
50.0 × 104 354.5 × 104 6.2 102.0
50.0 × 104 306.3 × 104 5.8 89.4
277.4 × 104 20.0 × 104 295.9 × 104 6.5 92.5
50.0 × 104 331.3 × 104 4.3 107.8
Human serum samples were 104 times diluted prior to the recovery test for the proposed method, while no dilution was required for ELISA.
time and the incubation time on the CL responses. After careful investigation, HRP–anti-TRF at 40 ng mL−1 was chosen to coat the microplate. The volume ratio of probe to sample was chosen to be 50 L/50 L for competitive CLIA based on CRET. In addition, 1.5 h of incubation time and blocking time were used in the further study since they provided optimal assay performance. 3.5. Specificity of CRET-based immunoassay To estimate the nonspecific interaction between the donor and the acceptor, a control experiment using BSA-conjugated ACNPs was conducted. As shown in Fig. 3A, following the increasing amount of TRF, the CL signals obtained in the control experiment consisted with the control value and almost kept constant, implying the nonspecific interaction was negligible in this protocol. The specificity of the proposed CRET-based immunoassay was also investigated by comparing the signals from TRF, HSA, human IgA, human IgM and human IgG at 100 ng mL−1 . As presented in Fig. 3B, the CL signals for other protein were all close to the blank value, and much less than that of TRF, indicating that the CRETbased immunoassay approach was not tended to be interfered by these biomolecules. 3.6. Analytical performance and real samples application Under the optimal conditions, a five-point calibration curve of CL intensity versus TRF concentration was achieved in the range of 20–400 ng mL−1 (Fig. 4). The regression equation could be expressed as I (a.u.) = 2.660 C (ng mL−1 ) + 1475.7 (I represents the CL intensity; C represents the concentration of TRF; n = 5) with a correlation coefficient of 0.9972. The limit of detection (LOD) for TRF was 20 ng mL−1 . The results in Table S1 of Supplementary Data demonstrated that the LOD of this method was lower than that of the standard CL ELISA (50 ng mL−1 ), and the reproducibility of this proposed method was also comparable with that of CL ELISA.
In order to further evaluate the reliability and application potential of the developed CRET-based competitive immunoassay, the levels of TRF in three healthy human sera were evaluated. Serum samples were diluted appropriately prior to assay to ensure that the levels of TRF were in the linear ranges. The TRF levels were detected to be 3.0, 2.6 and 2.7 mg mL−1 in the three human sera, which consisted with the clinical reference [29]. Known quantities of TRF standards were spiked into the diluted samples to perform the recovery tests. The results listed in Table 1 show acceptable recoveries of 88.0–110.5%, and the RSDs were all less than 7.84%. Also, the assay results obtained from the proposed method were compared with those from the classic ELISA method. As seen in Table 1, the agreement between the two methods was satisfactory. 4. Conclusions In summary, ACNPs were synthesized and used as the novel energy acceptor to develop a CRET-based immunoassay for biomolecules detection with low cost, simple manipulation and high specificity. In this protocol, the ACNP is adopted to label TRF and act as the acceptor for CRET system. Combined with HRPcatalyzed luminol emitter as the donor, a CRET-based competitive immunoassay is established for TRF detection. The proposed CRET strategy is very simple and does not need an external light excitation source in comparison with the conventional FRET approach. Compared with other CRET approaches using fluorophore emitters as the acceptors, the present protocol does not suffer from spectral overlapping of CL and FL, and can employ a simple light detection device for biomolecules detection. Furthermore, as the energy quencher for CRET, ACNP shows higher energy transfer efficiency than other nano-scaled quenchers such as GO. Thus, the proposed ACNPs hold great promise for very simple and highly efficient resonance energy transfer. We anticipate that this approach can be applied for detection of other biomarkers and provide a promising inspiration in other bioassay designs.
