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Chemiluminescence Detection of a Protein through the Aptamer Controlled Catalysis of a Porphyrin Probe Wenying Li,‡ab Qingfeng Zhang,‡ab Huipeng Zhou,a Jian Chen,a Yongxin Li,a Cuiyun Zhang,a and Cong Yuab*
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China b
University of the Chinese Academy of Sciences, Beijing 100049, P. R. China
*Corresponding author E-mail: [email protected] Fax: +86-431-85262710
‡These authors contributed equally to this work
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Abstract Sensitive and selective protein detection based on the aptamer controlled noncovalent porphyrin probe self-assembly is reported for the first time. Vascular endothelial growth factor (VEGF) is a predominant biomarker in cancer angiogenesis. In this work, a positively charged porphyrin probe (Mn-PyP) was prepared. Using it as a catalyst, a label-free chemiluminescence (CL) turn-on approach for sensitive VEGF detection is developed. Mn-PyP could catalyze the luminol CL reaction. The VEGF aptamer could induce aggregation of Mn-PyP. As a result, the Mn-PyP catalyzed CL reaction is efficiently suppressed. Upon the addition of VEGF, the specific binding of VEGF to the aptamer weakens the interactions between the aptamer and Mn-PyP. The Mn-PyP monomers are released, a turn-on CL signal is thus detected. Our method is quite sensitive, 50 pM of VEGF could be easily detected. It is also very selective against other proteins. Our assay provides an aptamer based efficient way for protein quantification.
Keywords chemiluminescence, porphyrin, VEGF, protein, aptamer, self-assembly
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Introduction Proangiogenic factors have attracted significant interest because angiogenesis is essential for cancer growth and metastasis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a predominant regulator of both physiologic and pathologic angiogenesis.1,2 Overexpression of VEGF would lead to the formation of a vascular network, which could provide oxygen and nutrients for further growth and metastasis of tumor.3-6 From this point of view, VEGF can be used as a biomarker for the sensing of angiogenic activity in cancer.
The VEGF family comprises five members including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PGF).7 VEGF-A, which is commonly referred to as VEGF, exists in several homodimeric isoforms as a result of the alternative splicing of mRNA. The major expressed monomer variants consist of 121, 145, 165, 189, and 206 amino acid residues. VEGF165 is the dominating isoform and is commonly overexpressed in a variety of human tumors.6,8 Therefore, VEGF165 was selected as the model protein in this work.
To date, a number of aptamer-based VEGF detection techniques, such as the electrochemical9-11 and fluorometric methods,12-15 have been developed. Aptamers are DNA or RNA oligonucleotides which have high specific affinity to their targets. They usually have shorter sequences and can be synthesized chemically.16 Thus, they 3
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have been widely used in biosensing new technology developments. However, many aptamer-based methods require complex covalent labeling, which is laborious, expensive and time-consuming. More importantly, the covalent labeling may weaken the affinity of aptamers to their targets. Therefore, it is highly desirable to develop a label-free aptamer-based method for sensitive protein detection.
Chemiluminescence (CL) has been widely utilized for the development of powerful analytical techniques because of its relatively high sensitivity and low cost. The reaction of luminol with hydrogen peroxide (H2O2) is one of the most commonly used CL reactions. Some metal containing porphyrins exhibit very good catalytic activity.17 It was reported that the manganese porphyrin shows better catalytic performance than the other metal containing porphyrins.17,18 In this work, a positively charged manganese(III) meso-tetrakis(N-methylpyridinum-4-yl)porphyrin (Mn-PyP, Figure 1) was prepared. Using it as a catalyst, a label-free CL turn-on approach for VEGF detection is developed.
The overall assay strategy is illustrated in Scheme 1. (1) In an aqueous buffer solution, Mn-PyP mainly exists in the monomeric form. It could efficiently catalyze the luminol CL reaction. (2) Aggregation of the porphyrin probe could significantly reduce its catalytic activity.19 The VEGF aptamer (a polyanion) contains multiple negatively charged phosphate groups. When the VEGF aptamer is added to the solution of Mn-PyP, strong electrostatic and hydrophobic interactions between the 4
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aptamer and Mn-PyP result in aggregation of the Mn-PyP probe. As a result, the Mn-PyP catalyzed CL reaction is efficiently suppressed. (3) Upon the addition of VEGF, the specific affinity of VEGF to the aptamer weakens the interactions between the aptamer and Mn-PyP. The Mn-PyP probe exists as the monomeric form. A turn-on CL signal is detected, and a novel VEGF sensing method is therefore established. Our method is label-free, simple, fast, and inexpensive.
