Anal Bioanal Chem DOI 10.1007/s00216-013-7611-9
RESEARCH PAPER
Chemiluminescent detection of cell apoptosis enzyme by gold nanoparticle-based resonance energy transfer assay Xiangyi Huang & Yiran Liang & Lingao Ruan & Jicun Ren
Received: 3 October 2013 / Revised: 27 December 2013 / Accepted: 30 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract We report a new chemiluminescence resonance energy transfer (CRET) technique, using gold nanoparticles (AuNPs) as efficient energy acceptor, for homogeneous measurement of cell apoptosis enzyme with high sensitivity. In the design of the CRET system, we chose the highly sensitive chemiluminescence (CL) reaction between luminol and hydrogen peroxide catalysed by horseradish peroxidase (HRP) because the CL spectrum of luminol (λmax 425 nm) partially overlaps the visible absorption bands of AuNPs. In this system, the peptide substrate (DEVD) of caspase 3 was linked to the AuNP surface by Au–S linkage. HRP was attached to the AuNP surface by means of a bridge formed by the streptavidin–biotin reaction. CRET occurred as a result of formation of AuNP–peptide–biotin–streptavidin–HRP complexes. The CL of luminol was significantly reduced, because of the quenching effect of AuNPs. The quenched CL was recovered after cleavage of DEVD by caspase 3, an enzyme involved in the apoptotic process. Experimental conditions were systematically investigated. Under the optimum conditions the increase of the CL signal was linearly dependent on caspase 3 concentration within the concentration range 25 pmol L−1 to 800 pmol L−1 and the detection limit of caspase 3 was as low as 20 pmol L−1, one order of magnitude lower than for FRET sensors based on graphene oxides. Our method was successfully used to detect drug-induced apoptosis of cells. This approach is expected to be extended to other assays, i.e., using other enzymes,
Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli. Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7611-9) contains supplementary material, which is available to authorized users. X. Huang : Y. Liang : L. Ruan : J. Ren (*) College of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China e-mail: [email protected]
analytes, CL substances, and even other nanoparticles (e.g., quantum dots and graphene). Keywords Gold nanoparticles . Chemiluminescence resonance energy transfer . Luminol . Peptide . Caspase 3
Introduction Apoptosis is an evolutionarily conserved and highly regulated process that results in cell death [1]. Deregulation of cell apoptosis can ultimately lead to cancers, autoimmune diseases, neurodegenerative diseases, and many other diseases. The process of apoptosis is controlled by a wide variety of intracellular and extracellular factors. Among apoptotic proteases, caspase 3 has been identified as a key mediator of apoptosis of mammalian cells. Therefore, highly sensitive and specific detection of caspase 3 activation is important. It is well known that caspase 3 specifically cleaves the Nterminus of the tetrapeptide Asp–Glu–Val–Asp (DEVD), and this tetrapeptide group of DEVD has been used to develop caspase-specific probes used in many methods for analysis of caspase 3 activity or apoptosis imaging at the single-cell level [2], including fluorescence resonance energy transfer (FRET)based fluorimetry [3–7], colorimetry [8], electrochemistry [9], chemiluminescence assay [10], bioluminescence detection [11], and atomic force microscopy [12]. Among these methods, FRET has been widely applied, because this homogeneous assay can be used to measure the dynamics of caspase 3 activation and for real-time monitoring of cellular apoptosis. Recently, nanoparticle-mediated sensors for FRET have also been developed [13–17]. Energy transfer-based multiplexed assay of proteases has been developed by using gold nanoparticle and quantum dot conjugates on a surface [14]. Boeneman et al. reported quantum dotsbased FRET for monitoring caspase 3 proteolysis [15]. An intracellular protease sensor had been demonstrated
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for caspase 3 activation imaging in live cells by using FRET and graphene oxide as energy acceptor [17]. In addition, AuNPs are very attractive acceptors because of their highly molar extinction coefficient and their ability to serve as biocompatible structures for intracellular targeting. The AuNPs-based FRET system was composed of a core particle surrounded by AuNP satellites linked via a specific peptide with the caspase 3 recognition sequence (DEVD); this enabled continuously imaging of the progress of caspase 3 activity in live cells [18]. Chemiluminescence resonance energy transfer (CRET) involves nonradiative (dipole–dipole) transfer of energy from a chemiluminescent donor to a suitable acceptor. In contrast with FRET, CRET occurs by oxidation of a luminescent substrate without an excitation source. Thus, the signal-tonoise ratio and sensitivity of the detection can be improved, owing to the absence of sample autofluorescence. Recently such nanoparticles as quantum dots and graphene have been successfully used as acceptors in CRET for homogeneous assay [19–26]. AuNPs should be an efficient energy acceptor in CRET, similar to FRET, because of their extremely high molar absorption coefficient and absorption spectra covering whole visible spectrum range, which overlap the CL spectra of some CL compounds, for example luminol. However, there are only a few reports of use of AuNPs as energy acceptors in CRET-based techniques [27–30]. Our group developed a new CRET technique by using AuNPs as efficient long-range energy acceptors in sandwich immunoassays [27]. Qin et al. reported AuNPs sensing based on CRET for detection of biomolecules and achieved a detection limit of 15 pmol L−1 for thrombin [29]. Waud et al. measured caspase 3 by using CRET between green fluorescent protein and aequorin [10]. Although many methods have been developed for detection of caspase 3, to the best of our knowledge, no method has been reported for detection of caspase 3 by nanoparticle-based CRET. In this study we built a chemiluminescent detection system for homogeneous measurement of cellular apoptosis enzyme activity by coupling of AuNPs-based resonance energy transfer. In this system, a 12-mer peptide containing the caspase 3 recognition sequence (DEVD) was linked to the AuNP surface mainly by Au–S linkage [31]. HRP was attached to AuNP surface by means of a bridge formed by the streptavidin–biotin reaction. The CRET occurred by formation of AuNP– peptide–biotin–streptavidin–HRP complexes. Because the sensitivity of the proposed CRET-based method is an order of magnitude higher than those of similar graphene oxide based FRET sensors [17], it has excellent potential for use in biomolecule recognition and protein detection.