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Acknowledgments This project was financially supported by the Natural Science Foundation of China (21035005 and 21175111), Natural Science Foundation of Chongqing (CSTC2013jjB0096), the Fundamental Research Funds for the Central Universities (XDJK2012A002 and XDJK2013A025), and Program for Innovative Research Team in University of Chongqing (2013). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.02.018. References [1] X.Y. Huang, L. Li, H.F. Qian, C.Q. Dong, J.C. Ren, Angewandte ChemieInternational Edition 45 (2006) 5140–5143. [2] X.Y. Huang, J.C. Ren, Trends in Analytical Chemistry 40 (2012) 77–89. [3] R. Freeman, X.Q. Liu, I. Willner, Journal of the American Chemical Society 133 (2011) 11597–11604. [4] X.Q. Liu, R. Freeman, E. Golub, I. Willner, ACS Nano 5 (2011) 7648– 7655. [5] H.Q. Wang, Y.Q. Li, J.H. Wang, Q. Xu, X.Q. Li, Y.D. Zhao, Analytica Chimica Acta 610 (2008) 68–73. [6] G.X. Qin, S.L. Zhao, Y. Huang, J. Jiang, F.G. Ye, Analytical Chemistry 84 (2012) 2708–2712. [7] S.L. Zhao, J.W. Liu, Y. Huang, Y.M. Liu, Chemical Communications 48 (2012) 699–701.
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Amorphous carbon nanoparticle used as novel resonance energy transfer acceptor for chemiluminescent immunoassay of transferrin Hongfei Gao, Wenwen Wang, Zhenxing Wang, Jing Han, Zhifeng Fu ∗ Key Laboratory of Luminescence and Real-Time Analysis (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
h i g h l i g h t s
g r a p h i c a l
• Amorphous
carbon nanoparticles (ACNPs) were prepared with a very simple protocol. • The ACNP was employed as a novel energy acceptor for CRET immunosensing platform. • The ACNPs showed higher energy transfer efficiency than graphene oxide. • The proposed platform showed low cost, simple manipulation and high specificity.
Amorphous carbon nanoparticle was synthesized and used as a novel energy acceptor to develop a CRETbased immunoassay platform for biomolecule (TRF as the model) detection.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 7 July 2013 Received in revised form 31 January 2014 Accepted 12 February 2014 Available online 15 February 2014 Keywords: Amorphous carbon nanoparticles Immunoassay Chemiluminescence resonance energy transfer Transferrin
Amorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of chemiluminescence (CL) were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a chemiluminescence resonance energy transfer (CRET)-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP–anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP–TRF for HRP–anti-TRF, thus led to the decreased occurrence of CRET. A linear range of 20–400 ng mL−1 and a limit of detection of 20 ng mL−1 were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Chemiluminescence (CL) resonance energy transfer (CRET) involving a nonradiative dipole–dipole transfer of energy from a donor to an acceptor has attracted increasing attention since its
∗ Corresponding author. Tel.: +86 23 6825 0184; fax: +86 23 6825 1048. E-mail address: [email protected] (Z. Fu). http://dx.doi.org/10.1016/j.aca.2014.02.018 0003-2670/© 2014 Elsevier Ltd. All rights reserved.