Experimental Section Materials Oligonucleotides used in this study were synthesized and purified with PAGE by Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences of the oligonucleotides are as follows: VEGF165 aptamer 1: 5′-TTGTCCCGTCTTCCAGACAAGAGTGCAGGGA-3′ VEGF165 aptamer 2: 5′-CAATTGGGCCCGTCCGTATGGTGGGTTTTTTTTTTTC AATTGGGCCCGTCCGTATGGTGGGT-3′
Recombinant human VEGF165 and PDGF-BB were purchased from Beijing Zhong Ke Wu Yuan Biotechnology Co., Ltd. (Beijing, China). Lysozyme and trypsin were purchased from Beijing Dingguo Biotechnology Co., Ltd (Beijing, China). Bovine serum albumin (BSA) was purchased from Bio Basic Inc. (Markham, Ontario, Canada). Luminol was purchased from Acros Organics (Geel, Belgium). H2O2 (30%, v/v) was obtained from Beijing Chemical Works (Beijing, China). Nuclease S1 was 5
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purchased
from
Sangon
Biotechnology
Co.,
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(Shanghai,
China).
Poly(vinylsulfonic acid, sodium salt) (PVS) was purchased from Aldrich (St. Louis, MO, USA). The albumin/IgG removal kit was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). The concentration of PVS used in the current investigation is the concentration of the repeating unit. The concentration of the aptamers used in the current investigation is the concentration of the nucleobase.
Instrumentation UV/Vis absorption spectra were obtained with a Cary 50 Bio spectrophotometer (Varian Inc., CA, USA). Quartz cuvettes with 10 mm path length and 2 mm window width were used. A Chemiluminescence Analyzer System (Remax Analytical Instrument Co. Ltd., Xi’An, China) was used to collect the CL emission of luminol oxidation by H2O2.
Synthesis of meso-5,10,15,20-tetrakis(4-N-methyl-pyridiniumyl)porphyrinato manganese(III) pentachloride (Mn-PyP) Mn-PyP was synthesized according to literature methods (Scheme S1, supporting information).20 Briefly, porphyrin tetratosylate (101 mg, 0.074 mmol) and manganese(II) chloride tetrahydrate (16 mg, 0.081 mmol) were added to 15 mL methanol. The mixture was refluxed with stirring under N2 atmosphere. UV/Vis absorption spectroscopy was used to monitor the reaction, and it was stopped when the Soret band completely shifted from 422 to 463 nm. Subsequently, the tosylate 6
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anion was exchanged by chloride ion on 5 g anion exchange resin (DOWEX 1x8-200) for 24 h with slow stirring. The solution was filtered and the resin was washed with methanol. The methanol solution was concentrated to 1 – 2 mL and the product was precipitated by the addition of Et2O. The precipitate was filtered, washed with Et2O, dried and dissolved in 4 mL water. Finally, the manganese salt was allowed to precipitate at 4 ºC, and the aqueous phase was filtered and then evaporated. After drying under vacuum, Mn-PyP was obtained as a purple solid. The product was characterized by ESI-MS (Figure S1). The ESI-MS analysis provided a m/z value of 182.9, whereas the calculated m/z value for [C44H36MnN8]4+ was 182.8, which confirmed the identity and purity of the probe.
Assay procedures The VEGF luminol CL assay was performed at an ambient temperature. Briefly, 980 µL Tris-HCl solution (20 mM, pH 9.0) containing 50 nM VEGF165 aptamer 1 (or 80 nM VEGF165 aptamer 2), 10 nM Mn-PyP, 25 µM luminol, 2 mM H2O2, 0.05% Triton X-100 and VEGF of different concentrations were mixed. Then, 20 µL H2O2 stock solution (100 mM) was quickly added. The CL signal at 1 min was collected by the MCDR-A System. The PMT voltage was set at 500 V. The biological fluid analysis was conducted in the sample mixtures containing 0.1%, 1% human serum, or 1% human serum with albumin and IgG removed (Figures S9 – S11). The procedures were the same as described above.
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Results and Discussion Properties of the porphyrin probe (Mn-PyP) The UV/Vis absorption spectra of the metal-free porphyrin (PyP) and Mn-PyP were obtained. Figure S2 shows that both probes have two characteristic absorption bands: the Soret band at about 400 – 500 nm [a strong transition from the ground state to the second excited state (S0 → S2)], and the Q band in the region of 500 – 750 nm [a weak transition from the ground state to the first excited state (S0 → S1)].21 Compared with PyP, both the Soret band and the Q band of Mn-PyP show obvious red-shift. Meanwhile the number of absorption peaks of Q band reduce significantly.20 The results clearly suggest that the metalation of porphyrin was successful and the manganese porphyrin was obtained.