Materials and methods Materials Caspase 3 was from R&D Systems (USA). Luminol and streptavidin–horseradish peroxidase (streptavidin–HRP) were from Sigma–Aldrich Chemical (Milwaukee, USA). The 12mer peptide (CALNNDEVDGK(biotin)G), designed as reported elsewhere [31], was purchased from GL Biochem (Shanghai, China). Hydrogen tetrachloroaurate(III) hydrate (HAuCl4) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Bovine serum albumin (BSA) and glucose oxidase (GOX) were from BBI (UK). Uricase was obtained from Worthington (USA). Low-density lipoprotein from human plasma (LDL) was purchased from Invitrogen (USA). Lidamycin (LDM) was a product of Shanghai Laiyi Center for Biopharmaceutical R&D (China). Cell lysis buffer P0013 was from Beyotime Institute of Biotechnology (Nantong, China). Ultra-pure water (18.2 MΩ) was obtained from a Millipore (Bedford, MA, USA) Simplicity System. All materials were of analytical grade and used without further purification. Preparation of gold nanoparticles AuNPs were synthesized by reduction of the HAuCl4 solution with sodium citrate in accordance with the procedure described in Ref. [32]. The concentration of AuNPs was calculated by use of the method described in Ref. [33]. Absorption spectra were collected by use of a UV/Vis-3501 spectrophotometer, and the sizes of AuNPs were determined by use of a JEM-2100HR transmission electron microscope (TEM; Jeol, Japan). Conjugation of peptides and gold nanoparticles The 12-mer peptide (CALNNDEVDGK(biotin)G) used in this study was designed as reported elsewhere [31]. The thiol group in the side chain of the N-terminal cysteine (C) is able to bond covalently to the gold surface. Briefly, 12-mer peptidecapped gold nanoparticles were prepared by mixing citrate AuNPs and peptide stock solution in 1:200 mole ratio and the resulting mixture was left to react for overnight at 4 °C. The AuNP–peptide conjugates were purified by centrifugation at 5,000 rpm for 30 min to remove excess peptide. Control conjugation reactions performed to confirm whether labelling was successful included incubation with no peptide and replacing the peptide with 5 mg mL−1 BSA. Resonance light-scattering correlation spectroscopic (RLSCS) analysis The principle of RLSCS is similar to that of fluorescence correlation spectroscopy (FCS), and is based on the fact that
Chemiluminescence detection of cell apoptosis enzyme
the resonance light-scattering intensity of AuNPs in a very small illuminated volume (less than 1 fL) can be correlated to obtain information the processes that cause fluctuations of the resonance light-scattering (RLS) intensity of the nanoparticles [34]. Similar to FCS, RLSCS measures the intensity of scattered light and the characteristic diffusion time of such metal nanoparticles as AuNPs, in addition to other information obtained by use of the autocorrelation function. In this study, RLSCS was used to characterize AuNPs and AuNP–peptide conjugates. RLSCS measurements were performed at room temperature on a laboratory-built RLSCS system. Details of the experimental equipment can be found elsewhere [34]. Cell culture and drug-induced apoptosis PANC-1 cells were maintained in vitro in DMEM highglucose medium (Gibco, CA, USA) supplemented with 10 % (v/v) foetal bovine serum (FBS) (Gibco). Cells were incubated at 37 °C in a humidified incubator with 5 % CO2. To induce apoptosis, cells were incubated for the desired time (20 h) with 0.5 μg mL−1, 5.0 μg mL−1, or 50 μg mL−1 LDM [35]. Cells not treated with LDM were used as control group. Cell lysis Approximately 3×106 PANC-1 cells were collected, and the medium was removed. Cell lysis buffer (200 μL; 20 mmol L−1 Tris buffer containing 150 mmol L−1 NaCl and 1 % Triton X-100, pH 7.5) was added to the collected cells, and the mixture was incubated at 4 °C for 10 min. The solution was then centrifuged at 12,000 rpm for 5 min and the precipitate was discarded. Finally, the cell lysate was used for subsequent experiments. CRET-based analysis of caspase 3 CRET-based analysis of caspase 3 was conducted as follows. Typically, 1,980 μL peptide-conjugated AuNPs suspension and 20 μL streptavidin–HRP were mixed and incubated for 30 min at 25 °C. AuNP-S-HRP conjugate solution (180 μL), prepared as described above, was placed in a series of 1-mL Eppendorf tubes containing 20 μL of a solution of caspase 3 standard and different concentrations of cell lysate. The mixed solutions were then incubated for 30 min at 37 °C. Subsequently, 200 μL 10 mmol L−1 HEPES solution (pH 7.5) containing 2.0×10−4 mol L−1 luminol was added. The resulting mixture was poured into a 1 cm path-length quartz cell. Finally 200 μL 50 mmol L−1 sodium hydrogen carbonate solution (pH 9.90) containing 2.5 × 10−3 mol L−1 H2O2 and 4 × 10−5 mol L−1 para-iodophenol was added to the quartz cuvette. In this work the luminol–hydrogen peroxide CL reaction catalysed by streptavidin–HRP was adopted, and paraiodophenol was acted as CL enhancer. When CL reactions
were initiated after addition of CL reagents, CL spectra were recorded immediately with an F-380 spectrometer (Tianjin Gangdong SCI and Technical Development, China).
Results and discussion Principle and design of CRET We chose the luminol–hydrogen peroxide CL reaction catalysed by HRP, with para-iodophenol as enhancer, because this is one of the most sensitive CL reactions. More importantly, AuNPs have good absorption properties in the visible region [33], which makes AuNPs a suitable energy acceptor and/or quencher in CRET-based assays. Figure S1 (Electronic Supplementary Material) shows the CL spectrum of luminol (λmax 425–435 nm) and the UV–vis absorption spectra of AuNPs suspension (λmax 519.0 nm, 526.3 nm, and 529.7 nm). Although the maximum emission peak of luminol does not overlap the maximum absorption peak of the AuNPs, the AuNPs still have strong quenching properties because of their large molar extinction coefficients near 425 nm [27]. Thus CRET is possible between the streptavidin–HRPcatalysed luminol (donor) and biotin-peptides–conjugated AuNPs (acceptor). The principle of CRET is illustrated in Scheme 1. AuNPs were linked with peptide–biotin (AuNP–peptide–biotin) by Au–S linkage, and HRP was conjugated with streptavidin. When the two solutions were mixed, HRP-labelled streptavidin interacted with the AuNP–peptide-conjugated biotin and the AuNP–peptide–biotin–streptavidin–HRP complexes were formed. In the absence of caspase 3, when CL reagents were added, formation of the AuNP–peptide–biotin– streptavidin–HRP complex brought the CL molecules and the AuNPs into close proximity. Consequently, CRET occurred and the CL signal was quenched by AuNPs. In the presence of caspase 3, the complex was cleaved because of the specific reaction between caspase 3 and DEVD peptides. In this case, CL emission of luminol was increased because of cleavage of the DEVD substrate. The CL increase (i.e., the overall enzyme cleavage response) therefore depended on the number of dissociative streptavidin–HRP complexes formed. Characterization of AuNPs and AuNP–peptide conjugates In this work, three different sizes of AuNPs were synthesized for further experiments. Comparison of AuNPs of different sizes enabled us to investigate experimentally the effect on quenching efficiency of acceptors of different diameters. Figure S2 (Electronic Supplementary Material) shows TEM images of the AuNPs. It can be seen that the as-prepared AuNPs are spherical in shape, with diameters of 18±3 nm, 27±5 nm, and 36±8 nm. After modification with the peptides,
X. Huang et al. Scheme 1 Schematic illustration of AuNP-based resonance energy transfer for CL detection of caspase 3
the maximum absorption peak red-shifted from 519.0 nm to 522.5 nm for 18-nm AuNPs, from 526.3 nm to 528.2 nm for 27-nm AuNPs, and from 529.7 nm to 532.0 nm for 36-nm AuNPs (Fig. S3, Electronic Supplementary Material), which was indicative of formation of bioconjugates and was in accordance with results published elsewhere [31]. It also implied the peptides conjugated to AuNPs and formed complexes, because the peptide–AuNP complexes were stable in HEPES buffer whereas AuNPs were unstable and readily aggregated in HEPES buffer. The complexes were because the thiol group in the side chain of the 12-mer peptide’s Nterminal cysteine (C) is able to bond covalently to the gold surface. This reaction may be in addition to that of the Nterminal primary amine, because amino groups are also known to interact strongly with gold surfaces [36]. Moreover, Gordillo and co-workers observed that the presence of a positively charged ammonium group in the vicinity of the thiol significantly accelerated the kinetics of adsorption of thiols on to citrate-stabilized gold nanoparticles [37]. RLSCS analysis was used to further characterize AuNPs and AuNP–peptide conjugates. Figure 1 shows RLSCS curves of AuNPs and AuNP–peptide conjugates. The characteristic diffusion time of free AuNPs (λ(27)max 526.3 nm) before reaction with the peptide was 1.27 ms. When the AuNPs reacted with the peptides by Au–S bonding, the characteristic diffusion time of the mixture increased to 1.73 ms. The clear increase of the diffusion time shown by the RLSCS curves also suggests the peptides were conjugated to AuNPs and formed complexes.