a b s t r a c t
concept was proposed by Ren’s group in 2006 [1]. Compared with conventional fluorescence resonance energy transfer (FRET), CRET occurs by the oxidation of a luminescent substrate without an excitation source, which results in a lower background signal and an improved sensitivity for bioassay [2]. Consequently, CRET opens up broad prospects for bioassay applications. Up to now, two main kinds of energy acceptors have been applied to CRET for the purpose of achieving efficient resonance energy transfer. The first kind of energy acceptor is fluorophore
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material. Some fluorophores such as quantum dot (QD) [3–5] and fluorescein [6–8] can be excited by the CL donors via energy transfer and then eradiate their fluorescence (FL) signals. Monitoring the optical signal at a given wavelength enables quantitative detection of the analyte. Recently, Zhao et al. [9,10] employed CdTe QD as energy acceptor in luminol–NaBrO–QD system. Zhang et al. developed a sensitive CRET-based aptasensor involving donor luminol and acceptor fluorescein [11]. However, these CRET approaches using fluorophores require a sophisticated and expensive apparatus to simultaneously detect the multiple emissions from the donor and acceptor at different wavelength, and sometimes suffer from overlapping of the emission spectra of CL donor and FL acceptor. The second kind of energy acceptor for CRET is non-FL quencher that can highly efficient quench the CL from the donor. Generally, this kind of acceptor has strong light absorption and readily absorbs the energy from the CL donor but does not emit luminescence. This mode only requires a simple CL detector to collect the decreased CL signal for quantification purpose. For instance, owing to their high molar absorptivity and wide range of absorption wavelength that overlap CL spectra of certain CL compounds such as luminol, gold nanoparticles (AuNPs) have been used as luminescence quenchers in CRET [12–15]. More recently, some carbon materials such as graphene [16,17], graphene oxide (GO) [18–21] and carbon nanotubes [22,23] have been proved to be a super-quencher of a CL donor in CRET, because of the nonradiative transfer of electrons from the CL excited states to the system of these materials. For example, Lee et al. [16] used graphene as a super-quencher to construct CRET system for homogeneous immunoassay of C-reactive protein. Bi et al. [18] utilized the excellent quenching efficiency of GO with the long-range nano-scaled energy transfer property to construct a CL sensing platform for biorecognition event detection. An amplified CL turn-on sensing platform using single-walled carbon nanotubes as highly efficient quenching acceptors for ultrasensitive DNA detection was also developed by Huang et al. [22]. From these examples, it has been demonstrated that a simple and inexpensive instrument can be used for highly sensitive detection just by monitoring the change of emission intensity from the CL donor when this kind of quencher was employed as the energy acceptor. Whereas, so far there are merely several above-mentioned nanomaterials applied as efficient energy quenchers. Therefore, further efforts are required to seeking more novel CRET acceptors with highly efficient energy quenching to expand the potential applications of this mode. In this investigation, we synthesized novel amorphous carbon nanoparticles (ACNPs) that are expected to be employed as the new energy quenching acceptor for CRET. The prepared ACNPs show some attractive properties such as ease of synthesis, simplicity of conjugation chemistry, good biocompatibility and excellent water solubility. Moreover, the ACNPs show higher energy transfer efficiency than other nano-scaled acceptor for CRET assay. A competitive immunoassay based on CRET by using ACNPs was constructed to assay transferrin (TRF), an important iron-binding protein involved in anemia and inflammation diseases.
2. Experimental 2.1. Materials and apparatus TRF, HRP labeled mouse monoclonal antibody for TRF, human serum albumin (HSA), human IgA (HIgA), human IgM (HIgM) and human IgG (HIgG) were all purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (China). BSA was obtained from