The aggregation properties of Mn-PyP was studied. Figure 2-a shows that the absorbance of Mn-PyP increased with increasing Mn-PyP concentration. The maximum absorbance of Mn-PyP (at 463 nm) was found to be directly proportional to Mn-PyP concentration in the range of 0.1 – 10 µM (Figure 2-b). Deviations from the Lambert-Beer law have been ascribed to self-aggregation of the water-soluble small molecular probes.22,23,29 Therefore, the results indicate that Mn-PyP mainly existed in
the monomeric form under the experimental conditions (≤ 10 µM). Since Mn-PyP contains four positively charged pyridinium groups, the strong electrostatic repulsive interactions between the Mn-PyP probe molecules diminish their tendency of self-aggregation, which is consistent with the previously reported literature 8
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works.24,25
Polyanion-induced Mn-PyP aggregation The polyanion-induced aggregation of Mn-PyP was studied. Poly(vinyl sulfonate) (PVS) was selected. Aggregation of porphyrin is commonly accompanied by hypochromicity.26 The addition of PVS could reduce the intensity of both the Soret and Q-band absorption of Mn-PyP as a result of the induced aggregation. A gradual decrease of the absorbance of Mn-PyP was observed with the addition of increasing PVS concentration from 0 to 15 µM (Figure S3-a). The results suggest that PVS could induce aggregation of the positively charged Mn-PyP. In addition, further increase of the PVS concentration (> 20 µM) caused absorbance recovery of Mn-PyP (Figure S3-b). The results indicate that further increase of the PVS concentration would “dilute” the bound porphyrin molecules (or reduce the average number of bound porphyrin molecules on each PVS chain), which is in good agreement with the previous reported literature studies.27,28
The effect of assay solution pH value on the PVS-induced Mn-PyP aggregation was investigated (Figure S4). A gradual increase of the changes of Mn-PyP absorbance at 463 nm (absorbancewithout PVS – absorbancewith PVS) was observed with the gradual increase of the pH value from 5.0 to 9.0. The results indicate that a higher pH value is more conducive for the PVS-induced Mn-PyP aggregation. Further increase of the solution pH value was not attempted since the aptamer and VEGF may be slowly 9
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hydrolyzed under very basic assay conditions. A buffer pH value of 9.0 was therefore used for the following experiments.
It was reported that the manganese porphyrin could catalyze the luminol CL reaction. Figure 3 shows that in the absence of Mn-PyP, the mixture of 25 µM luminol and 2 mM H2O2 showed very little CL signal increase. Upon the addition of 10 nM Mn-PyP, strong CL signal increase was observed. And when 60 nM PVS was introduced, the CL signal decreased very significantly within 1 min. The results clearly suggest that Mn-PyP could very efficiently catalyze the luminol CL reaction. However, with the addition of PVS, the CL signal decreased significantly since PVS induced aggregation of Mn-PyP, the catalytic activity of Mn-PyP was thus effectively inhibited.
Optimization of the Mn-PyP catalyzed CL reaction The concentrations of luminol and H2O2 were optimized to get the best sensing performance. Figure S5-a shows that at a fixed Mn-PyP concentration of 10 nM, the integrated CL intensity ratio (CLwith Mn-PyP/CLwithout Mn-PyP) increased gradually with the gradual increase of luminol concentration from 2.5 to 25 µM. A maximum CL value was obtained at the luminol concentration of 25 µM. When the luminol concentration was further increased, the integrated CL intensity ratio decreased. Thus, 25 µM luminol was selected in the following assays. Similarly, Figure S5-b shows that when 2 mM H2O2 was introduced, a maximum CL value was obtained. 2 mM 10
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H2O2 was therefore selected.
Aptamer-induced Mn-PyP aggregation The VEGF aptamer (an anionic polymer) could induce aggregation of Mn-PyP.29-32 A number of VEGF165 aptamers have been reported. Because RNA aptamers are considerably less chemically stable and more expensive than DNA aptamers, DNA aptamers were selected in this work. Two DNA aptamers (VEGF165 aptamer 1 and
aptamer 2) were selected. VEGF165 is a homodimeric protein. It has two receptor binding domains and two heparin binding domains.33 VEGF165 aptamer 1 provides a 1 : 1 binding with VEGF165. It binds to the interface of VEGF165.12,34 VEGF165 aptamer 2 is composed of a 10-mer thymidine linker and two monomeric aptamers, which can recognize the VEGF165 heparin binding domain.35
Figure S6 shows that the absorbance of Mn-PyP decreased gradually with the addition of increasing VEGF165 aptamer 1 concentration from 0 to 15 µM. Figure 4 shows that the integrated CL intensity decreased gradually with the gradual increase of the VEGF165 aptamer 1 concentration from 0 to 50 nM. When 50 nM of VEGF165 aptamer 1 was introduced, minimum integrated CL intensity was obtained. Further increase of the aptamer concentration caused no further decrease in CL intensity. The results suggest that the aptamer could induce aggregation of Mn-PyP and quench the CL reaction catalyzed by Mn-PyP.
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To investigate the possible quenching mechanism, control experiments were conducted. Figure S7 shows that after the digestion of VEGF165 aptamer 1 with nuclease S1, the integrated CL intensity recovered almost completely. The results further support the notion that the aptamer-induced Mn-PyP aggregation played a major role in the quenching of the CL reaction catalyzed by Mn-PyP.