caspase 3 (results not shown). CL reaction buffer containing 50 mmol L−1 sodium hydrogen carbonate solution (pH 9.90, containing 2.5×10−3 mol L−1 H2O2 and 4.0×10−5 mol L−1 para-iodophenol) was used in this study, and 4.0 × 10−4 mol L−1 luminol was used by adding in HEPES buffer (10 mmol L−1, pH 7.5). Control experiments with BSA-AuNPs (without conjugation of peptides) were performed to test whether caspase 3 affects the luminol CL. As presented in Fig. S4 (Electronic Supplementary Material), the CL intensity of luminol in the control experiment remained almost unchanged with increasing amount of caspase 3, indicating no increasing or quenching effect of caspase 3 on this CL reaction. We established that the specific reaction between caspase 3 and DEVD was almost complete within 30 min (Fig. S5, Electronic Supplementary Material), so incubation for 30 min was used in this study.
CRET and analysis of caspase The pH of the CL reaction buffer and the concentrations of luminol, H2O2, and para-iodophenol were studied systematically to establish the optimum conditions for CL detection of
Fig. 1 Resonance light-scattering correlation spectroscopy curves obtained from 27-nm AuNPs (a) and AuNP–peptide conjugates (b). Concentrations of AuNP and AuNP–peptide were both 2.0×10−10 mol L−1
Chemiluminescence detection of cell apoptosis enzyme
The number of peptides attached to one AuNP affects the stability of the assembly and the quenching efficiency. Therefore, the peptide to AuNP ratio was optimized. Figure S6 (Electronic Supplementary Material) shows CL spectra obtained by use of different peptide-to-AuNP ratios. The CL signal increased when ratio of peptides to AuNPs was increased from 40 to 2,000, indicating that the lower the peptide-to-AuNP ratio, the higher the quenching efficiency and the larger the CL increase, which resulted in a wider detection range and greater sensitivity. However, the AuNP– peptide conjugates were instable in the buffer when a low peptide-to-AuNP ratio was used. Taking into consideration both the stability of AuNP–peptide conjugates and quenching efficiency, a peptide-to-AuNP ratio of 200 was used in subsequent experiments. The CRET sensor formed depends on the concentrations of streptavidin–HRP and AuNP–peptide conjugates. Thus, reducing the concentration of streptavidin–HRP may lead to a more sensitive assay. In practice, the minimum concentration of streptavidin–HRP may be limited by the photo detectors. Up to a specific value, increasing the concentration of the streptavidin–HRP conjugates leads to broadening of the dynamic range of the assay. Therefore, the concentration of streptavidin–HRP was studied systematically to determine the optimum conditions for the CRET detection of caspase 3. Figure 2 shows the relationship between the concentration of caspase 3 and the relative CL intensity (I/I0) for different concentrations of streptavidin–HRP. I0 was the CL intensity without caspase 3 and I was the CL intensity with caspase 3. The higher relative CL intensity means more peptides cleaved and more dissociative streptavidin–HRP was formed. The
Fig. 2 Relationship between the concentration of caspase 3 and relative CL intensity (I/I0) for different concentrations of streptavidin–HRP. The concentration of streptavidin–HRP was 2.5 nmol L−1 (a), 1.25 nmol L−1 (b), and 5.0 nmol L−1 (c). The concentration of the 18-nm AuNP–peptide conjugate was 2.0 nmol L−1. The concentrations of luminol, H2O2, and para-iodophenol were 2.0×10−4 mol L−1, 2.5×10−3 mol L−1, and 4.0× 10−5 mol L−1, respectively
relative CL intensity for 1.0 nmol L−1 caspase 3 increased from 2.40 to 2.64 when the concentration of streptavidin– HRP was increased from 1.25 nmol L−1 to 2.5 nmol L−1, then decreased from 2.64 to 2.13 when the concentration of streptavidin–HRP was increased from 2.5 nmol L−1 to 5.0 nmol L −1 . This might be because the number of streptavidin–HRP complexes coupling to a single AuNP increases with increasing streptavidin–HRP concentration, leading to lower quenching efficiency. However, the effects of self-absorbance might be stronger when the concentration of streptavidin–HRP is lower. The relative CL intensity was the highest when the concentration of streptavidin–HRP was 2.5 nmol L−1 because of relatively high quenching efficiency and lower self-absorbance. Hence this concentration was used in subsequent experiments. Similarly, the concentration of the AuNP–peptide conjugates must be optimized for CRET detection of caspase 3. Figure 3 shows the relationship between the concentration of caspase 3 and relative CL intensity (I/I0) for different concentrations of AuNP–peptide conjugates. The relative CL intensity for 0.75 nmol L−1 caspase 3 increased from 1.46 to 2.34 when the concentration of AuNP–peptide conjugates was increased from 1.0 nmol L−1 to 2.0 nmol L−1, then decreased from 2.34 to 1.92 when the concentration was increased from 2.0 nmol L−1 to 4.0 nmol L−1. This might be because the number of streptavidin–HRP complexes coupling to a single AuNP decreased with increasing concentration of AuNP– peptide conjugates. Although self-absorbance occurred for higher concentrations of AuNP–peptide conjugates, relative CL intensity decreased. Relative CL intensity was highest when the concentration of AuNP–peptide conjugates was 2.0 nmol L −1 . This may be because the number of streptavidin–HRP complexes coupling to a single AuNP is
Fig. 3 Relationship between the concentration of caspase 3 and relative CL intensity for different concentrations of AuNP–peptide conjugates. The concentration of 18-nm AuNP–peptide conjugates was 2.0 nmol L−1 (a), 4.0 nmol L−1 (b), 1.0 nmol L−1 (c). The concentration of streptavidin– HRP was 2.5 nmol L−1. Other conditions as for Fig. 2
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appropriate and self-absorbance by the AuNPs is reduced. This concentration was therefore used in subsequent experiments. It has been found that distance-dependent quenching efficiency in FRET is highly dependent on nanoparticle size [38]. The effect of acceptors of different diameter on quenching efficiency was investigated experimentally. As shown in Fig. 4, the relative CL intensity for 0.75 nmol L−1 caspase 3 was 2.34, 3.20, and 3.27 for 18-nm, 27-nm, and 36-nm AuNP– peptide conjugates, respectively. This variation of quenching efficiency may be mainly because the extinction coefficient at 425 nm of 36-nm AuNPs (4.9×109 cm−1 mol−1 L) is larger than those of 27-nm and 18-nm AuNPs (2.1×109 cm−1 mol−1 L and 6.5×108 cm−1 mol−1 L, respectively), so 36-nm AuNPs have stronger quenching ability. On the basis of the principle of CRET-based sensing, the amount of caspase 3 was detected by using the CRET system consisting of luminol (donor), and peptide-AuNP (acceptor). As shown in Fig. 5, the CL intensity increased gradually with increasing amount of caspase 3. The CL increase was related to the concentration of caspase 3. When more caspase 3 was added, more streptavidin–HRP would be cleaved from the AuNP–peptide–biotin–streptavidin–HRP complexes, thus achieving spacing suitable for prevention of CRET, which would, in turn, lead to an increase of CL intensity (Scheme 1). As shown in Fig. 5, a linear relationship between caspase 3 concentration (X) and the CL intensity (Y) was obtained in the range 25 pmol L−1 to 800 pmol L−1 for caspase 3. The linear regression equation was Y=1,100.3+210.4 X, and the correlation coefficient (R) was 0.994. The detection limit for caspase 3 was 20 pmol L−1, which is one order of magnitude lower than those of FRET sensors based on graphene oxides [17].