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Beijing Dingguo Biotechnology Co., Ltd. (China). Sodium chloroacetate (ClCH2 COONa) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Aladdin Chemistry Co., Ltd. (China). N-Hydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) were provided by Sigma–Aldrich Chemical Co., Ltd. (USA). SuperBlock® T20 used as blocking buffer was obtained from Thermo Fisher Scientific Inc. (USA). The ELISA kit for TRF was provided by R&D Systems Inc. (USA). Phosphate buffer saline (PBS) at 0.10 M and pH 7.0 was used as dilution buffer for antigens and antibodies. CL substrate solution for HRP was composed of solution 1 containing 5.0 × 10−5 M luminol (Sigma, USA) and 5.0 × 10−5 M p-iodophenol (PIP) (Aladdin, China) in 0.10 M PBS at pH 8.5, and solution 2 containing 1.0 × 10−2 M H2 O2 (Chengdu Kelong Chemical Reagent Co., Ltd., China). The serum samples were kindly provided by three healthy adult volunteers. All other chemicals were of analytical reagent grade and used without further purification. All solutions were prepared using ultra-pure water (18.2 M) purified by an ELGA PURELAB classic system. The polystyrene 96-well high-affinity microplate was provided by Greiner Bio-One Biochemical Co., Ltd. (Germany). The CL measurements were performed with a MPI-A CL analyzer (Xi’an Remax Electroni Science & Technology Co., Ltd., China) equipped with a photomultiplier operated at −800 V. The ultraviolet visible (UV–vis) absorption spectra were obtained from a UV-2450 UV–vis spectrophotometer (Shimadzu, Japan). The photoluminescence spectrum of luminol was obtained from a F-7000 fluorescence spectrometer (Hitachi, Japan). The FT-IR spectrum was measured by a Bruker IFS66 spectrometer (Bruker, Germany). The morphologies of ACNPs were examined on a F-7500 transmission electron microscope (Hitachi, Japan). X-ray photoelectron spectroscopy (XPS) spectrum was recorded with an ESCALAB-250 X-ray photoelectron spectroscope (Thermo, USA). The X-ray diffraction analysis was conducted by a DMAX-2500 diffractometer using a Cu K␣-1 radiation source (Rigaku, Japan). 2.2. Preparation of ACNPs ACNPs were synthesized by acidolysis of the candle soot. Generally, the crude candle soot was collected by putting a clean glass plate on the top of a smoldering candle. After soaked with acetone overnight and centrifuged to discard the supernatant, the precipitate was dried at 60 ◦ C. Fifty milligrams of the resultant solid was mixed with 15 mL of 5.0 M HNO3 , followed by refluxing at 140 ◦ C for 16 h. After cooled down to room temperature (RT), the solution obtained was centrifuged at 12,000 rpm for 10 min to remove the supernatant. Afterward, black precipitate was washed twice with acetone and dried at 60 ◦ C. The obtained ACNPs were then stored at RT for further use. 2.3. Preparation of ACNP–TRF conjugate For preparation of ACNP–TRF conjugate, firstly, 1.0 mg of ACNPs was dispersed in 1.0 mL of ultra-pure water, followed by addition of 50 mg of NaOH and 50 mg of ClCH2 COONa to transform hydroxyl and carbonyl groups into carboxylic groups. After sonication for 3 h at RT, the resultant mixture was neutralized with 2.0 M HCl aqueous solution and centrifuged at 8000 rpm for 10 min to discard the supernatant. Secondly, TRF was covalently bound to the COOH group of the carboxylated ACNPs to synthesize ACNP–TRF composite. Typically, carboxylated ACNPs were mixed with 0.40 M EDC and 0.10 M NHS in 1.0 mL of MES buffer (pH 5.2) and incubated at RT for 1 h to activate the carboxylate groups. After that, the resulting mixture was washed twice by centrifugation with 0.10 M PBS at pH 7.0 to remove excess EDC and NHS moleculars. The precipitates were then added into 1.0 mL of PBS and sonicated for
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Fig. 1. (A) TEM images of ACNPs. (B) XRD of ACNPs. (C) (a) CL spectrum of luminol and UV–vis absorption spectra of (b) ACNPs, (c) GO and (d) graphene under the same concentration (20 g mL−1 ). (D) FT-IR spectrum of ACNPs.
about 10 min to harvest a homogeneous dispersion. Subsequently, 100 L of 1.0 mg mL−1 TRF was mixed with the resultant mixture followed by stirring overnight at 4 ◦ C. After the reaction, BSA solution (1.0 mg mL−1 ) was added as a blocking agent, and the reaction continued for another 1 h. Finally, the ACNP–TRF was purified and washed by centrifugation thrice at 7000 rpm for 10 min with PBS buffer. The resulted conjugate was re-dispersed in 1.0 mL of PBS at pH 7.0, and stored at 4 ◦ C until use. 2.4. Assay procedure Typically, a polystyrene 96-well microplate was coated with 100 L of 40 ng mL−1 HRP–anti-TRF dispersed in 0.10 M Tris–HCl buffer at pH 8.0 overnight at 4 ◦ C. Subsequently, the plate was washed thrice with 300 L of washing buffer (PBS containing 0.05% Tween 20) to discard the unbound antibodies. And that, the microplate was blocked with 150 L of blocking buffer per well for 1.5 h at 37 ◦ C, followed by washing thrice with washing buffer. The mixture containing 50 L of ACNP–TRF (50 g mL−1 ) and 50 L of TRF at different concentrations was delivered into the well and incubated for 1.5 h at 37 ◦ C for competitive immunoreactions. After thorough washing, 80 L of CL substrate (solution 1) was pipetted into the well, and the peak height of the total emission was collected at 60 s after injection of solution 2.