VEGF165 detection Figure 5-a shows that in the presence of 50 nM VEGF165 aptamer 1 and 10 nM Mn-PyP, the integrated CL intensity increased gradually with the addition of increasing VEGF165 concentration from 0 to 25 nM. A good linear relationship was obtained at a VEGF165 concentration range from 0 to 15 nM (Figure 5-b). The linear correlation equation is Y = 562.6C + 2108 (correlation coefficient R2 = 0.996), where “C” is the concentration of VEGF165 in nM and “Y” is the integrated CL intensity. The assay could easily detect 500 pM VEGF165. VEGF165 aptamer 2 was also tested. Figures 6 and S8 show that in the presence of 80 nM VEGF165 aptamer 2 and 10 nM Mn-PyP, the integrated CL intensity increased gradually with increasing VEGF165 concentration. As low as 50 pM VEGF165 could be easily detected, which is among the best DNA aptamer based VEGF165 assay methods reported.10,12-14 The results also indicate that the assay could be more sensitive by employing a bidentate aptamer (VEGF165 aptamer 2).36,37
Selectivity studies 12
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The selectivity of the assay was investigated. A number of proteins with different molecular weights and pI values, including PDGF-BB, thrombin, trypsin, BSA and collagenase, were selected. Both VEGF165 aptamer 1 and aptamer 2 were tested. Figure 7 shows that under the same experimental conditions, VEGF165 could induce a significant increase in CL intensity. In contrast, the other proteins of the same concentration could hardly induce obvious increase in CL intensity. The results clearly suggest that our assay is highly selective for VEGF165 detection.
VEGF165 detection in biological fluid The assay was further tested in complex sample mixtures. Sample mixtures containing diluted human serum (0.1% or 1%), or human serum (1%) with albumin and IgG removed were tested. Figures S9 – S11 show that the more VEGF165 was spiked, the larger the integrated CL intensity was obtained. These results show that our assay could be used in complex sample mixtures.
Conclusions In summary, aptamer based controlled self-assembly of a porphyrin probe for protein detection is reported for the first time. A positively charged porphyrin probe (Mn-PyP) was prepared. Using it as a catalyst, a label-free CL turn-on approach for VEGF detection is demonstrated. Mn-PyP monomer could efficiently catalyze the luminol CL reaction. VEGF aptamer could induce aggregation of Mn-PyP. As a result, the Mn-PyP catalyzed CL reaction is efficiently quenched. Upon the addition 13
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of VEGF, the specific affinity of VEGF to the aptamer weakens the interactions between the aptamer and Mn-PyP. The Mn-PyP monomers are released, a CL turn-on signal is thus detected. This assay has several distinct advantages: (1) Mn-PyP can be easily prepared, and the aptamer is commercial available. All materials used are fairly inexpensive. (2) It is based on the CL “turn-on” mode, which could reduce the probability of false positive signal. (3) Our method is label-free, and the experimental procedures are fairly simple. (4) Our assay is quite sensitive. 50 pM of VEGF could be easily detected. The assay provides a new strategy for protein quantification.
Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2011CB911002), the National Natural Science Foundation of China (21275139 and 21405151), the Pillar Program of Changchun Municipal Bureau of Science and Technology (14KG062), the Jilin Provincial Strategic Economic Infrastructure Adjustment fund (2014Y077), and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201505).
Associated Content Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 14
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Author Information *Corresponding Author E-mail: [email protected] Fax: (+86)-431-85262710
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Figure 1. Structure of the porphyrin probe (Mn-PyP).
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Scheme 1. Schematic Illustration of the porphyrin probe (Mn-PyP) based VEGF sensing strategy.
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Figure 2. Changes in the UV/vis absorption intensity with Mn-PyP concentration (0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5, and 10 µM, respectively). Assay conditions: 20 mM Tris-HCl, pH 9.0.
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Figure 3. Changes in the integrated CL intensity. Line 1: without Mn-PyP. Line 2: with the addition of 10 nM Mn-PyP. Line 3: with the addition of 10 nM Mn-PyP and 60 nM PVS. Assay conditions: 25 µM luminol, 2 mM H2O2, 20 mM Tris-HCl, pH 9.0.
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Figure 4. (a) Changes in the integrated CL intensity with VEGF165 aptamer 1 concentration (0, 10, 20, 30, 40, and 50 nM, respectively). (b) Plot of the changes in CL intensity at 1 min against VEGF165 aptamer 1 concentration. Assay conditions: 25 µM luminol, 2 mM H2O2, 10 nM Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Figure 5. (a) Changes in the integrated CL intensity with VEGF165 concentration (0, 0.5, 2.5, 5, 7.5, 10.0, 15.0, 20, and 25 nM, respectively). (b) Plot of the changes in the integrated CL intensity at 1 min against VEGF165 concentration. Assay conditions: 25 µM luminol, 2 mM H2O2, 50 nM VEGF165 aptamer 1, 10 nM Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Figure 6. Changes in the integrated CL intensity with VEGF165 concentration (0, 50, 100, 500, 1000, and 2500 pM, respectively). Assay conditions: 25 µM luminol, 2 mM H2O2, 80 nM VEGF165 aptamer 2, 10 nM Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Figure 7. Selectivity studies. The proteins were A) PDGF-BB, B) thrombin, C) trypsin, D) BSA, and E) collagenase. (a) 50 nM VEGF165 aptamer 1 was used. Protein concentration: 15 nM each. (b) 80 nM VEGF165 aptamer 2 was used. Protein concentration: 1 nM each. Integrated CL – Integrated CL0 = Integrated CLwith protein – Integrated CLwithout
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Chemiluminescence Detection of a Protein through the Aptamer Controlled Catalysis of a Porphyrin Probe Wenying Li,‡ab Qingfeng Zhang,‡ab Huipeng Zhou,a Jian Chen,a Yongxin Li,a Cuiyun Zhang,a and Cong Yuab*
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China b
University of the Chinese Academy of Sciences, Beijing 100049, P. R. China
*Corresponding author E-mail: [email protected] Fax: +86-431-85262710
‡These authors contributed equally to this work
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Abstract Sensitive and selective protein detection based on the aptamer controlled noncovalent porphyrin probe self-assembly is reported for the first time. Vascular endothelial growth factor (VEGF) is a predominant biomarker in cancer angiogenesis. In this work, a positively charged porphyrin probe (Mn-PyP) was prepared. Using it as a catalyst, a label-free chemiluminescence (CL) turn-on approach for sensitive VEGF detection is developed. Mn-PyP could catalyze the luminol CL reaction. The VEGF aptamer could induce aggregation of Mn-PyP. As a result, the Mn-PyP catalyzed CL reaction is efficiently suppressed. Upon the addition of VEGF, the specific binding of VEGF to the aptamer weakens the interactions between the aptamer and Mn-PyP. The Mn-PyP monomers are released, a turn-on CL signal is thus detected. Our method is quite sensitive, 50 pM of VEGF could be easily detected. It is also very selective against other proteins. Our assay provides an aptamer based efficient way for protein quantification.