Fig. 4 Relationship between the concentration of caspase 3 and relative CL intensity for different sizes of AuNPs. The sizes of AuNPs were 36 nm (a), 27 nm (b), and 18 nm (c), respectively. The concentration of streptavidin–HRP was 2.5 nmol L −1 . The concentrations were 2.0 nmol L−1, 0.6 nmol L−1, and 0.25 nmol L−1 for 18 nm, 27 nm, and 36 nm AuNP–peptide conjugates, respectively. Other conditions as for Fig. 2
Fig. 5 CL spectra obtained by use of the proposed CRET system in the presence of caspase 3 at different concentrations. The inset shows the linear relationship between CL intensity and caspase 3 concentration. The concentration of the 36 nm AuNP–peptide conjugate was 0.25 nmol L−1. The concentration of streptavidin–HRP was 2.5 nmol L−1. Other conditions as for Fig. 2
The relative standard deviation (RSD) of this method was 3.0 % (obtained by analysis of seven standard solutions each containing 0.10 nmol L−1 of caspase 3). These results demonstrated that the CRET system consisting of biofunctionalized AuNPs and HRP–streptavidin is a promising approach for highly sensitive detection of proteins. Figure S7 (Electronic Supplementary Material) shows the specificity of the proposed CRET system containing peptide– AuNP conjugates and HRP–streptavidin. The relative CL intensity remained almost unchanged in the presence of BSA, HSA, GOX, IgG, LDL, thrombin, uricase, and LDM, indicating the probe did not respond to these proteins and LDM at the concentration tested. The concentrations of most of these proteins (50 nmol L−1) were approximately 50 times
Fig. 6 Relative CL intensity for cell lysate from normal cells (without LDM, control sample) and from apoptotic cells induced with different concentrations of LDM. The concentration of streptavidin–HRP was 2.5 nmol L−1. Other conditions as for Fig. 2
Chemiluminescence detection of cell apoptosis enzyme
that of caspase 3 (1 nmol L−1). The good selectivity is attributed to the high specificity of caspase 3–DEVD, including the peptide reaction used. These results suggested that the proposed CRET system could be used for highly sensitive detection of caspase 3 with high specificity. CRET assay of drug-induced apoptosis In this study, LDM, as model drug, was used to induce apoptosis of human pancreatic cancer cells. LDM is an acid protein containing 110 amino acid residues and a chromophore with an enediyne structure [39]. It has significant anticancer activity because of its ability to damage DNA by radical-mediated hydrogen abstraction [40]. LDM also has marked cytotoxicity against cancers in vitro and in vivo [41]. This method was used for discrimination of normal cells from LDM-induced apoptotic cells, on the basis of their different apoptotic protease, especially caspase 3, content. The method used for induction of apoptosis by LDM was described in the Experimental section. Levels of apoptosis detected by use of this CRET assay were similar to those detected by other methods, for example fluorescence correlation spectroscopy [35]. Drug levels were similar to those usually used in cell-based experiments [17]. A strategy for CRET assay of apoptosis is shown in Scheme 1. Briefly, LDM-induced apoptotic and normal cell lysates were diluted tenfold with buffer. Their CL signals were then detected by use of this method. As shown in Fig. 6, compared with that for normal cell lysate (control group), relative CL intensity was 1.46, 2.64, and 2.78 for lysates of cells treated with 0.5 μg mL−1, 5.0 μg mL−1, and 50 μg mL−1 LDM, respectively. Even in the presence of 0.5 μg mL−1 LDM, the relative CL intensity for lysate from cells treated with the drug was clearly different from that for control cells. Although cell lysate is very complicated, AuNP-based CRET successfully enabled detection of the apoptotic status of cells and assay of caspase 3 in apoptotic cell lysate. To further validate the reproducibility of the CRET method, we repeated the experiments by conducting several independent assays on different days; the RSDs of relative CL intensity were approximately 3.8 % to 9.8 %. These results indicate our method enables reliable assay of drug-induced apoptosis.
Conclusion In conclusion, we have developed a highly sensitive method for analysis of cell apoptosis enzyme by using AuNP-based CRET. The CRET system was the bridge formed by the biotin–streptavidin reaction between AuNP–peptide–biotin and streptavidin–HRP; this brought the CL molecules and the AuNPs into close proximity and led to the occurrence of CRET and quenching of the CL signal by the AuNPs. The
presence of caspase 3 in the system resulted in termination of CRET and the recovery of the CL signal, which enabled specific quantitative monitoring of the caspase 3 by use of the CRET system. Recovery of the CL signal may be explained by the specific reaction between caspase 3 and DEVD-containing peptides, and the formation of more dissociative streptavidin–HRP. Our method was successfully used to detect drug-induced apoptosis of cells. Advantages of this sensing approach include the pairing of CL emission with a highly efficient acceptor, facile attachment to AuNPs, high sensitivity and specificity, small amounts of enzyme and substrate, and the possibility of in-vitro assays. Moreover, this approach is expected to be extended to other assays, that is, use of other enzyme, analytes, CL substances, and even other nanoparticles (e.g., quantum dots and graphene). Acknowledgments This work was supported financially by the National Science Foundation of China (20705019, 21135004, and 21327004).