(b), indicating that they were probably a suitable couple of donor/acceptor and could be applied for CRET system. Meanwhile, FT-IR spectroscopy (Fig. 1D) of the ACNPs showed the absorption peaks at around 3400 cm−1 ( OH) and 1650 cm−1 (C O), suggesting that ACNPs can be used for bioconjugation by covalently binding carboxylated ACNPs with amine groups in biomolecules. XPS was also used to characterize the original synthesized ACNPs. As seen in Fig. S1 of Supplementary Data, the binding energy of the core electrons for the C 1s line at 284.6 eV was attributed to sp2 C C, while the peaks located at the binding energies of 287.2 and 288.8 eV were assigned to C O, O C O, respectively [24]. Therefore, ACNPs containing a plenty of bonds proned to occurrence of electronic transference. We anticipated that the ACNPs can serve as the energy quenchers in CRET assay. In addition, we also evaluated the number of TRF molecules conjugated to one single ACNP with a protocol reported by Sardesai et al. [25]. The number of TRF in the obtained ACNP–TRF conjugate dispersion was evaluated to be 5.0 × 1016 by the fluorescence emission at 340 nm. The viscosity of the ACNP–TRF dispersion was measured to be 1.015 after it was diluted for 10 times, thus the number of ACNPs in the undiluted dispersion was calculated to be 1.8 × 1015 . Therefore, the TRF/ACNP ratio was estimated to be about 28:1. 3.2. Principle of CRET-based immunoassay
3. Results and discussion 3.1. Characterization of ACNPs The TEM images (Fig. 1A) of the ACNPs showed that they were spheres with an average diameter of 40 nm. Amorphous nature of the particles was demonstrated by the results of powder XRD data (Fig. 1B), in which no peak of crystalline origin was found. UV–vis absorption spectra were recorded as shown in Fig. 1C. It was noted that the CL emission spectrum of luminol (a) was largely overlapped by the absorption spectrum of this material
The principle of the designed CRET-based immunoassay was illustrated in Fig. 2. In this protocol, luminol emitter was chosen as the CRET donor and participated in the HRP-catalyzed luminol/H2 O2 CL reaction. The as-synthesized ACNP was adopted as the acceptor to label the TRF antigen (ACNP–TRF). With the absence of ACNP–TRF, the intensity of luminol emission catalyzed by the HRP-labeled immunocomplex reached a maximum value (control value) since CRET did not occur. When only ACNP–TRF participated into the immunoreaction, the donor and the acceptor were brought to close proximity to enable the occurrence
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Fig. 2. Schematic illustrations of (A) the CRET process from the luminol donor to the ACNP acceptor and (B) the interrupted CRET process in a competitive immunoassay.
of CRET. Thus, the CL signal was the weakest (blank value) because of the transfer of the most energy from the donor to the acceptor and thus the strongest quenching of luminol CL. For a competitive immunoassay, TRF at different concentration competed with ACNP–TRF at a fixed amount for HRP–anti-TRF immobilized on the microplate. Therefore the CL intensity increased with the increasing amounts of TRF because of the decreased acceptors coupled with the donors, and thus the decreased occurrence of CRET. The relationship between the quenching of CL intensity and the TRF concentration showed a good linearity, indicating ACNPs can be used to design a CRET-based quantitative immunoassay. To demonstrate the CRET process occurred in the immunoassay, the CL reaction catalyzed by HRP was investigated by adding different amounts of unconjugated ACNPs. As shown in Fig. S6 of Supplementary