Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 164–168
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A carbon nanotubes based fluorescent aptasensor for highly sensitive detection of adenosine deaminase activity and inhibitor screening in natural extracts Kun Hu ∗ , Yong Huang, Sheng’e wang, Shulin Zhao ∗ Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), College of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
a r t i c l e
i n f o
Article history: Received 19 December 2013 Received in revised form 24 February 2014 Accepted 27 February 2014 Available online 12 March 2014 Keywords: Aptasensor Carbon nanotubes Adenosine deaminase Fluorescence Inhibition
a b s t r a c t A carbon nanotubes (CNTs) based fluorescent aptasensor was developed for adenosine deaminase (ADA) activity detection and inhibitor screening by using adenosine (AD) as the substrate. This sensing system consists of CNTs, AD, split anti-AD aptamer fragment and dye-labeled aptamer fragment. In the absence of ADA, two aptamer fragments bind simultaneously with AD to form an AD-aptamer complex. This ADaptamer complex cannot adsorb onto CNTs, and has high fluorescence intensity. When ADA is introduced into this system, ADA can convert AD into inosine, which has not affinity to the split anti-AD aptamer fragment. Thus, the split anti-AD aptamer fragments were adsorbed onto CNTs via strong – stacking interactions, resulting in the quenching of the fluorescence of the dye-labeled aptamer fragment. The proposed aptasensor can detect ADA activity from 0.005 to 0.2 U/mL with a low detection limit of 0.002 U/mL. Moreover, it has been also demonstrated that this CNTs-based fluorescence aptasensor is suitable for ADA inhibitor screening from traditional Chinese medicine (TCM). Considering the superior sensitivity and specificity, the proposed CNTs-based fluorescent aptasensor can be expected to provide a simple, cost-effective and sensitive platform for the detection of ADA activity and screening of potential drugs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Adenosine deaminase (ADA), a key hydrolytic enzyme for purine metabolism, can catalyze the conversion of adenosine (AD) to inosine by the removal of an amino group [1]. It plays a key role in the differentiation and maturation of the lymphoid system. Moreover, accumulating evidence indicate that the dysfunction of ADA in human body is closely related to a number of important diseases, such as tuberculosis, sarcoidosis, cancer and severe combined immunodeficiency (SCID) [2,3]. The significance of ADA in pathology makes it an important target for drug development and diseases detection. Traditional methods including the measuring ammonia produced [4], high-performance liquid chromatography (HPLC) [5], and colorimetric assay [6] have been described to be effective for monitoring ADA activity. Although each method has its advantages, many reported techniques still suffer the
∗ Corresponding authors. Tel.: +86 773 2120958; fax: +86 773 2120958. E-mail addresses: [email protected] (K. Hu), [email protected] (S. Zhao). http://dx.doi.org/10.1016/j.jpba.2014.02.027 0731-7085/© 2014 Elsevier B.V. All rights reserved.
drawbacks, such as time-intensive, laborious, and low sensitivity. Thus, the search for simple and sensitive assay of ADA is ongoing. With rapid development in the field of DNA biotechnology, the oligonucleotides have emerged as attractive recognition units for monitoring enzymes activities. A series of DNA-based probes have been developed for sensitive activity assays of various enzymes, such as DNA methyltransferases [7], endonucleases [8], RNase H [9] and DNA ligase [10]. In recent years, the aptamers have received tremendous attention in sensing applications because of their relative ease of isolation and modification, high affinity and specificity toward targets, and resistance against denaturation [11]. Up to now, several methods based on the use of aptamers as recognition units have been developed for the quantitative determination of ADA activity. These include the electrochemical aptasensor [12], colorimetric aptasensor [13] and fluorescence sensor [14]. Although these techniques can be quite powerful, a simpler and more sensitive method for ADA detection is still required. The carbon nanomaterials have a significant role to play in new developments in each of the biosensor size domains. This significance arises as nanomaterials can help address some of the key issues in the development of all biosensors [15]. For
K. Hu et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 164–168
example, a series of graphene oxide-based aptasensors have been developed for diverse biomolecules detection [16–18], the carbon nanoparticles and carbon nanodots have also extensively been used to develop biosensors [19,20]. Recently, carbon nanotubes (CNTs) have been extensively studied due to their unique optoelectronic properties and excellent biocompatibility [19]. Specifically, the extraordinary fluorescence quenching property of CNTs has been employed to develop nanosensors for diverse biomolecules in homogeneous solution. For example, the CNTs has used as a biosensing platform for the detection of DNA based on the quenching effect on the dye-labeled DNA probe [21]. Similarly, the CNTs-based aptasensors were also used for sensitive detection of various biomolecules [22,23]. However, to the best of our knowledge, no study has been reported the use of CNTs for homogeneous assay of ADA assay. In the present work, we developed a sensitive and selective fluorescent aptasensor based on multi-walled carbon nanotubes (MWCNTs) using AD as the substrate for ADA activity detection and inhibitor screening. This aptasensor relies on the high fluorescence quenching property of MWCNTs and the different interaction ability of aptamer, AD-aptamer complex with MWCNTs. Compared with the traditional fluorescence resonance energy transfer (FRET)-based aptasensor [14], the proposed assay only requires the labeling of the oligonucleotide probe with one dye, which is very simple and cost-effective. Moreover, this aptasensor exhibits high sensitivity and specificity toward ADA over other non-specific enzymes, with a detection limit of 0.002 U/mL for ADA. In addition, the suitability of this MWCNT-based fluorescence aptasensor for ADA inhibitor screening from TCM has also been demonstrated. 