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RESEARCH PAPER
Chemiluminescent detection of cell apoptosis enzyme by gold nanoparticle-based resonance energy transfer assay Xiangyi Huang & Yiran Liang & Lingao Ruan & Jicun Ren
Received: 3 October 2013 / Revised: 27 December 2013 / Accepted: 30 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract We report a new chemiluminescence resonance energy transfer (CRET) technique, using gold nanoparticles (AuNPs) as efficient energy acceptor, for homogeneous measurement of cell apoptosis enzyme with high sensitivity. In the design of the CRET system, we chose the highly sensitive chemiluminescence (CL) reaction between luminol and hydrogen peroxide catalysed by horseradish peroxidase (HRP) because the CL spectrum of luminol (λmax 425 nm) partially overlaps the visible absorption bands of AuNPs. In this system, the peptide substrate (DEVD) of caspase 3 was linked to the AuNP surface by Au–S linkage. HRP was attached to the AuNP surface by means of a bridge formed by the streptavidin–biotin reaction. CRET occurred as a result of formation of AuNP–peptide–biotin–streptavidin–HRP complexes. The CL of luminol was significantly reduced, because of the quenching effect of AuNPs. The quenched CL was recovered after cleavage of DEVD by caspase 3, an enzyme involved in the apoptotic process. Experimental conditions were systematically investigated. Under the optimum conditions the increase of the CL signal was linearly dependent on caspase 3 concentration within the concentration range 25 pmol L−1 to 800 pmol L−1 and the detection limit of caspase 3 was as low as 20 pmol L−1, one order of magnitude lower than for FRET sensors based on graphene oxides. Our method was successfully used to detect drug-induced apoptosis of cells. This approach is expected to be extended to other assays, i.e., using other enzymes,
Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli. Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7611-9) contains supplementary material, which is available to authorized users. X. Huang : Y. Liang : L. Ruan : J. Ren (*) College of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China e-mail: [email protected]
analytes, CL substances, and even other nanoparticles (e.g., quantum dots and graphene). Keywords Gold nanoparticles . Chemiluminescence resonance energy transfer . Luminol . Peptide . Caspase 3
Introduction Apoptosis is an evolutionarily conserved and highly regulated process that results in cell death [1]. Deregulation of cell apoptosis can ultimately lead to cancers, autoimmune diseases, neurodegenerative diseases, and many other diseases. The process of apoptosis is controlled by a wide variety of intracellular and extracellular factors. Among apoptotic proteases, caspase 3 has been identified as a key mediator of apoptosis of mammalian cells. Therefore, highly sensitive and specific detection of caspase 3 activation is important. It is well known that caspase 3 specifically cleaves the Nterminus of the tetrapeptide Asp–Glu–Val–Asp (DEVD), and this tetrapeptide group of DEVD has been used to develop caspase-specific probes used in many methods for analysis of caspase 3 activity or apoptosis imaging at the single-cell level [2], including fluorescence resonance energy transfer (FRET)based fluorimetry [3–7], colorimetry [8], electrochemistry [9], chemiluminescence assay [10], bioluminescence detection [11], and atomic force microscopy [12]. Among these methods, FRET has been widely applied, because this homogeneous assay can be used to measure the dynamics of caspase 3 activation and for real-time monitoring of cellular apoptosis. Recently, nanoparticle-mediated sensors for FRET have also been developed [13–17]. Energy transfer-based multiplexed assay of proteases has been developed by using gold nanoparticle and quantum dot conjugates on a surface [14]. Boeneman et al. reported quantum dotsbased FRET for monitoring caspase 3 proteolysis [15]. An intracellular protease sensor had been demonstrated
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for caspase 3 activation imaging in live cells by using FRET and graphene oxide as energy acceptor [17]. In addition, AuNPs are very attractive acceptors because of their highly molar extinction coefficient and their ability to serve as biocompatible structures for intracellular targeting. The AuNPs-based FRET system was composed of a core particle surrounded by AuNP satellites linked via a specific peptide with the caspase 3 recognition sequence (DEVD); this enabled continuously imaging of the progress of caspase 3 activity in live cells [18]. Chemiluminescence resonance energy transfer (CRET) involves nonradiative (dipole–dipole) transfer of energy from a chemiluminescent donor to a suitable acceptor. In contrast with FRET, CRET occurs by oxidation of a luminescent substrate without an excitation source. Thus, the signal-tonoise ratio and sensitivity of the detection can be improved, owing to the absence of sample autofluorescence. Recently such nanoparticles as quantum dots and graphene have been successfully used as acceptors in CRET for homogeneous assay [19–26]. AuNPs should be an efficient energy acceptor in CRET, similar to FRET, because of their extremely high molar absorption coefficient and absorption spectra covering whole visible spectrum range, which overlap the CL spectra of some CL compounds, for example luminol. However, there are only a few reports of use of AuNPs as energy acceptors in CRET-based techniques [27–30]. Our group developed a new CRET technique by using AuNPs as efficient long-range energy acceptors in sandwich immunoassays [27]. Qin et al. reported AuNPs sensing based on CRET for detection of biomolecules and achieved a detection limit of 15 pmol L−1 for thrombin [29]. Waud et al. measured caspase 3 by using CRET between green fluorescent protein and aequorin [10]. Although many methods have been developed for detection of caspase 3, to the best of our knowledge, no method has been reported for detection of caspase 3 by nanoparticle-based CRET. In this study we built a chemiluminescent detection system for homogeneous measurement of cellular apoptosis enzyme activity by coupling of AuNPs-based resonance energy transfer. In this system, a 12-mer peptide containing the caspase 3 recognition sequence (DEVD) was linked to the AuNP surface mainly by Au–S linkage [31]. HRP was attached to AuNP surface by means of a bridge formed by the streptavidin–biotin reaction. The CRET occurred by formation of AuNP– peptide–biotin–streptavidin–HRP complexes. Because the sensitivity of the proposed CRET-based method is an order of magnitude higher than those of similar graphene oxide based FRET sensors [17], it has excellent potential for use in biomolecule recognition and protein detection.