Data, even though 50 g mL−1 unconjugated ACNPs were added into the CL reaction system, only a very slight decrease of 6.2% was observed. This result demonstrated that the unconjugated ACNPs could not efficiently quench the CL signal when the ACNP was far from the CL emitter. Only after the HRPcatalyzed donor and the acceptor (ACNP) were brought to close proximity by the immunoreaction, the CL signal showed obvious decrease. It was found that the prepared ACNPs absorbed strongly at the CL emission band of HRP-catalyzed luminol system (Fig. 1C). Moreover, the ACNPs show high molar extinction coefficient at 425 nm which was calculated to be 1.5 × 107 L cm−1 M−1 according to Beer’s law. These facts suggest that the quenching of CL signal should be mainly caused by the resonance energy transfer
phenomenon resulting from the immunoreaction [15,16,26]. However, work remains to be done before exact mechanism can be stated. 3.3. The quenching efficiency of ACNPs As shown in Fig. 1C, compared with other carbon nanomaterials such as GO and graphene, which have recently received increasing attention due to its remarkable electron transport and accepting ability [27,28], the ACNPs (b) showed absorption value five and twice times higher than those of GO (c) and graphene (d) at 425 nm, respectively, implying that ACNP may be a more powerful CL quencher. Up to date, GO had been widely reported to act as a super quencher of CL donor and fluorophore in resonance energy transfer, owing to it excellent quenching efficiency [18–21]. The quenching efficiencies of ACNPs and GO were compared in the same CRET protocol. As seen in Fig. 3A, the maximum quenching efficiency was only 33.4% in the GO-based CRET system, while that of ACNPsbased CRET system was 52.8%, demonstrating that ACNP was a more powerful CL quencher. 3.4. Optimization of detection conditions Some condition parameters were compared to obtain optimal performance for CL immunoassay (CLIA). Figs. S2–S5 in Supplementary Data showed the effect of the concentration of HRP–anti-TRF, the volume ratio of probe (ACNP–TRF) to sample (TRF), the blocking
Fig. 3. (A) The relationship between the concentration of TRF and the CL intensity using ACNP–TRF, GO–TRF and BSA-conjugated ACNPs, respectively. (B) Specificity of the proposed CRET-based immunoassay. The concentrations of HSA, HIgA, HIgM, HIgG and TRF were all 100 ng mL−1 .
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Fig. 4. (A) The CL responses of the CRET-based immunoassay for analyzing the target TRF at the concentrations of (a) 0 (blank), (b) 20, (c) 50, (d) 100, (e) 200, (f) 400 ng mL−1 and (g) in the absence of ACNP–TRF and TRF (control). (B) Calibration curve for TRF detection with the concentration of 20, 50, 100, 200 and 400 ng mL−1 . Error bars are standard deviation of five repetitive measurements.
Table 1 Recoveries of TRF spiked in the human serum samples obtained by the proposed CRET-based immunoassay and the ELISA kit. The proposed method (n = 5)a Sample number −1
Initial (ng mL ) Added (ng mL−1 ) Found (ng mL−1 ) RSD (%) Recovery (%) a
1
2 a
302.2 20.0 320.2 6.0 90.0
a
50.0 350.6 7.8 96.8
ELISA (n = 5) 3
258.7 20.0 276.3 4.6 88.0
1 a
50.0 303.5 6.5 89.6
271.3 20.0 293.4 5.1 110.5
2
303.5 × 10 20.0 × 104 322.4 × 104 3.8 94.5 4
50.0 324.1 3.4 105.6
3
261.6 × 10 20.0 × 104 283.6 × 104 7.7 110.0 4
50.0 × 104 354.5 × 104 6.2 102.0
50.0 × 104 306.3 × 104 5.8 89.4
277.4 × 104 20.0 × 104 295.9 × 104 6.5 92.5
50.0 × 104 331.3 × 104 4.3 107.8
Human serum samples were 104 times diluted prior to the recovery test for the proposed method, while no dilution was required for ELISA.