2. Experiment 2.1. Chemical and reagents The adenosine specific aptamer was splited into two singlestranded oligonucleotides (Apt-1 and Apt-2) [24], and their sequences were shown below: Apt-1: 5 -ACCTGGGGGAGTAT-3 ; and Apt-2: 5 -ATGCGGAGGAAGGT-3 . The Apt-1 was modified at 5 ends with 6-carboxyfluo rescein (FAM). And these aptamers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, Shanghai, China) and purified using high performance liquid chromatography. Multi-walled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanotech Port Co. Ltd. (Shenzhen, Guangdong, China). Adenosine, adenosine deaminase and inosine were purchased from Amresco Co., Ltd. (Solon, OH, USA). Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, DE, USA). Traditional Chinese medicine (TCM) was obtained from Guilin Pharmaceuticals Group of China (Guilin, Guangxi, China). Milli-Q water (18.2 M cm) was used throughout this work. All other reagents used in this work were of analytical grade. 2.2. Pretreatment of MWCNTs The commercial MWCNTs were purified and oxidized according to the procedure described by He and Bayachou [25]. The MWCNTs (200 mg) was first refluxed in HNO3 (150 mL, 2.0 mol/L) for two days. After being kept overnight, the suspension was centrifuged at 14,000 rpm for 30 min, and the clear solution was removed. The purified precipitates were further oxidized by 40 mL of HNO3 /H2 SO4 (VHNO3 : VH2 SO4 = 1 : 3) solution in an ultrasonic bath for 4 h. Then, the suspension was diluted 10-fold with water and allowed to stand for 12 h at room temperature. After removal of the clear solution over the precipitates, the remaining suspension was filtered through a 0.45 m filtration membrane and then
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washed with water to obtain a neutral pH. The resulting precipitates were dried in a vacuum drier about 12 h to remove the water. The obtained MWCNTs with a carboxyl group were re-dispersed in water to give a final concentration at 5 mg/mL. 2.3. Preparation of natural extracts Air-dried TCM was ground into a fine powder with a pulverizer. 5.0 g of powder was extracted with 50 mL of isopropanol in an ultrasonic cleaning bath for 0.5 h at 60 ◦ C, and this process was repeated three times. The solvent was evaporated using rotary evaporator at 40 ◦ C. Finally, these extracts were dried in vacuum drier for about 5.0 h. The natural extracts were dissolved with 2-propanol aqueous solution giving a final concentration of 5.0 mg/mL. Other natural extracts were prepared in the same way. 2.4. ADA activity detection The Apt-1 (10 L, 1 M) was mixed with Apt-2 (10 L, 1 M), and then 10 L of 5 mg/mL MWCNTs was added into the above solution in Tris–HCl buffer (10 mM, pH = 7.4), and allowed to incubate for 15 min. The fixed concentration of adenosine (1.0 M) was treated with different activity of ADA (0, 0.002, 0.005, 0.01, 0.015, 0.05, 0.1, 0.15, 0.20, and 0.30 U/mL), and the resulting solution was added to the aptasensor/MWCNTs complex solution in Tris–HCl buffer (10 mM, pH = 7.4). After 50 min of incubation at 37 ◦ C, the fluorescence emission spectra the solution were measured with a LS-55 spectrofluorometer (Perkin-Elmer, USA) at 520 nm with excitation at 480 nm. All experiments were repeated three times. Each sample was measured five times. 2.5. Inhibition screening procedures For comparison of the inhibition ability of test compounds, different concentration EHNA or 0.20 mg/mL of each TCM was mixed with 1.0 M adenosine solution before the addition of 0.20 U/mL ADA, and incubated 50 min at 37 ◦ C. Other assay steps were the same as that of the ADA activity assay. 3. Results and discussion 3.1. Assay principle Fig. 1 depicts the principle of the MWCNTs-based fluorescence aptasensor for ADA activity detection and inhibitor screening. This sensing system consists of two anti-AD aptamer fragments (FAMlabeled Apt-1 and Apt-2), AD and MWCNTs. In the absence of ADA, AD binds with both FAM-labeled Apt-1 and Apt-2 to form the AD/aptamer complex that cannot bind to MWCNTs stably. In this case, the FAM dye exhibits high background fluorescence (Fig. 1a). When ADA is introduced into the system, ADA can convert AD into inosine, which has not affinity with two anti-AD aptamer fragments. Then, the free anti-AD aptamer fragments can be adsorbed onto MWCNTs by means of strong – stacking interactions between nucleotide bases and the MWCNT sidewalls [21], resulting in the quenching of the fluorescence of the FAM dye (Fig. 1b). The fluorescence intensity should decrease with increased amount of ADA. In addition, since the conversion of AD into inosine by the ADA-catalyzed is restrained in the presence of inhibitors, the proposed aptasensor can be adapted to screen the inhibitors of ADA (Fig. 1c). 3.2. Feasibility study To investigate the feasibility of the proposed sensing strategy, the fluorescence spectra under different conditions were measured,
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K. Hu et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 164–168
Fig. 1. The principle of the MWCNT-based fluorescence aptasensor for adenosine deaminase activity and inhibitor screening.