Keywords chemiluminescence, porphyrin, VEGF, protein, aptamer, self-assembly
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Introduction Proangiogenic factors have attracted significant interest because angiogenesis is essential for cancer growth and metastasis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a predominant regulator of both physiologic and pathologic angiogenesis.1,2 Overexpression of VEGF would lead to the formation of a vascular network, which could provide oxygen and nutrients for further growth and metastasis of tumor.3-6 From this point of view, VEGF can be used as a biomarker for the sensing of angiogenic activity in cancer.
The VEGF family comprises five members including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PGF).7 VEGF-A, which is commonly referred to as VEGF, exists in several homodimeric isoforms as a result of the alternative splicing of mRNA. The major expressed monomer variants consist of 121, 145, 165, 189, and 206 amino acid residues. VEGF165 is the dominating isoform and is commonly overexpressed in a variety of human tumors.6,8 Therefore, VEGF165 was selected as the model protein in this work.
To date, a number of aptamer-based VEGF detection techniques, such as the electrochemical9-11 and fluorometric methods,12-15 have been developed. Aptamers are DNA or RNA oligonucleotides which have high specific affinity to their targets. They usually have shorter sequences and can be synthesized chemically.16 Thus, they 3
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have been widely used in biosensing new technology developments. However, many aptamer-based methods require complex covalent labeling, which is laborious, expensive and time-consuming. More importantly, the covalent labeling may weaken the affinity of aptamers to their targets. Therefore, it is highly desirable to develop a label-free aptamer-based method for sensitive protein detection.
Chemiluminescence (CL) has been widely utilized for the development of powerful analytical techniques because of its relatively high sensitivity and low cost. The reaction of luminol with hydrogen peroxide (H2O2) is one of the most commonly used CL reactions. Some metal containing porphyrins exhibit very good catalytic activity.17 It was reported that the manganese porphyrin shows better catalytic performance than the other metal containing porphyrins.17,18 In this work, a positively charged manganese(III) meso-tetrakis(N-methylpyridinum-4-yl)porphyrin (Mn-PyP, Figure 1) was prepared. Using it as a catalyst, a label-free CL turn-on approach for VEGF detection is developed.
The overall assay strategy is illustrated in Scheme 1. (1) In an aqueous buffer solution, Mn-PyP mainly exists in the monomeric form. It could efficiently catalyze the luminol CL reaction. (2) Aggregation of the porphyrin probe could significantly reduce its catalytic activity.19 The VEGF aptamer (a polyanion) contains multiple negatively charged phosphate groups. When the VEGF aptamer is added to the solution of Mn-PyP, strong electrostatic and hydrophobic interactions between the 4
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aptamer and Mn-PyP result in aggregation of the Mn-PyP probe. As a result, the Mn-PyP catalyzed CL reaction is efficiently suppressed. (3) Upon the addition of VEGF, the specific affinity of VEGF to the aptamer weakens the interactions between the aptamer and Mn-PyP. The Mn-PyP probe exists as the monomeric form. A turn-on CL signal is detected, and a novel VEGF sensing method is therefore established. Our method is label-free, simple, fast, and inexpensive.
Experimental Section Materials Oligonucleotides used in this study were synthesized and purified with PAGE by Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences of the oligonucleotides are as follows: VEGF165 aptamer 1: 5′-TTGTCCCGTCTTCCAGACAAGAGTGCAGGGA-3′ VEGF165 aptamer 2: 5′-CAATTGGGCCCGTCCGTATGGTGGGTTTTTTTTTTTC AATTGGGCCCGTCCGTATGGTGGGT-3′
Recombinant human VEGF165 and PDGF-BB were purchased from Beijing Zhong Ke Wu Yuan Biotechnology Co., Ltd. (Beijing, China). Lysozyme and trypsin were purchased from Beijing Dingguo Biotechnology Co., Ltd (Beijing, China). Bovine serum albumin (BSA) was purchased from Bio Basic Inc. (Markham, Ontario, Canada). Luminol was purchased from Acros Organics (Geel, Belgium). H2O2 (30%, v/v) was obtained from Beijing Chemical Works (Beijing, China). Nuclease S1 was 5
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purchased
from
Sangon
Biotechnology
Co.,
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Ltd.