Materials and methods Materials Caspase 3 was from R&D Systems (USA). Luminol and streptavidin–horseradish peroxidase (streptavidin–HRP) were from Sigma–Aldrich Chemical (Milwaukee, USA). The 12mer peptide (CALNNDEVDGK(biotin)G), designed as reported elsewhere [31], was purchased from GL Biochem (Shanghai, China). Hydrogen tetrachloroaurate(III) hydrate (HAuCl4) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Bovine serum albumin (BSA) and glucose oxidase (GOX) were from BBI (UK). Uricase was obtained from Worthington (USA). Low-density lipoprotein from human plasma (LDL) was purchased from Invitrogen (USA). Lidamycin (LDM) was a product of Shanghai Laiyi Center for Biopharmaceutical R&D (China). Cell lysis buffer P0013 was from Beyotime Institute of Biotechnology (Nantong, China). Ultra-pure water (18.2 MΩ) was obtained from a Millipore (Bedford, MA, USA) Simplicity System. All materials were of analytical grade and used without further purification. Preparation of gold nanoparticles AuNPs were synthesized by reduction of the HAuCl4 solution with sodium citrate in accordance with the procedure described in Ref. [32]. The concentration of AuNPs was calculated by use of the method described in Ref. [33]. Absorption spectra were collected by use of a UV/Vis-3501 spectrophotometer, and the sizes of AuNPs were determined by use of a JEM-2100HR transmission electron microscope (TEM; Jeol, Japan). Conjugation of peptides and gold nanoparticles The 12-mer peptide (CALNNDEVDGK(biotin)G) used in this study was designed as reported elsewhere [31]. The thiol group in the side chain of the N-terminal cysteine (C) is able to bond covalently to the gold surface. Briefly, 12-mer peptidecapped gold nanoparticles were prepared by mixing citrate AuNPs and peptide stock solution in 1:200 mole ratio and the resulting mixture was left to react for overnight at 4 °C. The AuNP–peptide conjugates were purified by centrifugation at 5,000 rpm for 30 min to remove excess peptide. Control conjugation reactions performed to confirm whether labelling was successful included incubation with no peptide and replacing the peptide with 5 mg mL−1 BSA. Resonance light-scattering correlation spectroscopic (RLSCS) analysis The principle of RLSCS is similar to that of fluorescence correlation spectroscopy (FCS), and is based on the fact that
Chemiluminescence detection of cell apoptosis enzyme
the resonance light-scattering intensity of AuNPs in a very small illuminated volume (less than 1 fL) can be correlated to obtain information the processes that cause fluctuations of the resonance light-scattering (RLS) intensity of the nanoparticles [34]. Similar to FCS, RLSCS measures the intensity of scattered light and the characteristic diffusion time of such metal nanoparticles as AuNPs, in addition to other information obtained by use of the autocorrelation function. In this study, RLSCS was used to characterize AuNPs and AuNP–peptide conjugates. RLSCS measurements were performed at room temperature on a laboratory-built RLSCS system. Details of the experimental equipment can be found elsewhere [34]. Cell culture and drug-induced apoptosis PANC-1 cells were maintained in vitro in DMEM highglucose medium (Gibco, CA, USA) supplemented with 10 % (v/v) foetal bovine serum (FBS) (Gibco). Cells were incubated at 37 °C in a humidified incubator with 5 % CO2. To induce apoptosis, cells were incubated for the desired time (20 h) with 0.5 μg mL−1, 5.0 μg mL−1, or 50 μg mL−1 LDM [35]. Cells not treated with LDM were used as control group. Cell lysis Approximately 3×106 PANC-1 cells were collected, and the medium was removed. Cell lysis buffer (200 μL; 20 mmol L−1 Tris buffer containing 150 mmol L−1 NaCl and 1 % Triton X-100, pH 7.5) was added to the collected cells, and the mixture was incubated at 4 °C for 10 min. The solution was then centrifuged at 12,000 rpm for 5 min and the precipitate was discarded. Finally, the cell lysate was used for subsequent experiments. CRET-based analysis of caspase 3 CRET-based analysis of caspase 3 was conducted as follows. Typically, 1,980 μL peptide-conjugated AuNPs suspension and 20 μL streptavidin–HRP were mixed and incubated for 30 min at 25 °C. AuNP-S-HRP conjugate solution (180 μL), prepared as described above, was placed in a series of 1-mL Eppendorf tubes containing 20 μL of a solution of caspase 3 standard and different concentrations of cell lysate. The mixed solutions were then incubated for 30 min at 37 °C. Subsequently, 200 μL 10 mmol L−1 HEPES solution (pH 7.5) containing 2.0×10−4 mol L−1 luminol was added. The resulting mixture was poured into a 1 cm path-length quartz cell. Finally 200 μL 50 mmol L−1 sodium hydrogen carbonate solution (pH 9.90) containing 2.5 × 10−3 mol L−1 H2O2 and 4 × 10−5 mol L−1 para-iodophenol was added to the quartz cuvette. In this work the luminol–hydrogen peroxide CL reaction catalysed by streptavidin–HRP was adopted, and paraiodophenol was acted as CL enhancer. When CL reactions
were initiated after addition of CL reagents, CL spectra were recorded immediately with an F-380 spectrometer (Tianjin Gangdong SCI and Technical Development, China).
Results and discussion Principle and design of CRET We chose the luminol–hydrogen peroxide CL reaction catalysed by HRP, with para-iodophenol as enhancer, because this is one of the most sensitive CL reactions. More importantly, AuNPs have good absorption properties in the visible region [33], which makes AuNPs a suitable energy acceptor and/or quencher in CRET-based assays. Figure S1 (Electronic Supplementary Material) shows the CL spectrum of luminol (λmax 425–435 nm) and the UV–vis absorption spectra of AuNPs suspension (λmax 519.0 nm, 526.3 nm, and 529.7 nm). Although the maximum emission peak of luminol does not overlap the maximum absorption peak of the AuNPs, the AuNPs still have strong quenching properties because of their large molar extinction coefficients near 425 nm [27]. Thus CRET is possible between the streptavidin–HRPcatalysed luminol (donor) and biotin-peptides–conjugated AuNPs (acceptor). The principle of CRET is illustrated in Scheme 1. AuNPs were linked with peptide–biotin (AuNP–peptide–biotin) by Au–S linkage, and HRP was conjugated with streptavidin. When the two solutions were mixed, HRP-labelled streptavidin interacted with the AuNP–peptide-conjugated biotin and the AuNP–peptide–biotin–streptavidin–HRP complexes were formed. In the absence of caspase 3, when CL reagents were added, formation of the AuNP–peptide–biotin– streptavidin–HRP complex brought the CL molecules and the AuNPs into close proximity. Consequently, CRET occurred and the CL signal was quenched by AuNPs. In the presence of caspase 3, the complex was cleaved because of the specific reaction between caspase 3 and DEVD peptides. In this case, CL emission of luminol was increased because of cleavage of the DEVD substrate. The CL increase (i.e., the overall enzyme cleavage response) therefore depended on the number of dissociative streptavidin–HRP complexes formed. Characterization of AuNPs and AuNP–peptide conjugates In this work, three different sizes of AuNPs were synthesized for further experiments. Comparison of AuNPs of different sizes enabled us to investigate experimentally the effect on quenching efficiency of acceptors of different diameters. Figure S2 (Electronic Supplementary Material) shows TEM images of the AuNPs. It can be seen that the as-prepared AuNPs are spherical in shape, with diameters of 18±3 nm, 27±5 nm, and 36±8 nm. After modification with the peptides,
X. Huang et al. Scheme 1 Schematic illustration of AuNP-based resonance energy transfer for CL detection of caspase 3
the maximum absorption peak red-shifted from 519.0 nm to 522.5 nm for 18-nm AuNPs, from 526.3 nm to 528.2 nm for 27-nm AuNPs, and from 529.7 nm to 532.0 nm for 36-nm AuNPs (Fig. S3, Electronic Supplementary Material), which was indicative of formation of bioconjugates and was in accordance with results published elsewhere [31]. It also implied the peptides conjugated to AuNPs and formed complexes, because the peptide–AuNP complexes were stable in HEPES buffer whereas AuNPs were unstable and readily aggregated in HEPES buffer. The complexes were because the thiol group in the side chain of the 12-mer peptide’s Nterminal cysteine (C) is able to bond covalently to the gold surface. This reaction may be in addition to that of the Nterminal primary amine, because amino groups are also known to interact strongly with gold surfaces [36]. Moreover, Gordillo and co-workers observed that the presence of a positively charged ammonium group in the vicinity of the thiol significantly accelerated the kinetics of adsorption of thiols on to citrate-stabilized gold nanoparticles [37]. RLSCS analysis was used to further characterize AuNPs and AuNP–peptide conjugates. Figure 1 shows RLSCS curves of AuNPs and AuNP–peptide conjugates. The characteristic diffusion time of free AuNPs (λ(27)max 526.3 nm) before reaction with the peptide was 1.27 ms. When the AuNPs reacted with the peptides by Au–S bonding, the characteristic diffusion time of the mixture increased to 1.73 ms. The clear increase of the diffusion time shown by the RLSCS curves also suggests the peptides were conjugated to AuNPs and formed complexes.