and the experimental results are shown in Fig. 2. The fluorescence spectrum of the FAM-labeled Apt-1/Apt-2 mixture without MWCNTs showed strong fluorescence emission owing to the presence of the FAM dye (curve a). Upon the addition of MWCNTs into the FAM-labeled Apt-1/Apt-2 mixture, the fluorescence of the FAM was significantly quenched (curve e). This observation indicated strong adsorption of the FAM-labeled Apt-1 strand onto MWCNTs, and high fluorescence quenching efficiency of MWCNTs. When AD is introduced into the FAM-labeled Apt-1/Apt-2/MWCNTs mixture, the fluorescence intensity of the FAM increased significantly (curve b), suggesting that the AD-aptamer complex has been formed and released from MWCNTs surface. However, when AD complex with ADA was introduced into the FAM-labeled Apt-1/Apt-2/MWCNTs mixture, the fluorescence decreased significantly compared with that in curve b (curve d). This fluorescence decrease was attributed to that ADA catalyzed the conversion of AD into inosine that could not bind with the anti-AD aptamer fragments, and then the free anti-AD aptamer fragments were adsorbed onto MWCNTs. Further introduction of a model ADA inhibitor, EHNA, into the FAM-labeled Apt-1/Apt-2/AD/ADA/MWCNTs mixture, the fluorescence of the
FAM increased compared with that in curve d (curve c), indicating the inhibition of ADA by EHNA. The above results demonstrated that the designed aptasensor could be used for ADA activity detection and inhibitor screening. 3.3. Optimization of assay conditions To obtain high sensitivity, the amount of MWCNTs was first optimized. As shown in Fig. S1, the fluorescence intensity of the FAM-labeled Apt-1 fragment decreased gradually with the increase of the MWCNTs concentration, and reached the minimum value when 50 g/mL MWCNTs was employed. Under this condition, more than 90% of original fluorescence was quenched. Therefore, 50 g/mL was selected as the optimal MWCNTs concentration. Moreover, the effects of AD concentration were also investigated. The fluorescence intensity increased dramatically with the AD concentration up to 1.0 M and then reached an equilibration over 1.0 M (Fig. S2). Thus, the AD concentration of 1.0 M was selected for the subsequent assay. 3.4. Assay of ADA activity
Fig. 2. Fluorescence emission spectra of DNA probe (Apt-1 and Apt-2, each 50 nM) at different conditions: (a) DNA probe in Tris–HCl; (b) DNA probe + AD (1 M) + 50 g/mL MWCNTs; (c) ADA (0.20 U/mL) + inhibitor (20 nM) + AD (1 M) + DNA probe + MWCNTs (50 g/mL); (d) AD (1 M) + ADA (0.20 U/mL) + DNA probe + 50 g/mL MWCNTs; and (e) DNA probe + MWCNTs (50 g/mL).
To confirm the ability of the proposed aptasensor to detect target enzyme, a series of different concentrations of ADA were measured under the optimal conditions. Before ADA activity assay was examined, the effect on the ADA catalyzes product-inosine on the fluorescence intensity change of MWCNTs aptasensor was investigated (Fig. S3). It is clearly seen that the MWCNTs aptasensor has nearly no response to inosine (100.0 M). Fig. 3 depicts the fluorescence spectra of the proposed aptasensor upon analyzing different concentrations of ADA. As the concentration of ADA increases, the fluorescence intensity decrease gradually. This is consistent with the enhanced conversion of AD into inosine and the adsorption of more amounts of the FAM-labeled Apt-1 on the surface of MWCNTs. The derived calibration curve is shown in the inset of Fig. 3. As can be seen, a good linear relationship between fluorescence intensity and ADA concentration from 0.005 to 0.20 U/mL was obtained. The regression equation was obtained as follows: F = −697.92C + 187.94, R = 0.9963, where F and C are the fluorescence intensity and ADA activity (U/mL), respectively. The detection limits with the MWCNT-based fluorescent aptasensor was 0.002 U/mL, which was about two orders of magnitude lower than that of the electrochemical measurements (0.2 U/mL)
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Fig. 3. The fluorescence emission of the MWCNTs aptasensor with 1.0 M AD at different activity of ADA (from a to i): 0, 0.002, 0.005, 0.01, 0.015, 0.05, 0.1, 0.15, 0.20, and 0.30 U mL−1 . Inset: the plot of the fluorescence intensity changes versus the activity of ADA.
Fig. 5. The fluorescence emission of the MWCNTs aptasensor with 1.0 M AD, 0.20 U/mL ADA at different concentrations of EHNA (from a to g): 0, 0.5, 5.0, 10.0, 15.0, 20.0, and 25.0 nM. Inset: the inhibition plot of ADA in the presence of different concentrations of inhibitor EHNA.