(Shanghai,
China).
Poly(vinylsulfonic acid, sodium salt) (PVS) was purchased from Aldrich (St. Louis, MO, USA). The albumin/IgG removal kit was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). The concentration of PVS used in the current investigation is the concentration of the repeating unit. The concentration of the aptamers used in the current investigation is the concentration of the nucleobase.
Instrumentation UV/Vis absorption spectra were obtained with a Cary 50 Bio spectrophotometer (Varian Inc., CA, USA). Quartz cuvettes with 10 mm path length and 2 mm window width were used. A Chemiluminescence Analyzer System (Remax Analytical Instrument Co. Ltd., Xi’An, China) was used to collect the CL emission of luminol oxidation by H2O2.
Synthesis of meso-5,10,15,20-tetrakis(4-N-methyl-pyridiniumyl)porphyrinato manganese(III) pentachloride (Mn-PyP) Mn-PyP was synthesized according to literature methods (Scheme S1, supporting information).20 Briefly, porphyrin tetratosylate (101 mg, 0.074 mmol) and manganese(II) chloride tetrahydrate (16 mg, 0.081 mmol) were added to 15 mL methanol. The mixture was refluxed with stirring under N2 atmosphere. UV/Vis absorption spectroscopy was used to monitor the reaction, and it was stopped when the Soret band completely shifted from 422 to 463 nm. Subsequently, the tosylate 6
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anion was exchanged by chloride ion on 5 g anion exchange resin (DOWEX 1x8-200) for 24 h with slow stirring. The solution was filtered and the resin was washed with methanol. The methanol solution was concentrated to 1 – 2 mL and the product was precipitated by the addition of Et2O. The precipitate was filtered, washed with Et2O, dried and dissolved in 4 mL water. Finally, the manganese salt was allowed to precipitate at 4 ºC, and the aqueous phase was filtered and then evaporated. After drying under vacuum, Mn-PyP was obtained as a purple solid. The product was characterized by ESI-MS (Figure S1). The ESI-MS analysis provided a m/z value of 182.9, whereas the calculated m/z value for [C44H36MnN8]4+ was 182.8, which confirmed the identity and purity of the probe.
Assay procedures The VEGF luminol CL assay was performed at an ambient temperature. Briefly, 980 µL Tris-HCl solution (20 mM, pH 9.0) containing 50 nM VEGF165 aptamer 1 (or 80 nM VEGF165 aptamer 2), 10 nM Mn-PyP, 25 µM luminol, 2 mM H2O2, 0.05% Triton X-100 and VEGF of different concentrations were mixed. Then, 20 µL H2O2 stock solution (100 mM) was quickly added. The CL signal at 1 min was collected by the MCDR-A System. The PMT voltage was set at 500 V. The biological fluid analysis was conducted in the sample mixtures containing 0.1%, 1% human serum, or 1% human serum with albumin and IgG removed (Figures S9 – S11). The procedures were the same as described above.
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Results and Discussion Properties of the porphyrin probe (Mn-PyP) The UV/Vis absorption spectra of the metal-free porphyrin (PyP) and Mn-PyP were obtained. Figure S2 shows that both probes have two characteristic absorption bands: the Soret band at about 400 – 500 nm [a strong transition from the ground state to the second excited state (S0 → S2)], and the Q band in the region of 500 – 750 nm [a weak transition from the ground state to the first excited state (S0 → S1)].21 Compared with PyP, both the Soret band and the Q band of Mn-PyP show obvious red-shift. Meanwhile the number of absorption peaks of Q band reduce significantly.20 The results clearly suggest that the metalation of porphyrin was successful and the manganese porphyrin was obtained.