caspase 3 (results not shown). CL reaction buffer containing 50 mmol L−1 sodium hydrogen carbonate solution (pH 9.90, containing 2.5×10−3 mol L−1 H2O2 and 4.0×10−5 mol L−1 para-iodophenol) was used in this study, and 4.0 × 10−4 mol L−1 luminol was used by adding in HEPES buffer (10 mmol L−1, pH 7.5). Control experiments with BSA-AuNPs (without conjugation of peptides) were performed to test whether caspase 3 affects the luminol CL. As presented in Fig. S4 (Electronic Supplementary Material), the CL intensity of luminol in the control experiment remained almost unchanged with increasing amount of caspase 3, indicating no increasing or quenching effect of caspase 3 on this CL reaction. We established that the specific reaction between caspase 3 and DEVD was almost complete within 30 min (Fig. S5, Electronic Supplementary Material), so incubation for 30 min was used in this study.
CRET and analysis of caspase The pH of the CL reaction buffer and the concentrations of luminol, H2O2, and para-iodophenol were studied systematically to establish the optimum conditions for CL detection of
Fig. 1 Resonance light-scattering correlation spectroscopy curves obtained from 27-nm AuNPs (a) and AuNP–peptide conjugates (b). Concentrations of AuNP and AuNP–peptide were both 2.0×10−10 mol L−1
Chemiluminescence detection of cell apoptosis enzyme
The number of peptides attached to one AuNP affects the stability of the assembly and the quenching efficiency. Therefore, the peptide to AuNP ratio was optimized. Figure S6 (Electronic Supplementary Material) shows CL spectra obtained by use of different peptide-to-AuNP ratios. The CL signal increased when ratio of peptides to AuNPs was increased from 40 to 2,000, indicating that the lower the peptide-to-AuNP ratio, the higher the quenching efficiency and the larger the CL increase, which resulted in a wider detection range and greater sensitivity. However, the AuNP– peptide conjugates were instable in the buffer when a low peptide-to-AuNP ratio was used. Taking into consideration both the stability of AuNP–peptide conjugates and quenching efficiency, a peptide-to-AuNP ratio of 200 was used in subsequent experiments. The CRET sensor formed depends on the concentrations of streptavidin–HRP and AuNP–peptide conjugates. Thus, reducing the concentration of streptavidin–HRP may lead to a more sensitive assay. In practice, the minimum concentration of streptavidin–HRP may be limited by the photo detectors. Up to a specific value, increasing the concentration of the streptavidin–HRP conjugates leads to broadening of the dynamic range of the assay. Therefore, the concentration of streptavidin–HRP was studied systematically to determine the optimum conditions for the CRET detection of caspase 3. Figure 2 shows the relationship between the concentration of caspase 3 and the relative CL intensity (I/I0) for different concentrations of streptavidin–HRP. I0 was the CL intensity without caspase 3 and I was the CL intensity with caspase 3. The higher relative CL intensity means more peptides cleaved and more dissociative streptavidin–HRP was formed. The
Fig. 2 Relationship between the concentration of caspase 3 and relative CL intensity (I/I0) for different concentrations of streptavidin–HRP. The concentration of streptavidin–HRP was 2.5 nmol L−1 (a), 1.25 nmol L−1 (b), and 5.0 nmol L−1 (c). The concentration of the 18-nm AuNP–peptide conjugate was 2.0 nmol L−1. The concentrations of luminol, H2O2, and para-iodophenol were 2.0×10−4 mol L−1, 2.5×10−3 mol L−1, and 4.0× 10−5 mol L−1, respectively
relative CL intensity for 1.0 nmol L−1 caspase 3 increased from 2.40 to 2.64 when the concentration of streptavidin– HRP was increased from 1.25 nmol L−1 to 2.5 nmol L−1, then decreased from 2.64 to 2.13 when the concentration of streptavidin–HRP was increased from 2.5 nmol L−1 to 5.0 nmol L −1 . This might be because the number of streptavidin–HRP complexes coupling to a single AuNP increases with increasing streptavidin–HRP concentration, leading to lower quenching efficiency. However, the effects of self-absorbance might be stronger when the concentration of streptavidin–HRP is lower. The relative CL intensity was the highest when the concentration of streptavidin–HRP was 2.5 nmol L−1 because of relatively high quenching efficiency and lower self-absorbance. Hence this concentration was used in subsequent experiments. Similarly, the concentration of the AuNP–peptide conjugates must be optimized for CRET detection of caspase 3. Figure 3 shows the relationship between the concentration of caspase 3 and relative CL intensity (I/I0) for different concentrations of AuNP–peptide conjugates. The relative CL intensity for 0.75 nmol L−1 caspase 3 increased from 1.46 to 2.34 when the concentration of AuNP–peptide conjugates was increased from 1.0 nmol L−1 to 2.0 nmol L−1, then decreased from 2.34 to 1.92 when the concentration was increased from 2.0 nmol L−1 to 4.0 nmol L−1. This might be because the number of streptavidin–HRP complexes coupling to a single AuNP decreased with increasing concentration of AuNP– peptide conjugates. Although self-absorbance occurred for higher concentrations of AuNP–peptide conjugates, relative CL intensity decreased. Relative CL intensity was highest when the concentration of AuNP–peptide conjugates was 2.0 nmol L −1 . This may be because the number of streptavidin–HRP complexes coupling to a single AuNP is
Fig. 3 Relationship between the concentration of caspase 3 and relative CL intensity for different concentrations of AuNP–peptide conjugates. The concentration of 18-nm AuNP–peptide conjugates was 2.0 nmol L−1 (a), 4.0 nmol L−1 (b), 1.0 nmol L−1 (c). The concentration of streptavidin– HRP was 2.5 nmol L−1. Other conditions as for Fig. 2
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appropriate and self-absorbance by the AuNPs is reduced. This concentration was therefore used in subsequent experiments. It has been found that distance-dependent quenching efficiency in FRET is highly dependent on nanoparticle size [38]. The effect of acceptors of different diameter on quenching efficiency was investigated experimentally. As shown in Fig. 4, the relative CL intensity for 0.75 nmol L−1 caspase 3 was 2.34, 3.20, and 3.27 for 18-nm, 27-nm, and 36-nm AuNP– peptide conjugates, respectively. This variation of quenching efficiency may be mainly because the extinction coefficient at 425 nm of 36-nm AuNPs (4.9×109 cm−1 mol−1 L) is larger than those of 27-nm and 18-nm AuNPs (2.1×109 cm−1 mol−1 L and 6.5×108 cm−1 mol−1 L, respectively), so 36-nm AuNPs have stronger quenching ability. On the basis of the principle of CRET-based sensing, the amount of caspase 3 was detected by using the CRET system consisting of luminol (donor), and peptide-AuNP (acceptor). As shown in Fig. 5, the CL intensity increased gradually with increasing amount of caspase 3. The CL increase was related to the concentration of caspase 3. When more caspase 3 was added, more streptavidin–HRP would be cleaved from the AuNP–peptide–biotin–streptavidin–HRP complexes, thus achieving spacing suitable for prevention of CRET, which would, in turn, lead to an increase of CL intensity (Scheme 1). As shown in Fig. 5, a linear relationship between caspase 3 concentration (X) and the CL intensity (Y) was obtained in the range 25 pmol L−1 to 800 pmol L−1 for caspase 3. The linear regression equation was Y=1,100.3+210.4 X, and the correlation coefficient (R) was 0.994. The detection limit for caspase 3 was 20 pmol L−1, which is one order of magnitude lower than those of FRET sensors based on graphene oxides [17].