[12]. These results indicated that the MWCNTs-based fluorescence aptasensor could detect ADA activity with high sensitivity. To evaluate the assay selectivity, the responses of the aptasensor to the target ADA and several non-specific enzymes including xanthine oxidase (XOD), DNA topoisomerase II, and thrombin was tested (Fig. 4). It was found that the target ADA led to a significant decrease in the fluorescence intensity, while no apparent fluorescence change was observed in the assay for non-specific enzymes. These results clearly demonstrated that the high specificity of proposed MWCNTs-based fluorescence aptasensor.
the Michaelis–Menten constant (Km ) for the ADA enzymatic reaction was estimated to be 71.6 M, which was in an acceptable agreement with that of the reported absorbance assay methods (89 M) [26,27]. This result indicated that no significant change on enzyme property was observed in the proposed sensing system.
3.5. Enzyme kinetics study To further confirm the validity of the MWCNTs-based fluorescence aptasensor for ADA activity detection, the kinetic behavior of ADA was studied by the present method. In these experiments, 0.20 U/mL ADA with different concentrations of AD substrate ranging from 0.2 to 1.0 M was assayed. According to Michaelis–Menten and Lineweaver–Burk equation, a doublereciprocal plot was constructed, and is shown in Fig. S4. The curve provided a maximum initial velocity (Vmax ) of 3.8 nmol min−1 , and
3.6. Inhibitor screening To explore the potential of the proposed aptasensor to screen ADA inhibitor, a known inhibitor of ADA, EHNA was first selected as a model for the inhibition study. Fig. 5 shows the fluorescence spectra of the sensing system upon analyzing 0.20 U/mL ADA in the presence of different concentrations of EHNA. As the concentration of EHEA increase, the fluorescence of the system was intensified, and the inhibition of ADA activity was enhanced. Fig. 5, insert, shows the derived calibration curve between the fluorescence intensity and the EHEA concentration. From this calibration curve, the IC50 value of EHEA was estimated to be 12 nM (R = 0.9951), which was in good agreement with previous reports [12,28]. Then, we further applied this MWCNTs-based fluorescence aptasensor to screen ADA inhibitors in ten types of TCMs. Before inhibition studies, the effect of the natural extracts from these TCMs on the fluorescence intensity of the MWCNTs-based fluorescence aptasensor was tested, and the experimental results showed that almost no fluorescence change of the aptasensor occurred in the presence of natural extracts (Fig. S5). This demonstrated no interference of natural extracts on the inhibition assays. After that, the inhibition of natural extracts from ten types of TCMs was assayed, and the results are summarized in Table 1. It can be seen that only Rhizoma chuanxiong was identified to be positive for ADA inhibition. Table 1 Extracts library used for inhibitor screening.
Fig. 4. Selective of MWCNTs based ADA (1) aptasensor over thrombin (2), xanthine oxidase (3), and DNA topoisomerase II (4). Where I0 and I correspond to the fluorescence intensity before and after adding enzyme, respectively. The activity of ADA is 0.20 U/mL and other nontarget enzyme is 2.0 U/mL.
TCM
Inhibition (%)
TCM
Inhibition (%)
Aloe barbadensis Radix sophorae tonkinensis
0
43.6
Rhizoma corydalis Herba sedi Rhizoma dioscoreae
0
Rhizoma chuanxiong Rhizoma curcumae aeruginosae Rhizoma coptidis Semen ginkgo Fructus schisandrae
0
0 0
0
0 0 0
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This result was consistent with that of our previous report [28]. These results indicated that the proposed MWCNT-based fluorescent aptasensor was suitable for screening the ADA inhibitors from TCMs. 4. Conclusion In summary, we have developed a MWCNTs-based fluorescent aptasensor for rapid, sensitive, and selective detection of ADA activity and screening of inhibitor. This assay approach has several important advantages. Firstly, the assay is homogeneous, “mixand-read”, and does not require separation and washing steps, which is very simple and convenient. Second, the sensitivity of proposed assay is about two orders of magnitude higher than that of the reported electrochemical methods. Thirdly, owing to the high specificity of aptamer, this approach exhibits high selectivity for ADA. Finally, this aptasensor is suitable for screening ADA inhibitor from TCMs. Considering these qualities, the developed fluorescent aptasensor not only provides a sensing platform for enzyme activity detection, but also holds great potential in inhibitor screening. Acknowledgments The authors gratefully acknowledge the financial support provided by National Natural Science Foundation of China (nos. 21175030, 21305021, 21165004), the Guangxi Natural Science Foundation of China (nos. 2013GXNSFBA019044, 2013GXNSFBA019044, 2013GXNSFBA019038), IRT1225, and “Bagui Scholar Program of Guangxi”. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.02.027. References [1] E.J. Conway, R. Cooke, The deaminases of adenosine and adenylic acid in blood and tissues, Biochem. J. 33 (1939) 479–492. [2] B.K. Gupta, V. Bharat, D. Bandyopadhyay, Role of adenosine deaminase estimation in differentiation of tuberculous and non-tuberculous exudative pleural effusions, J. Clin. Med. Res. 2 (2010) 79–84. [3] M.S. Hershfied, Genotype is an important determinant of phenotype in adenosine deaminase deficiency, Curr. Opin. Immunol. 15 (2003) 571–577. [4] J. Linden, Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 775–787. [5] M.K. Paul, V. Grover, A.K. Mukhopadhyay, Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase, J. Chromatogr. B 822 (2005) 146–153. [6] P. Vielh, M.J. Castellazzi, A colorimetric assay for serial determination of adenosine deaminase activity in small lymphocyte populations, J. Immunol. Methods 73 (1984) 313–320.