time and the incubation time on the CL responses. After careful investigation, HRP–anti-TRF at 40 ng mL−1 was chosen to coat the microplate. The volume ratio of probe to sample was chosen to be 50 L/50 L for competitive CLIA based on CRET. In addition, 1.5 h of incubation time and blocking time were used in the further study since they provided optimal assay performance. 3.5. Specificity of CRET-based immunoassay To estimate the nonspecific interaction between the donor and the acceptor, a control experiment using BSA-conjugated ACNPs was conducted. As shown in Fig. 3A, following the increasing amount of TRF, the CL signals obtained in the control experiment consisted with the control value and almost kept constant, implying the nonspecific interaction was negligible in this protocol. The specificity of the proposed CRET-based immunoassay was also investigated by comparing the signals from TRF, HSA, human IgA, human IgM and human IgG at 100 ng mL−1 . As presented in Fig. 3B, the CL signals for other protein were all close to the blank value, and much less than that of TRF, indicating that the CRETbased immunoassay approach was not tended to be interfered by these biomolecules. 3.6. Analytical performance and real samples application Under the optimal conditions, a five-point calibration curve of CL intensity versus TRF concentration was achieved in the range of 20–400 ng mL−1 (Fig. 4). The regression equation could be expressed as I (a.u.) = 2.660 C (ng mL−1 ) + 1475.7 (I represents the CL intensity; C represents the concentration of TRF; n = 5) with a correlation coefficient of 0.9972. The limit of detection (LOD) for TRF was 20 ng mL−1 . The results in Table S1 of Supplementary Data demonstrated that the LOD of this method was lower than that of the standard CL ELISA (50 ng mL−1 ), and the reproducibility of this proposed method was also comparable with that of CL ELISA.
In order to further evaluate the reliability and application potential of the developed CRET-based competitive immunoassay, the levels of TRF in three healthy human sera were evaluated. Serum samples were diluted appropriately prior to assay to ensure that the levels of TRF were in the linear ranges. The TRF levels were detected to be 3.0, 2.6 and 2.7 mg mL−1 in the three human sera, which consisted with the clinical reference [29]. Known quantities of TRF standards were spiked into the diluted samples to perform the recovery tests. The results listed in Table 1 show acceptable recoveries of 88.0–110.5%, and the RSDs were all less than 7.84%. Also, the assay results obtained from the proposed method were compared with those from the classic ELISA method. As seen in Table 1, the agreement between the two methods was satisfactory. 4. Conclusions In summary, ACNPs were synthesized and used as the novel energy acceptor to develop a CRET-based immunoassay for biomolecules detection with low cost, simple manipulation and high specificity. In this protocol, the ACNP is adopted to label TRF and act as the acceptor for CRET system. Combined with HRPcatalyzed luminol emitter as the donor, a CRET-based competitive immunoassay is established for TRF detection. The proposed CRET strategy is very simple and does not need an external light excitation source in comparison with the conventional FRET approach. Compared with other CRET approaches using fluorophore emitters as the acceptors, the present protocol does not suffer from spectral overlapping of CL and FL, and can employ a simple light detection device for biomolecules detection. Furthermore, as the energy quencher for CRET, ACNP shows higher energy transfer efficiency than other nano-scaled quenchers such as GO. Thus, the proposed ACNPs hold great promise for very simple and highly efficient resonance energy transfer. We anticipate that this approach can be applied for detection of other biomarkers and provide a promising inspiration in other bioassay designs.
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Acknowledgments This project was financially supported by the Natural Science Foundation of China (21035005 and 21175111), Natural Science Foundation of Chongqing (CSTC2013jjB0096), the Fundamental Research Funds for the Central Universities (XDJK2012A002 and XDJK2013A025), and Program for Innovative Research Team in University of Chongqing (2013). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.02.018. References [1] X.Y. Huang, L. Li, H.F. Qian, C.Q. Dong, J.C. Ren, Angewandte ChemieInternational Edition 45 (2006) 5140–5143. [2] X.Y. Huang, J.C. Ren, Trends in Analytical Chemistry 40 (2012) 77–89. [3] R. Freeman, X.Q. Liu, I. Willner, Journal of the American Chemical Society 133 (2011) 11597–11604. [4] X.Q. Liu, R. Freeman, E. Golub, I. Willner, ACS Nano 5 (2011) 7648– 7655. [5] H.Q. Wang, Y.Q. Li, J.H. Wang, Q. Xu, X.Q. Li, Y.D. Zhao, Analytica Chimica Acta 610 (2008) 68–73. [6] G.X. Qin, S.L. Zhao, Y. Huang, J. Jiang, F.G. Ye, Analytical Chemistry 84 (2012) 2708–2712. [7] S.L. Zhao, J.W. Liu, Y. Huang, Y.M. Liu, Chemical Communications 48 (2012) 699–701.
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