The aggregation properties of Mn-PyP was studied. Figure 2-a shows that the absorbance of Mn-PyP increased with increasing Mn-PyP concentration. The maximum absorbance of Mn-PyP (at 463 nm) was found to be directly proportional to Mn-PyP concentration in the range of 0.1 – 10 µM (Figure 2-b). Deviations from the Lambert-Beer law have been ascribed to self-aggregation of the water-soluble small molecular probes.22,23,29 Therefore, the results indicate that Mn-PyP mainly existed in
the monomeric form under the experimental conditions (≤ 10 µM). Since Mn-PyP contains four positively charged pyridinium groups, the strong electrostatic repulsive interactions between the Mn-PyP probe molecules diminish their tendency of self-aggregation, which is consistent with the previously reported literature 8
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works.24,25
Polyanion-induced Mn-PyP aggregation The polyanion-induced aggregation of Mn-PyP was studied. Poly(vinyl sulfonate) (PVS) was selected. Aggregation of porphyrin is commonly accompanied by hypochromicity.26 The addition of PVS could reduce the intensity of both the Soret and Q-band absorption of Mn-PyP as a result of the induced aggregation. A gradual decrease of the absorbance of Mn-PyP was observed with the addition of increasing PVS concentration from 0 to 15 µM (Figure S3-a). The results suggest that PVS could induce aggregation of the positively charged Mn-PyP. In addition, further increase of the PVS concentration (> 20 µM) caused absorbance recovery of Mn-PyP (Figure S3-b). The results indicate that further increase of the PVS concentration would “dilute” the bound porphyrin molecules (or reduce the average number of bound porphyrin molecules on each PVS chain), which is in good agreement with the previous reported literature studies.27,28
The effect of assay solution pH value on the PVS-induced Mn-PyP aggregation was investigated (Figure S4). A gradual increase of the changes of Mn-PyP absorbance at 463 nm (absorbancewithout PVS – absorbancewith PVS) was observed with the gradual increase of the pH value from 5.0 to 9.0. The results indicate that a higher pH value is more conducive for the PVS-induced Mn-PyP aggregation. Further increase of the solution pH value was not attempted since the aptamer and VEGF may be slowly 9
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hydrolyzed under very basic assay conditions. A buffer pH value of 9.0 was therefore used for the following experiments.
It was reported that the manganese porphyrin could catalyze the luminol CL reaction. Figure 3 shows that in the absence of Mn-PyP, the mixture of 25 µM luminol and 2 mM H2O2 showed very little CL signal increase. Upon the addition of 10 nM Mn-PyP, strong CL signal increase was observed. And when 60 nM PVS was introduced, the CL signal decreased very significantly within 1 min. The results clearly suggest that Mn-PyP could very efficiently catalyze the luminol CL reaction. However, with the addition of PVS, the CL signal decreased significantly since PVS induced aggregation of Mn-PyP, the catalytic activity of Mn-PyP was thus effectively inhibited.
Optimization of the Mn-PyP catalyzed CL reaction The concentrations of luminol and H2O2 were optimized to get the best sensing performance. Figure S5-a shows that at a fixed Mn-PyP concentration of 10 nM, the integrated CL intensity ratio (CLwith Mn-PyP/CLwithout Mn-PyP) increased gradually with the gradual increase of luminol concentration from 2.5 to 25 µM. A maximum CL value was obtained at the luminol concentration of 25 µM. When the luminol concentration was further increased, the integrated CL intensity ratio decreased. Thus, 25 µM luminol was selected in the following assays. Similarly, Figure S5-b shows that when 2 mM H2O2 was introduced, a maximum CL value was obtained. 2 mM 10
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H2O2 was therefore selected.
Aptamer-induced Mn-PyP aggregation The VEGF aptamer (an anionic polymer) could induce aggregation of Mn-PyP.29-32 A number of VEGF165 aptamers have been reported. Because RNA aptamers are considerably less chemically stable and more expensive than DNA aptamers, DNA aptamers were selected in this work. Two DNA aptamers (VEGF165 aptamer 1 and
aptamer 2) were selected. VEGF165 is a homodimeric protein. It has two receptor binding domains and two heparin binding domains.33 VEGF165 aptamer 1 provides a 1 : 1 binding with VEGF165. It binds to the interface of VEGF165.12,34 VEGF165 aptamer 2 is composed of a 10-mer thymidine linker and two monomeric aptamers, which can recognize the VEGF165 heparin binding domain.35
Figure S6 shows that the absorbance of Mn-PyP decreased gradually with the addition of increasing VEGF165 aptamer 1 concentration from 0 to 15 µM. Figure 4 shows that the integrated CL intensity decreased gradually with the gradual increase of the VEGF165 aptamer 1 concentration from 0 to 50 nM. When 50 nM of VEGF165 aptamer 1 was introduced, minimum integrated CL intensity was obtained. Further increase of the aptamer concentration caused no further decrease in CL intensity. The results suggest that the aptamer could induce aggregation of Mn-PyP and quench the CL reaction catalyzed by Mn-PyP.
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To investigate the possible quenching mechanism, control experiments were conducted. Figure S7 shows that after the digestion of VEGF165 aptamer 1 with nuclease S1, the integrated CL intensity recovered almost completely. The results further support the notion that the aptamer-induced Mn-PyP aggregation played a major role in the quenching of the CL reaction catalyzed by Mn-PyP.