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Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A carbon nanotubes based fluorescent aptasensor for highly sensitive detection of adenosine deaminase activity and inhibitor screening in natural extracts Kun Hu ∗ , Yong Huang, Sheng’e wang, Shulin Zhao ∗ Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), College of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
a r t i c l e
i n f o
Article history: Received 19 December 2013 Received in revised form 24 February 2014 Accepted 27 February 2014 Available online 12 March 2014 Keywords: Aptasensor Carbon nanotubes Adenosine deaminase Fluorescence Inhibition
a b s t r a c t A carbon nanotubes (CNTs) based fluorescent aptasensor was developed for adenosine deaminase (ADA) activity detection and inhibitor screening by using adenosine (AD) as the substrate. This sensing system consists of CNTs, AD, split anti-AD aptamer fragment and dye-labeled aptamer fragment. In the absence of ADA, two aptamer fragments bind simultaneously with AD to form an AD-aptamer complex. This ADaptamer complex cannot adsorb onto CNTs, and has high fluorescence intensity. When ADA is introduced into this system, ADA can convert AD into inosine, which has not affinity to the split anti-AD aptamer fragment. Thus, the split anti-AD aptamer fragments were adsorbed onto CNTs via strong – stacking interactions, resulting in the quenching of the fluorescence of the dye-labeled aptamer fragment. The proposed aptasensor can detect ADA activity from 0.005 to 0.2 U/mL with a low detection limit of 0.002 U/mL. Moreover, it has been also demonstrated that this CNTs-based fluorescence aptasensor is suitable for ADA inhibitor screening from traditional Chinese medicine (TCM). Considering the superior sensitivity and specificity, the proposed CNTs-based fluorescent aptasensor can be expected to provide a simple, cost-effective and sensitive platform for the detection of ADA activity and screening of potential drugs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Adenosine deaminase (ADA), a key hydrolytic enzyme for purine metabolism, can catalyze the conversion of adenosine (AD) to inosine by the removal of an amino group [1]. It plays a key role in the differentiation and maturation of the lymphoid system. Moreover, accumulating evidence indicate that the dysfunction of ADA in human body is closely related to a number of important diseases, such as tuberculosis, sarcoidosis, cancer and severe combined immunodeficiency (SCID) [2,3]. The significance of ADA in pathology makes it an important target for drug development and diseases detection. Traditional methods including the measuring ammonia produced [4], high-performance liquid chromatography (HPLC) [5], and colorimetric assay [6] have been described to be effective for monitoring ADA activity. Although each method has its advantages, many reported techniques still suffer the
∗ Corresponding authors. Tel.: +86 773 2120958; fax: +86 773 2120958. E-mail addresses: [email protected] (K. Hu), [email protected] (S. Zhao). http://dx.doi.org/10.1016/j.jpba.2014.02.027 0731-7085/© 2014 Elsevier B.V. All rights reserved.
drawbacks, such as time-intensive, laborious, and low sensitivity. Thus, the search for simple and sensitive assay of ADA is ongoing. With rapid development in the field of DNA biotechnology, the oligonucleotides have emerged as attractive recognition units for monitoring enzymes activities. A series of DNA-based probes have been developed for sensitive activity assays of various enzymes, such as DNA methyltransferases [7], endonucleases [8], RNase H [9] and DNA ligase [10]. In recent years, the aptamers have received tremendous attention in sensing applications because of their relative ease of isolation and modification, high affinity and specificity toward targets, and resistance against denaturation [11]. Up to now, several methods based on the use of aptamers as recognition units have been developed for the quantitative determination of ADA activity. These include the electrochemical aptasensor [12], colorimetric aptasensor [13] and fluorescence sensor [14]. Although these techniques can be quite powerful, a simpler and more sensitive method for ADA detection is still required. The carbon nanomaterials have a significant role to play in new developments in each of the biosensor size domains. This significance arises as nanomaterials can help address some of the key issues in the development of all biosensors [15]. For
K. Hu et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 164–168
example, a series of graphene oxide-based aptasensors have been developed for diverse biomolecules detection [16–18], the carbon nanoparticles and carbon nanodots have also extensively been used to develop biosensors [19,20]. Recently, carbon nanotubes (CNTs) have been extensively studied due to their unique optoelectronic properties and excellent biocompatibility [19]. Specifically, the extraordinary fluorescence quenching property of CNTs has been employed to develop nanosensors for diverse biomolecules in homogeneous solution. For example, the CNTs has used as a biosensing platform for the detection of DNA based on the quenching effect on the dye-labeled DNA probe [21]. Similarly, the CNTs-based aptasensors were also used for sensitive detection of various biomolecules [22,23]. However, to the best of our knowledge, no study has been reported the use of CNTs for homogeneous assay of ADA assay. In the present work, we developed a sensitive and selective fluorescent aptasensor based on multi-walled carbon nanotubes (MWCNTs) using AD as the substrate for ADA activity detection and inhibitor screening. This aptasensor relies on the high fluorescence quenching property of MWCNTs and the different interaction ability of aptamer, AD-aptamer complex with MWCNTs. Compared with the traditional fluorescence resonance energy transfer (FRET)-based aptasensor [14], the proposed assay only requires the labeling of the oligonucleotide probe with one dye, which is very simple and cost-effective. Moreover, this aptasensor exhibits high sensitivity and specificity toward ADA over other non-specific enzymes, with a detection limit of 0.002 U/mL for ADA. In addition, the suitability of this MWCNT-based fluorescence aptasensor for ADA inhibitor screening from TCM has also been demonstrated. 