Fig. 4 Relationship between the concentration of caspase 3 and relative CL intensity for different sizes of AuNPs. The sizes of AuNPs were 36 nm (a), 27 nm (b), and 18 nm (c), respectively. The concentration of streptavidin–HRP was 2.5 nmol L −1 . The concentrations were 2.0 nmol L−1, 0.6 nmol L−1, and 0.25 nmol L−1 for 18 nm, 27 nm, and 36 nm AuNP–peptide conjugates, respectively. Other conditions as for Fig. 2
Fig. 5 CL spectra obtained by use of the proposed CRET system in the presence of caspase 3 at different concentrations. The inset shows the linear relationship between CL intensity and caspase 3 concentration. The concentration of the 36 nm AuNP–peptide conjugate was 0.25 nmol L−1. The concentration of streptavidin–HRP was 2.5 nmol L−1. Other conditions as for Fig. 2
The relative standard deviation (RSD) of this method was 3.0 % (obtained by analysis of seven standard solutions each containing 0.10 nmol L−1 of caspase 3). These results demonstrated that the CRET system consisting of biofunctionalized AuNPs and HRP–streptavidin is a promising approach for highly sensitive detection of proteins. Figure S7 (Electronic Supplementary Material) shows the specificity of the proposed CRET system containing peptide– AuNP conjugates and HRP–streptavidin. The relative CL intensity remained almost unchanged in the presence of BSA, HSA, GOX, IgG, LDL, thrombin, uricase, and LDM, indicating the probe did not respond to these proteins and LDM at the concentration tested. The concentrations of most of these proteins (50 nmol L−1) were approximately 50 times
Fig. 6 Relative CL intensity for cell lysate from normal cells (without LDM, control sample) and from apoptotic cells induced with different concentrations of LDM. The concentration of streptavidin–HRP was 2.5 nmol L−1. Other conditions as for Fig. 2
Chemiluminescence detection of cell apoptosis enzyme
that of caspase 3 (1 nmol L−1). The good selectivity is attributed to the high specificity of caspase 3–DEVD, including the peptide reaction used. These results suggested that the proposed CRET system could be used for highly sensitive detection of caspase 3 with high specificity. CRET assay of drug-induced apoptosis In this study, LDM, as model drug, was used to induce apoptosis of human pancreatic cancer cells. LDM is an acid protein containing 110 amino acid residues and a chromophore with an enediyne structure [39]. It has significant anticancer activity because of its ability to damage DNA by radical-mediated hydrogen abstraction [40]. LDM also has marked cytotoxicity against cancers in vitro and in vivo [41]. This method was used for discrimination of normal cells from LDM-induced apoptotic cells, on the basis of their different apoptotic protease, especially caspase 3, content. The method used for induction of apoptosis by LDM was described in the Experimental section. Levels of apoptosis detected by use of this CRET assay were similar to those detected by other methods, for example fluorescence correlation spectroscopy [35]. Drug levels were similar to those usually used in cell-based experiments [17]. A strategy for CRET assay of apoptosis is shown in Scheme 1. Briefly, LDM-induced apoptotic and normal cell lysates were diluted tenfold with buffer. Their CL signals were then detected by use of this method. As shown in Fig. 6, compared with that for normal cell lysate (control group), relative CL intensity was 1.46, 2.64, and 2.78 for lysates of cells treated with 0.5 μg mL−1, 5.0 μg mL−1, and 50 μg mL−1 LDM, respectively. Even in the presence of 0.5 μg mL−1 LDM, the relative CL intensity for lysate from cells treated with the drug was clearly different from that for control cells. Although cell lysate is very complicated, AuNP-based CRET successfully enabled detection of the apoptotic status of cells and assay of caspase 3 in apoptotic cell lysate. To further validate the reproducibility of the CRET method, we repeated the experiments by conducting several independent assays on different days; the RSDs of relative CL intensity were approximately 3.8 % to 9.8 %. These results indicate our method enables reliable assay of drug-induced apoptosis.
Conclusion In conclusion, we have developed a highly sensitive method for analysis of cell apoptosis enzyme by using AuNP-based CRET. The CRET system was the bridge formed by the biotin–streptavidin reaction between AuNP–peptide–biotin and streptavidin–HRP; this brought the CL molecules and the AuNPs into close proximity and led to the occurrence of CRET and quenching of the CL signal by the AuNPs. The
presence of caspase 3 in the system resulted in termination of CRET and the recovery of the CL signal, which enabled specific quantitative monitoring of the caspase 3 by use of the CRET system. Recovery of the CL signal may be explained by the specific reaction between caspase 3 and DEVD-containing peptides, and the formation of more dissociative streptavidin–HRP. Our method was successfully used to detect drug-induced apoptosis of cells. Advantages of this sensing approach include the pairing of CL emission with a highly efficient acceptor, facile attachment to AuNPs, high sensitivity and specificity, small amounts of enzyme and substrate, and the possibility of in-vitro assays. Moreover, this approach is expected to be extended to other assays, that is, use of other enzyme, analytes, CL substances, and even other nanoparticles (e.g., quantum dots and graphene). Acknowledgments This work was supported financially by the National Science Foundation of China (20705019, 21135004, and 21327004).
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