VEGF165 detection Figure 5-a shows that in the presence of 50 nM VEGF165 aptamer 1 and 10 nM Mn-PyP, the integrated CL intensity increased gradually with the addition of increasing VEGF165 concentration from 0 to 25 nM. A good linear relationship was obtained at a VEGF165 concentration range from 0 to 15 nM (Figure 5-b). The linear correlation equation is Y = 562.6C + 2108 (correlation coefficient R2 = 0.996), where “C” is the concentration of VEGF165 in nM and “Y” is the integrated CL intensity. The assay could easily detect 500 pM VEGF165. VEGF165 aptamer 2 was also tested. Figures 6 and S8 show that in the presence of 80 nM VEGF165 aptamer 2 and 10 nM Mn-PyP, the integrated CL intensity increased gradually with increasing VEGF165 concentration. As low as 50 pM VEGF165 could be easily detected, which is among the best DNA aptamer based VEGF165 assay methods reported.10,12-14 The results also indicate that the assay could be more sensitive by employing a bidentate aptamer (VEGF165 aptamer 2).36,37
Selectivity studies 12
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The selectivity of the assay was investigated. A number of proteins with different molecular weights and pI values, including PDGF-BB, thrombin, trypsin, BSA and collagenase, were selected. Both VEGF165 aptamer 1 and aptamer 2 were tested. Figure 7 shows that under the same experimental conditions, VEGF165 could induce a significant increase in CL intensity. In contrast, the other proteins of the same concentration could hardly induce obvious increase in CL intensity. The results clearly suggest that our assay is highly selective for VEGF165 detection.
VEGF165 detection in biological fluid The assay was further tested in complex sample mixtures. Sample mixtures containing diluted human serum (0.1% or 1%), or human serum (1%) with albumin and IgG removed were tested. Figures S9 – S11 show that the more VEGF165 was spiked, the larger the integrated CL intensity was obtained. These results show that our assay could be used in complex sample mixtures.
Conclusions In summary, aptamer based controlled self-assembly of a porphyrin probe for protein detection is reported for the first time. A positively charged porphyrin probe (Mn-PyP) was prepared. Using it as a catalyst, a label-free CL turn-on approach for VEGF detection is demonstrated. Mn-PyP monomer could efficiently catalyze the luminol CL reaction. VEGF aptamer could induce aggregation of Mn-PyP. As a result, the Mn-PyP catalyzed CL reaction is efficiently quenched. Upon the addition 13
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of VEGF, the specific affinity of VEGF to the aptamer weakens the interactions between the aptamer and Mn-PyP. The Mn-PyP monomers are released, a CL turn-on signal is thus detected. This assay has several distinct advantages: (1) Mn-PyP can be easily prepared, and the aptamer is commercial available. All materials used are fairly inexpensive. (2) It is based on the CL “turn-on” mode, which could reduce the probability of false positive signal. (3) Our method is label-free, and the experimental procedures are fairly simple. (4) Our assay is quite sensitive. 50 pM of VEGF could be easily detected. The assay provides a new strategy for protein quantification.
Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2011CB911002), the National Natural Science Foundation of China (21275139 and 21405151), the Pillar Program of Changchun Municipal Bureau of Science and Technology (14KG062), the Jilin Provincial Strategic Economic Infrastructure Adjustment fund (2014Y077), and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201505).
Associated Content Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 14
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Author Information *Corresponding Author E-mail: [email protected] Fax: (+86)-431-85262710
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Figure 1. Structure of the porphyrin probe (Mn-PyP).
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Scheme 1. Schematic Illustration of the porphyrin probe (Mn-PyP) based VEGF sensing strategy.
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Figure 2. Changes in the UV/vis absorption intensity with Mn-PyP concentration (0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5, and 10 µM, respectively). Assay conditions: 20 mM Tris-HCl, pH 9.0.
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Figure 3. Changes in the integrated CL intensity. Line 1: without Mn-PyP. Line 2: with the addition of 10 nM Mn-PyP. Line 3: with the addition of 10 nM Mn-PyP and 60 nM PVS. Assay conditions: 25 µM luminol, 2 mM H2O2, 20 mM Tris-HCl, pH 9.0.
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Figure 4. (a) Changes in the integrated CL intensity with VEGF165 aptamer 1 concentration (0, 10, 20, 30, 40, and 50 nM, respectively). (b) Plot of the changes in CL intensity at 1 min against VEGF165 aptamer 1 concentration. Assay conditions: 25 µM luminol, 2 mM H2O2, 10 nM Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Figure 5. (a) Changes in the integrated CL intensity with VEGF165 concentration (0, 0.5, 2.5, 5, 7.5, 10.0, 15.0, 20, and 25 nM, respectively). (b) Plot of the changes in the integrated CL intensity at 1 min against VEGF165 concentration. Assay conditions: 25 µM luminol, 2 mM H2O2, 50 nM VEGF165 aptamer 1, 10 nM Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Figure 6. Changes in the integrated CL intensity with VEGF165 concentration (0, 50, 100, 500, 1000, and 2500 pM, respectively). Assay conditions: 25 µM luminol, 2 mM H2O2, 80 nM VEGF165 aptamer 2, 10 nM Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Figure 7. Selectivity studies. The proteins were A) PDGF-BB, B) thrombin, C) trypsin, D) BSA, and E) collagenase. (a) 50 nM VEGF165 aptamer 1 was used. Protein concentration: 15 nM each. (b) 80 nM VEGF165 aptamer 2 was used. Protein concentration: 1 nM each. Integrated CL – Integrated CL0 = Integrated CLwith protein – Integrated CLwithout
protein.
Assay conditions: 25 µM luminol, 2 mM H2O2, 10 nM
Mn-PyP, 20 mM Tris-HCl, pH 9.0.
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Graphic for TOC only
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