2. Experiment 2.1. Chemical and reagents The adenosine specific aptamer was splited into two singlestranded oligonucleotides (Apt-1 and Apt-2) [24], and their sequences were shown below: Apt-1: 5 -ACCTGGGGGAGTAT-3 ; and Apt-2: 5 -ATGCGGAGGAAGGT-3 . The Apt-1 was modified at 5 ends with 6-carboxyfluo rescein (FAM). And these aptamers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, Shanghai, China) and purified using high performance liquid chromatography. Multi-walled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanotech Port Co. Ltd. (Shenzhen, Guangdong, China). Adenosine, adenosine deaminase and inosine were purchased from Amresco Co., Ltd. (Solon, OH, USA). Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, DE, USA). Traditional Chinese medicine (TCM) was obtained from Guilin Pharmaceuticals Group of China (Guilin, Guangxi, China). Milli-Q water (18.2 M cm) was used throughout this work. All other reagents used in this work were of analytical grade. 2.2. Pretreatment of MWCNTs The commercial MWCNTs were purified and oxidized according to the procedure described by He and Bayachou [25]. The MWCNTs (200 mg) was first refluxed in HNO3 (150 mL, 2.0 mol/L) for two days. After being kept overnight, the suspension was centrifuged at 14,000 rpm for 30 min, and the clear solution was removed. The purified precipitates were further oxidized by 40 mL of HNO3 /H2 SO4 (VHNO3 : VH2 SO4 = 1 : 3) solution in an ultrasonic bath for 4 h. Then, the suspension was diluted 10-fold with water and allowed to stand for 12 h at room temperature. After removal of the clear solution over the precipitates, the remaining suspension was filtered through a 0.45 m filtration membrane and then
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washed with water to obtain a neutral pH. The resulting precipitates were dried in a vacuum drier about 12 h to remove the water. The obtained MWCNTs with a carboxyl group were re-dispersed in water to give a final concentration at 5 mg/mL. 2.3. Preparation of natural extracts Air-dried TCM was ground into a fine powder with a pulverizer. 5.0 g of powder was extracted with 50 mL of isopropanol in an ultrasonic cleaning bath for 0.5 h at 60 ◦ C, and this process was repeated three times. The solvent was evaporated using rotary evaporator at 40 ◦ C. Finally, these extracts were dried in vacuum drier for about 5.0 h. The natural extracts were dissolved with 2-propanol aqueous solution giving a final concentration of 5.0 mg/mL. Other natural extracts were prepared in the same way. 2.4. ADA activity detection The Apt-1 (10 L, 1 M) was mixed with Apt-2 (10 L, 1 M), and then 10 L of 5 mg/mL MWCNTs was added into the above solution in Tris–HCl buffer (10 mM, pH = 7.4), and allowed to incubate for 15 min. The fixed concentration of adenosine (1.0 M) was treated with different activity of ADA (0, 0.002, 0.005, 0.01, 0.015, 0.05, 0.1, 0.15, 0.20, and 0.30 U/mL), and the resulting solution was added to the aptasensor/MWCNTs complex solution in Tris–HCl buffer (10 mM, pH = 7.4). After 50 min of incubation at 37 ◦ C, the fluorescence emission spectra the solution were measured with a LS-55 spectrofluorometer (Perkin-Elmer, USA) at 520 nm with excitation at 480 nm. All experiments were repeated three times. Each sample was measured five times. 2.5. Inhibition screening procedures For comparison of the inhibition ability of test compounds, different concentration EHNA or 0.20 mg/mL of each TCM was mixed with 1.0 M adenosine solution before the addition of 0.20 U/mL ADA, and incubated 50 min at 37 ◦ C. Other assay steps were the same as that of the ADA activity assay. 3. Results and discussion 3.1. Assay principle Fig. 1 depicts the principle of the MWCNTs-based fluorescence aptasensor for ADA activity detection and inhibitor screening. This sensing system consists of two anti-AD aptamer fragments (FAMlabeled Apt-1 and Apt-2), AD and MWCNTs. In the absence of ADA, AD binds with both FAM-labeled Apt-1 and Apt-2 to form the AD/aptamer complex that cannot bind to MWCNTs stably. In this case, the FAM dye exhibits high background fluorescence (Fig. 1a). When ADA is introduced into the system, ADA can convert AD into inosine, which has not affinity with two anti-AD aptamer fragments. Then, the free anti-AD aptamer fragments can be adsorbed onto MWCNTs by means of strong – stacking interactions between nucleotide bases and the MWCNT sidewalls [21], resulting in the quenching of the fluorescence of the FAM dye (Fig. 1b). The fluorescence intensity should decrease with increased amount of ADA. In addition, since the conversion of AD into inosine by the ADA-catalyzed is restrained in the presence of inhibitors, the proposed aptasensor can be adapted to screen the inhibitors of ADA (Fig. 1c). 3.2. Feasibility study To investigate the feasibility of the proposed sensing strategy, the fluorescence spectra under different conditions were measured,
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Fig. 1. The principle of the MWCNT-based fluorescence aptasensor for adenosine deaminase activity and inhibitor screening.
and the experimental results are shown in Fig. 2. The fluorescence spectrum of the FAM-labeled Apt-1/Apt-2 mixture without MWCNTs showed strong fluorescence emission owing to the presence of the FAM dye (curve a). Upon the addition of MWCNTs into the FAM-labeled Apt-1/Apt-2 mixture, the fluorescence of the FAM was significantly quenched (curve e). This observation indicated strong adsorption of the FAM-labeled Apt-1 strand onto MWCNTs, and high fluorescence quenching efficiency of MWCNTs. When AD is introduced into the FAM-labeled Apt-1/Apt-2/MWCNTs mixture, the fluorescence intensity of the FAM increased significantly (curve b), suggesting that the AD-aptamer complex has been formed and released from MWCNTs surface. However, when AD complex with ADA was introduced into the FAM-labeled Apt-1/Apt-2/MWCNTs mixture, the fluorescence decreased significantly compared with that in curve b (curve d). This fluorescence decrease was attributed to that ADA catalyzed the conversion of AD into inosine that could not bind with the anti-AD aptamer fragments, and then the free anti-AD aptamer fragments were adsorbed onto MWCNTs. Further introduction of a model ADA inhibitor, EHNA, into the FAM-labeled Apt-1/Apt-2/AD/ADA/MWCNTs mixture, the fluorescence of the
FAM increased compared with that in curve d (curve c), indicating the inhibition of ADA by EHNA. The above results demonstrated that the designed aptasensor could be used for ADA activity detection and inhibitor screening. 3.3. Optimization of assay conditions To obtain high sensitivity, the amount of MWCNTs was first optimized. As shown in Fig. S1, the fluorescence intensity of the FAM-labeled Apt-1 fragment decreased gradually with the increase of the MWCNTs concentration, and reached the minimum value when 50 g/mL MWCNTs was employed. Under this condition, more than 90% of original fluorescence was quenched. Therefore, 50 g/mL was selected as the optimal MWCNTs concentration. Moreover, the effects of AD concentration were also investigated. The fluorescence intensity increased dramatically with the AD concentration up to 1.0 M and then reached an equilibration over 1.0 M (Fig. S2). Thus, the AD concentration of 1.0 M was selected for the subsequent assay. 3.4. Assay of ADA activity
Fig. 2. Fluorescence emission spectra of DNA probe (Apt-1 and Apt-2, each 50 nM) at different conditions: (a) DNA probe in Tris–HCl; (b) DNA probe + AD (1 M) + 50 g/mL MWCNTs; (c) ADA (0.20 U/mL) + inhibitor (20 nM) + AD (1 M) + DNA probe + MWCNTs (50 g/mL); (d) AD (1 M) + ADA (0.20 U/mL) + DNA probe + 50 g/mL MWCNTs; and (e) DNA probe + MWCNTs (50 g/mL).
To confirm the ability of the proposed aptasensor to detect target enzyme, a series of different concentrations of ADA were measured under the optimal conditions. Before ADA activity assay was examined, the effect on the ADA catalyzes product-inosine on the fluorescence intensity change of MWCNTs aptasensor was investigated (Fig. S3). It is clearly seen that the MWCNTs aptasensor has nearly no response to inosine (100.0 M). Fig. 3 depicts the fluorescence spectra of the proposed aptasensor upon analyzing different concentrations of ADA. As the concentration of ADA increases, the fluorescence intensity decrease gradually. This is consistent with the enhanced conversion of AD into inosine and the adsorption of more amounts of the FAM-labeled Apt-1 on the surface of MWCNTs. The derived calibration curve is shown in the inset of Fig. 3. As can be seen, a good linear relationship between fluorescence intensity and ADA concentration from 0.005 to 0.20 U/mL was obtained. The regression equation was obtained as follows: F = −697.92C + 187.94, R = 0.9963, where F and C are the fluorescence intensity and ADA activity (U/mL), respectively. The detection limits with the MWCNT-based fluorescent aptasensor was 0.002 U/mL, which was about two orders of magnitude lower than that of the electrochemical measurements (0.2 U/mL)
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Fig. 3. The fluorescence emission of the MWCNTs aptasensor with 1.0 M AD at different activity of ADA (from a to i): 0, 0.002, 0.005, 0.01, 0.015, 0.05, 0.1, 0.15, 0.20, and 0.30 U mL−1 . Inset: the plot of the fluorescence intensity changes versus the activity of ADA.
Fig. 5. The fluorescence emission of the MWCNTs aptasensor with 1.0 M AD, 0.20 U/mL ADA at different concentrations of EHNA (from a to g): 0, 0.5, 5.0, 10.0, 15.0, 20.0, and 25.0 nM. Inset: the inhibition plot of ADA in the presence of different concentrations of inhibitor EHNA.
[12]. These results indicated that the MWCNTs-based fluorescence aptasensor could detect ADA activity with high sensitivity. To evaluate the assay selectivity, the responses of the aptasensor to the target ADA and several non-specific enzymes including xanthine oxidase (XOD), DNA topoisomerase II, and thrombin was tested (Fig. 4). It was found that the target ADA led to a significant decrease in the fluorescence intensity, while no apparent fluorescence change was observed in the assay for non-specific enzymes. These results clearly demonstrated that the high specificity of proposed MWCNTs-based fluorescence aptasensor.
the Michaelis–Menten constant (Km ) for the ADA enzymatic reaction was estimated to be 71.6 M, which was in an acceptable agreement with that of the reported absorbance assay methods (89 M) [26,27]. This result indicated that no significant change on enzyme property was observed in the proposed sensing system.
3.5. Enzyme kinetics study To further confirm the validity of the MWCNTs-based fluorescence aptasensor for ADA activity detection, the kinetic behavior of ADA was studied by the present method. In these experiments, 0.20 U/mL ADA with different concentrations of AD substrate ranging from 0.2 to 1.0 M was assayed. According to Michaelis–Menten and Lineweaver–Burk equation, a doublereciprocal plot was constructed, and is shown in Fig. S4. The curve provided a maximum initial velocity (Vmax ) of 3.8 nmol min−1 , and
3.6. Inhibitor screening To explore the potential of the proposed aptasensor to screen ADA inhibitor, a known inhibitor of ADA, EHNA was first selected as a model for the inhibition study. Fig. 5 shows the fluorescence spectra of the sensing system upon analyzing 0.20 U/mL ADA in the presence of different concentrations of EHNA. As the concentration of EHEA increase, the fluorescence of the system was intensified, and the inhibition of ADA activity was enhanced. Fig. 5, insert, shows the derived calibration curve between the fluorescence intensity and the EHEA concentration. From this calibration curve, the IC50 value of EHEA was estimated to be 12 nM (R = 0.9951), which was in good agreement with previous reports [12,28]. Then, we further applied this MWCNTs-based fluorescence aptasensor to screen ADA inhibitors in ten types of TCMs. Before inhibition studies, the effect of the natural extracts from these TCMs on the fluorescence intensity of the MWCNTs-based fluorescence aptasensor was tested, and the experimental results showed that almost no fluorescence change of the aptasensor occurred in the presence of natural extracts (Fig. S5). This demonstrated no interference of natural extracts on the inhibition assays. After that, the inhibition of natural extracts from ten types of TCMs was assayed, and the results are summarized in Table 1. It can be seen that only Rhizoma chuanxiong was identified to be positive for ADA inhibition. Table 1 Extracts library used for inhibitor screening.
Fig. 4. Selective of MWCNTs based ADA (1) aptasensor over thrombin (2), xanthine oxidase (3), and DNA topoisomerase II (4). Where I0 and I correspond to the fluorescence intensity before and after adding enzyme, respectively. The activity of ADA is 0.20 U/mL and other nontarget enzyme is 2.0 U/mL.
TCM
Inhibition (%)
TCM
Inhibition (%)
Aloe barbadensis Radix sophorae tonkinensis
0
43.6
Rhizoma corydalis Herba sedi Rhizoma dioscoreae
0
Rhizoma chuanxiong Rhizoma curcumae aeruginosae Rhizoma coptidis Semen ginkgo Fructus schisandrae
0
0 0
0
0 0 0
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This result was consistent with that of our previous report [28]. These results indicated that the proposed MWCNT-based fluorescent aptasensor was suitable for screening the ADA inhibitors from TCMs. 4. Conclusion In summary, we have developed a MWCNTs-based fluorescent aptasensor for rapid, sensitive, and selective detection of ADA activity and screening of inhibitor. This assay approach has several important advantages. Firstly, the assay is homogeneous, “mixand-read”, and does not require separation and washing steps, which is very simple and convenient. Second, the sensitivity of proposed assay is about two orders of magnitude higher than that of the reported electrochemical methods. Thirdly, owing to the high specificity of aptamer, this approach exhibits high selectivity for ADA. Finally, this aptasensor is suitable for screening ADA inhibitor from TCMs. Considering these qualities, the developed fluorescent aptasensor not only provides a sensing platform for enzyme activity detection, but also holds great potential in inhibitor screening. Acknowledgments The authors gratefully acknowledge the financial support provided by National Natural Science Foundation of China (nos. 21175030, 21305021, 21165004), the Guangxi Natural Science Foundation of China (nos. 2013GXNSFBA019044, 2013GXNSFBA019044, 2013GXNSFBA019038), IRT1225, and “Bagui Scholar Program of Guangxi”. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.02.027. References [1] E.J. Conway, R. Cooke, The deaminases of adenosine and adenylic acid in blood and tissues, Biochem. J. 33 (1939) 479–492. [2] B.K. Gupta, V. Bharat, D. Bandyopadhyay, Role of adenosine deaminase estimation in differentiation of tuberculous and non-tuberculous exudative pleural effusions, J. Clin. Med. Res. 2 (2010) 79–84. [3] M.S. Hershfied, Genotype is an important determinant of phenotype in adenosine deaminase deficiency, Curr. Opin. Immunol. 15 (2003) 571–577. [4] J. Linden, Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 775–787. [5] M.K. Paul, V. Grover, A.K. Mukhopadhyay, Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase, J. Chromatogr. B 822 (2005) 146–153. [6] P. Vielh, M.J. Castellazzi, A colorimetric assay for serial determination of adenosine deaminase activity in small lymphocyte populations, J. Immunol. Methods 73 (1984) 313–320.
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